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Testing Methods for Fertilizers(2014)

Incorporated Administrative Agency
Food and Agricultural Materials Inspection
CenterIntroduction“The Official Methods of Analysis of Fertilizers” stipulated by the Ministry of Agriculture,
Forestry and Fisheries is the only analysis method to assess main components and harmful
components in fertilizers in Japan and contributes to maintaining quality and securing safetyof fertilizers. However no new revision has been issued since “The Official Methods ofAnalysis of Fertilizers 1992” was issued. Some quarters such as fertilizer manufacturers andinspection instruction agencies have requested a revised edition of “The Official Methods ofAnalysis of Fertilizers” since new kinds of fertilizers and its new components were added into
the public standard, and analysis instruments and technologies have progressed during theperiod.
Incorporated Administrative Agency, Food and Agricultural Materials Inspection Center(FAMIC) has revised the Official Methods of Analysis of Fertilizers by introducing theanalysis conditions and the analysis methods, etc. which meet the latest situation. Additionally,
FAMIC tried to study how to introduce the analysis methods or the new analysis instruments
to cope with new effective components or harmful components and new fertilizers which are
not documented in the Official Methods of Analysis of Fertilizers, and established new testing
methods. At the same time, FAMIC conducted a validity test according to the requirements of
ISO/IEC 17025 and opened the results and new testing methods which were discussed andapproved by “the Technical Committee for Fertilizers etc.” including outside experts on
FAMIC’s web site in 2008 as “The Testing Methods for Fertilizers 2008”. Since then, thecontents have been annually added and updated. In this year, two testing methods such as
nitrous acid (High Performance Liquid Chromatography), whose validity were checked at asingle laboratory in 2013, were merged as reference methods. In addition, four testing
methods such as melamine (High Performance Liquid Chromatography) were formallymerged from reference methods and the “Testing Methods for Fertilizes 2014” in which 57
items (components, etc.) and 94 testing methods were incorporated was opened on FAMIC’s
web site.
In addition, the procedure to validate characteristics of testing methods is appended as an
Appendix this year. The Appendix explains the procedure for performance evaluation of
testing methods which will be listed in the Testing Methods of Fertilizers, and furthermore
evaluation criteria prepared based on the validation test results of new testing methods
hitherto conducted and the performance investigation results of existing methods, etc. isshown in the Appendix.
Although the “Testing Methods for Fertilizes” cannot substitute “the Official Methods ofAnalysis of Fertilizers” which is stipulated as an official method in an official notice of the
Ministry of Agriculture, Forestry and Fisheries, reagents and instruments used are stipulated
in JIS standards, the validity of the testing method is checked by referencing IUPAC protocols
and it is listed as an analysis method whose validity is checked in “The Management
Handbook of Heavy Metal in Sludge Fertilizers” published by the Ministry of Agriculture,
Forestry and Fisheries in August 2010. Therefore FAMIC would like people engaged in thequality control and analysis of fertilizes to use this as a practical document.
December, 2014
Incorporated Administrative Agency
Food and Agricultural Materials Inspection Center
General Director
Makoto Kimura

2

The members of The Technical Committee for the Testing Methods for Fertilizers(As of March, 2014)(Without honorifics)
Mayumi AIZAKI

Saitama Prefectural Agriculture and Forestry Comprehensive
Research Center

Masashi UWASAWA

Japan Fertilizer and Feed Inspection Association

Akira KAWASAKI

Incorporated Administrative Agency: National Institute forAgro-Environmental Sciences

Narihiro NAITO

Incorporated Administrative Agency: National Agriculture andFood Research Organization

Munetomo NAKAMURA Japan Food Research Laboratories
Akira NOGUCHI

Incorporated Educational Institution: Nihon University

Kazuyuki YAZIMACorporate Juridical Person: Niigata Prefectural Institute of Public
Health and Environmental Sciences

Akemi YASUIIncorporated Administrative Agency: National Agriculture andFood Research Organization

3

Contents
Introduction
1. General Rules
1.1 Common Items
1.2 Validity check of testing methods

……2
……8
……8
…..11

2.

Handling of samples
2.1 Storage of samples
2.2 Preparation of test samples
2.2.1 Pre-drying
2.2.2 Reduction (Separation)
2.2.3 Grinding

…..12
…..12
…..13
…..14
…..16
…..17

3.

General tests
3.1 Moisture
3.1.a Loss on drying method with drying apparatus
3.1.b Loss on drying method by moisture analyzers
3.2 Ash content
3.2.a Ignition residue method
3.3 pH
3.3.a Glass electrode method
3.4 Electrical conductivity
3.4.a Measurement method with an electrical conductivity meter
3.5 Granularity
3.5.a Dry-type sieving testing method
3.6 Oil content
3.6.a Diethyl ether extraction method

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…..18
…..18
…..22
…..25
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…..32
…..32

4.

Main components, guaranteed components, etc.
4.1 Nitrogen4.1.1 Total nitrogen4.1.1.a Kjeldahl method
4.1.1.b Combustion method
4.1.1.c Devarda’s alloy – Kjeldahl method
4.1.1.d Reduced iron– Kjeldahl method
4.1.1.e Calculation with ammoniac nitrogen and nitrate nitrogen
4.1.2 Ammoniac nitrogen
4.1.2.a Distillation method
4.1.2.b Formaldehyde method
4.1.3 Nitrate nitrogen
4.1.3.a Devarda’s alloy - distillation method
4.1.3.b Reduced iron- distillation method
4.1.3.c Phenol sulfuric acid method
4.2 Phosphate
4.2.1 Total phosphate
4.2.1.a Ammonium vanadomolybdate absorptiometric analysis
4.2.1.b Quinoline gravimetric analysis
4.2.2 Soluble phosphoric acid
4.2.2.a Ammonium vanadomolybdate absorptiometric analysis
4.2.2.b Quinoline gravimetric analysis

…..34
…..34
…..34
…..34
…..40
…..46
…..51
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…..67
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…..82
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…..82
…..88
…..91
…..91
…..96

4

4.2.3 Citrate soluble phosphoric acid…..99
4.2.3.a Ammonium vanadomolybdate absorptiometric analysis…..99
4.2.3.b Quinoline gravimetric analysis….103
4.2.4 Water-soluble phosphoric acid….106
4.2.4.a Ammonium vanadomolybdate absorptiometric analysis….106
4.2.4.b Ammonium vanadomolybdate absorptiometric analysis….111(Fluid mixed fertilizers containing phosphonic acid or phosphite)
4.2.4.c Quinoline gravimetric analysis….115
4.3 Potassium….118
4.3.1 Total potassium….118
4.3.1.a Flame atomic absorption spectrometry or flame photometry….118
4.3.1.b Sodium tetraphenylborate gravimetric analysis….122
4.3.2 Citrate soluble potassium….125
4.3.2.a Flame atomic absorption spectrometry or flame photometry….125
4.3.2.b Sodium tetraphenylborate gravimetric analysis….128
4.3.2.c Sodium tetraphenylborate volumetric analysis….131
4.3.3 Water-soluble potassium….134
4.3.3.a Flame atomic absorption spectrometry or flame photometry….134
4.3.3.b Sodium tetraphenylborate gravimetric analysis….138
4.3.3.c Sodium tetraphenylborate volumetric analysis….142
4.4 Silicic acid….145
4.4.1 Soluble silicic acid….145
4.4.1.a Potassium fluoride method….145
4.4.1.b Potassium fluoride method (Silica gel fertilizers, etc.)….149
4.4.1.c Potassium fluoride method (Fertilizers containing silica gel fertilizers)….153
4.4.1.d Perchloric acid method….158
4.4.2 Water-soluble silicic acid….161
4.4.2.a Potassium fluoride method….161
4.5 Lime, calcium and alkalinity….164
4.5.1 Total lime….164
4.5.1.a Flame atomic absorption spectrometry….164
4.5.2 Soluble lime….167
4.5.2.a Flame atomic absorption spectrometry….167
4.5.3 Water-soluble calcium….170
4.5.3.a Flame atomic absorption spectrometry….170
4.5.4 Alkalinity….173
4.5.4.a Ethylenediamine tetraacetate method….173
4.5.4.b Calculation from soluble lime and soluble magnesia….179
4.6 Magnesia….180
4.6.1 Soluble magnesia….180
4.6.1.a Flame atomic absorption spectrometry….180
4.6.2 Citrate soluble magnesia….183
4.6.2.a Flame atomic absorption spectrometry….183
4.6.3 Water-soluble magnesia….187
4.6.3.a Flame atomic absorption spectrometry….187
4.7 Manganese….190
4.7.1 Soluble manganese….190
4.7.1.a Flame atomic absorption spectrometry….190
4.7.2 Citrate soluble manganese….193
4.7.2.a Flame atomic absorption spectrometry….193
5

4.7.3 Water-soluble manganese
4.7.3.a Flame atomic absorption spectrometry
4.8 Boron
4.8.1 Citrate soluble boron
4.8.1.a Azomethine-H method
4.8.2 Water-soluble boron
4.8.2.a Azomethine-H method
4.9 Zinc4.9.1 Total zinc
4.9.1.a Flame atomic absorption spectrometry
4.9.1.b ICP Atomic Emission Spectrometry <Reference method>
4.9.2 Water-soluble zinc
4.9.2.a Flame atomic absorption spectrometry
4.10 Copper
4.10.1 Total copper
4.10.1.a Flame atomic absorption spectrometry
4.10.1.b ICP Atomic Emission Spectrometry <Reference method>
4.10.2 Water-soluble copper
4.10.2.a Flame atomic absorption spectrometry
4.11 Organic carbon and carbon-nitrogen ratio
4.11.1 Organic carbon
4.11.1.a Dichromate oxidation
4.11.1.b Combustion method
4.11.2 Carbon-nitrogen ratio
4.12 Sulfur
4.12.1 Total sulfur content
4.12.1.a Titration analysis <Reference method>
4.12.1.b Barium chloride gravimetric analysis
4.12.1.c Transmitted light analysis <Reference method>
4.13 Iron
4.13.1 Water-soluble iron4.13.1.a Flame atomic absorption spectrometry
4.14 Molybdenum
4.14.1 Water-soluble molybdenum
4.14.1.a Sodium thiocyanate absorptiometric analysis
5.

….196
….196
….199
….199
….199
….202
….202
….205
….205
….205
….208
….212
….212
….215
….215
….215
….219
….223
….223
….226
….226
….226
….229
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….234
….234
….234
….236
….240
….244
….244
….244
….247
….247
….247

Harmful components….250
5.1 Mercury….250
5.1.a Cold vapor atomic absorption spectrometry….250
5.2 Arsenic….254
5.2.a Hydride generation atomic absorption spectrometry….254
5.2.b Silver diethyl dithiocarbamate absorptiometric analysis….260
5.3 Cadmium….264
5.3.a Flame atomic absorption spectrometry….264
5.3.b ICP Atomic Emission Spectrometry <Reference method>….268
5.4 Nickel….272
5.4.a Flame atomic absorption spectrometry….272
5.4.b ICP Atomic Emission Spectrometry <Reference method>….276
5.5 Chromium….280
5.5.a Flame atomic absorption spectrometry (Fertilizers containing organic matters)….280
6

5.5.b Flame atomic absorption spectrometry(Fertilizers not containing organic matters)
5.5.c Flame atomic absorption spectrometry (Calcined sludge fertilizer)
5.5.d ICP Atomic Emission Spectrometry <Reference method>
5.6 Lead
5.6.a Flame atomic absorption spectrometry
5.6.b ICP Atomic Emission Spectrometry <Reference method>
5.7 Sulfamic acid (amidosulfuric acid)5.7.a Ion Chromatography <Reference method>
5.8 Sulfurized cyanide (ammonium thiocyanate)
5.8.a Ion Chromatography <Reference method>
5.8.b High Performance Liquid Chromatography <Reference method>
5.9 Nitrous acid
5.9.a High Performance Liquid Chromatography <Reference method>

….284
….288
….292
….296
….296
….300
….304
….304
….308
….308
….312
….316
….316

6.

Testing relating to the other limitations
6.1 Dicyandiamide nitrogen
6.1.a High Performance Liquid Chromatography

….320
….320
….320

7.

Nitrification inhibitor
7.1 2-amino-4-chloro-6-methylpyrimidine (AM)
7.1.a High Performance Liquid Chromatography
7.2 1-amidino-2-thiourea (ASU)
7.2.a High Performance Liquid Chromatography
7.3 4-amino-1,2,4-triazole hydrochloride (ATC)7.3.a High Performance Liquid Chromatography
7.4 N-2,5-dichlorophenyl succinamic acid (DCS)
7.4.a High Performance Liquid Chromatography
7.5 Dicyandiamide (Dd)
7.5.a High Performance Liquid Chromatography
7.6 2-sulfanilamide thiazole (ST)7.6.a High Performance Liquid Chromatography

….324
….324
….324
….328
….328
….332
….332
….335
….335
….339
….339
….344
….344

8.

Others….349
8.1 Melamine and its degradation products….349
8.1.a Gas Chromatography/Mass Spectrometry <Reference method>….349
8.1.b High Performance Liquid Chromatography (Nitrolime) <Reference method>….355
8.1.c High Performance Liquid Chromatography(Fertilizers not containing organic matters)….359
8.2 Clopyralid and its degradation products….364
8.2.a High Performance Liquid Chromatography/Tandem Mass Spectrometry<Reference method>….364
8.3 Residue agrichemicals multi component analysis….371
8.3.a High Performance Liquid Chromatography/Tandem Mass Spectrometry ….371

Appendix.

The procedure to validate characteristics of testing methods
7

….379

1.

General Rules

1.1

Common Items(1) Applicable range
The Testing Methods for Fertilizers stipulate the official method of analysis of fertilizers andfertilizer materials. The type of samples in the tests is shown in the summary of respective test
items.
(2) General matters and terms in commona) General notices: General matters common to analysis are according to JIS K 0050.
b) Definition: The definitions of major terms used in the Testing Methods for Fertilizers
are according to JIS K 0211, JIS K 0214, JIS K 0215, JIS Z 8101-1, JIS Z 8101-2 or JIS
Z 8101-3.
c) Main components or major components: The main components or major components
in fertilizers in Table 1 are stipulated as components to be calculated by a public noticeof the Ministry of Agriculture, Forestry and Fisheries.
Table 1

Components to be calculated as main component
or major component in fertilizers

Main component
component
Phosphate
Potassium

or

major

Component to be calculated
Phosphorus pentoxide (P2 O5 )Potassium oxide (K 2 O)

Silicate
Magnesium
Manganese
Boron
Sulfur content

Silicon dioxide (SiO2 )Magnesium oxide (MgO)
Manganese oxide (MnO)
Boron oxide (B2 O3 )Sulfur trioxide (SO 3 )Calcium oxide (CaO)

Limed) Organic matters: Organic fertilizers, sludge fertilizers, compost, etc. Organic
compounds such as urea are excluded.
e) Laboratory sample: A sample transferred to a laboratory. Laboratory sample as
specified in JIS K 0211.
f) Test sample: A sample obtained from a laboratory sample after pretreatment such as
grinding. Test sample as specified in JIS K 0211.
g) Analytical sample: A sample measured from a laboratory sample or a test sample andto be used in one test.
h) Sample: A sample in the Test methods indicates a laboratory sample, a test sample or ananalytical sample.
i) Dilution of solution: “Transfer accurately a predetermined amount (to a vessel)” means
the procedure to measure any volume of solution with a measuring instrument specified
in JIS K 0050 (into a vessel).
Also, “dilute accurately a predetermined amount (with solvent or solution)” means theprocedure to measure any volume of solution with a measuring instrument specified in
JIS K 0050 into a volumetric flask of arbitrary volume and fill up to the markedline(with solvent or solution).(1)j) Preparation of a calibration curve: “Transfer A mL - B mL of the standard solution tovolumetric flasks step-by-step.” means the procedure to transfer a volume of 4 - 6 steps(2)in the range from A mL to B mL of the standard solution to respective volumetric8flasks step-by-step.
Prepare a calibration curve every time a test is conducted. Also, when the same test item
is measured under the same conditions for multiple samples continuously, measure thestandard solution at regular intervals to check the indicated value.
k) Notes, comments, figures, tables and formulae: Serial numbers for each test item
should be recorded in notes, comments, figures, tables and formulae.
l) Rounding numbers: Methods of rounding the numbers are according to JIS Z 8401.
m) Referential matters related to the validity of testing methods: Information related to
the validity of respective test methods such as quantification range (minimum andmaximum limits of quantification), mean recovery, repeatability and reproducibility is
described in a Comment, etc. Note that the numerical values such as Minimum Limit of
Quantification, etc. are not standards to be targeted but examples.
n) Absorptiometric analysis: General rules for absorptiometric analysis are according to
JIS K 0115.
o) Atomic absorption spectrometry: Atomic absorption spectrometry includes flame
atomic absorption spectrometry, electrically heated atomic absorption spectrometry(hereinafter referred to as “electrically heated atomic absorption spectrometry”) andother atomic absorption spectrometry. General matters common to these are according
to JIS K 0121.
p) Gas chromatography: General matters common to gas chromatography are according
to JIS K 0114.
q) Gas chromatography/Mass spectrometry: General matters common to gas
chromatography/Mass spectrometry are according to JIS K 0123.
r) Electrical conductivity measuring method: General matters common to electricalconductivity measuring methods are according to JIS K 0130.
s) Test sieving: General matters common to test sieving are according to JIS Z 8815.
t) High Performance Liquid Chromatography: General matters common to High
Performance Liquid Chromatography are according to JIS K 0124.
u) High Performance Liquid Chromatography/Mass Spectrometry: General matterscommon to High Performance Liquid Chromatography mass spectrometry areaccording to JIS K 0136.
v) ICP Atomic Emission Spectrometry: General matters common to ICP Atomic
Emission Spectrometry are according to JIS K 0116.
w) Ion Chromatography: General matters common to Ion Chromatography are according
to JIS K 0127.
Note (1) When the dilution factor is large, accuracy should be secured by procedures suchas repeating the dilution procedure.
(2) Set according to the specification and operation method of the measurement
instrument used. There is no need to include the minimum and the maximum
values.
(3) Watera) Water: Water used in the Testing Methods for Fertilizers herein is water of A2 specified
in JIS K 0557 or water that is confirmed not to affect a quantitation value. However,when otherwise specified in respective test items, use the specified water.
(4) Reagentsa) Reagents: When the reagent is JIS-specified, use one of highest quality among thosemarked with the JIS symbol; when none of the reagent is marked with the JIS symbol,
use one of quality that will not cause a problem in the test. Use reference materials forvolumetric analysis specified in JIS K 8005 for the standardization of titration solutions.
9b) Concentration of reagent solution: Unless otherwise specified, the mass concentration
is expressed as g/L or mg/L, while the molar concentration is expressed as mol/L or
mmol/L. The concentration of the standard solution is expressed as the mass in 1 mL
(mg/mL, µg/mL or ng/mL) except for the ion-selective electrode method.
c) Concentration in parenthesis shown after the name of reagent solution: It indicatesthat the solution is about that concentration except the standard solution. For example,
sodium hydroxide solution (0.1 mol/L) means that it is about 0.1 mol/L sodiumhydroxide solution. Also, the concentration shown in front of the name of solutionmeans that it is the accurate concentration. However, the concentration is generally
expressed as a round figure; calculate the factor separately.
d) Description of mixture solution: Mixture solutions are described as shown in 1) - 4).
1) Reagent + reagent: Describe as reagent name 1 - reagent name 2 (V1 + V2). In thiscase, V1 volume of reagent name 1 is mixed with V2 volume of reagent name 2.
Example: acetonitrile-water (1+1), hexane-ethyl acetate (2+1), methanol-buffer
solution (3+1)2) Reagent + water: Describe as reagent name 1 (V1+V2). In the case of reagentsdescribed in Table 1 in JIS K 0050, it means V1 volume of reagent name 1 is dilutedby mixing with V2 volume of water.
Example: hydrochloric acid (1+1), sulfuric acid (1+2), ammonia solution (1+3)
3) Solution + reagent: Describe as solution name a (concentration) - reagent name b[V1+V2]. In this case, it means V1 volume of solution name a of a certain
concentration is mixed with V2 volume of reagent name b.
Example: sodium hydroxide solution (4 g/L) - methanol [1+4]4) Diluted reagent + reagent: Describe as reagents name a (V1+V2) - reagent name b[V3+V4]. In this case, V3 volume of the solution in which V1 volume of reagent
name a described in Table 1 in JIS K 0050 diluted by mixing with V2 volume ofwater, is mixed with V4 volume of reagent name b.
Example: hydrochloric acid (1+100) -methanol [2+3]e) Water for reagent preparation: Use water in (3) a). However, when otherwise
specified in respective test items, use the specified water.
f) Reagent name: Unless otherwise specified, conform to the names by the chemical
nomenclature established by the Chemical Society of Japan [in accordance with theInternational Union of Pure and Applicable Chemistry (IUPAC) nomenclature of
inorganic chemistry and nomenclature of organic chemistry] and the names of JIS
reagents.
g) Handling of reagents and liquid waste, etc.: Handle with care and in compliance with
relevant laws and regulations.
(5) Apparatusa) Glass apparatus: Unless otherwise specified, use glass apparatus specified in JIS R
3503 and JIS R 3505. Also, when a heating procedure is involved, use borosilicate
glass-1 specified in JIS R 3503.
b) Desiccants for desiccators: Unless otherwise specified, use silica gel.
c) Porcelain crucibles and porcelain basins: Use ones specified in JIS R 1301 and JIS R
1302.
d) Platinum crucibles and platinum dishes: Use ones specified in JIS H 6201 and JIS H
6202.
e) Filter paper: Use that specified in JIS P 3801. However, the type of filter paper is
specified in respective test items.
f) Absorbance measurement (absorptiometric analysis) absorbance cells: Unlessotherwise specified, use ones of 10 mm in optical path length.
10

1.2

Validity check of testing methods

The Testing Methods for Fertilizers have been discussed and approved by the TechnicalCommittee for Fertilizers etc. The Testing Methods for Fertilizers will be revised such as byadding, modifying, or deleting test methods with the approval of the Technical Committee for
Fertilizers etc. due to the needs such as from progress in analysis techniques and changing
social situation.
Additionally, although validated test methods can be used instead of the test methods herein, a
final judgment should be determined with a stipulated method when a result is doubtful.
Comment

The procedure of the validity check of testing methods is shown in the
Appendix of this Testing Methods for Fertilizers. This procedure was madebased on 5.4.5 Validation of methods in JIS Q 17025 “General requirements
for the competence of testing and calibration laboratories” or 2.4 Tests forvalidation requirements in “Guidelines for the design and implementation ofsurveillance and monitoring and for the evaluation and publication of theresults” which was issued by the Ministry of Agriculture, Forestry and
Fisheries. Validated test methods are methods which are conducted according
to this procedure and confirmed conforming to the standards such as required
accuracy (trueness and precision), quantification range (maximum andminimum limits of quantitation) and so on.

11

2.

Handling of samples

2.1

Storage of samples(1) Summary
Put a sample in a container suitable for its characteristics and form and seal it tightly, and thenstore the sample at room or cold temperature. Care should be taken not to freeze it when it isstored at cold temperature.
(2) Apparatus and instruments: Apparatus and instruments are as shown below:a) Refrigerator: A refrigerator that can be adjusted to 1 ºC - 5 ºC.
b) Storage container for a sample: A storage container for a sample should be clean,strong and completely sealed airtight. In particular, in case it contains sludge for raw
materials, the container should be made of non-degradable, non-absorbable material.
Additionally, it should be airtight, water-proof, vapor-proof and non-corrosive.
(3) Procedure: Conduct storage as shown below.
a) Store a relatively stable sample in a tightly sealed container to avoid direct sunlight.
b) Store a sample in a desiccator, etc. by tightly sealing it if test results are affected bymoisture absorption.
c) Store a sample in a tightly sealed container in a dark place at 1 ºC - 5 ºC if it is easily
deteriorated by moisture.

12

2.2

Preparation of test samples(1) Summarya) Prepare a test sample by pre-drying, reducing, and grinding laboratory samples asnecessary.
b) Conduct pre-drying if a laboratory sample is moist and hard to grind.
c) A laboratory sample made from such fertilizers as a fluid fertilizer or a particle-fertilizer,
etc. that is sufficiently homogeneous can be used as a test sample.
d) If contamination by apparatus affects a test result, procedures such as pre-drying,
reduction and grinding are prohibited.
e) Note that part of a test sample should not scatter, nor should surrounding fine particles
or other alien substances be mixed with the test sample being prepared
References
1) JIS M 8100: Particulate materials - General rules for methods of sampling (1992)
2) JIS K 0060: Sampling method of industrial wastes (1992)

13

2.2.1 Pre-drying(1) Summary
Conduct pre-drying if a laboratory sample is moist and hard to grind. Measure the rate ofvolatile matter content and convert the analyzed value of respective tests to the content of alaboratory sample (actual article).

(2) Apparatus and instruments: Apparatus and instruments are shown below.
a) Drying apparatus: A drying apparatus that can be adjusted to the pre-drying
temperature at ± 2 ºC.
b) Sample drying dish: Measure the mass to the order of 0.1g in advance. Additionally,
use materials of a quality that do not affect the measurement of test components.
(3) Procedure: Conduct pre-drying as shown below.
a) Transfer 250 g - 1 kg of a laboratory sample to a sample drying dish, spread uniformly
and measure the mass to the order of 0.1g.
b) Place a sample drying dish containing a laboratory sample in a drying apparatus and dry(1).
c) Remove a sample drying dish from a drying apparatus and leave at rest at room
temperature until it is balanced with atmospheric temperature (2).
d) After leaving at rest, measure the mass of c) to the order of 0.1 g.
e) Calculate loss on drying in the pre-drying by the following formula (1). If necessary,calculate a conversion factor (actual article) by the following formula (2).
Loss on drying (% (mass fraction)) = ((A - B)/A) × 100 ………. (1)Conversion factor (actual article) = B/A………… (2)A: Mass (g) of the sampled laboratory sample
B: Mass (g) of the laboratory sample after drying
Note (1) Examples of drying temperature and drying time: About 70 hours at 40 ºC, noless than 5 hours at 65 ºC.
(2) An example of time to leave at rest: About 20 minutes
Reference
1) Mariko AIZAWA, Yuji SHIRAI, Yasushi SUGIMURA, Yuichi TAKAHASHI, Jun OKI,
Yukio FUKUCHI and Norio HIKICHI: Evaluation of Pre-drying Procedure to Prepare
Test Samples from Sludge Fertilizer, Research Report of Fertilizer Vol.1, 2008, (122 128)

14

(4)

Flow sheet for pre-drying: The flow sheet for the pre-drying of a moist laboratory
sample is shown below.
250 g – 1 kg ofLaboratory sample
DryingTransfer to a sample drying dish and spread uniformly.
Measure the mass to the order of 0.1 g
Example: About 70 hours at 40 ºC, no less than 5 hours at 65 ºC

Standing to coolRoom temperature

Measurement

Measure the mass to the order of 0.1 g

Figure The flow sheet for pre-drying

15

2.2.2 Reduction (Separation)(1) Summary
In order to distinguish a test sample from a sample for granularity test and physicalcharacteristics test, etc., reduce (separate) a laboratory sample with an increment reduction
method, a riffle sampler method or a conical quartering method.
(2) Apparatusa) Scoop for increment reduction: A scoop for increment reduction specified in theattached chart 1 of JIS M 8100.
b) Riffle sampler: A riffle sampler specified in the attached chart 3 of JIS M 8100.
(3)
a)
b)
c)

Procedure: Conduct reduction (separation) as shown below.
Increment reduction method: Conduct as indicated in 6.5.2 of JIS M 8100.
Riffle sampler method: Conduct as indicated in 6.5.3 of JIS M 8100.
Conical quartering method: Conduct as indicated in 6.5.4 of JIS M 8100.

16

2.2.3 Grinding(1) Summary
In order to prepare a homogeneous test sample, grind a laboratory sample with an adequate
grinder until it completely passes through the designated granularity.
(2) Apparatus and instruments: Apparatus and instruments are as shown below.
a) Grinder: Use a grinder (2) of a type and competence suitable for the granularity and thephysical characteristics (1) of a laboratory sampleb) Primary grinder: A grinder (3) that can primarily grind a large lump.
c) Cutter machine: A cutter that can cut long stems, etc.
d) Sieve: A sieve for the test specified in JIS Z 8801-1 or JIS Z 8801-2 or equivalents.
Note (1) The physical characteristics of a laboratory sample are defined by their solidity,
toughness, specific gravity and adhesiveness.
(2) A centrifuging type grinder, a cutting mill, a vibration mill type grinder, etc.
(3) A blender with an attachable blade, etc.
(3) Procedure: Conduct grinding as shown below.
(3.1) Fertilizers except ones specified in (3.2): Conduct as specified in 6.4 of JIS M 8100
and as shown below.
a) Break or cut a laboratory sample with a primary grinder or a cutting machine as
necessary.
b) Grind with a grinding machine until it completely passes through a sieve of 500 µm - 1
mm aperture.
c) Mix ground samples to make the test sample.
(3.2) Fused phosphate fertilizer, calcined phosphate fertilizer, silica fertilizer, calcareous
fertilizer, magnesia fertilizer, manganese fertilizers, etc.: Conduct as specified in 6.4
of JIS M 8100 and as shown below.
a) Grind a laboratory sample with a vibration mill type grinder.
b) Transfer the ground laboratory sample to a sieve of 212 µm apertures.
c) Incline the sieve about 20 degrees, supporting it with one hand or a bent arm, and tap
the sieve frame with the other hand at the rate of about 120 times per minute. During theprocedure, place the sieve in a horizontal position at the rate of 4 times per minute,rotate it 90 degrees and tap the sieve frame firmly one or two times.
d) When fine powder attaches to the back side of a sieve screen, remove it gently from theback side to make minus sieve.
e) Regarding the plus sieve of a sample, make them pass through by repeating theprocedure in a) - d).
f) Combine and mix the sample passed to make the test sample.
Comment 1 The procedures in b) - d) are the procedures in 6.1.3 (1.4) of JIS Z 8815.

17

3.

General tests

3.1

Moisture

3.1.a Loss on drying method with drying apparatus(1) Summary
Use drying apparatus under conditions suitable for the kind of fertilizers to be measured toheat analytical samples to measure loss on drying. This test method corresponds to loss onheating in the Official Methods of Analysis of Fertilizers (1992).
(2) Apparatus and instruments: Use apparatus and instruments as shown below:a) Drying apparatus: Drying apparatus that can be adjusted to the test temperature ± 2 ºC.
b) Ground-in stoppered weighing bottles (1): Low-form weighing bottles, 50 mm × 30
mm, specified in JIS R 3503. Dry by heating in advance in a drying apparatus at 75 ºC 130 ºC, stand to cool in a desiccator, and measure the mass to the order of 1 mg.
Note (1) Aluminum weighing dishes described in the Handbook of the Feed Analysis
Standards -2009- can also be used.
(3) Measurement: Conduct measurement as shown below.
a) Transfer 2 g-5 g of an analytical sample to a ground-in stoppered weighing bottle,
spread so that the thickness is no more than 10 mm, and measure the mass to the orderof 1 mg.
b) Place the ground-in stoppered weighing bottle containing the analytical sample in adrying apparatus at 100 ºC ± 2 ºC, and heat for 5 hours.(2)c) After heating, fit the stopper into the ground-in stoppered weighing bottle, andimmediately transfer to a desiccator to let it stand to cool.
d) After cooling, remove the ground-in stoppered weighing bottle from the desiccator, andmeasure the mass to the order of 1 mg.
e) Calculate loss on drying in the analytical sample by the following formula (1) asmoisture. If necessary, calculate the conversion factor (dry matter) by the following
formula (2):Loss on drying (% (mass fraction)) = ((A - B)/A) × 100……… (1)

Conversion factor (dry matter) = A/B……………………………….. (2)A:
B:

Mass (g) of the sampled analytical sample
Mass (g) of the analytical sample after drying

Note (2) Heat simultaneously the slightly moved or removed stopper of the ground-in
stoppered weighing bottle.
Comment 1 When pre-drying is conducted, calculate the moisture of the laboratory
sample (actual article) by the following formula:

Total moisture (% (mass fraction)) in the laboratory sample (actual article) = A + B ×((100 - A)/100)

18

A: Loss on drying (% (mass fraction)) of the laboratory sample (actual article)
by the pre-drying procedure
B: Loss on drying (% (mass fraction)) in the analytical sample by moisturemeasurement
Comment 2 Use drying conditions in Table 1 for fertilizers of the types shown below:

Table 1
Type of fertilizers

Drying conditions

Sampling amount ofDrying temperature
analytical samples

Drying time

Superphosphate, concentrated
superphosphate fertilizer, or
fertilizers containing these

About 5 g

100 ºC ± 2 ºC

3 hours

Ammonium sulfate, sodium
nitrate, and potassium salts

2 g- 5 g

130 ºC ± 2 ºC

Until a constantweight is achieved.

Urea and urea-containing
fertilizers

About 5 g

75 ºC ± 2 ºC

4 hours

Comment 3 For samples containing volatile matters, subtract the volatile matter content
by the following a) and b) from loss on drying to obtain moisture.
a) Fertilizers containing guano or diammonium hydrogen phosphate, etc.: Determine total
nitrogen in the test sample, and in the analytical sample after the drying procedure;
convert the difference between the quantitation values into ammonia (NH3) to make thevolatile matter content.
b) Potassium hydrogen carbonate: Determine carbon dioxide in the test sample, and in theanalytical sample after the drying procedure; the difference between the quantitation
values is the volatile matter content.
Comment 4 Table 2 shows results from a simultaneous analysis with the same samples(proficiency testing and external quality control testing) hosted by the Japan
Fertilizer Quality Assurance Committee analyzed by the robust method.

19

Table 2 Results and analysis results from a simultaneous analysis with the same samples
for moisture hosted by the Japan Fertilizer Quality Assurance Committee1)
2)

4)

Year

SampleNumber oflaboratories

Median Μ

NIQR

(%)3)

(%)3)

RSDrob(%)

2006

High analysis compound
fertilizer

147

1.70

0.30

17.6

2007

Organic compound
fertilizer

146

4.99

0.35

7.0

2008

High analysis compound
fertilizer

145

2.87

0.24

8.5

2009

Ordinary compound
fertilizer

145

3.53

0.15

4.2

2010

High analysis compound
fertilizer

143

1.58

0.41

26.0

2011

High analysis compound
fertilizer

137

2.03

0.09

12.0

2013

High analysis compound
fertilizer

136

2.93

0.84

28.7

5)

1) Proficiency testing and external quality control testing2) Median (M) agrees with the mean in normal distribution.
3) Mass fraction4) Robust standard deviation (NIQR) agrees with standard deviation in normal distribution.
5) RSDrob stands for relative standard deviation obtained from the robust methodby the formula shown below:
RSDrob = (NIQR/M) × 100

References
1) Masayoshi KOSHINO: Second Revision of The Methods of Analysis of Fertilizers(Details), p.20 - 23, Yokendo, Tokyo (1988)
2) Society for the Study of Feed Analysis Standards：Methods of Analysis of Feeds andFeed Additives-2009-I, p.37 - 39, Incorporated Administrative Agency Food andAgricultural Materials Inspection Center, Saitama (2009)

20

(4)

Flow sheet for moisture: The flow sheet for moisture in fertilizers is shown below:
2 g -5 ganalytical sample
Heating

Transfer to a ground-in stoppered weighing bottle, spread so that the
thickness is no more than 10 mm. Measure the mass to the order of 1 mg.
100 ºC ± 2 ºC, 5 hours

Standing to cool

Desiccator

Measurement

Measure the mass to the order of 1 mg.

Figure Example of the flow sheet for moisture in fertilizers
by loss on drying with drying apparatus.

21

3.1.bLoss on drying method by moisture analyzers(1) Summary
The test method is applicable to sludge fertilizers, compost, and organic fertilizers, etc.
Moisture in an analytical sample is measured by a moisture analyzer in the heat drying
method.
(2) Instruments: Instruments are as shown below:a) Moisture analyzer: A moisture analyzer consisted of a heat source to heat an analyticalsample (halogen lamp, infrared heater, ceramic heater, etc.) and a built-in balance (1)with calibration function.
Note (1) There is a method to calibrate with calibration weights or a method to calibrate
automatically with built-in weights.
(3)

a)
b)
c)
d)

Measurement: Conduct measurement as shown below. However, conduct in advance acomparative test with 3.1.a Loss on heating with drying apparatus using sludge
fertilizers, compost, and organic fertilizers, etc., to confirm that there is no difference inthe quantitation value of moisture.
Transfer about 5 g of an analytical sample to a weighing dish, spread so that the
thickness is no more than 10 mm, and measure the mass to the order of 1 mg.
Heat at 100 ºC (2), until a constant weight is achieved.
After the end of heating (2), measure the mass to the order of 1 mg.
Calculate loss on drying in the analytical sample by the following formula (1) asmoisture. If necessary, calculate the conversion factor (actual article) by the following
formula (2):Loss on drying (% (mass fraction)) = ((A - B) / A) × 100
Conversion factor (dry matter) = A/B

(1)

(2)

A: Mass (g) of the sampled analytical sample
B: Mass (g) of the analytical sample after drying
Note (2) The setup of the drying program and the determination parameter for the end ofheating (constant weight) is according to the specification and the operation
method of the moisture analyzer used.
Comment 1 When pre-drying is conducted, calculate the moisture of the laboratory
sample (actual article) by the following formula:
Total moisture (% (mass fraction)) in the laboratory sample (actual article) = A + B ×((100 - A)/100)
A: Loss on drying (% (mass fraction)) of the laboratory sample (actual article)
by the pre-drying procedure
B: Loss on drying (% (mass fraction)) in the analytical sample by moisturemeasurement
Comment 2 Table 1 shows results from replicate testing conducted with organic fertilizers,
compost and sludge fertilizers.
22Additionally, Table 2 shows results and analysis results from a collaborative
study for test method validation.
Table 1

Replicate testing on moisture in sludge fertilizers, etc., with a moisture analyzer.
Mean1)

Standard deviation

Relative standard
deviation

(%)2)

(%)2)

(%)

Sewage sludge fertilizer

24.40

0.29

1.2

Human waste sludge fertilizer A

5.50

0.03

0.6

Human waste sludge fertilizer B

13.63

0.09

0.7

Industrial sludge fertilizer A

13.39

0.11

0.8

Industrial sludge fertilizer B

90.61

0.14

0.2

Composted sludge fertilizer A

33.92

0.20

0.6

Composted sludge fertilizer B

63.14

0.21

0.3

Fish meal

6.27

0.01

0.2

Steamed leather meal

8.03

0.11

1.4

Rape seed meal and powderedrape seed meal

8.85

0.03

0.3

Rice bran meal and powderedrice bran meal

9.05

0.01

0.1

Byproduct organic fertilizer ofvegetable origin

6.26

0.07

1.0

Organic fertilizer mixture

6.47

0.02

0.3

Compost

10.19

0.04

0.4

Name of fertilizer

1) Mean of triplicates.
2) Mass fraction

23

Table 2

Results and analysis results from a collaborative study
for the validation of the moisture test method.
4)

RSDr

6)

RSDR

(%)

3)

(%)

(%)

3)

(%)

21.93

0.32

1.4

0.47

2.1

8

13.36

0.14

1.1

0.37

2.8

Industrial sludge
fertilizer

9

34.28

0.21

0.6

0.50

1.5

Calcined sludge
fertilizer

9

38.75

0.59

1.5

0.59

1.5

Composted sludge
fertilizer

9

27.1

0.26

0.9

0.60

2.2

Number of

Mean2)

laboratories1)

(%)

3)

Sewage sludge
fertilizer

9

Human waste sludge
fertilizer

Sample name

sr

5)

sR

7)

1) Number of laboratories used in analysis
2) Mean (n= number of laboratories  number of samples (2))3) Mass fraction
4) Repeatability standard deviation
5) Repeatability relative standard deviation
6) Reproducibility standard deviation
7) Reproducibility relative standard deviation

References
1) Takeshi UCHIYAMA and Chiyo SAKASEGAWA: Validation of a Heating Method
Using a Moisture Analyzer for Moisture Content in Sludge Fertilizer. Research Reportof Fertilizer Vol.1, 2008, (1- 5)
2) Takeshi UCHIYAMA and Yuji SHIRAI: Determination of Moisture Content in Sludge
Fertilizer by a Heating Method Using Moisture Analyzer: A Collaborative Study.
Research Report of Fertilizer Vol.1, 2008, (6 - 11)3) Satono AKIMOTO and Sakiko TAKAHASHI: Validation of a Heating Method Using a
Moisture Analyzer for Moisture Content in Sludge Fertilizer. Research Report of
Fertilizer Vol.2, 2009, (1 - 5)
(4)

Flow sheet for moisture: The flow sheet for moisture in sludge fertilizers, compost,
and organic fertilizers, etc. is shown below:
About 5 ganalytical sample
Heating

Transfer to a weighing dish, spread so that the thickness is
no more than 10 mm. Measure the mass to the order of 1 mg.
100 ºCEnd of drying

Constant weight

Measurement

Measure the mass to the order of 1 mg

Figure Flow sheet for moisture in sludge fertilizers, compost, and organic
fertilizers, etc. by the heat drying method with a moisture analyzer.
24

3.2
3.2.a

Ash content
Ignition residue method(1) Summary
The method is applicable to organic fertilizers and fertilizers containing organic matters.
The method ignites an analytical sample with an electric furnace and measures residue on
ignition.
(2) Apparatus and instruments: Apparatus and Instruments are shown below.
a) Electric furnace: An electric furnace that can be adjusted to the temperature 550 ºC ± 5ºC.
b) Crucible: After heating a porcelain crucible for chemical analysis specified in JIS R
1301 with an electric furnace at 550 ºC ± 5 ºC, stand to cool in a desiccator in advanceand measure the mass to the order of 1 mg.
(3) Measurement: Conduct measurement as shown below.
a) Transfer about 2 g of an analytical sample into a crucible, and measure the mass to theorder of 1 mg.
b) Place it into an electric furnace, heat gently until carbonized (1).
c) Heat at 550 ºC ± 5 ºC for no less than 4 hours.
d) After heating, move the crucible into a desiccator and let it stand to cool.
e) After standing to cool, remove the crucible from the desiccator and measure the mass tothe order of 1 mg.
f) Calculate the residue on ignition in the analytical sample by the following formula to
make ash content.
Residue on ignition (% (mass fraction)) = (B/A) × 100
A: Mass (g) of the sampled analytical sample
B: Mass (g) of the ignited analytical sample
Note (1) Example of carbonizing procedure: Heat at about 250 ºC until smoke no longer
form.
(4) Flow sheet for ash content: The flow sheet for ash content in fertilizers is shown below.
About 2 g ofanalytical sample

Transfer to a crucible. Measure the mass to the order of 1 mg.

CharredHeating gently with an electric furnace

Incineration

No less than 4 hours at 550 ºC ± 5 ºC

Standing to cool

Desiccator

Measurement

Measure the mass to the order of 1mg

Figure Flow sheet for ash content in fertilizers.

25

3.3
3.3.a

pH
Glass electrode method(1) Summary
Measure with a pH meter using a glass electrode(2) Reagent: Reagents are as shown below.
a) Oxalate pH standard solution: Oxalate pH standard solution class 2 traceable to a
specified reference material (national measurement standard) according to Article 134of the Measurement Act.
b) Phthalate pH standard solution: Phthalate pH standard solution class 2 traceable to a
specified reference material (national measurement standard) according to Article 134of the Measurement Act.
c) Neutral phosphate pH standard solution: Neutral phosphate pH standard solution
class 2 traceable to a specified reference material (national measurement standard)
according to Article 134 of the Measurement Act.
d) Borate pH standard solution: Borate pH standard solution class 2 traceable to a
specified reference material (national measurement standard) according to Article 134of the Measurement Act.
e) Carbonate pH standard solution: Carbonate pH standard solution class 2 traceable toa specified reference material (national measurement standard) according to Article 134of the Measurement Act.
Comment 1 Respective pH standard solutions stored for long time should not be usedsince the pH value may change during storage period. In particular, note thatborate pH standard solution and carbonate pH standard solution easily absorb
carbon dioxide in the air, so that the pH values deteriorate. The pH standard
solution that was used once or left exposed to the air should not be used.
(3) Instruments: Instruments are shown below.
a) pH meter: Use type II specified in JIS Z 8802.
Comment 2 Conduct the calibration of a pH meter as indicated in JIS Z 8802. Actual
calibration operation is according to the operation procedure of the pH meterused for measurement.
When the pH of a sample solution is no more than 7, use neutral phosphate
pH standard solution and oxalate pH standard solution, or phthalate pH
standard solution. When it exceeds 7, use neutral phosphate pH standard
solution and borate pH standard solution, or carbonate pH standard solution.
(4) Test procedures(4.1) Preparation of sample solution: Conduct preparation of sample solution as shown
below.
(4.1.1) Fertilizers except inorganic fertilizersa) Transfer a predetermined amount of an analytical sample (1) into a ground-in stopper
flask and add water 5 - 10 times the volume.
b) Mix with a magnetic stirrer, filter with Type 3 filter paper to make the sample solution.
Note (1)In the case of a moist laboratory sample, it is recommended to use a sample thatis not pre-dried.
26

(4.1.2) Inorganic fertilizersa) Transfer a predetermined amount of an analytical sample (1) into a ground-in stopper
flask and add water 100 times the volume.
b) Mix with a magnetic stirrer, filter with Type 3 filter paper to make the sample solution.
Comment 3 The procedure in (4.1.1) is the same as 3.4.a (4.1). Additionally, the sample
solution prepared in 4.2.4.a (4.1) can be used instead of the sample solution
prepared by (4.1.2).
(4.2) Measurement: Conduct the measurement as indicated in JIS Z 8802 and as shown
below. Actual calibration operation is according to the operation procedure of the pH
meter used for measurement.
a) Wash the read station of a calibrated pH meter repeatedly no less than 3 times withwater and wipe out with clean and soft paper, etc.
b) Transfer a sample solution into a beaker (2), dip the read station in the solution andmeasure the pH value.
Note (2) It is necessary to transfer sufficient volume of sample solution to keep ameasurement value stable.
Comment 4 If a pH meter has a temperature correction dial or a digital switching, measure
the pH value after adjusting the graduation of the pH meter with thetemperature of a sample.
Reference
1) JIS Z 8002: Methods for determination of pH of aqueous solutions (2011)(5)

Flow sheet for pH value: The flow sheet for pH value in fertilizers is shown below.
Predetermined volume
Ground-in stopper flask.
of analytical sample← Water 5 -10 times or 100 times the volume
MixtureFiltration
Measurement

Type 3 filter paper
pH meterFigure Flow sheet for pH in fertilizers.

27

3.4
3.4.a

Electrical conductivity
Measurement method with an electrical conductivity meter(1) Summary
Measure the electrical conductivity of organic fertilizers such as compost or sludge fertilizers
with an electrical conductivity meter.
(2) Reagents: Reagents are shown below.
a) Potassium chloride: Grind potassium chloride used for measurement of electricalconductivity specified in JIS K 0130 with an agate mortar to powder and heat for 4
hours at 500 ºC ± 5 ºC, and then stand to cool in a desiccator.
b) Potassium chloride standard solution (1): Measure predetermined volume (2) ofpotassium chloride of a) on a weighing dish, dissolve in a small amount of water,transfer it into a 1,000-mL volumetric flask , and add up to the marked line with water.
Note (1) Store potassium chloride standard solution in a polyethylene or borosilicate glass
bottle and seal the bottle.
(2) The volume that is recommended for an instrument or a cell used.
Comment 1 Potassium chloride standard solution used once or left in the air should not be
used.
(3) Instruments: Instruments are shown below.
a) Electrical conductivity meter: An electrical conductivity meter specified in JIS K
0130
Comment 2 Check the indicated value as shown in 6.2 in JIS K 0130 as necessary. Actual
procedure to check is according to the operation procedure of the electricalconductivity meter used for measurement.
(4) Test procedure(4.1) Preparation of sample solution: Conduct the preparation of sample solution as shown
below.
a) Transfer the predetermined volume of an analytical sample (3) into a ground-in stopper
flask and add water 10 times the equivalent volume of dry matter (4).
b) Mix with a magnetic stirrer, filter with Type 3 filter paper to make the sample solution.
Note (3) In the case of a moist laboratory sample, it is recommended to use a sample thatis not pre-dried.
(4) If the sample solution becomes hard to measure because it is gelled by the
influence of flocculants in sludge fertilizer, etc., increase the volume of water
added. However this fact should be expressed in the test result.
Comment 3

The procedure in (4.1) is the same as 3.3.a (4.1.1).

(4.2) Measurement: Conduct the measurement as indicated in JIS K 0132 and as shown
below. Actual measurement operation is according to the operation procedure of theelectrical conductivity meter used for measurement.
a) Wash the read station of an electrical conductivity meter repeatedly no less than 3 times
with water.
28b) Transfer a sample solution into a beaker (5), dip the read station and measure electricalconductivity.
Note (5) It is necessary to transfer sufficient volume of sample solution to keep themeasurement value stable.
Reference
1) JIS K 0130: General rules for electrical conductivity measuring method (2008)(5)

Flow sheet for electrical conductivity: The flow sheet for electrical conductivity isshowed below.
Predetermined volume
Ground-in stopper flask.
of analytical sample← Water ,10 times the volume
MixtureFiltration
Measurement

Type 3 filter paper
Electrical conductivity meter

Figure Flow sheet for electrical conductivity in fertilizers.

29

3.5
3.5.a

Granularity
Dry-type sieving testing method(1) Summary
Measure the particle diameter distribution of particulates and powdery fertilizers with adry-type sieving analysis.
(2) Apparatus: Apparatus are shown below.
a) Sieve: A sieve for testing specified in JIS Z 8815b) Clogging removal brush: A brush which is adequately hard for the apertures and does
not damage the sieving screen.
c) Weighing dish: A container that can contain about 250 g of a sample. Measure the mass
to the order of 0.1g in advance.
(3)

Dry-type sieving analysis procedure: Conduct sieving analysis corresponding to theaperture size of a sieve used as indicated in JIS Z 8815 and as shown below.
(3.1) More than 1mm and no more than 4mma) Stack a large aperture sieve on an acceptor so that the large sieve is on top.
b) Measure the total mass (2) of a laboratory sample or a separated test sample (1) to theorder of 0.1g and transfer it to the sieve at the top section.
c) After putting a stopper on it, hold the stacked sieves with both hands, and vibrate (3)them back and forth at about 70 mm amplitude along a unidirectional and horizontal
plane at about 60 times per minute.
d) Transfer respective plus and minus sieves to a weighing dish (4).
Note (1) Conduct the separation of a sample as indicated in 2.2.2(3).
(2) The minimum volume of a separated test sample is 250 g.
(3) Conduct more circular motion at the rate of about 3 revolutions per minute asnecessary.
(4) Turning over the back side of a sieve, remove the clogged particles from thesieve screen with a clogging removal brush and combine them with the plus
sieve.
(3.2) Less than and equal to 1mma) Stack a large aperture sieve on an acceptor so that the large sieve is on top.
b) Measure the total mass (2) of a laboratory sample, a separated test sample (1) or minus
sieve of (3.1) c) to the order of 0.1g and transfer it to the sieve at the top section.
c) After putting a stopper on it, incline the stacked sieves about 20 degrees, supportingwith one hand or a bent arm, and tap the sieve frame with the other hand at the rate ofabout 120 times per a minute.
d) During the procedure in c), place the sieve in a horizontal position at the rate of 4 times
per minute, rotate it 90 degrees and tap the sieve frame hard one or two times.
e) Transfer respective plus and minus sieves (5) to a weighing dish (4).
Note (5) When fine powder attaches to the back side of a sieve screen, remove them
gently from the back side with a clogging removal brush and combine them withthe minus sieve(4)

Measurement of granularity distribution: Calculate the granularity distribution in ananalytical sample as shown below.
a) Measure respective mass of plus and minus sieves to the order of 0.1g.
30b) Calculate “plus sieve percentage” and “integrated minus sieve percentage” with thefollowing formula and round the results to the first decimal place.
c) Confirm that the sum of the mass of the plus sieve and the mass of the minus sieve withthe smallest aperture is in the range of ± 2 % of the mass of sample measured in (3.1)b)or (3.2)b).
Mass percentage of plus sieve or minus sieve (%) (R) = (A/T) × 100
A: Mass of plus sieve or minus sieve (g)T: Sum of the mass of plus and minus sieve (g)References
1) JIS Z 8815: Test sieving - General requirements (1994).
2) JIS K 0069: Test methods for sieving of chemical products (1992).
(5)

Flow sheet of granularity: The flow sheet of granularity of particulate or powdery
fertilizers is shown below.

.
Laboratory sample
or Test sample

Measure the mass to the order of 0.1g

Dry-type sieving
Mass measurement ofrespective plus sieveand minus sieve
FigureMeasure the mass to the order of 0.1g

Flow sheet for granularity of particulate or powdery fertilizers

31

3.6

Oil content

3.6.a Diethyl ether extraction method(1) Summary
The method is applicable to organic fertilizers.
Extract an analytical sample with diethyl ether using a Soxhlet extractor to measure theextract obtained.
The oil content contains not only fat but also fat-soluble pigments (carotenoid, chlorophyll,etc.), wax, and free fatty acids, etc..
(2) Reagents: Reagents are as shown below.
a) Diethyl ether: A JIS Guaranteed Reagent specified in JIS K 8951 or a reagent ofequivalent quality.
(3) Apparatus and instruments: Apparatus and instruments are shown below.
a) Drying apparatus: Drying apparatus that can be adjusted to the test temperature ± 2 ºCb) Soxhlet extractor: An inter-changeable Soxhlet extractor, cooling apparatus andweighing bottles (Example: JIS R 3503, attached figure 71)c) Water bath: A water bath that can be adjusted to about 60 ºCd) Weighing bottle: A flat bottle flask connectable to a Soxhlet extractor. After heatingwith a drying apparatus at 100 ºC - 105 ºC in advance, stand to cool in a desiccator andmeasure the mass to the order of 1mg.
e) Cylindrical filter paper: A cylindrical filter paper made of cellulose. Example: 22 mm
external diameter, 20 mm internal diameter, 90 mm total length (1).
(4)
Measurement: Conduct measurement as shown belowa) Weigh 2 g - 5 g of an analytical sample to the order of 1mg, and transfer it into acylindrical filter paper.
b) Place absorbent cotton on the upper end of an analytical sample (2), as if gently pushingit, and heat it at 100 ºC – 105 ºC for 2 hours.
c) As soon as heating is complete, move the cylindrical filter paper to a desiccator andstand to cool.
d) After standing to cool, transfer it into a Soxhlet extractor and connect it to a coolingapparatus.
e) Transfer adequate volume of diethyl ether (3) into a weighing bottle, connect it to theSoxhlet extractor and heat (4) it for 8 hours to extract.
f) Recover the diethyl ether (5).
g) Disconnect the Soxhlet extractor from the weighing bottle and vaporize the diethyl ether(6).
h) Heat the weighing bottle (7) at 100 ºC - 105 ºC for 3 hours.
i) As soon as heating is complete, move the weighing bottle to the desiccator and stand tocool.
j) After standing to cool, remove the weighing bottle from the desiccator and measure themass to the order of 1mg.
k) Calculate oil content with the following formula.
Oil content (% (mass fraction)) = (B/A) × 100
A: Mass of a sampled analytical sample (g)B: Mass of extract of diethyl ether (g)Note (1) Select a scale according to the volume of a Soxhlet extractor.
32

(2) The purpose is to prevent overflow at the upper end of an analytical sample.
(3) The amount of diethyl ether depends on the volume of a weighing bottle.
(4) Adjust the temperature for diethyl ether to circulate 16 - 20 times per hour.
(Target temperature is about 60 ºC)(5) Remove the cylindrical filter paper from the Soxhlet extractor. In the case of acock attached Soxhlet extractor, open the cock and recover it.
(6) It is dangerous if diethyl ether resides in a weighing bottle when the bottle istransferred to a drying apparatus.
(7) Wipe the outside of a weighing bottle since there is a risk of garbage or stain
sticking to it.
References
1) Japan Oil Chemist ‘Society: Standard Method for the Analysis of Fats, Oils and RelatedMaterials (2003), 1.5 Oil content p.1 - 2, Incorporated Foundation Japan Oil Chemist‘Society, Tokyo (2009)
2) Feed Analysis Standard Task Force: Feed Analysis Method/Handbook -2009 - I, p.37 39, Incorporated Administrative Agency, Food and Agriculture Materials Inspection
Center, Saitama (2009)(5)

Flow sheet of oil content: Flow sheet of oil content in organic fertilizers is shown
below.
2 g- 5 ganalytical sample
Pre-drying

Weigh to the order of 1mg to a cylindrical filter paper
100 ºC - 105 ºC, for 2 hours

Soxhlet extractor
Extraction

Diethyl ether, heating, for 8 hours

Weighing bottle
Heating

100 ºC - 105 ºC, for 5 hours

Standing to cool

Desiccator

Measurement

Measure the mass to the order of 1mg

Figure Flow sheet for oil content in organic fertilizers.

33

4.

Main components, guaranteed components, etc.

4.1

Nitrogen4.1.1 Total nitrogen4.1.1.a Kjeldahl method(1) Summary
This test method is applicable to fertilizers containing no nitrate nitrogen.
Add sulfuric acid, potassium sulfate and copper (II) sulfate pentahydrate to an analyticalsample, pretreat by Kjeldahl method to change total nitrogen (T-N) to ammonium ion, and add
sodium hydroxide solution to subject to steam distillation. Measure separated ammonium ion
by neutralization titration, to obtain total nitrogen (T-N) in the analytical sample. This test
method corresponds to the sulfuric acid method in the Official Methods of Analysis of
Fertilizers (1992).
(2) Reagents: Reagents are as shown below:a) 0.1 mol/L - 0.2 mol/L sodium hydroxide solution(1): Transfer about 30 mL of water toa polyethylene bottle, dissolve about 35 g of sodium hydroxide specified in JIS K 8576
by adding in small portions while cooling, seal tightly and leave at rest for 4-5 days.
Transfer 5.5 mL -11 mL of the supernatant to a ground-in stoppered storage container,
and add 1,000 mL of water containing no carbonic acid.
Standardization: Dry sulfamic acid reference material for volumetric analysis
specified in JIS K 8005 by leaving at rest in a desiccator at no more than 2 kPa for about
48 hours, then transfer about 2.5 g to a weighing dish, and measure the mass to the orderof 0.1 mg. Dissolve in a small amount of water, transfer to a 250-mL volumetric flask,
and add water up to the marked line.(1) Transfer a predetermined amount of the solution
to a 200-mL - 300-mL Erlenmeyer flask, add a few drops of bromothymol blue solution(0.1 mg/100 mL) as an indicator, and titrate with 0.1 mol/L - 0.2 mol/L sodiumhydroxide solution until the color of the solution becomes green. Calculate the factor of
0.1 mol/L - 0.2 mol/L sodium hydroxide solution by the following formula:
Factor of 0.1 mol/L - 0.2 mol/L sodium hydroxide solution (f1)
= (W1 × A × 0.01/97.095) × (V1/V2) × (1,000/V3) × (1/C1)
W1: Mass (g) of sulfamic acid sampled
A: Purity (% (mass fraction)) of sulfamic acidV1: Volume (mL) of sulfamic acid solution transferred
V2: Constant volume (250 mL) of sulfamic acid solution
V3: Volume (mL) of 0.1 mol/L - 0.2 mol/L sodium hydroxide solution needed fortitration
C1: Set concentration (mol/L) of 0.1 mol/L - 0.2 mol/L sodium hydroxide solutionb) Sulfuric acid: A JIS Guaranteed Reagent specified in JIS K 8951 or a reagent ofequivalent quality.
c) 0.25 mol/L sulfuric acid(1) (2): Add about 14 mL of sulfuric acid to a beaker containing
100 mL of water in advance, stir well, and add water to make 1,000 mL.
Standardization: Transfer a predetermined amount (3) of 0.25 mol/L sulfuric acid to a
200-mL - 300-mL Erlenmeyer flask, add a few drops of methyl red-methylene blue
mixture solution, and titrate with 0.1 mol/L -0.2 mol/L sodium hydroxide solution until
the color of the solution becomes gray-green.(4) Calculate the volume of 0.1 mol/L - 0.2
34mol/L sodium hydroxide solution equivalent to 1 mL of 0.25 mol/L sulfuric acid by thefollowing formula (1). Or, calculate the factor of 0.25 mol/L sulfuric acid by thefollowing formula (2):Volume (B) of 0.1 mol/L -0.2 mol/L sodium hydroxide solution equivalent to 1 mL
of 0.25 mol/L sulfuric acid= V4/V5…… (1)
Factor of 0.25 mol/L sulfuric acid (f2)
= (f1 × C1 × V4/V5)/(C2 × 2)…… (2)

V4: Volume (mL) of 0.1 mol/L -0.2 mol/L sodium hydroxide solution needed fortitration
V5: Volume (mL) of 0.25 mol/L sulfuric acid subjected to standardization
C1: Set concentration (mol/L) of 0.1 mol/L - 0.2 mol/L sodium hydroxide solution
C2: Set concentration (0.25 mol/L) of 0.25 mol/L sulfuric acidd) Boric acid solution (40 g/L): Dissolve 40 g of boric acid specified in JIS K 8863 inwater to make 1,000 mL.
e) Catalyst (5): Mix potassium sulfate specified in JIS K 8962 and copper (II) sulfate
pentahydrate (6) specified in JIS K 8983 in the ratio of 9 to 1.
f) Sodium hydroxide solution (200 g/L - 500 g/L) (1): Dissolve 100 g - 250 g of sodiumhydroxide specified in JIS K 8576 in water to make 500 mL.
g) Bromothymol blue solution (0.1 g/100 mL): Dissolve 0.1 g of bromothymol blue
specified in JIS K 8842 in 20 mL of ethanol (95) specified in JIS K 8102, and add water
to make 100 mL.
h) Methyl red solution (0.1 g/100 mL): Dissolve 0.1 g of methyl red specified in JIS K
8896 in 100 mL of ethanol (95) specified in JIS K 8102.
i) Methylene blue solution (0.1 g/100 mL): Dissolve 0.1 g of methylene blue specified in
JIS K 8897 in 100 mL of ethanol (95) specified in JIS K 8102.
j) Methyl red - methylene blue mixture solution: To 2 volumes of methyl red solution(0.1 g/100 mL), add 1 volume of methylene blue solution (0.1 g/100 mL).
k) Bromocresol green solution (0.5 g/100 mL): Dissolve 0.5 g of bromocresol green
specified in JIS K 8840 in 100 mL of ethanol (95) specified in JIS K 8102.
l) Methyl red - bromocresol green mixture solution: To methyl red solution (0.1 g/100mL), add equal volume of bromocresol green solution (0.5 g/100 mL).
Note (1) This is an example of preparation; prepare an amount as appropriate.
(2) This corresponds to the standard sulfuric acid solution 0.5 M (1/2 sulfuric acid)solution in the Official Methods of Analysis of Fertilizers (1992).
(3) 5 mL -10 mL(4) The endpoint is reached when the color becomes gray-green via dark blue fromblue-purple.
(5) A tablet is commercially available.
(6) Crush into powder as appropriate.
(3)
a)
b)
c)

Apparatus and instruments: Apparatus and instruments are as shown below.
Steam distillation apparatus
Digestion flask: Kjeldahl flask
Distillation flask: A Kjeldahl flask or round bottom flask that can be connected to asteam distillation apparatus.
35

(4) Test procedures(4.1) Kjeldahl method: Conduct digestion as shown below.
a) Weigh 0.5 g - 5 g of an analytical sample to the order of 1 mg, and put it in a 300-mL 500-mL digestion flask.
b) Add 5 g - 10 g of catalyst, and further add 20 mL - 40 mL of sulfuric acid, shake to mix
and heat gently.
c) After bubbles cease to form, heat until white smoke of sulfuric acid evolves.
d) Ignite until organic matters are completely digested (7).
e) After standing to cool, add a small amount of water, mix well by shaking, transfer to a
250-mL - 500-mL volumetric flask with water (8), and further mix by shaking.
f) After standing to cool, add water up to the marked line to make the digestion solution.
Note (7) When the solution has finished changing color, heat further for no less than 2hours.
(8) When the entire sample solution volume is used in measurement, it is notnecessary to transfer it to a volumetric flask.
Comment 1 The procedure in (4.1) is the same as that in (4.1) a) - f) in 4.2.1.a.
Comment 2 In the case of fish meal containing amino acids that are not easily digested,
use 0.5 g -1 g analytical sample, 10 g catalyst and 30 mL - 40 mL sulfuricacid.
Comment 3 In the case of nitrolime, moisten by adding a small amount of water before
the procedure in (4.1) b). Care should be taken because bubbles are producedby the addition of sulfuric acid.
(4.2) Distillation: Conduct distillation as shown below. Specific distillation procedures areaccording to the operation method of the steam distillation apparatus used inmeasurement.
a) Transfer a predetermined amount(9) of 0.25 mol/L sulfuric acid to an acceptor,(10) add a
few drops of methyl red - methylene blue mixture solution, and connect this acceptor toa steam distillation apparatus. Or, transfer a predetermined amount(9) of boric acidsolution (40 g/L) to an acceptor,(10) add a few drops of methyl red - bromocresol green
mixture solution, and connect this acceptor to a steam distillation apparatus.
b) Transfer a predetermined amount of the digestion solution to a 300-mL distillation flask,
add a proper amount of sodium hydroxide solution (200 g/L - 500 g/L) (11), andimmediately connect this distillation flask to the steam distillation apparatus.
c) Send steam to the distillation flask to heat the solution in the distillation flask, and distill
at a distillation rate of 5 mL/min - 7 mL/min.
d) Stop distilling when the distillate has reached 120 mL - 160 mL.
e) Wash the part of the steam distillation apparatus that came in contact with the solution
in the acceptor with a small amount of water, and pool the washing with the distillate.
Note (9) 5 mL - 20 mL(10) As an acceptor, use a 200-mL - 300-mL Erlenmeyer flask or a 200-mL - 300-mL
beaker with which the distillate outlet of the steam distillation apparatus can be
immersed in 0.25 mol/L sulfuric acid or boric acid solution (40 g/L).
(11) An amount sufficient to make the solution strong alkalinity. A blue color willappear.
(4.3) Measurement: Conduct measurement as shown below.
(4.3.1) When 0.25 mol/L sulfuric acid is used in (4.2):
36a) Titrate the distillate with 0.1 mol/L - 0.2 mol/L sodium hydroxide solution until thecolor of the solution becomes gray-green (4).
b) Calculate the total nitrogen (T-N) in the analytical sample by the following formula:
Total nitrogen (T-N) (% (mass fraction)) in the analytical sample= (B × V6 - V7) × C1 × f1 × (V8/V9) × (14.007/W2) × (100/1,000)= (B × V6 - V7) × C1 × f1 × (V8/V9) × (1.4007/W2)
B: Volume of 0.1 mol/L -0.2 mol/L sodium hydroxide solution equivalent to 1
mL of 0.25 mol/L sulfuric acidV6: Volume (mL) of 0.25 mol/L sulfuric acid transferred to the acceptor in (4.2)a)
V7: Volume (mL) of 0.1 mol/L - 0.2 mol/L sodium hydroxide solution needed fortitration
C1: Set concentration (mol/L) of 0.1 mol/L - 0.2 mol/L sodium hydroxide solution
f1: Factor of 0.1 mol/L - 0.2 mol/L sodium hydroxide solution
V8: Predetermined volume (mL) of the digestion solution in (4.1) e)
V9: Transferred amount (mL) of the digestion solution subjected to distillation in(4.2) b)
W2: Mass (g) of the analytical sample(4.3.2) When boric acid solution (40 g/L) is used in (4.2):a) Titrate the distillate with 0.25 mol/L sulfuric acid until the color of the solution becomeslight red (12).
b) Calculate the total nitrogen (T-N) in the analytical sample by the following formula:
Total nitrogen (T-N) (% (mass fraction)) in the analytical sample= V10 × C2×2× f2 × (V11/V12) × (14.007/W2) × (100/1,000)= V10 × C2 × f2 × (V11/V12) × (2.8014/W2)
V10: Volume (mL) of 0.25 mol/L sulfuric acid needed for titration
C2: Set concentration (mol/L) of 0.25 mol/L sulfuric acidf2: Factor of 0.25 mol/L sulfuric acidV11: Predetermined volume (mL) of the digestion solution in (4.1) e)
V12: Transferred amount (mL) of the digestion solution subjected to distillation in(4.2) b)
W2: Mass (g) of the analytical sample
Note (12) The endpoint is reached when the color changes from green to light red.

Comment 4 The titration procedures in (2) a) Standardization, (2) c) Standardizationand (4.3) can be conducted by an automatic titrator. The setup of the titration
program, the determination parameter for the endpoint and vessels such as
acceptors are according to the specification and the operation method of theautomatic titrator used.
Comment 5 The nitrogen content in the analytical sample can be measured by using an
automatic nitrogen analyzer (Kjeldahl method) instead of the test procedurein (4). The setup of the program and the parameter of the analyzer as well as
vessels etc. are according to the specification and the operation method of theautomatic nitrogen analyzer used. However, conduct in advance a37

comparative test with the test procedure in (4) using fertilizers containing no
nitrate nitrogen, to confirm that there is no difference in the quantitation valueof total nitrogen.
Comment 6 Table 1 shows results from a simultaneous analysis with the same samples(proficiency testing and external quality control testing) hosted by the Japan
Fertilizer Quality Assurance Committee analyzed by the robust method.
Table 1 Results and analysis results from a simultaneous analysis with the same samples
for total nitrogen hosted by the Japan Fertilizer Quality Assurance Committee1)
Year

SampleNumber of Median Μ2)laboratories(%)3)

NIQR4)(%)3)

RSDrob5)(%)

High analysis compound
158
14.60
0.13
0.9
fertilizer
Organic compound
2007
145
8.74
0.07
0.8
fertilizer
High analysis compound
2010
132
14.11
0.11
0.8
fertilizer
1) Proficiency testing and external quality control testing2) Median (M) agrees with the mean in normal distribution.
3) Mass fraction4) Robust standard deviation (NIQR) agrees with standard deviation in normal distribution.
5) RSDrob stands for relative standard deviation obtained from the robust methodby the formula shown below:
RSDrob = (NIQR/M) × 100
2006

References
1) Masayoshi KOSHINO: Second Revision of The Methods of Analysis of Fertilizers(Details), p.27 - 31, Yokendo, Tokyo (1988)
2) Society for the Study of Feed Analysis Standards: Methods of Analysis of Feeds andFeed Additives-2009-I, p.28 - 33, Incorporated Administrative Agency Food andAgricultural Materials Inspection Center, Saitama (2009)
3) Takashi KUBOTA, Tomoko OSHIDA, Kozue YANAI, Yuzuru INOUE, Seiji MATSUI,Takaharu MATSUMOTO, Eiichi ISHIKURO and Akemi YASUI: Improvement of theConditions for the Determination of Total Nitrogen in Fish Meal in Kjeldahl Method
and Its Comparison with Dumas Method, Bunsekikagaku, 60, 67 - 74 (2011)

38

(5)

Flow sheet for total nitrogen: The flow sheet for total nitrogen in fertilizers is shown
below.
0.5 g - 5 g
Weigh to the order of 1 mg into a 300-mL digestion flask.
analytical sample← 5 g - 10 g catalyst←20 mL - 40 mL sulfuric acid
Heating
GentlyHeating

After foam no longer evolves, ignite
until organic matters are completely digested.

Standing to cool← small amount of waterTransfer
250-mL - 500-mL volumetric flask, waterStanding to coolRoom temperature← Water (up to the marked line)
Aliquot
300-mL distillation flask(predetermined volume)← Sodium hydroxide solution (200 g/L - 500 g/L)
Receiver: 200-mL - 300 - mL Erlenmeyer flask or beakerA predetermined amount of 0.25 mol/L sulfuric acid andSteam distillationa few drops of methyl red-methylene blue mixture solution,
apparatus
or boric acid solution (40 g/L), several drops ofmethyl red - bromocresol green mixture solution
Steam distillation

Distillation rate: 5 mL/min -7 mL/min

Stop distilling
120 mL – 160 mL distillate← Water (wash the part of the distillation apparatus
that came in contact with the solution in the receiver)
0.1 mol/L-0.2 mol/L sodium hydroxide solution (until the
Titration
solution becomes gray-green), or
0.25 mol/L sulfuric acid (until the solution becomes light red)
Figure Flow sheet (example) for total nitrogen in fertilizers.

394.1.1.b Combustion method(1) Summary
This test method is applicable to fertilizers.
Thermally decompose nitrogen compounds in an analytical sample using a total nitrogen
analyzer by the combustion method to produce nitrogen gas and nitroxide gas. Reduce thenitroxide gas to nitrogen, and measure the nitrogen gas content with a thermal conductivity
detector. This test method is also referred to as a modified Dumas’ method.
(2) Instruments: Instruments are as shown below:a) Total nitrogen analyzer by the combustion method: A total nitrogen analyzer
configured on the basis of the principle of the combustion method (modified Dumas’
method).
1) Turn on the total nitrogen analyzer by the combustion method (1), and adjust so thatstable indicated values can be obtained.
(i) Combustion gas: Oxygen having purity no less than 99.99 % (volume percentage)(ii) Carrier gas: Helium having purity no less than 99.99 % (volume percentage)(3)

Measurement: Conduct measurement as shown below. However, confirm in advanceusing an analytical sample that there is no difference from the measured value of total
nitrogen obtained according to 4.1.1.a, 4.1.1.c, 4.1.1.d or 4.1.1.e.
a) Measurement conditions for the total nitrogen analyzer by the combustion
method: Set up the measurement conditions for the total nitrogen analyzer considering
the following:
Combustion temperature: No less than 870 ºCb) Calibration curve preparation
1) Turn on the total nitrogen analyzer by the combustion method (1), and adjust so thatstable indicated values can be obtained.
2) Weigh a predetermined amount of the standard for calibration curves (2) to the orderof 0.1 mg into a combustion vessel.
3) Insert the combustion vessel into the total nitrogen analyzer by the combustion
method, and read the indicated value.
4) Conduct the procedure in 3) for another combustion vessel for a blank test, and readthe indicated value.
5) Prepare a curve for the relationship between the nitrogen content and the indicatedvalue of the standard for calibration curves and the blank test for calibration curves.
c) Sample measurement
1) Weigh a predetermined amount of an analytical sample to the order of 0.1 mg into a
combustion vessel.
2) Insert the combustion vessel containing the analytical sample to the total nitrogen
analyzer by the combustion method, and read the indicated value.
3) Obtain the nitrogen content from the calibration curve, and calculate total nitrogen in
the analytical sample.
Note (1) The setup of the program and the parameter of the analyzer are according to thespecification and the operation method of the total nitrogen analyzer by the
combustion method used.
(2) Standard for calibration curves: DL-Aspartic acid (purity no less than 99 %(mass fraction)), EDTA (purity no less than 99 % (mass fraction)), hippuric acid(purity no less than 98 % (mass fraction)) or other reagents having equivalent
purity recommended by the total nitrogen analyzer by the combustion method
used.
40

Comment 1 Sample an analytical sample from a test sample prepared in 2.2.3 Grinding(3) Procedure (3.1) b) by grinding with a mill until it completely passes
through a sieve of 500 µm aperture. Additionally, the sampling amount of ananalytical sample is as shown in Table 1. Additionally, set the sampling
amount of analytical samples considering the estimated content of total
nitrogen in the test sample and the measurement range of the total nitrogen
analyzer by the combustion method.
Table 1 Sampling amount of analytical samples
Type of fertilizers
Sampling amount (g)Mixed fertilizers and designated blended fertilizers

0.02 - 0.5

Organic fertilizers and compost

0.05 - 0.5

Sludge fertilizers

0.05 - 0.5

Comment 2 Compound fertilizers, designated blended fertilizers and nitrolime may havehigh contents of phosphate (P2O5), alkali metals (Na, K), alkaline earth metals(Ca, Mg), etc., causing contamination of packing or damage in quartz parts,
etc. To avoid their influences, it is recommended to add tungsten oxide(elemental analysis reagent or heat-treated reagent) to completely cover the
analytical sample.
Comment 3 When a sample with a low content of organic compounds, such as mixed
fertilizers and designated blended fertilizers etc., and thus with low
combustion efficiency is measured, it is recommended to add sucrose to theanalytical sample so that the carbon content will be comparable to thestandard for calibration curves. Additionally, confirm in advance that sucrose
to be used has a nitrogen content that does not affect the measured value of
total nitrogen of the analytical sample.
Comment 4 Table 2 shows results from replicate testing conducted with mixed fertilizers,
designated blended fertilizers, nitrogenous fertilizers, fluid fertilizers, organic
fertilizers, and compost and sludge fertilizers.
Additionally, Table 3 shows results and analysis results from a collaborative
study for test method validation.
Additionally, the minimum limit of quantification of this test method is about
0.01 % (mass fraction) for fluid fertilizers for home gardening, and 0.05 %(mass fraction) for the other fertilizers.

41

Table 2 Replicate testing on total nitrogen in fertilizers by the combustion method.
Type of fertilizer

Mixed fertilizers

Numberofsamples

Mean 1)(%)
Minimum

3)Standard deviation

2)

(%)

Maximum

4)

Minimum

3)

Relative standard

2)

(%)

Maximum

4)

Minimum

3)

Maximum

15

3.92

18.30

0.01

0.33

0.1

2.0

7

5.20

11.06

0.02

0.17

0.3

1.5

8

15.65

46.56

0.01

0.05

0.03

0.2

6

0.60

16.08

0.002

0.03

0.1

0.4

Organic fertilizers
and compost

5

1.09

10.83

0.01

0.04

0.1

0.7

Sludge fertilizers

5

0.61

8.09

0.004

0.05

0.1

3.1

Designated blended
fertilizers
Nitrogenous
fertilizers
Fluid fertilizers

1) Mean of triplicates.
2) Mass fraction3) Minimum of values obtained from samples.
4) Maximum of values obtained from samples

42

4)

Table 3 Analysis results of results from a collaborative study
for the validation of the total nitrogen test method.
sR

6)

RSDR

(%)

(%)3)

(%)

0.07

0.8

0.25

2.7

18.34

0.06

0.3

0.45

2.5

12

14.06

0.12

0.9

0.42

3.0

Nitrolime

8

19.96

0.07

0.4

0.17

0.8

Fish meal

10

8.34

0.04

0.4

0.10

1.3

Steamed wool waste

11

13.42

0.10

0.7

0.26

2.0

11

6.21

0.07

1.1

0.25

4.0

13

6.20

0.02

0.3

0.09

1.4

Composted sludge fertilizer B

12

2.36

0.01

0.6

0.04

1.8

Human waste sludge fertilizer

11

4.44

0.02

0.4

0.06

1.3

Industrial sludge fertilizer

11

8.06

0.03

0.4

0.07

0.9

Calcined sludge fertilizer

13

0.80

0.02

2.8

0.03

4.3

Sample nameCompound fertilizer (containing nitrate

nitrogen)Compound fertilizer (containing urea)
Designated blended fertilizer (containing

organic fertilizer)

Rape seed meal and powdered rape seedmeal
Composted sludge fertilizer A

1)
2)
3)
4)
5)
6)
7)

4)

RSDr

(%)3)

(%)3)

11

9.32

11

Number oflaboratories

Mean

2)

sr

5)

7)

1)

Number of laboratories used in analysis
Mean (n= number of laboratories x number of samples (2))
Mass fraction
Repeatability standard deviation
Repeatability relative standard deviation
Reproducibility standard deviation
Reproducibility relative standard deviation

References
1) Mariko AIZAWA, Yasushi SUGIMURA, Yuichi TAKAHASHI, Jun OKI, Yukio
FUKUCHI, Yuji SHIRAI and Norio HIKICHI: Validation of a Combustion Method for
Determination of Total Nitrogen Content in Sludge Fertilizer. Research Report of
Fertilizer Vol.1, 2008, (12 - 17)2) Mariko AIZAWA and Yuji SHIRAI: Determination of Total Nitrogen content in Sludge
Fertilizer by a Combustion Method: A Collaborative Study. Research Report of
Fertilizer Vol.1, 2008, (18 - 24)
3) Mariko AIZAWA and Yuji SHIRAI: Validation of a Combustion Method for
Determination of Total Nitrogen Content in Organic Fertilizer. Research Report of
Fertilizer Vol.2, 2009, (6 - 11)4) Mariko AIZAWA and Yuji SHIRAI: Validation of a Combustion Method for
Determination of Total Nitrogen Content in Inorganic Fertilizer. Research Report of
Fertilizer Vol.3, 2010, (1 - 10)5) Mariko AIZAWA, Yuko SEKINE and Yuji SHIRAI: Determination of Total Nitrogen
Content in Fertilizer by a Combustion Method: A Collaborative Study. Research Reportof Fertilizer Vol.3, 2010, (11 - 18)
436) Kazumi UCHIYAMA and Yoshio MAEBASHI: Effective Analysis of Organic Trace
Element, p.99, Mimizuku-sha, Tokyo (2008)(4)

Flow sheet for total nitrogen: The flow sheet for total nitrogen in fertilizers is shown
below:
Analytical sample

Weigh to the order of 1 mg into a combustion vessel

Total nitrogen
analyzer by the
combustion method
Figure Flow sheet for total nitrogen in fertilizers by the combustion method.

Reference: Chromatograms of the standard for calibration curves and an analytical sample
are shown below:

1) Standard for calibration curves (DL-aspartic acid)442) Analytical sample (sludge fertilizer)
Reference figures

Chromatograms of total nitrogen.

Measurement conditions for total nitrogen analyzer by the combustion method
Combustion gas: Highly pure oxygen, purity no less than 99.9999 % (volume fraction),flow rate 200 mL/min
Carrier gas: Highly pure helium, purity no less than 99.9999 % (volume fraction), flow rate
80 mL/min
Separation column: A silica gel stainless column
Detector: Thermal conductivity detector (TCD)
Measurement cycle: Purge time = 60 seconds, circulation combustion time = 200 seconds,
measurement time = 100 seconds
Current value of Detector: 160 mA
Temperature conditions: Reaction furnace temperature: 870 ºC
Reduction furnace temperature: 600 ºC
Column oven temperature: 70 ºC
Detector temperature: 100 ºC

45

4.1.1.c Devarda’s alloy - Kjeldahl method(1) Summary
The method is applicable to the fertilizers that contain nitrate nitrogen (N-N) and guarantee
total nitrogen.
Add hydrochloric acid (1+1) and tin (II) chloride dihydrate to an analytical sample and further
add devarda’s alloy to reduce nitrate nitrogen (N-N), and then add sulfuric acid (1+1), pretreat
by Kjeldahl method to change total nitrogen (T-N) to ammonium ion and add sodiumhydroxide to subject to steam distillation. Measure separated ammonium ion by neutralization
titration to obtain total nitrogen (T-N) in the analytical sample. This test method corresponds
to the sulfuric acid method in the Official Methods of Analysis of Fertilizers (1992).
(2) Reagents: Reagents are as shown below:a) 0.1mol/L - 0.2 mol/L sodium hydroxide solution(1): Transfer about 30 mL of water toa polyethylene bottle, dissolve about 35 g of sodium hydroxide specified in JIS K 8576
by adding in small portions while cooling, seal tightly and leave at rest for 4 - 5 days.
Transfer 5.5 mL - 11 mL of the supernatant to a ground-in stoppered storage container,
and add 1,000 mL of water containing no carbonic acid.
Standardization: Dry sulfamic acid reference material for volumetric analysis
specified in JIS K 8005 by leaving at rest in a desiccator at no more than 2 kPa for about
48 hours, then transfer about 2.5 g to a weighing dish, and measure the mass to the orderof 0.1 mg. Dissolve in a small amount of water, transfer to a 250-mL volumetric flask,
and add water up to the marked line (1). Transfer a predetermined amount of the solution
to a 200-mL - to 300-mL Erlenmeyer flask, add a few drops of bromothymol blue
solution (0.1 mg/100 mL) as an indicator, and titrate with 0.1 mol/L - 0.2 mol/L sodiumhydroxide solution until the color of the solution becomes green. Calculate the factor of
0.1 mol/L - 0.2 mol/L sodium hydroxide solution by the following formula:
Factor of 0.1 mol/L - 0.2 mol/L sodium hydroxide solution (f1)
= (W1 × A × 0.01/97.095) × (V1/V2) × (1,000/V3) × (1/C1)
W1:
A:
V1:
V2:
V3:

Mass (g) of sulfamic acid sampled
Purity (% (mass fraction)) of sulfamic acid
Volume (mL) of sulfamic acid solution transferred
Constant volume (250 mL) of sulfamic acid solution
Volume (mL) of 0.1 mol/L-0.2 mol/L sodium hydroxide solution needed fortitration
C1: Set concentration (mol/L) of 0.1 mol/L - 0.2 mol/L sodium hydroxide
solutionb) Sulfuric acid: A JIS Guaranteed Reagent specified in JIS K 8951 or a reagent ofequivalent quality.
c) 0.25 mol/L sulfuric acid(1) (2): Add about 14 mL of sulfuric acid to a beaker containing
100 mL of water added in advance, stir well, and add water to make 1,000 mL.
Standardization: Transfer a predetermined amount (3) of 0.25 mol/L sulfuric acid to a
200-mL - 300-mL Erlenmeyer flask, add a few drops of methyl red-methylene blue
mixture solution, and titrate with 0.1 mol/L - 0.2 mol/L sodium hydroxide solution until
the color of the solution becomes gray-green(4). Calculate the volume of 0.1 mol/L - 0.2
mol/L sodium hydroxide solution equivalent to1 mL of 0.25 mol/L sulfuric acid, by thefollowing formula (1). Or, calculate the factor of 0.25 mol/L sulfuric acid by thefollowing formula (2):46Volume (B) of 0.1 mol/L - 0.2 mol/L sodium hydroxide solution equivalent to 1 mL
of 0.25 mol/L sulfuric acid= V4/V5………. (1)
Factor of 0.25 mol/L sulfuric acid (f2)
= (f1 × C1 × V4/V5) / (C2 × 2)……….. (2)V4: Volume (mL) of 0.1 mol/L - 0.2 mol/L sodium hydroxide solution needed fortitration
V5: Volume (mL) of 0.25 mol/L sulfuric acid subjected to standardization
C1: Set concentration (mol/L) of 0.1 mol/L - 0.2 mol/L sodium hydroxide solution
C2: Set concentration (0.25 mol/L) of 0.25 mol/L sulfuric acidd) Boric acid solution (40 g/L): Dissolve 40 g of boric acid specified in JIS K 8863 inwater to make 1,000 mL.
e) Hydrochloric acid: A JIS Guaranteed Reagent specified in JIS K 8180 or a reagent ofequivalent quality.
f) Tin (II) chloride dihydrate: A JIS Guaranteed Reagent specified in JIS K 8136 or areagent of mercury analysis grade or equivalent quality.
g) Devarda’s alloy: A reagent of nitrogen analysis grade specified in JIS K 8653 or areagent of equivalent quality.
h) Sodium hydroxide solution (200 g/L - 500 g/L)(1): Dissolve 100 g - 250 g of sodiumhydroxide specified in JIS K 8576 in water to make 500 mL.
i) Bromothymol blue solution (0.1 g/100 mL): Dissolve 0.1 g of bromothymol blue
specified in JIS K 8842 in 20 mL of ethanol (95) specified in JIS K 8102, add water tomake 100 mL.
j) Methyl red solution (0.1 g/100 mL): Dissolve 0.1 g of methyl red specified in JIS K
8896 in 100 mL of ethanol (95) specified in JIS K 8102.
k) Methylene blue solution (0.1 g/100 mL): Dissolve 0.1 g of methylene blue specified in
JIS K 8897 in 100 mL of ethanol (95) specified in JIS K 8102.
l) Methyl red - methylene blue mixture solution: To 2 volumes of methyl red solution(0.1 g/100 mL), add 1 volume of methylene blue solution (0.1 g/100 mL).
m) Bromocresol green solution (0.5 g/100 mL): Dissolve 0.5 g of bromocresol green
specified in JIS K 8840 in 100 mL of ethanol (95) specified in JIS K 8102.
n) Methyl red - bromocresol green mixture solution: To methyl red solution (0.1 g/100mL), add equal volume of bromocresol green solution (0.5 g/100 mL).
Note (1) This is an example of preparation; prepare an amount as appropriate.
(2) This corresponds to the standard sulfuric acid solution 0.5 M (1/2 sulfuric acid)solution in the Official Methods of Analysis of Fertilizers (1992).
(3) 5 mL - 10 mL(4) The endpoint is reached when the color becomes gray-green via dark blue fromblue-purple.
(3)
a)
b)
c)

Apparatus and instruments: Apparatus and instruments are as shown below:
Steam distillation apparatus
Digestion flask: Kjeldahl flask
Distillation flask: A Kjeldahl flask or round bottom flask that can be connected to asteam distillation apparatus.

(4)

Test procedures47

(4.1) Reduction and Kjeldahl method: Conduct reduction and digestion as shown below:a) Weigh 0.5 g - 1 g (no more than the equivalents of N-N 50 mg) of an analytical sample
to the order of 1 mg, and put it in a 300-mL- 500-mL digestion flask (5)b) Add 60 mL of hydrochloric acid (1+1) and 2 g of tin (II) chloride dihydrate, and shaketo mix and leave at rest for about 20 minutes.
c) Add 3.5 g of devarda’s alloy and leave at rest for about 40 minutes while sometimes
shaking to mix.
d) Add 70 mL of sulfuric acid (1+1) and one boiling stone as necessary, and heat at low
temperature (6).
e) As soon as white smoke start evolving, strengthen heating gradually and further
continue heating for about 90 minutes.
f) After standing to cool, add 100 mL - 200 mL of water, mix well by shaking, transfer to a
250-mL - 500-mL volumetric flask with water, and further mix by shaking (7).
g) After standing to cool, add water up to the marked line to make the digestion solution.
Note (5) In the case of direct distillation, a 500-mL Kjeldahl flask connectable to a steam
distillation apparatus is preferable.
(6) If the bubbles foam strongly and excessively, suspend heating for a little while.
(7) It is not necessary to fill up if a whole sample solution is used for themeasurement.
(4.2) Distillation: Conduct distillation as shown below. Specific distillation procedures areaccording to the operation method of the steam distillation apparatus used inmeasurement.
a) Transfer a predetermined amount(8) of 0.25 mol/L sulfuric acid to an acceptor(9), add a
few drops of methyl red - methylene blue mixture solution, and connect this acceptor toa steam distillation apparatus. Or, transfer a predetermined amount(8) of boric acidsolution (40 g/L) to an acceptor(9), add a few drops of methyl red - bromocresol green
mixture solution, and connect this acceptor to a steam distillation apparatus.
b) Transfer a predetermined amount of the digestion solution to a 300-mL distillation flask,
add a proper amount of sodium hydroxide solution (200 g/L – 500 g/L) (10), andimmediately connect this distillation flask to the steam distillation apparatus.
c) Send steam to the distillation flask to heat the solution in the distillation flask, and distill
at a distillation rate of 5 mL/min - 7 mL/min.
d) Stop distilling when the distillate reaches 120 mL - 160 mL.
e) Wash the part of the steam distillation apparatus that came in contact with the solution
in the acceptor with a small amount of water, and pool the washing with the distillate.
Note (8) 5 mL - 20 mL(9) As an acceptor, use a 200-mL - 300mL Erlenmeyer flask or a 200-mL - 300-mL
beaker with which the distillate outlet of the steam distillation apparatus can be
immersed in 0.25 mol/L sulfuric acid or boric acid solution (40 g/L).
(10) A sufficient amount to make the solution strong alkalinity. A blue color willappear.
(4.3) Measurement: Conduct measurement as shown below.
(4.3.1) When 0.25 mol/L sulfuric acid is used in (4.2),a) Titrate the distillate with 0.1 mol/L - 0.2 mol/L sodium hydroxide solution until thecolor of the solution becomes gray-green (4).
b) Calculate the total nitrogen (T-N) in the analytical sample by the following formula:48

Total nitrogen (T-N) (% (mass fraction)) in the analytical sample= (B × V6 - V7) × C1 × f1 × (V8/V9) × (14.007/W2) × (100/1,000)= (B × V6 - V7) × C1 × f1 × (V8/V9) × (1.4007/W2)
B: Volume of 0.1 mol/L - 0.2 mol/L sodium hydroxide solution equivalent to 1
mL of 0.25 mol/L sulfuric acidV6: Volume (mL) of 0.25 mol/L sulfuric acid transferred to the acceptor in (4.2)a)
V7: Volume (mL) of 0.1 mol/L - 0.2 mol/L sodium hydroxide solution needed fortitration
C1: Set concentration (mol/L) of 0.1 mol/L - 0.2 mol/L sodium hydroxide solution
f1: Factor of 0.1 mol/L-0.2 mol/L sodium hydroxide solution
V8: Predetermined volume (mL) of the digestion solution in (4.1) g)
V9: Transferred amount (mL) of the digestion solution subjected to distillation in(4.2) b)
W2: Mass (g) of the analytical sample(4.3.2) When boric acid solution (40 g/L) is used in (4.2),a) Titrate the distillate with 0.25 mol/L sulfuric acid until the color of the solution becomeslight red (11).
b) Calculate the total nitrogen (T-N) in the analytical sample by the following formula:
Total nitrogen (T-N) (% (mass fraction)) in the analytical sample= V10 × C2 × 2×f2 × (V11/V12) × (14.007/W2) × (100/1,000)= V10 × C2 × f2 × (V11/V12) × (2.8014/W2)
V10: Volume (mL) of 0.25 mol/L sulfuric acid needed for titration
C2: Set concentration (mol/L) of 0.25 mol/L sulfuric acidf2: Factor of 0.25 mol/L sulfuric acidV11: Predetermined volume (mL) of the digestion solution in (4.1) g)
V12: Transferred amount (mL) of the digestion solution subjected to distillation in(4.2) b)
W2: Mass (g) of the analytical sample
Note (11) The endpoint is reached when the color changes from green to light red.
Comment 1 The titration procedures in (2) a) Standardization, (2) c) Standardizationand (4.3) can be conducted by an automatic titrator. The setup of the titration
program, the determination parameter for the endpoint and vessels such as
acceptors are according to the specification and the operation method of theautomatic titrator used.
Reference
1) Masayoshi KOSHINO: Second Revision of The Methods of Analysis of Fertilizers(Details), p.31-33, Yokendo, Tokyo (1988)

49

(5)

Flow sheet for total nitrogen: The flow sheet for total nitrogen in fertilizers is shown
below:
0.5 g – 1 ganalytical sample
Weigh to the order of 1 mg into a 300-mL - 500-mL digestion flask.
← 60 mL of hydrochloric acid (1+1)← 2 g of tin (II) chloride dihydrate
Leaving at rest
About 20 minutes← 3.5 g devardar's alloy
Leaving at rest
About 40 minutes← 70 mL sulfuric acid (1+1)← 1 boiling stone
Heat at low temperature, and as soon as white smoke evolves,
Heating
strengthen heating gradually and further heat for about 90 minutes.
Standing to cool
← 100 mL - 200 mL Water
Transfer
250-mL - 500-mL volumetric flask, waterStanding to coolRoom temperature← Water (up to the marked line)
Aliquot
300-mL distillation flask(predetermined volume)← Sodium hydroxide (200 g/L - 500 g/L)
Receiver: 200-mL - 300-mL Erlenmeyer flask or beaker
Steam distillation
A predetermined amount of 0.25 mol/L sulfuric acid and a few drops ofapparatus
methyl red-methylene blue mixture solution, or boric acid solution (40 g/L),
several drops of methyl red - bromocresol green mixture solution
Steam distillation

Distillation rate: 5 mL/min - 7 mL/min

Stop distilling
120 mL – 160 mL distillate← Water (wash the part of the distillation apparatus
that came in contact with the solution in the receiver)
0.1 mol/L - 0.2 mol/L sodium hydroxide solution until the color of the
Titration
solution becomes gray-green, or
0.25 mol/L sulfuric acid (until the solution becomes light red)
Figure Flow sheet (example) for total nitrogen in fertilizers.

50

4.1.1.d Reduced iron - Kjeldahl method(1) Summary
The method is applicable to the fertilizers that contain nitrate nitrogen (N-N) and guarantee
total nitrogen.
Add water, reduced iron and sulfuric acid (1+1) to an analytical sample to reduce nitrate
nitrogen (N-N) and heat at low temperature, and then add sulfuric acid and pretreat by
Kjeldahl method to change total nitrogen (T-N) to ammonium ion, and add sodium hydroxide
to subject to steam distillation. Measure separated ammonium ion by neutralization titration to
obtain total nitrogen (T-N) in the analytical sample. This test method corresponds to thesulfuric acid method in the Official Methods of Analysis of Fertilizers (1992).
(2) Reagents: Reagents are as shown below:a) 0.1 mol/L - 0.2 mol/L sodium hydroxide solution(1): Transfer about 30 mL of water toa polyethylene bottle, dissolve about 35 g of sodium hydroxide specified in JIS K 8576
by adding in small portions while cooling, seal tightly and leave at rest for 4-5 days.
Transfer 5.5 mL - 11 mL of the supernatant to a ground-in stoppered storage container,
and add 1,000 mL of water containing no carbonic acid.
Standardization: Dry sulfamic acid reference material for volumetric analysis
specified in JIS K 8005 by leaving at rest in a desiccator at no more than 2 kPa for about
48 hours, then transfer about 2.5 g to a weighing dish, and measure the mass to the orderof 0.1 mg. Dissolve in a small amount of water, transfer to a 250-mL volumetric flask,
and add water up to the marked line.(1) Transfer a predetermined amount of the solution
to a 200-mL - 300-mL Erlenmeyer flask, add a few drops of bromothymol blue solution(0.1 mg/100 mL) as an indicator, and titrate with 0.1 mol/L - 0.2 mol/L sodiumhydroxide solution until the color of the solution becomes green. Calculate the factor of
0.1 mol/L - 0.2 mol/L sodium hydroxide solution by the following formula:
Factor of 0.1 mol/L - 0.2 mol/L sodium hydroxide solution (f1)
= (W1 × A × 0.01/97.095) × (V1/V2) × (1,000/V3) × (1/C1)
W1:
A:
V1:
V2:
V3:
C1 :

Mass (g) of sulfamic acid sampled
Purity (% (mass fraction)) of sulfamic acid
Volume (mL) of sulfamic acid solution transferred
Constant volume (250 mL) of sulfamic acid solution
Volume (mL) of 0.1 mol/L - 0.2 mol/L sodium hydroxide solution needed
for titration
Set concentration (mol/L) of 0.1 mol/L - 0.2 mol/L sodium hydroxide
solutionb) Sulfuric acid: A JIS Guaranteed Reagent specified in JIS K 8951 or a reagent ofequivalent quality.
c) 0.25 mol/L sulfuric acid(1) (2): Add about 14 mL of sulfuric acid to a beaker containing
100 mL of water in advance, stir well, and add water to make 1,000 mL.
Standardization: Transfer a predetermined amount (3) of 0.25 mol/L sulfuric acid to a
200-mL - 300-mL Erlenmeyer flask, add a few drops of methyl red-methylene blue
mixture solution, and titrate with 0.1 mol/L - 0.2 mol/L sodium hydroxide solution until
the color of the solution becomes gray-green.(4) Calculate the volume of 0.1 mol/L - 0.2
mol/L sodium hydroxide solution equivalent to 1 mL of 0.25 mol/L sulfuric acid by thefollowing formula (1). Or, calculate the factor of 0.25 mol/L sulfuric acid by thefollowing formula (2):51

Volume (B) of 0.1 mol/L - 0.2 mol/L sodium hydroxide solution equivalent to 1 mL
of 0.25 mol/L sulfuric acid= V4/V5…… (1)
Factor of 0.25 mol/L sulfuric acid (f2)
= (f1 × C1 × V4/V5)/ (C2 × 2)…… (2)

V4: Volume (mL) of 0.1 mol/L - 0.2 mol/L sodium hydroxide solution needed fortitration
V5: Volume (mL) of 0.25 mol/L sulfuric acid subjected to standardization
C1: Set concentration (mol/L) of 0.1 mol/L - 0.2 mol/L sodium hydroxide
solution
C2: Set concentration (0.25 mol/L) of 0.25 mol/L sulfuric acidd) Boric acid solution (40 g/L): Dissolve 40 g of boric acid specified in JIS K 8863 inwater to make 1,000 mL.
e) Reduced iron: Nitrogen content is no more than 0.005 % (mass fraction)f) Catalyst (5): Mix potassium sulfate specified in JIS K 8962 and copper (II) sulfate
pentahydrate (6) specified in JIS K 8983 in the ratio of 9 to 1.
g) Sodium hydroxide solution (200 g/L - 500 g/L)(1): Dissolve 100 g - 250 g of sodiumhydroxide specified in JIS K 8576 in water to make 500 mL.
h) Bromothymol blue solution (0.1 g/100 mL): Dissolve 0.1 g of bromothymol blue
specified in JIS K 8842 in 20 mL of ethanol (95) specified in JIS K 8102, and add water
to make 100 mL.
i) Methyl red solution (0.1 g/100 mL): Dissolve 0.1 g of methyl red specified in JIS K
8896 in 100 mL of ethanol (95) specified in JIS K 8102.
j) Methylene blue solution (0.1 g/100 mL): Dissolve 0.1 g of methylene blue specified in
JIS K 8897 in 100 mL of ethanol (95) specified in JIS K 8102.
k) Methyl red - methylene blue mixture solution: To 2 volumes of methyl red solution(0.1 g/100 mL), add 1 volume of methylene blue solution (0.1 g/100 mL).
l) Bromocresol green solution (0.5 g/100 mL): Dissolve 0.5 g of bromocresol green
specified in JIS K 8840 in 100 mL of ethanol (95) specified in JIS K 8102.
m) Methyl red - bromocresol green mixture solution: To methyl red solution (0.1 g/100mL), add equal volume of bromocresol green solution (0.5 g/100 mL).
Note (1) This is an example of preparation; prepare an amount as appropriate.
(2) This corresponds to the standard sulfuric acid solution 0.5 M (1/2 sulfuric acid)solution in the Official Methods of Analysis of Fertilizers (1992).
(3) 5 mL - 10 mL(4) The endpoint is reached when the color becomes gray-green via dark blue fromblue-purple.
(5) A tablet is commercially available.
(6) Crush into powder as appropriate.
(3)
a)
b)
c)

Instruments: Instruments are as shown below:
Steam distillation apparatus
Digestion flask: Kjeldahl flask
Distillation flask: A Kjeldahl flask or a round bottom flask that can be connected to asteam distillation apparatus.
(4) Test procedures(4.1) Reduction and Kjeldahl method: Conduct reduction and digestion as shown below:
52a) Weigh 0.5 g - 1 g of an analytical sample to the order of 1 mg, and put it in a 300-mL 500-mL digestion flask.
b) Add 30 mL of water and mix well.
c) As soon as 5 g of reduced iron and 30mL of sulfuric acid (1+1) are added, insert a longstem funnel to a digestion flask and shake to mix gently while cooling the outside of thecontainer under flowing water(7).
d) Leave at rest for about 5 minutes (8), boil in a low flame for about 15 minutes.
e) After standing to cool, add 5 g - 10 g of catalyst, 30 mL of sulfuric acid and, if
necessary, one boiling stone, heat gradually until water evaporates and white smoke ofsulfuric acid evolves(9).
f) Ignite until it is completely digested (10).
g) After standing to cool, add a small amount of water, mix well by shaking, transfer to a
250-mL - 500-mL volumetric flask with water, and further mix by shaking.
h) After standing to cool, add water up to the marked line to make the digestion solution.

Note (7) A sudden reaction generates heat, and unreacted nitric acid vaporizes or digests
to make nitrogen oxide etc. through which process losses occur easily. Carefuland efficient operation should be taken(8) Until a sudden reaction is settled.
(9) If the bubbles foam strongly and excessively, suspend heating for a little while.
(10) After the color of the solution stops changing, heat for no less than 2 hours.
(4.2) Distillation: Conduct distillation as shown below. Specific distillation procedures areaccording to the operation method of the steam distillation apparatus used inmeasurement.
a) Transfer a predetermined amount (11) of 0.25 mol/L sulfuric acid to an acceptor (12), add a
few drops of methyl red - methylene blue mixture solution, and connect this acceptor toa steam distillation apparatus. Or, transfer a predetermined amount (11) of boric acidsolution (40 g/L) to an acceptor (12), add a few drops of methyl red - bromocresol green
mixture solution, and connect this acceptor to a steam distillation apparatus.
b) Transfer a predetermined amount of the digestion solution to a 300-mL distillation flask,
add a proper amount of sodium hydroxide solution (200 g/L - 500 g/L) (13), andimmediately connect this distillation flask to the steam distillation apparatus.
c) Send steam to the distillation flask to heat the solution in the distillation flask, and distill
at a distillation rate of 5 mL /min - 7 mL /min.
d) Stop distilling when the distillate reaches 120 mL - 160 mL.
e) Wash the part of the steam distillation apparatus that came in contact with the solution
in the acceptor with a small amount of water, and pool the washing with the distillate.
Note (11) 5 mL - 20 mL(12) As an acceptor, use a 200-mL – 300-mL Erlenmeyer flask or a 200-mL - 300-mL
beaker with which the distillate outlet of the steam distillation apparatus can be
immersed in 0.25 mol/L sulfuric acid or boric acid solution (40 g/L).
(13) A sufficient amount to make the solution strong alkalinity. A blue or reddish
brown color will appear.
(4.3) Measurement: Conduct measurement as shown below.
(4.3.1) When 0.25 mol/L sulfuric acid is used in (4.2),a) Titrate the distillate with 0.1 mol/L - 0.2 mol/L sodium hydroxide solution until thecolor of the solution becomes gray-green (4).
53

b) Calculate the total nitrogen (T-N) in the analytical sample by the following formula:
Total nitrogen (T-N) (% (mass fraction)) in the analytical sample= (B × V6 - V7) × C1 × f1 × (V8/V9) × (14.007/W2) × (100/1,000)= (B × V6 - V7) × C1 × f1 × (V8/V9) × (1.4007/W2)
B: Volume of 0.1 mol/L - 0.2 mol/L sodium hydroxide solution equivalent to 1
mL of 0.25 mol/L sulfuric acidV6: Volume (mL) of 0.25 mol/L sulfuric acid transferred to the acceptor in (4.2)a)
V7: Volume (mL) of 0.1 mol/L - 0.2 mol/L sodium hydroxide solution needed fortitration
C1: Set concentration (mol/L) of 0.1 mol/L - 0.2 mol/L sodium hydroxide solution
f1: Factor of 0.1 mol/L - 0.2 mol/L sodium hydroxide solution
V8: Predetermined volume (mL) of the digestion solution in (4.1) e)
V9: Transferred amount (mL) of the digestion solution subjected to distillation in(4.2) b)
W2: Mass (g) of the analytical sample(4.3.2) When boric acid solution (40 g/L) is used in (4.2),a) Titrate the distillate with 0.25 mol/L sulfuric acid until the color of the solution becomeslight red (14).
b) Calculate the total nitrogen (T-N) in the analytical sample by the following formula:
Total nitrogen (T-N) (% (mass fraction)) in the analytical sample= V10 × C2 × 2×f2 × (V11/V12) × (14.007/W2) × (100/1,000)= V10 × C2 × f2 × (V11/V12) × (2.8014/W2)
V10: Volume (mL) of 0.25 mol/L sulfuric acid needed for titration
C2: Set concentration (mol/L) of 0.25 mol/L sulfuric acidf2: Factor of 0.25 mol/L sulfuric acidV11: Predetermined volume (mL) of the digestion solution in (4.1) e)
V12: Transferred amount (mL) of the digestion solution subjected to distillation in(4.2) b)
W2: Mass (g) of the analytical sample
Note (14) The endpoint is reached when the color changes from green to light red.
Comment 1 The titration procedures in (2) a) Standardization, (2) c) Standardizationand (4.3) can be conducted by an automatic titrator. The setup of the titration
program, the determination parameter for the endpoint and vessels such as
acceptors are according to the specification and the operation method of theautomatic titrator used.
Reference
1) Masayoshi KOSHINO: Second Revision of The Methods of Analysis of Fertilizers(Details), p.33 - 34, Yokendo, Tokyo (1988)

54

(5)

Flow sheet for total nitrogen: The flow sheet for total nitrogen in fertilizers is shown
below:
0.5 g - 1 g
Weigh to the order of 1 mg into a 300-mL digestion flask.
analytical sample← 30 mL water← 5 g reduced iron
← 30 mL sulfuric acid (1+1)
Immediately insert a long stem funnel to the flask and shake to mix
Mixturewhile cooling outside the container with flowing water.
Leaving at rest
Heating

For about 5 min (Until a sudden reaction is settled.)
Boil in a low flame for about 15 minutes.

Standing to cool
← 5 g - 10 g catalyst← 30 mL sulfuric acidHeat gradually until water evaporates and the white smoke
Heatingof sulfuric acid evolves and ignite to digest completely
Standing to cool
← A small amount of waterTransfer
250-mL - 500-mL volumetric flask, waterStanding to coolRoom temperature← Water (up to the marked line)
Aliquot
Distillation flask(predetermined volume)← Sodium hydroxide (200 g/L - 500 g/L)
Receiver: 200-mL - 300-mL Erlenmeyer flask or beaker
Steam distillation
A predetermined amount of 0.25 mol/L sulfuric acid and a few drops ofapparatus
methyl red-methylene blue mixture solution, or boric acid solution (40 g/L),
several drops of methyl red - bromocresol green mixture solution
Steam distillation

Distillation rate: 5 mL/min - 7 mL/min

Stop distilling
120 mL – 160 mL distillate← Water (wash the part of the distillation apparatus
that came in contact with the solution in the receiver)0.1mol/L - 0.2 mol/L sodium hydroxide solution (until the
Titration
solution becomes gray-green), or
0.25 mol/L sulfuric acid (until the solution becomes light red)
Figure Flow sheet (example) for total nitrogen in fertilizers.

55

4.1.1.e Calculation with ammoniac nitrogen and nitrate nitrogen(1) Summary
The calculation is applicable to the fertilizers that contain ammoniac nitrogen (A-N) andnitrate nitrogen (N-N), and that does not contain the fertilizers guaranteeing total nitrogen(T-N).
Calculate total nitrogen (T-N) by adding the ammoniac nitrogen (A-N) obtained in 4.1.2 to thenitrate nitrogen (N-N) obtained in 4.1.3.
(2) The calculation of total nitrogena) Calculate the total nitrogen (T-N) in the analytical sample by the following formula:
Total nitrogen (T-N) (% (mass fraction)) in the analytical sample= (A-N) + (N-N)
A-N: Ammoniac nitrogen (% (mass fraction)) in the analytical sample (1) obtainedby 4.1.2
N-N: Nitrate nitrogen (% (mass fraction)) in the analytical sample (1) obtained by
4.1.3
Note (1) A-N and N-N use raw data without rounding numerical value

56

4.1.2 Ammoniac nitrogen
4.1.2.a Distillation method(1) Summary
The testing method is applicable to the fertilizers that contain ammonium salt. However, insome cases, it is not applicable to the fertilizers that contain such compounds as nitrolime thatdigests by heating.
Add water to an analytical sample, further add sodium hydroxide solution or magnesiumoxide to make the solution alkalinity and subject it to steam distillation. Measure separated
ammonium ion by neutralization titration to obtain ammoniac nitrogen (A-N) in the analyticalsample.
(2) Reagents: Reagents are as shown below:a) 0.1 mol/L - 0.2 mol/L sodium hydroxide solution(1): Transfer about 30 mL of water toa polyethylene bottle, dissolve about 35 g of sodium hydroxide specified in JIS K 8576
by adding in small portions while cooling, seal tightly and leave at rest for 4 - 5 days.
Transfer 5.5 mL - 11 mL of the supernatant to a ground-in stoppered storage container,
and add 1,000 mL of water containing no carbonic acid.
Standardization: Dry sulfamic acid reference material for volumetric analysis
specified in JIS K 8005 by leaving at rest in a desiccator at no more than 2 kPa for about
48 hours, then transfer about 2.5 g to a weighing dish, and measure the mass to the orderof 0.1 mg. Dissolve in a small amount of water, transfer to a 250-mL volumetric flask,
and add water up to the marked line.(1) Transfer a predetermined amount of the solution
to a 200-mL - 300-mL Erlenmeyer flask, add a few drops of bromothymol blue solution(0.1 mg/100 mL) as an indicator, and titrate with 0.1 mol/L - 0.2 mol/L sodiumhydroxide solution until the color of the solution becomes green. Calculate the factor of
0.1 mol/L - 0.2 mol/L sodium hydroxide solution by the following formula:
Factor of 0.1 mol/L - 0.2 mol/L sodium hydroxide solution (f1)
= (W1 × A × 0.01/97.095) × (V1/V2) × (1,000/V3) × (1/C1)
W1: Mass (g) of sulfamic acid sampled
A: Purity (% (mass fraction)) of sulfamic acidV1: Volume (mL) of sulfamic acid solution transferred
V2: Constant volume (250 mL) of sulfamic acid solution
V3: Volume (mL) of 0.1 mol/L - 0.2 mol/L sodium hydroxide solution needed fortitration
C1: Set concentration (mol/L) of 0.1 mol/L - 0.2 mol/L sodium hydroxide solutionb) Magnesium oxide: A JIS Guaranteed Reagent specified in JIS K 8432 or a reagent ofequivalent quality.
c) Sulfuric acid: A JIS Guaranteed Reagent specified in JIS K 8951 or a reagent ofequivalent quality.
d) 0.25 mol/L sulfuric acid(1) (2): Add about 14 mL of sulfuric acid to a beaker containing
100 mL of water in advance, stir well, and add water to make 1,000 mL.
Standardization: Transfer a predetermined amount(3) of 0.25 mol/L sulfuric acid to a
200-mL - 300-mL Erlenmeyer flask, add a few drops of methyl red-methylene blue
mixture solution, and titrate with 0.1 mol/L - 0.2 mol/L sodium hydroxide solution until
the color of the solution becomes gray-green.(4) Calculate the volume of 0.1 mol/L - 0.2
mol/L sodium hydroxide solution equivalent to 1 mL of 0.25 mol/L sulfuric acid by the57

following formula (1). Or, calculate the factor of 0.25 mol/L sulfuric acid by thefollowing formula (2):Volume (B) of 0.1 mol/L - 0.2 mol/L sodium hydroxide solution equivalent to 1 mL
of 0.25 mol/L sulfuric acid= V4/V5…….. (1)
Factor of 0.25 mol/L sulfuric acid (f2)
= (f1 × C1 × V4/V5)/ (C2 × 2)…….. (2)V4: Volume (mL) of 0.1 mol/L - 0.2 mol/L sodium hydroxide solution needed fortitration
V5: Volume (mL) of 0.25 mol/L sulfuric acid subjected to standardization
C1: Set concentration (mol/L) of 0.1 mol/L - 0.2 mol/L sodium hydroxide solution
C2: Set concentration (0.25 mol/L) of 0.25 mol/L sulfuric acide) Boric acid solution (40 g/L): Dissolve 40 g of boric acid specified in JIS K 8863 inwater to make 1,000 mL.
f) Sodium hydroxide solution (200 g/L - 500 g/L)(1): Dissolve 100 g - 250 g of sodiumhydroxide specified in JIS K 8576 in water to make 500 mL.
g) Bromothymol blue solution (0.1 g/100 mL): Dissolve 0.1 g of bromothymol blue
specified in JIS K 8842 in 20 mL of ethanol (95) specified in JIS K 8102, add water tomake 100 mL.
h) Methyl red solution (0.1 g/100 mL): Dissolve 0.1 g of methyl red specified in JIS K
8896 in 100 mL of ethanol (95) specified in JIS K 8102.
i) Methylene blue solution (0.1 g/100 mL): Dissolve 0.1 g of methylene blue specified in
JIS K 8897 in 100 mL of ethanol (95) specified in JIS K 8102.
j) Methyl red - methylene blue mixture solution: To 2 volumes of methyl red solution(0.1 g/100 mL), add 1 volume of methylene blue solution (0.1 g/100 mL).
k) Bromocresol green solution (0.5 g/100 mL): Dissolve 0.5 g of bromocresol green
specified in JIS K 8840 in 100 mL of ethanol (95) specified in JIS K 8102.
l) Methyl red - bromocresol green mixture solution: To methyl red solution (0.1 g/100mL), add equal volume of bromocresol green solution (0.5 g/100 mL).
Note (1) This is an example of preparation; prepare an amount as appropriate.
(2) This corresponds to the standard sulfuric acid solution 0.5 M (1/2 sulfuric acid)solution in the Official Methods of Analysis of Fertilizers (1992).
(3) 5 mL - 10 mL(4) The endpoint is reached when the color becomes gray-green via dark blue fromblue-purple.
(3) Apparatus and Instruments: Instruments are as shown below:a) Rotary shaker: A rotary shaker that can rotate a 500-mL volumetric flask upside down
at 30 - 40 revolutions/min.
b) Steam distillation apparatusc) Distillation flask: A Kjeldahl flask or a round bottom flask that can be connected to asteam distillation apparatus.
(4) Test procedures(4.1) Sample solution preparation: Prepare a sample solution as shown below:
58a) Weigh 0.25 g - 2g (5) (the equivalents of 20 mg – 100 mg as N) of an analytical sample
to the order of 1 mg, and put it in a 300-mL - 500-mL distillation flaskb) Add about 25 mL of water to make the sample solution.
Note (5) The sampling amount of the analytical sample is 5 g when there is less nitrogencontent in the fertilizers such as a home garden-use fertilizer.

Comment 1 When it is a fertilizer which contains uric ammonium acid, humus acidammonium or nitrate nitrogen, etc., or when it is not a fertilizer in which
phosphate, ammonium and magnesium coexist, conduct the procedure in(4.1) a) - c) in 4.2.4.a and transfer a predetermined amount of suspension (theequivalents of 20 mg – 100 mg as N) to a 300-mL - 500-mL distillation flask
to make the sample solution.
(4.2) Distillation: Conduct distillation as shown below. Specific distillation procedures areaccording to the operation method of the steam distillation apparatus used inmeasurement.
a) Transfer a predetermined amount(5) of 0.25 mol/L sulfuric acid to an acceptor (6), add a
few drops of methyl red - methylene blue mixture solution, and connect this acceptor toa steam distillation apparatus. Or, transfer a predetermined amount (5) of boric acidsolution (40 g/L) to an acceptor (6), add a few drops of methyl red - bromocresol green
mixture solution, and connect this acceptor to a steam distillation apparatus.
b) Add a proper amount (7) of sodium hydroxide solution (200 g/L - 500 g/L) (8) to the
distillation flask, and connect this distillation flask to a steam distillation apparatus.
c) Send steam to the distillation flask to heat the solution in the distillation flask, and distill
at a distillation rate of 5 mL/min - 7 mL/min.
d) Stop distilling when the distillate reaches 120 mL - 160 mL.
e) Wash the part of the steam distillation apparatus that came in contact with the solution
in the acceptor with a small amount of water, and pool the washing with the distillate.
Note (5) 5 mL - 20 mL(6) As an acceptor, use a 200-mL - 300-mL Erlenmeyer flask or a 200-mL - 300-mL
beaker with which the distillate outlet of the steam distillation apparatus can be
immersed in 0.25 mol/L sulfuric acid or boric acid solution (40 g/L).
(7) A sufficient amount to make the solution strong alkalinity.
(8) Add a small amount of silicone oil as necessary.
Comment 2 When the sample contains organic matters or urea, add no less than 2 g ofmagnesium oxide instead of sodium hydroxide solution.
(4.3) Measurement: Conduct measurement as shown below.
(4.3.1) When 0.25 mol/L sulfuric acid is used in (4.2)a) Titrate the distillate with 0.1 mol/L - 0.2 mol/L sodium hydroxide solution until thecolor of the solution becomes gray-green (4).
b) Calculate the ammoniac nitrogen (A-N) in the analytical sample by the following
formula:Ammoniac nitrogen (A-N) (% (mass fraction)) in the analytical sample= (B × V6 - V7) × C1 × f1 × (14.007/W2) × (100/1,000)= (B × V6 - V7) × C1 × f1 × (1.4007/W2)
59

B: Volume of 0.1 mol/L - 0.2 mol/L sodium hydroxide solution equivalent to 1
mL of 0.25 mol/L sulfuric acidV6: Volume (mL) of 0.25 mol/L sulfuric acid transferred to the acceptor in (4.2)a)
V7: Volume (mL) of 0.1 mol/L - 0.2 mol/L sodium hydroxide solution needed fortitration
C1: Set concentration (mol/L) of 0.1 mol/L - 0.2 mol/L sodium hydroxide solution
f1: Factor of 0.1 mol/L - 0.2 mol/L sodium hydroxide solution
W2: Mass (g) of the analytical sample(4.3.2) When boric acid solution (40 g/L) is used in (4.2),
a)Titrate the distillate with 0.25 mol/L sulfuric acid until the color of the solution
becomes light red (9).
b) Calculate the ammoniac nitrogen (A-N) in the analytical sample by the following
formula:Ammoniac nitrogen (A-N) (% (mass fraction)) in the analytical sample= V8 × C2 × 2×f2 × (14.007/W2) × (100/1,000)= V10 × C2 × f2 × (2.8014/W2)
V8: Volume (mL) of 0.25 mol/L sulfuric acid needed for titration
C2: Set concentration (mol/L) of 0.25 mol/L sulfuric acidf2: Factor of 0.25 mol/L sulfuric acid
W2: Mass (g) of the analytical sample
Note (9) The endpoint is reached when the color changes from green to light red.
Comment 3 If it is hard to confirm the endpoint due to the carbon dioxide resulting fromcarbonate in the extract when magnesium oxide is used, it is recommended toboil the extract for 1-2 minute(s) after distilling and cool, and then titrate.
Comment 4 The titration procedures in (2) a) Standardization, (2) d) Standardizationand (4.3) can be conducted by an automatic titrator. The setup of the titration
program, the determination parameter for the endpoint and vessels such as
acceptors are according to the specification and the operation method of theautomatic titrator used.
Comment 5 Table 1 shows results from a simultaneous analysis with the same samples(proficiency testing and external quality control testing) hosted by the Japan
Fertilizer Quality Assurance Committee analyzed by the robust method.

60

Table 1

Results and analysis results from simultaneous analysis with the same samples
for ammonium-nitrogen hosted by the Japan Fertilizer Quality Assurance Committee1)
2)

Year

SampleNumber of Median Μlaboratories
(%)3)

4)

5)

NIQR

RSDrob(%)3)(%)

Organic compound
143
6.20
0.09
1.4
fertilizer
High analysis
2008
147
12.56
0.16
1.2
compound fertilizer
Ordinary compound
2009
138
5.56
0.07
1.3
fertilizer
High analysis
2011
130
13.50
0.17
1.3
compound fertilizer
2012
Fluid mixed fertilizer
120
2.41
0.04
1.8
High analysis
2013
130
9.92
0.75
7.5
compound fertilizer
1) Proficiency testing and external quality control testing2) Median (M) agrees with the mean in normal distribution.
3) Mass fraction4) Robust standard deviation (NIQR) agrees with standard deviation in normal distribution.
5) RSDrob stands for relative standard deviation obtained from the robust methodby the formula shown below:
RSDrob = (NIQR/M) × 100
2007

Reference
1) Masayoshi KOSHINO: Second Revision of The Methods of Analysis of Fertilizers(Details), p.36 - 37, Yokendo, Tokyo (1988)

61

(5) Flow sheet for ammoniac nitrogen: The flow sheet for ammoniac nitrogen in fertilizers
is shown below.
0.25 g - 2 g
Weigh the equivalents of 20 mg – 100 mg as N
analytical sample
to the order of 1 mg, and put it in a 300-mL distillation flask← About 25 mL water← Water (up to the marked line)← Sodium hydroxide (200 g/L - 500 g/L)
(Magnesium oxide no less than 2 g when it contains organic matters)← A small amount of defoaming agent as necessary
Receiver: 200-mL - 300-mL Erlenmeyer flask or beaker
Steam distillation
A predetermined amount of 0.25 mol/L sulfuric acid and a few drops ofapparatus
methyl red-methylene blue mixture solution, or boric acid solution (40 g/L),
several drops of methyl red - bromocresol green mixture solution
Steam distillation

Distillation rate: 5 mL/min - 7 mL/min

Stop distilling
120 mL – 160 mL distillate← Water (wash the part of the distillation apparatus
that came in contact with the solution in the receiver)
0.1 mol/L - 0.2 mol/L sodium hydroxide solution (until the
Titration
solution becomes gray-green), or
0.25 mol/L sulfuric acid (until the solution becomes light red)
Figure Flow sheet (example) for ammoniac nitrogen in fertilizers.

624.1.2.b Formaldehyde method(1) Summary
The testing method is applicable to the fertilizers which do not contain a large amount of floraand fauna sample.
After adding potassium chloride solution to an analytical sample to extract ammonium ion,
add aluminum chloride solution, drop potassium hydroxide solution and precipitate phosphateand excessive aluminum to make the sample solution. Adjust the sample solution to slight
acidity, add formaldehyde solution and measure ammonium ion by neutralization titration to
obtain ammoniac nitrogen (A-N) in the analytical sample.
(2) Reagents: Reagents are as shown below:a) 0.1 mol/L - 0.2 mol/L sodium hydroxide solution(1): Transfer about 30 mL of water toa polyethylene bottle, dissolve about 35 g of sodium hydroxide specified in JIS K 8576
by adding in small portions while cooling, seal tightly and leave at rest for 4 - 5 days.
Transfer 5.5 mL - 11 mL of the supernatant to a ground-in stoppered storage container,
and add 1,000 mL of water containing no carbonic acid.
Standardization: Dry sulfamic acid reference material for volumetric analysis
specified in JIS K 8005 by leaving it at rest in a desiccator at no more than 2 kPa for
about 48 hours, then transfer about 2.5 g to a weighing dish, and measure the mass tothe order of 0.1 mg. Dissolve this in a small amount of water, transfer to a 250-mL
volumetric flask, and add water up to the marked line (1). Transfer a predeterminedamount of the solution to a 200-mL - 300-mL Erlenmeyer flask, add a few drops of
bromothymol blue solution (0.1 mg/100 mL) as an indicator, and titrate with 0.1 mol/L 0.2 mol/L sodium hydroxide solution until the color of the solution becomes green.
Calculate the factor of 0.1 mol/L - 0.2 mol/L sodium hydroxide solution by thefollowing formula:
Factor of 0.1mol/L - 0.2 mol/L sodium hydroxide solution (f)= (W × A × 0.01/97.095) × (V1/V2) × (1,000/V3) × (1/C)
W: Mass (g) of sulfamic acid sampled
A: Purity (% (mass fraction)) of sulfamic acidV1: Volume (mL) of sulfamic acid solution transferred
V2: Constant volume (250 mL) of sulfamic acid solution
V3: Volume (mL) of 0.1 mol/L - 0.2 mol/L sodium hydroxide solution needed fortitration
C: Set concentration (mol/L) of 0.1 mol/L - 0.2 mol/L sodium hydroxide solutionb) Potassium chloride solution (1 mol/L) (1): Dissolve 75 g of potassium chloride
specified in JIS K 8121 in water to make 1,000 mL.
c) Aluminum chloride solution (1 mol/L) (1): Dissolve 240 g of aluminum chloride
specified in JIS K 8114 in water to make 1,000 mL.
d) Potassium hydroxide solution (170 g/L) (1): Dissolve 170 g of potassium hydroxide inwater to make 1,000 mL.
e) Formaldehyde solution: Add one volume of water to one volume of the 36 % (massfraction) – 38 % (mass fraction) formaldehyde specified in JIS K 8872.
f) Hydrochloric acid: A JIS Guaranteed Reagent specified in JIS K 8180 or a reagent ofequivalent quality.
g) Bromothymol blue solution (0.1 g/100 mL): Dissolve 0.1 g of bromothymol blue
specified in JIS K 8842 in 20 mL of ethanol (95) specified in JIS K 8102, add water tomake 100 mL.
63h) Methyl red solution (0.1 g/100 mL): Dissolve 0.1 g of methyl red specified in JIS K
8896 in 100 mL of ethanol (95) specified in JIS K 8102.
i) Thymol blue solution (1 g/100 mL): Dissolve 1 g of thymol blue (sodium salt) in 20
mL of ethanol (95) specified in JIS K 8102.
Note (1) This is an example of preparation. Prepare necessary amount of solution.
Comment 1 Thymol blue can be dissolved if it is sodium salt. The thymol blue specified
in JIS K 8643 is slightly hard to dissolve in ethanol and hard to dissolve inwater. Therefore add about 2.15 mL of sodium hydroxide solution (0.1 mol/L)
per 0.1 g of thymol blue to neutralize, and then prepare the thymol blue
solution (1 g/100 mL) through the same procedure as (2) i).
(3) Instruments: Instruments are as shown below:a) Rotary shaker: A rotary shaker that can rotate a 500-mL volumetric flask upside
down at 30 - 40 revolutions/min.
(4) Test procedures(4.1) Extraction: Conduct extraction as shown below.
(4.1.1) Ammonium saltsa) Weigh 5g of an analytical sample to the order of 1 mg, and put it in a 500-mL
volumetric flaskb) Add 400 mL of water and shake to mix at the rate of 30-40 revolutions/min for about 30
minutes.
c) Add water up to the marked line.
d) Filter with Type 3 filter paper to make the sample solution.
Comment 2

The procedure in (4.1) is the same as the procedure in (4.1) in 4.2.4.a.

(4.1.2) Mixed fertilizersa) Weigh 5 g of an analytical sample to the order of 1 mg, and put it in a 500-mL
volumetric flaskb) Add about 300 mL of hydrochloric acid (1 + 20) and shake to mix at the rate of 30 - 40
revolutions/min for about 30 minutes.
c) Add aluminum chloride solution (1 mol/L) (2) to the solution and add a few drops ofmethyl red solution (1mol/L) as an indicator. After that, add immediately potassiumhydroxide (170 g/L) while shaking the flask until the color of the solution changes tolight yellow (3).
d) Add water up to the marked line.
e) Filter with Type 3 filter paper to make the sample solution.
Note (2) Add 3 mL of aluminum chloride per 0.04 g of P or 0.1 g of P2O5 in the sample
solution.
(3) Form precipitate of aluminum hydroxide and aluminum phosphate to separate
the phosphate.
Comment 3 In the procedure of (4.1.1) a) and (4.1.2) a), it is also allowed to weigh 2.5gof an analytical sample to the order of 1 mg, and put it in a 250-mL
volumetric flask
Comment 4 When it is not a fertilizer in which phosphate, ammonium and magnesium
coexist, about 400 mL of potassium chloride solution (1 mol/L) can be used64

instead of about 300 mL of hydrochloric acid (1+20) in the procedure of(4.1.2) b).
Comment 5 In the case of the mixed fertilizers containing bentonite, filter with Type 3
filter paper after shaking to mix using about 400 mL of potassium chloride
solution (1 mol/L) according to Comment 4 in (4.1.2) b) and transfer 50 ml 100 mL to a 250-mL volumetric flask, and then conduct the procedure in(4.1.2) c) - e).
(4.2) Measurement: Conduct measurement as shown below.
a) Transfer a predetermined volume (up to the equivalents of 50 mg as A-N) of sample
solution to a 300-mL Erlenmeyer flask (4).
b) Add water to the solution to make about 100 mL.
c) Add one or two drop(s) of methyl red solution (0.1 g/100 mL) and add hydrochloric
acid (1+200) until the color of the solution changes to light pink.
d) Add 10 mL of formaldehyde solution.
e) Add one or two drop(s) of thymol blue solution (1 g/100 mL) and titrate with 0.1 mol/L- 0.2 mol/L sodium hydroxide solution until the color of the solution changes to blue(5).
f) As a blank test, transfer 100 mL of water to another 300-mL Erlenmeyer flask andconduct the procedure in c) - e).
g) Calculate the ammoniac nitrogen (A-N) in the analytical sample by the following
formula:Ammoniac nitrogen (A-N) (% (mass fraction) in the analytical sample= (VS - VB) × C × f × (V1/V2) × (14.007/W) × (100/1,000)= (VS - VB) × C × f × (V1/V2) × (1.4007/W)
VS: Volume (mL) of 0.1 mol/L - 0.2 mol/L sodium hydroxide solution needed fortitrate in (4.2) e)
VB: Volume (mL) of 0.1 mol/L - 0.2 mol/L sodium hydroxide solution needed fortitration of the blank test in (4.2) f）
C: Setting concentration (mol/L) of 0.1 mol/L - 0.2 mol/L sodium hydroxide
solution
f: Factor of 0.1 mol/L - 0.2 mol/L sodium hydroxide solution
V1 Predetermined volume (mL) of the sample solution in (4.1.1) c) or (4.1.2) d)
V2: Transferred amount (mL) of the sample solution in (4.2) a)
W: Mass of sample solution (g)Note (4) The volume to be transferred should be up to 100 mL.
(5) The endpoint is reached when the solution changes from green to blue. It is easy
to observe the change of color under fluorescent light.
Comment 6 The titration procedures in (2) a) Standardization and (4.2) e) - f) can be
conducted by an automatic titrator. The setup of the titration program, thedetermination parameter for the endpoint and vessels such as acceptors areaccording to the specification and the operation method of the automatic
titrator used.
Comment 7 Table 1 shows results from a simultaneous analysis with the same samples(proficiency testing and external quality control testing) hosted by the Japan
Fertilizer Quality Assurance Committee analyzed by the robust method.

65

Table 1

Year

Results and analysis results from simultaneous analysis with the same samples
for ammonium-nitrogen hosted by the Japan Fertilizer Quality Assurance Committee1)
SampleNumber of Median Μ2)laboratories
(%)3)

4)

5)

NIQR

RSDrob(%)3)(%)

High analysis
150
14.60
0.10
0.9
compound fertilizer
High analysis
2010
107
11.51
0.17
1.5
compound fertilizer
1) Proficiency testing and external quality control testing2) Median (M) agrees with the mean in normal distribution.
3) Mass fraction4) Robust standard deviation (NIQR) agrees with standard deviation in normal distribution.
5) RSDrob stands for relative standard deviation obtained from the robust methodby the formula shown below:
RSDrob = (NIQR/M) × 100
2006

Reference
1) Masayoshi KOSHINO: Second Revision of The Methods of Analysis of Fertilizers(Details), p.39 - 42, Yokendo, Tokyo (1988)(5)

Flow sheet for ammoniac nitrogen: The flow sheet for ammoniac nitrogen in
fertilizers is shown below.
5 g analytical sample
Weigh to the order of 1 mg into a 500-mL volumetric flask(ammonium salts)← About 400 mL of water
Shaking
Rotary shaker (30 - 40 revolutions/minute), 30 minutes
5 g analytical sample
Weigh to the order of 1 mg into a 500-mL volumetric flask(compound fertilizer)← About 300 mL of hydrochloric acid (1 + 20)
Shaking to mix
Rotary shaker (30 - 40 revolutions/minute), 30 minutes← Aluminum chloride solution (1 mol/L)← One or two drop(s) of methyl red← Potassium hydroxide solution (170 g/L)
(Until the solution changes to light yellow)← Water (up to the marked line)
Filtration
Type 3 filter paper
Up to the equivalents of 50 mg as A-N,300-mL Erlenmeyer flask← Add water to make about 100 mL of solution← One or two drop(s) of methyl red← Hydrochloric acid (1+200) [light pink]← 10 mL of formaldehyde solution← One or two drop (s) of thymol blue solution (1 g/100 mL)
0.1 mol/L - 0.2 mol/L sodium hydroxide solution
Titration(until the solution becomes blue)
Aliquot

Figure The flow sheet (example) for ammoniac nitrogen in fertilizers is shown below.
664.1.3 Nitrate nitrogen
4.1.3.a Devarda’s alloy - distillation method(1) Summary
The testing method is applicable to fertilizers containing nitrate. However it is not applicable
to fertilizers containing nitrolime and organic matters that digest by heating and isolateammonia.
Add water to an analytical sample to dissolve ammoniac nitrogen (A-N) and nitrate nitrogen(N-N), and further add devarda’s alloy and sodium hydroxide to reduce nitrate nitrogen (N-N)
and separate ammonium ion by distillation. Measure the separated ammonium ion by
neutralization titration to obtain total amount of nitrogen (N-N+A-N) in the analytical sample.
Subtract separately obtained ammoniac nitrogen (A-N) to calculate nitrate nitrogen (N-N).
This test method corresponds to the devarda’s alloy method in the Official Methods ofAnalysis of Fertilizers (1992).
(2) Reagents: Reagents are as shown below:a) 0.1 mol/L - 0.2 mol/L sodium hydroxide solution(1): Transfer about 30 mL of water toa polyethylene bottle, dissolve about 35 g of sodium hydroxide specified in JIS K 8576
by adding in small portions while cooling, seal tightly and leave at rest for 4 - 5 days.
Transfer 5.5 mL - 11 mL of the supernatant to a ground-in stoppered storage container,
and add 1,000 mL of water containing no carbonic acid.
Standardization: Dry sulfamic acid reference material for volumetric analysis
specified in JIS K 8005 by leaving at rest in a desiccator at no more than 2 kPa for about
48 hours, then transfer about 2.5 g to a weighing dish, and measure the mass to the orderof 0.1 mg. Dissolve in a small amount of water, transfer to a 250-mL volumetric flask,
and add water up to the marked line (1). Transfer a predetermined amount of the solution
to a 200-mL - 300-mL Erlenmeyer flask, add a few drops of bromothymol blue solution(0.1 mg/100 mL) as an indicator, and titrate with 0.1 mol/L - 0.2 mol/L sodiumhydroxide solution until the color of the solution becomes green. Calculate the factor of
0.1 mol/L - 0.2 mol/L sodium hydroxide solution by the following formula:
Factor of 0.1 mol/L - 0.2 mol/L sodium hydroxide solution (f1)
= (W1 × A × 0.01/97.095) × (V1/V2) × (1,000/V3) × (1/C1)
W1: Mass (g) of sulfamic acid sampled
A: Purity (% (mass fraction)) of sulfamic acidV1: Volume (mL) of sulfamic acid solution transferred
V2: Constant volume (250 mL) of sulfamic acid solution
V3: Volume (mL) of 0.1 mol/L - 0.2 mol/L sodium hydroxide solution needed fortitration
C1: Set concentration (mol/L) of 0.1 mol/L - 0.2 mol/L sodium hydroxide solutionb) Sulfuric acid: A JIS Guaranteed Reagent specified in JIS K 8951 or a reagent ofequivalent quality.
c) 0.25 mol/L sulfuric acid(1) (2): Add about 14 mL of sulfuric acid to a beaker containing
100 mL of water in advance, stir well and add water to make 1,000 mL.
Standardization: Transfer a predetermined amount (3) of 0.25 mol/L sulfuric acid to a
200-mL - 300-mL Erlenmeyer flask, add a few drops of methyl red-methylene blue
mixture solution, and titrate with 0.1 mol/L - 0.2 mol/L sodium hydroxide solution until
the color of the solution becomes gray-green(4). Calculate the volume of 0.1 mol/L - 0.2
mol/L sodium hydroxide solution equivalent to 1 mL of 0.25 mol/L sulfuric acid by the67

following formula (1). Or, calculate the factor of 0.25 mol/L sulfuric acid by thefollowing formula (2):Volume (B) of 0.1 mol/L - 0.2 mol/L sodium hydroxide solution equivalent to 1 mL
of 0.25 mol/L sulfuric acid= V4/V5………. (1)
Factor of 0.25 mol/L sulfuric acid (f2)
= (f1 × C1 × V4/V5)/ (C2 × 2)………. (2)V4: Volume (mL) of 0.1 mol/L - 0.2 mol/L sodium hydroxide solution needed fortitration
V5: Volume (mL) of 0.25 mol/L sulfuric acid subjected to the standardization
C1: Set concentration (mol/L) of 0.1 mol/L - 0.2 mol/L sodium hydroxide solution
C2: Set concentration (0.25 mol/L) of 0.25 mol/L sulfuric acidd) Boric acid solution (40 g/L): Dissolve 40 g of boric acid specified in JIS K 8863 inwater to make 1,000 mL.
e) Sodium hydroxide solution (200 g/L - 500 g/L)(1): Dissolve 100 g - 250 g of sodiumhydroxide specified in JIS K 8576 in water to make 500 mL.
f) Devarda’s alloy: A reagent of nitrogen analysis grade specified in JIS K 8653 or areagent of equivalent qualityg) Bromothymol blue solution (0.1 g/100 mL): Dissolve 0.1 g of bromothymol blue
specified in JIS K 8842 in 20 mL of ethanol (95) specified in JIS K 8102, add water tomake 100 mL.
h) Methyl red solution (0.1 g/100 mL): Dissolve 0.1 g of methyl red specified in JIS K
8896 in 100 mL of ethanol (95) specified in JIS K 8102.
i) Methylene blue solution (0.1 g/100 mL): Dissolve 0.1 g of methylene blue specified in
JIS K 8897 in 100 mL of ethanol (95) specified in JIS K 8102.
j) Methyl red - methylene blue mixture solution: To 2 volumes of methyl red solution(0.1 g/100 mL), add 1 volume of methylene blue solution (0.1 g/100 mL).
k) Bromocresol green solution (0.5 g/100 mL): Dissolve 0.5 g of bromocresol green
specified in JIS K 8840 in 100 mL of ethanol (95) specified in JIS K 8102.
l) Methyl red - bromocresol green mixture solution: To methyl red solution (0.1 g/100mL), add equal volume of bromocresol green solution (0.5 g/100 mL).
Note (1) This is an example of preparation; prepare an amount as appropriate.
(2) This corresponds to the standard sulfuric acid solution 0.5 M (1/2 sulfuric acid)solution in the Official Methods of Analysis of Fertilizers (1992).
(3) 5 mL - 10 mL(4) The endpoint is reached when the color becomes gray-green via dark blue fromblue-purple.
(3) Instruments: Instruments are as shown below:a) Steam distillation apparatusb) Distillation flask: A Kjeldahl flask or a round bottom flask that can be connected to asteam distillation apparatus.
(4) Test procedures(4.1) Preparation of sample solution: Conduct preparation of sample solution as shown
below.
68a) Weigh 0.25 g - 1 g (5) (the equivalents of 20 mg - 100 mg as N) of an analytical sample
to the order of 1 mg, and put it in a 300-mL - 500-mL distillation flaskb) Add about 25 mL of water to make the sample solution.
Note (5) Conduct the procedure in Comment 1 when there is much nitrogen content in
the fertilizers such as simple salt fertilizers.
Comment 1 In the case of nitrate fertilizer, etc. containing much nitrogen content, weigh 2
g - 5g of an analytical sample to the order of 1 mg, put it into a 250- mL
volumetric flask, dissolve it in water, and further add water up to the marked
line. Put predetermined volume of suspension (the equivalents of 20 mg -100
mg as N) into a 300- mL - 500-mL distillation flask.
(4.2) Distillation: Conduct distillation as shown below. Specific distillation procedures areaccording to the operation method of the steam distillation apparatus used inmeasurement.
a) Transfer a predetermined amount (6) of 0.25 mol/L sulfuric acid to an acceptor (7), add a
few drops of methyl red - methylene blue mixture solution, and connect this acceptor toa steam distillation apparatus. Or, transfer a predetermined amount (6) of boric acidsolution (40 g/L) to an acceptor(7), add a few drops of methyl red - bromocresol green
mixture solution, and connect this acceptor to a steam distillation apparatus.
b) Add (10) no less than 3 mg of devarda’s alloy and adequate volume of sodium hydroxide(200 g/L - 500 g/L) (8) (9) and connect this distillation flask to the steam distillation
apparatus.
c) Send steam to the distillation flask to heat the solution in the distillation flask, and distill
at a distillation rate of 5 mL/min - 7 mL/min.
d) Stop distilling when the distillate reaches 120 mL - 160 mL.
e) Wash the part of the steam distillation apparatus that came in contact with the solution
in the acceptor with a small amount of water, and pool the washing with the distillate.
Note (6) 5 mL - 20 mL(7) As an acceptor, use a 200-mL - 300-mL Erlenmeyer flask or a 200-mL - 300-mL
beaker with which the distillate outlet of the steam distillation apparatus can be
immersed in 0.25 mol/L sulfuric acid or boric acid solution (40 g/L).
(8) Sudden reaction makes bubbles foam drastically and the bubbles overflow froma distillation flask. Therefore it is required to add alkali solution gradually andmix quietly.
(9) A sufficient amount to make the solution strong alkalinity.
(10) Add a small amount of silicone oil as necessary.
(4.3) Measurement: Conduct measurement as shown below.
(4.3.1) When 0.25 mol/L sulfuric acid is used in (4.2),a) Titrate the distillate with 0.1 mol/L - 0.2 mol/L sodium hydroxide solution until thesolution becomes gray-green (4).
b) Calculate the total amount of nitrogen (N-N+A-N) in the analytical sample by thefollowing formula:c) Subtract separately obtained ammoniac nitrogen (A-N) from the obtained total amountof nitrogen (N-N+A-N) to calculate nitrate nitrogen (N-N) (11) (12).
Total amount of nitrogen (N-N+A-N) (% (mass fraction)) in the analytical sample= (B × V6 - V7) × C1 × f1 × (14.007/W2) × (100/1,000)
69

= (B × V6 - V7) × C1 × f1 × (1.4007/W2)
B: Volume of 0.1 mol/L - 0.2 mol/L sodium hydroxide solution equivalent to 1
mL of 0.25 mol/L sulfuric acidV6: Volume (mL) of 0.25 mol/L sulfuric acid transferred to the acceptor in (4.2)a)
V7: Volume (mL) of 0.1 mol/L - 0.2 mol/L sodium hydroxide solution needed fortitration in (4.3) a)
C1: Set concentration (mol/L) of 0.1 mol/L - 0.2 mol/L sodium hydroxide
solution
f1: Factor of 0.1 mol/L - 0.2 mol/L sodium hydroxide solution
W2: Mass (g) of the analytical sample
Note (11) A-N and N-N use raw data without rounding the numerical value(12) When no ammoniac nitrogen (A-N) is contained, the total amount of nitrogen(N-N+A-N) calculated in (4.3) b) is regarded as nitrate nitrogen (N-N).
(4.3.2) When boric acid solution (40 g/L) is used in (4.2),a) Titrate the distillate with 0.25 mol/L sulfuric acid until the solution becomes light red(13).
b) Calculate the total amount of nitrogen (N-N+A-N) in the analytical sample by thefollowing formula:
Total amount of nitrogen (N-N+A-N) (% (mass fraction)) in the analytical sample= V10 × C2 × 2×f2 × (V11/V12) × (14.007/W2) × (100/1,000)= V10 × C2 × f2 × (V11/V12) × (2.8014/W2)
V10: Volume (mL) of 0.25 mol/L sulfuric acid needed for titration
C2: Set concentration (mol/L) of 0.25 mol/L sulfuric acidf2: Factor of 0.25 mol/L sulfuric acidV11: Predetermined volume (mL) of the sample solution in (4.1) b)
V12: Transferred amount (mL) of the digestion solution subjected to distillation in(4.2) b)
W2: Mass (g) of the analytical sample
Note (13) The endpoint is reached when the color changes from green to light red.
Comment 2 The titration procedures in (2) a) Standardization, (2) c) Standardizationand (4.3) can be conducted by an automatic titrator. The setup of the titration
program, the determination parameter for the endpoint and vessels such as
acceptors are according to the specification and the operation method of theautomatic titrator used.
Comment 3 Table 1 shows results from a simultaneous analysis with the same samples(proficiency testing and external quality control testing) hosted by the Japan
Fertilizer Quality Assurance Committee analyzed by the robust method.

70

Table 1

Year

Results and analysis results from simultaneous analysis with the same samples
1)
for nitrate nitrogen hosted by the Japan Fertilizer Quality Assurance Committee
SampleNumber of Median Μ2)
3)laboratories(%)

NIQR4)

RSDrob5)

(%)

3)

(%)

2009

Ordinary compound
fertilizer

84

3.60

0.12

3.3

2012

Fluid mixed fertilizer

77

2.18

0.09

4.1

1)
2)
3)
4)
5)

Proficiency testing and external quality control testingMedian (M) agrees with the mean in normal distribution.
Mass fraction
Robust standard deviation (NIQR) agrees with standard deviation in normal distribution.
RSDrob stands for relative standard deviation obtained from the robust methodby the formula shown below:
RSDrob = (NIQR/M) × 100

Reference
1) Masayoshi KOSHINO: Second Revision of The Methods of Analysis of Fertilizers(Details), p.49 - 50, Yokendo, Tokyo (1988)(5)

Flow sheet for nitrate nitrogen: The flow sheet for nitrate nitrogen in fertilizers isshown below.
0.25 g - 1 g
Weigh the equivalents of 20 mg - 100 mg as N to the order of 1 mg
analytical sample
into a 300-mL distillation flask.
← 25 mL of water← No less than 3 g of devarda's alloy← Sodium hydroxide solution (200 g/L - 500 g/L)
← A small amount of silicone oil as necessary
Receiver: 200-mL - 300-mL Erlenmeyer flask or beaker
Steam distillation
A predetermined amount of 0.25 mol/L sulfuric acid and a few drops ofapparatus
methyl red-methylene blue mixture solution, or boric acid solution (40 g/L),
several drops of methyl red - bromocresol green mixture solution
Steam distillation

Distillation rate: 5 mL/min - 7 mL/min

Stop distilling
120 mL - 160 mL distillate← Water (wash the part of the distillation apparatus
that came in contact with the solution in the receiver)
0.1 mol/L - 0.2 mol/L sodium hydroxide solution (until the
Titration
solution becomes gray-green), or
0.25 mol/L sulfuric acid (until the solution becomes light red)
Figure The flow sheet (example) for nitrate nitrogen in fertilizers

714.1.3.b Reduced iron - distillation method(1) Summary
The testing method is applicable to the fertilizer containing nitrate. However it is notapplicable to the fertilizers containing nitrolime and organic matters that digest by heating andisolate ammonia.
Add water to an analytical sample to dissolve ammoniac nitrogen (A-N) and nitrate nitrogen(N-N), and add reduced iron and sulfuric acid solution to reduce nitrate nitrogen (N-N) by
lightly boiling. And further add sodium hydroxide solution to distillate. Measure the separated
ammonium ion by neutralization titration to obtain total amount of nitrogen (N-N+A-N) in theanalytical sample. Subtract separately obtained ammoniac nitrogen (A-N) to calculate nitrate
nitrogen (N-N). This test method corresponds to the reduced iron method in the Official
Methods of Analysis of Fertilizers (1992).
(2) Reagents: Reagents are as shown below:a) 0.1 mol/L - 0.2 mol/L sodium hydroxide solution(1): Transfer about 30 mL of water toa polyethylene bottle, dissolve about 35 g of sodium hydroxide specified in JIS K 8576
by adding in small portions while cooling, seal tightly and leave at rest for 4 - 5 days.
Transfer 5.5 mL - 11 mL of the supernatant to a ground-in stoppered storage container,
and add 1,000 mL of water containing no carbonic acid.
Standardization: Dry sulfamic acid reference material for volumetric analysis
specified in JIS K 8005 by leaving at rest in a desiccator at no more than 2 kPa for about
48 hours, then transfer about 2.5 g to a weighing dish, and measure the mass to the orderof 0.1 mg. Dissolve in a small amount of water, transfer to a 250-mL volumetric flask,
and add water up to the marked line (1). Transfer a predetermined amount of the solution
to a 200-mL - 300-mL Erlenmeyer flask, add a few drops of bromothymol blue solution(0.1 mg/100 mL) as an indicator, and titrate with 0.1 mol/L - 0.2 mol/L sodiumhydroxide solution until the color of the solution becomes green. Calculate the factor of
0.1 mol/L - 0.2 mol/L sodium hydroxide solution by the following formula:
Factor of 0.1 mol/L - 0.2 mol/L sodium hydroxide solution (f1)
= (W1 × A × 0.01/97.095) × (V1/V2) × (1,000/V3) × (1/C1)
W1: Mass (g) of sulfamic acid sampled
A: Purity (% (mass fraction)) of sulfamic acidV1: Volume (mL) of sulfamic acid solution transferred
V2: Constant volume (250 mL) of sulfamic acid solution
V3: Volume (mL) of 0.1 mol/L - 0.2 mol/L sodium hydroxide solution needed fortitration
C1: Set concentration (mol/L) of 0.1 mol/L - 0.2 mol/L sodium hydroxide solutionb) Sulfuric acid: A JIS Guaranteed Reagent specified in JIS K 8951 or a reagent ofequivalent quality.
c) 0.25 mol/L sulfuric acid(1) (2): Add about 14 mL of sulfuric acid to a beaker containing
100 mL of water in advance, stir well, and add water to make 1,000 mL.
Standardization: Transfer a predetermined amount (3) of 0.25 mol/L sulfuric acid to a
200-mL - 300-mL Erlenmeyer flask, add a few drops of methyl red-methylene blue
mixture solution, and titrate with 0.1 mol/L - 0.2 mol/L sodium hydroxide solution until
the color of the solution becomes gray-green(4). Calculate the volume of 0.1 mol/L - 0.2
mol/L sodium hydroxide solution equivalent to 1 mL of 0.25 mol/L sulfuric acid by thefollowing formula (1). Or, calculate the factor of 0.25 mol/L sulfuric acid by thefollowing formula (2):72Volume (A) of 0.1 mol/L - 0.2 mol/L sodium hydroxide solution equivalent to 1 mL
of 0.25 mol/L sulfuric acid= V4/V5……….. (1)
Factor of 0.25 mol/L sulfuric acid (f2)
= (f1 × C1 × V4/V5)/ (C2 × 2)………... (2)V4: Volume (mL) of 0.1 mol/L - 0.2 mol/L sodium hydroxide solution needed fortitration
V5: Volume (mL) of 0.25 mol/L sulfuric acid subjected to standardization
C1: Set concentration (mol/L) of 0.1 mol/L - 0.2 mol/L sodium hydroxide solution
C2: Set concentration (0.25 mol/L) of 0.25 mol/L sulfuric acidd) Boric acid solution (40 g/L): Dissolve 40 g of boric acid specified in JIS K 8863 inwater to make 1,000 mL.
e) Sodium hydroxide solution (200 g/L - 500 g/L)(1): Dissolve 100 g - 250 g of sodiumhydroxide specified in JIS K 8576 in water to make 500 mL.
f) Reduced iron: Nitrogen content is no more than 0.005 % (mass fraction).
g) Bromothymol blue solution (0.1 g/100 mL): Dissolve 0.1 g of bromothymol blue
specified in JIS K 8842 in 20 mL of ethanol (95) specified in JIS K 8102, add water tomake 100 mL.
h) Methyl red solution (0.1 g/100 mL): Dissolve 0.1 g of methyl red specified in JIS K
8896 in 100 mL of ethanol (95) specified in JIS K 8102.
i) Methylene blue solution (0.1 g/100 mL): Dissolve 0.1 g of methylene blue specified in
JIS K 8897 in 100 mL of ethanol (95) specified in JIS K 8102.
j) Methyl red - methylene blue mixture solution: To 2 volumes of methyl red solution(0.1 g/100 mL), add 1 volume of methylene blue solution (0.1 g/100 mL).
k) Bromocresol green solution (0.5 g/100 mL): Dissolve 0.5 g of bromocresol green
specified in JIS K 8840 in 100 mL of ethanol (95) specified in JIS K 8102.
l) Methyl red - bromocresol green mixture solution: To methyl red solution (0.1 g/100mL), add equal volume of bromocresol green solution (0.5 g/100 mL).
Note (1) This is an example of preparation; prepare an amount as appropriate.
(2) This corresponds to the standard sulfuric acid solution 0.5 M (1/2 sulfuric acid)solution in the Official Methods of Analysis of Fertilizers (1992).
(3) 5 mL - 10 mL(4) The endpoint is reached when the color becomes gray-green via dark blue fromblue-purple.
(3) Instruments: Instruments are as shown below:a) Steam distillation apparatusb) Distillation flask: A Kjeldahl flask or a round bottom flask that can be connected to asteam distillation apparatus.
(4) Test procedures(4.1) Preparation of sample solution: Conduct preparation of sample solution as shown
below.
a) Weigh 0.25 g - 1g (5) (the equivalents of 20 mg -100 mg as N) of an analytical sample tothe order of 1 mg, and put it in a 300-mL - 500-mL distillation flaskb) Add about 30 mL of water and mix well.
73c) As soon as adding 5 g of reduced iron and 10 mL of sulfuric acid (1+1), insert a longstem funnel to a distillation flask and shake to mix gently while cooling the outside of
the container under flowing water(6).
d) After leaving at rest for about 5 minutes (7), heat gradually by low temperature and boil
in a low flame for about 15 minutes, and then stand to cool to make the sample solution.
Note (5) Conduct the procedure of Comment 1 when there is much nitrogen content in
the fertilizers such as simple salt fertilizers.
(6) Sudden reaction generates heat, and unreacted nitric acid vaporizes or digests tomake nitrogen oxide etc. through which process losses occur easily. Careful andefficient operation should be taken.
(7) Until strong reaction is settled.
Comment 1 In the case of nitrate fertilizers, etc. containing much nitrogen content, weigh
2 g - 5g of an analytical sample to the order of 1 mg, put it into a 250- mL
volumetric flask, dissolve it with water and add water up to the marked line.
Put predetermined volume of suspension (the equivalents of 20 mg - 100 mg
as N) into a 300- mL - 500-mL distillation flask.
(4.2) Distillation: Conduct distillation as shown below. Specific distillation procedures areaccording to the operation method of the steam distillation apparatus used inmeasurement.
a) Transfer a predetermined amount (8) of 0.25 mol/L sulfuric acid to an acceptor(9), add a
few drops of methyl red - methylene blue mixture solution, and connect this acceptor toa steam distillation apparatus. Or, transfer a predetermined amount(8) of boric acidsolution (40 g/L) to an acceptor (9) ,add a few drops of methyl red - bromocresol green
mixture solution, and connect this acceptor to a steam distillation apparatus.
b) Add(10) adequate volume of sodium hydroxide (200 g/L - 500 g/L)(10) and connect this
distillation flask to the steam distillation apparatus.
c) Send steam to the distillation flask to heat the solution in the distillation flask, and distill
at a distillation rate of 5 mL/min - 7 mL/min.
d) Stop distilling when the distillate reaches 120 mL - 160 mL.
e) Wash the part of the steam distillation apparatus that came in contact with the solution
in the acceptor with a small amount of water, and pool the washing with the distillate.
Note (8) 5 mL - 20 mL(9) As an acceptor, use a 200-mL - 300-mL Erlenmeyer flask or a 200-mL - 300-mL
beaker with which the distillate outlet of the steam distillation apparatus can be
immersed in 0.25 mol/L sulfuric acid or boric acid solution (40 g/L).
(10) A sufficient amount to make the solution strong alkalinity.
(4.3)
Measurement: Conduct measurement as shown below.
(4.3.1) When 0.25 mol/L sulfuric acid is used in (4.2) a),a) Titrate the distillate with 0.1 mol/L - 0.2 mol/L sodium hydroxide solution until thecolor of the solution becomes gray-green (4).
b) Calculate the total amount of nitrogen (N-N+A-N) in the analytical sample by thefollowing formula:c) Subtract separately obtained ammoniac nitrogen (A-N) from the obtained total amountof nitrogen (N-N+A-N) to calculate nitrate nitrogen (N-N) (11) (12).
Total amount of nitrogen (N-N+A-N) (% (mass fraction)) in the analytical sample74

= (B × V6 - V7) × C1 × f1 × (14.007/W2) × (100/1,000)= (B × V6 - V7) × C1 × f1 × (1.4007/W2)
B: Volume of 0.1 mol/L - 0.2 mol/L sodium hydroxide solution equivalent to 1
mL of 0.25 mol/L sulfuric acidV6: Volume (mL) of 0.25 mol/L sulfuric acid transferred to the acceptor in (4.2)a)
V7: Volume (mL) of 0.1 mol/L - 0.2 mol/L sodium hydroxide solution needed fortitration
C1: Set concentration (mol/L) of 0.1 mol/L - 0.2 mol/L sodium hydroxide solution
f1: Factor of 0.1 mol/L-0.2 mol/L sodium hydroxide solution
W2: Mass (g) of the analytical sample
Note (11) T-N and N-N use raw data without rounding numerical value(12) When no ammoniac nitrogen (A-N) is contained, total amount of nitrogen(N-N+A-N) calculated in (4.3) b) is regarded as nitrate nitrogen (N-N).
(4.3.2) When boric acid solution (40 g/L) is used in (4.2) a),a) Titrate the distillate with 0.25 mol/L sulfuric acid until the color of the solution becomeslight red (13).
b) Calculate the total amount of nitrogen (N-N+A-N) in the analytical sample by thefollowing formula:c) Subtract separately obtained ammoniac nitrogen (A-N) from the obtained total amountof nitrogen (N-N+A-N) to calculate nitrate nitrogen (N-N) (11) (12).
Total amount of nitrogen (N-N+A-N) (% (mass fraction)) in the analytical sample= V10 × C2 × 2×f2 × (14.007/W2) × (100/1,000)= V10 × C2 × f2 × (2.8014/W2)
V10: Volume (mL) of 0.25 mol/L sulfuric acid needed for titration
C2: Set concentration (mol/L) of 0.25 mol/L sulfuric acidf2: Factor of 0.25 mol/L sulfuric acid
W2: Mass (g) of the analytical sample
Note (13) The endpoint is reached when the color changes from green to light red.
Comment 2 The titration procedures in (2) a) Standardization, (2) c) Standardizationand (4.3) can be conducted by an automatic titrator. The setup of the titration
program, the determination parameter for the endpoint and vessels such as
acceptors are according to the specification and the operation method of theautomatic titrator used.
Comment 3 Table 1 shows results from a simultaneous analysis with the same samples(proficiency testing and external quality control testing) hosted by the Japan
Fertilizer Quality Assurance Committee analyzed by the robust method.

75

Table 1

Year

Results and analysis results from simultaneous analysis with the same samples
for nitrate nitrogen hosted by the Japan Fertilizer Quality Assurance Committee1)
SampleNumber of Median Μ2)laboratories
(%)3)

4)

NIQR
(%)3)

5)

RSDrob(%)

Ordinary compound
12
3.64
0.07
2.0
fertilizer
1) Proficiency testing and external quality control testing2) Median (M) agrees with the mean in normal distribution.
3) Mass fraction4) Robust standard deviation (NIQR) agrees with standard deviation in normal distribution.
5) RSDrob stands for relative standard deviation obtained from the robust methodby the formula shown below:
RSDrob = (NIQR/M) × 100
2009

Reference
1) Masayoshi KOSHINO: Second Revision of The Methods of Analysis of Fertilizers(Details), p.48 - 49, Yokendo, Tokyo (1988)

76

(5)

Flow sheet for nitrate nitrogen: The flow sheet for nitrate nitrogen in fertilizers isshown below.

0.5 g – 1 g
Weigh the equivalents of 20 mg - 100 mg of N to the order of 1 mg,
analytical sample
and put it into a 300-mL distillation flask.
←About 30 mL of water←5 g of reduced iron←About 10 mL of sulfuric acid (1+1)
Insert a long stem funnel immediately to a distillation flask and shake
Mixtureto mix gently while cooling the outside of the container under flowing water
Leaving at rest
Heating

For about 5 minutes (until a strong reaction is settled)
Boil in a low flame for about 15 minutes

Standing to cool
←Sodium hydroxide (200 g/L - 500 g/L)
Receiver: 200-mL - 300-mL Erlenmeyer flask or beaker
200-mL - 300-mL beaker. A predetermined amount of 0.25 mol/L sulfuric acid
Steam distillation
and a few drops of methyl red-methylene blue mixture solution,
apparatus
or boric acid solution (40 g/L),
several drops of methyl red - bromocresol green mixture solution
Steam distillation

Distillation rate: 5 mL/min - 7 mL/min

Stop distilling
120 mL - 160 mL distillate← Water (wash the part of the distillation apparatus
that came in contact with the solution in the receiver)
0.1 mol/L - 0.2 mol/L sodium hydroxide solution (until the
Titration
solution becomes gray-green), or
0.25 mol/L sulfuric acid (until the solution becomes light red)
Figure The flow sheet (example) for nitrate nitrogen in fertilizers

774.1.3.c Phenol sulfuric acid method(1) Summary
The testing method is applicable to the fertilizer containing nitrate. It is also applicable to thefertilizers containing such chemical compounds as urea, nitrolime and organic matters thatdigest by heating and isolate ammonia.
Add copper sulfate - silver nitrate solution, calcium hydroxide and basic magnesium
carbonate to an analytical sample, extract nitrate nitrogen (N-N) as well as removing chloride
and organic matters, and measure the absorbance of nitro phenol ammonium sulfate formedby the reaction with phenol sulfuric acid and ammonia solution.
(2) Reagents: Reagents are as shown below:a) Nitrate standard solution (N-N 5 mg/mL): Heat potassium nitrate (no less than 99.9(%) (mass fraction) in purity) at 110 ºC for no less than 1 hour, and after standing tocool in a desiccator, transfer 36.09 g to a weighing dish. Dissolve it in a small amount ofwater, move it into a 1,000-mL volumetric flask and add water up to the marked line (1).
b) Nitrate standard solution (N-N 0.05 mg/mL): Dilute predetermined volume of nitrate
standard solution (N-N 5 mg/mL) with water to prepare nitrate standard solution (N-N
0.05 mg/mL).
c) Copper sulfuric - silver sulfuric solution (1): Dissolve 5 g of copper (II) sulfate
pentahydrate specified JIS K 8983 in 900 mL of water, and dissolve while adding 4 g ofsilver sulfate specified in JIS K 8965 to make 1,000 mL (2).
d) Phenol sulfuric acid: Dissolve 15 g of phenol sulfuric acid specified in JIS K 8798 in
100 mL of sulfuric acid specified in JIS K 8965 while heating, and then let it stand tocool (2).
e) Ammonia solution: A JIS Guaranteed (NH3 28 % (mass fraction)) reagent specified in
JIS K 8085 or a reagent of equivalent quality.
f) Calcium hydroxide: A JIS Guaranteed reagent specified in JIS K 8575 or a reagent ofequivalent quality.
g) Basic magnesium carbonate: Basic magnesium carbonate that contains no nitrate
nitrogen.
Note (1) This is an example of preparation; prepare an amount as appropriate.
(2) Store in an amber bottle.
(3) Instruments: Instruments are as shown below:a) Rotary shaker: A rotary shaker that can rotate a 250-mL volumetric flask upside down
at 30 - 40 revolutions/min.
b) Spectrophotometer: A spectrophotometer specified in JIS K 0115c) Water bath: Water bath that can be adjusted to no less than 80 ºC(4) Test procedures(4.1) Extraction: Conduct extraction as shown below.
a) Weigh 1 g of an analytical sample to the order of 1 mg and transfer to a 250-mL
volumetric flask.
b) Add about 200 mL of copper sulfuric - silver sulfuric solution and shake to mix at 30 40 revolutions /min for about 20 minutes.
c) Add about 1 g of calcium hydroxide and about 1 mg of basic magnesium carbonate andshake to mix at 30 - 40 revolutions /min for about 10 minutes.
d) Add water up to the marked line.
e) Filter with Type 3 filter paper to make the sample solution (3).
78Note (3) As soon as sample solution is prepared, conduct the procedure in (4.2 a).
Comment 1 If the filtrate of (4.1) e) is colored, add no more than 0.5 g of active carbon
and filter with Type 3 filter paper to make the sample solution.
(4.2) Coloring: Conduct coloring as shown below.
a) Transfer a predetermined volume (the equivalents of 0.01 mg - 0.1 mg as N-N) into asmall evaporating dish (4).
b) Evaporate water until dry on a water bath at no less than 80 ºC.
c) After standing to cool, swiftly add 2 mL of phenol sulfuric acid (5) and then rotate theevaporating dish so that the whole residue comes in contact with the acid.
d) After leaving at rest for about 10 minutes, add 20 mL of water (6).
e) After standing to cool, transfer it with water to a 100- mL volumetric flask.
f) Add ammonia solution (1+2), until the color of solution changes to light yellow, toallow it to be weak alkalinity, and further add 3 mL of ammonia solution (1+3) (7).
g) After standing to cool, add water up to the marked line and leave at rest for 30 minutes.
Note (4) A round-bottom glass or porcelain evaporating dish is preferable.
(5) Add at the center of a small evaporating dish with Komagome pipet.
(6) If residue does not dissolve easily, grind it with a glass rod.
(7) As no color appears from a blank test solution for calibration curve preparation,
add almost the same volume of ammonia solution (1+2) as the nitrate standard
solution.
(4.3) Measurement: Conduct the measurement as indicated in JIS K 0115 and as shown
below. Specific measurement procedures are according to the operation method of thespectrophotometer used for the measurement.
a) Measurement conditions of spectrophotometer: Set up the measurement conditionsof spectrophotometer considering the following.
Detection wavelength: 410 nmb) Preparation of calibration curve
1) Transfer 1 mL - 10 mL of nitrate standard solution (N-N 0.05 mg/mL) to small
evaporating dishes (4) step-by-step.
2) Conduct the same procedure as (4.2) b) - g) to make the nitrate standard solution forthe calibration curve preparation.
3) Transfer 40 mL of water to a 100-mL volumetric flask, and shake to mix while gently
adding 2 mL of phenol sulfuric acid. Let it stand to cool and conduct the sameprocedure as (4.2) f) - g) to make the blank test solution for calibration curve.
4) Measure absorbance at wavelength 410 nm of the nitrate standard solution for thecalibration curve preparation using the blank test solution for the calibration curve
preparation as the control.
5) Prepare the calibration curve of the nitrate nitrogen (N-N) and absorbance of thenitrate standard solutions for the calibration curve preparation.
c) Measurement of a sample
1) Regarding the solution in (4.2) g), measure absorbance by the same procedure as b)4).
2) Obtain the nitrate nitrogen (N-N) content from the calibration curve and calculate
nitrate nitrogen (N-N) in the analytical sample.

79

Comment 2

Table 1

Year
2009
2012
1)
2)
3)
4)
5)

Table 1 shows results from a simultaneous analysis with the same samples(proficiency testing and external quality control testing) hosted by the Japan
Fertilizer Quality Assurance Committee analyzed by the robust method.

Results and analysis results from simultaneous analysis with the same samples
1)
for nitrate nitrogen hosted by the Japan Fertilizer Quality Assurance Committee
Number of Median Μ2) NIQR4)RSDrob5)
Samplelaboratories
(%)
(%)3)
(%)3)
Ordinary compound
26
3.67
0.09
2.3
fertilizer
Fluid mixed fertilizer

25

2.19

0.10

4.4

Proficiency testing and external quality control testingMedian (M) agrees with the mean in normal distribution.
Mass fraction
Robust standard deviation (NIQR) agrees with standard deviation in normal distribution.
RSDrob stands for relative standard deviation obtained from the robust methodby the formula shown below:
RSDrob = (NIQR/M) × 100

Reference
1) Masayoshi KOSHINO: Second Revision of The Methods of Analysis of Fertilizers(Details), p.52 - 55, Yokendo, Tokyo (1988)

80

(5)

Flow sheet for nitrate nitrogen: The flow sheet for nitrate nitrogen in fertilizers isshown below.
1 mg
Weigh to the order of 1 mg to a 250-mL volumetric flask
analytical sample← About 200 mL of copper sulfate - silver sulfate solution
Shaking to mix
Rotary shaker (30 - 40 revolutions/min) for 20 minutes← About 1 mg of calcium hydroxide← About 1 mg of basic magnesium carbonate
Shaking to mix
Rotary shaker (30 - 40 revolutions/min) for 10 minutes← Water (up to the marked line)
Filtration
Type 3 filter paper
Aliquot(predetermined volume)

Small evaporating dishEvaporation to dryness
80 ºC Water bath← 2 mL of phenol sulfuric acid
About 10 minutes after making phenol sulfuric acid
Leaving at rest
contact with residue← 20 mL of waterStanding to coolTransfer
100-mL volumetric flask← Ammonia solution (1+2)← Water (up to the marked line)
Leaving at rest
For about 30 minutes
Measurement

Spectrophotometer (410 mm)

Figure The flow sheet (example) for nitrate nitrogen in fertilizers

81

4.2

Phosphate

4.2.1 Total phosphate
4.2.1.a Ammonium vanadomolybdate absorptiometric analysis(1) Summary
This test method is applicable to fertilizers containing organic matters.
Add sulfuric acid, potassium sulfate and copper (II) sulfate pentahydrate to an analyticalsample. Pretreat by Kjeldahl method or incineration-hydrochloric acid boiling to convert total
phosphorus to phosphate ion, and measure the absorbance of phosphovanadomolybdate salt
formed by the reaction with ammonium vanadate (V), hexaammonium heptamolybdate andnitric acid, to obtain total phosphate (T-P2O5).
(2) Reagents: Reagents are as shown below:a) Sulfuric acid: A JIS Guaranteed Reagent specified in JIS K 8951 or a reagent ofequivalent quality.
b) Nitric acid: A JIS Guaranteed Reagent (HNO3 60 % (mass fraction)) specified in JIS K
8541 or a reagent of equivalent quality.
c) Ammonia solution: A JIS Guaranteed Reagent (NH3 28 % (mass fraction)) specified in
JIS K 8085 or a reagent of equivalent quality.
d) Catalyst(1): Mix potassium sulfate specified in JIS K 8962 and copper (II) sulfate
pentahydrate(2) specified in JIS K 8983 in the ratio of 9 to 1.
e) Coloring reagent solution(3) (4): Dissolve 1.12 g of ammonium vanadate (V)(5) specified
in JIS K 8747 in water, add 250 mL of nitric acid, then add 27 g of hexaammonium
heptamolybdate tetrahydrate(6) specified in JIS K 8905 dissolved in water, and further
add water to make 1,000 mL(7).
f) Phenolphthalein solution (1 g/100 mL): Dissolve 1 g of phenolphthalein specified in
JIS K 8799 in 100 mL of ethanol (95) specified in JIS K 8102.
g) Phosphate standard solution (P2O5 10 mg/mL)(3): Heat potassium dihydrogen
phosphate specified in JIS K 9007 at 105 ºC ± 2 ºC for about 2 hours, let it stand to coolin a desiccator, and weigh 19.17 g to a weighing dish. Dissolve in a small amount ofwater, transfer to a 1,000-mL volumetric flask, add 2 mL-3 mL of nitric acid, and add
water up to the marked line.
h) Phosphate standard solution (P2O5 0.5 mg/mL)(3): Transfer 50 mL of phosphatestandard solution (P2O5 10 mg/mL) to a 1,000-mL volumetric flask, add 2 mL - 3 mL ofnitric acid, and add water up to the marked line.
Note (1) A tablet is commercially available.
(2) Crush into powder as appropriate.
(3) This is an example of preparation; prepare an amount as appropriate.
(4) This corresponds to reagent “a” solution in the Official Methods of Analysis of
Fertilizers (1992).
(5) This corresponds to ammonium metavanadate in the Official Methods ofAnalysis of Fertilizers (1992).
(6) This corresponds to ammonium molybdate in the Official Methods of Analysis
of Fertilizers (1992).
(7) Store in an amber bottle.
(3) Apparatus and instruments: Apparatus and instruments are as follows:a) Spectrophotometer: A spectrophotometer specified in JIS K 0115.
b) Electric furnace: An electric furnace that can be adjusted to 550 ºC ± 5 ºC.
82c) Hot plate or sand bath: A hot plate whose surface temperature can be adjusted up to
250 ºC. Adjust the amounts of gas and silica sand of a sand bath so that the sand bathtemperature can be set to 250 ºC.
d) Digestion flask: Kjeldahl flask(4) Test procedures(4.1) Sample solution preparation: Prepare a sample solution as shown below:(4.1.1) Kjeldahl methoda) Weigh 0.5 g - 5 g of an analytical sample to the order of 1 mg, and put it in a 300-mL
digestion flask.
b) Add 5 g - 10 g of catalyst, and further add 20 mL - 40 mL of sulfuric acid, shake to mix
and heat gently.
c) After bubbles cease to form, heat until white smoke of sulfuric acid evolves.
d) Ignite until organic matters are completely digested (8).
e) After standing to cool, add a small amount of water, mix well by shaking, transfer to a
250-mL - 500-mL volumetric flask with water, and further mix by shaking.
f) After standing to cool, add water up to the marked line.
g) Filter with Type 3 filter paper to make the sample solution.
Note (8) After the change in the color of the solution ends, heat additionally for no lessthan 2 hours.
(4.1.2) Incineration-hydrochloric acid boilinga) Weigh 5 g of an analytical sample to the order of 1 mg, and transfer to a 200-mL 300-mL tall beaker.
b) Put the tall beaker in an electric furnace, and heat gently to char (9).
c) Ignite at 550 ºC ± 5 ºC for no less than 4 hours to incinerate.
d) After standing to cool, moisten the residue with a small amount of water, gradually add
about 10 mL of hydrochloric acid, and further add water to make 20 mL.
e) Cover the tall beaker with a watch glass, and heat on a hot plate or a sand bath to boil
for about 5 minutes.
f) After standing to cool, transfer to a 250-mL - 500-mL volumetric flask.
g) Add water up to the marked line.
h) Filter with Type 3 filter paper to make the sample solution.
Note (9) Example of the charring procedure: Heat at about 250 ºC until smoke is nolonger formed.
Comment 1 The procedures in (4.1.1) are the same as in (4.1) in 4.2.1.b. Also, theprocedures in (4.1.1) a) - f) are the same as in (4.1) in 4.1.1.a.
Comment 2 The procedures for the sample solution in (4.1.2) are the same as in (4.1) in
4.3.1.a, and 4.5.1.a.
Comment 3 The sample solution prepared in (4.1) a) - h) in 4.9.1.a can also be used.
(4.2) Coloring: Conduct coloring as follows:a) Transfer a predetermined amount of the sample solution (the equivalents of 0.5 mg - 6
mg as P2O5) to a 100-mL volumetric flask.
b) Add 1-2 drop(s) of phenolphthalein solution (1 g/100 mL), and neutralize by adding
ammonia solution (1+1) until the color of the solution becomes light red-purple (10).
c) Add nitric acid (1+10) until the light red-purple color of the solution disappears to makeit slightly acidic, and add a proper amount of water (11).
83d) Add 20 mL of coloring reagent solution, and further add water up to the marked line,
and then leave at rest for about 30 minutes.
Note (10) It is not necessary to add phenolphthalein solution (1 g/100 mL) when
determination can be done by the color change of copper ion (light blue →blue-purple).
(11) Without the addition of water, precipitate may be produced when coloring
reagent solution is added.
(4.3) Measurement: Conduct measurement according to JIS K 0115 and as shown below.
Specific measurement procedures are according to the operation method of thespectrophotometer used in measurement.
a) Measurement conditions for the spectrophotometer: Set up the measurement
conditions for the spectrophotometer considering the following:
Detection wavelength: 420 nmb) Calibration curve preparation
1) Transfer 1 mL - 12 mL of phosphate standard solution (P2O5 0.5 mg/mL) to 100-mL
volumetric flasks step-by-step.
2) Add a proper amount of water (11), and conduct the same procedure as (4.2) d) tomake the P2O5 0.5 mg/100 mL - 6 mg/100 mL phosphate standard solutions for thecalibration curve preparation.
3) Conduct the same procedures as 2) for another 100-mL volumetric flask to make theblank test solution for the calibration curve preparation.
4) Measure absorbance at a wavelength of 420 nm of the phosphate standard solutions
for the calibration curve preparation using the blank test solution for the calibration
curve preparation as the control (12).
5) Prepare the calibration curve of the phosphate concentration and absorbance of thephosphate standard solutions for the calibration curve preparation.
c) Sample measurement
1) Regarding the solution in (4.2) d), measure absorbance by the same procedure as b)4) (12).
2) Obtain the phosphate (P2O5) content from the calibration curve, and calculate total
phosphate (T-P2O5) in the analytical sample.
Note (12) Measure within 6 hours after adding the coloring reagent solution in theprocedure in (4.2) d).
Comment 4 Table 1 shows results from a simultaneous analysis with the same samples(proficiency testing and external quality control testing) hosted by the Japan
Fertilizer Quality Assurance Committee analyzed by the robust method.

84

Table 1

Results and analysis results from simultaneous analysis with the same samples
for nitrate nitrogen hosted by the Japan Fertilizer Quality Assurance Committee1)
4)5)
Number of Median Μ2)
NIQR
RSDrobYear
Samplelaboratories
(%)
(%)3)
(%)3)
Organic compound
2007
140
10.35
0.10
1.0
fertilizer
1) Proficiency testing and external quality control testing2) Median (M) agrees with the mean in normal distribution.
3) Mass fraction4) Robust standard deviation (NIQR) agrees with standard deviation in normal distribution.
5) RSDrob stands for relative standard deviation obtained from the robust methodby theby
formulathe formula
shownshown
below:below:
RSDrob = (NIQR/M) × 100

References
1) Masayoshi KOSHINO: Second Revision of The Methods of Analysis of Fertilizers(Details)，p.108 - 114，Yokendo, Tokyo (1988)
2) Kimie KATO, Masayuki YOSHIMOTO and Yuji SHIRAI: Systematization of
Determination Methods of Major Components in Sludge Fertilizer, Compost andOrganic Fertilizer, Research Report of Fertilizer, Vol.3 2010. (107 - 116)

85

(5)

Flow sheet for total phosphate: The flow sheets for total phosphate in fertilizers areshown below:
0.5 g - 5 g
Weigh to the order of 1 mg into a 300-mL Kjeldahl flask.
analytical sample← About 10 g catalyst← 20 mL - 40 mL sulfuric acid
Heating
GentlyHeating

After bubbles cease to form, ignite
until organic matters are completely digested.
Standing to coolRoom temperature← A small amount of waterTransfer
250-mL - 500-mL volumetric flask, waterStanding to coolRoom temperature←Water (up to the marked line)
Filtration
Type 3 filter paper
Sample solution
Figure 1 Flow sheet for total phosphate in fertilizers (1).
(Sample solution preparation by Kjeldahl method)
5g
analytical sample
Charring
Incineration

Weigh to the order of 1 mg into a 200-mL - 300-mL tall beaker.
Heat gently
550 ºC ± 5 ºC, no less than 4 hours

Standing to coolRoom temperature← A small amount of water, moisten the residue← About 10 mL of hydrochloric acid
← Water (up to about 100 mL)
Heating
Cover with a watch glass, and boil for 5 minutes.
Standing to coolRoom temperature

Transfer
250-mL - 500-mL volumetric flask, water← Water (up to the marked line)
Filtration
Type 3 filter paper
Sample solution
Figure 2 Flow sheet for total phosphate in fertilizers (2).
(Sample solution preparation by incineration-hydrochloric acid boiling)86Sample solution
Aliquot(predetermined amount)

100-mL volumetric flask← 1-2 drop(s) of phenolphthalein solution (1 g/100 mL)← Ammonia solution (1+1) [neutralization]← Nitric acid (1+10) [slightly acidic]← A proper amount of water← 20 mL of coloring reagent solution← Water (up to the marked line)
Leaving at rest

About 30 minutes

Measurement

Spectrophotometer (420 nm)

Figure 3 Flow sheet for total phosphate in fertilizers (3).
(Measurement procedure)87

4.2.1.b Quinoline gravimetric analysis(1) Summary
This test method is applicable to fertilizers containing organic matters.
It is suitable for the fertilizers with relatively a high content of phosphate. Add sulfuric acid,potassium sulfate and copper (II) sulfate pentahydrate to an analytical sample. Pretreat by
Kjeldahl method to convert total phosphate (T-P2O5) to phosphate ion, and measure the massof quinonium phosphomolybdate formed by the reaction with quinoline, molybdic acid andnitric acid to obtain total phosphate (T-P2O5).
(2) Reagents: Reagents are as shown below:a) Sulfuric acid: A JIS Guaranteed Reagent specified in JIS K 8951 or a reagent ofequivalent quality.
b) Nitric acid: A JIS Guaranteed Reagent (HNO3 60 % (mass fraction)) specified in JIS K
8541 or a reagent of equivalent quality.
c) Sodium molybdate solution: Dissolve 70 g of sodium molybdate dihydrate in 150 mL
of water.
d) Quinoline solution: Add 5 mL of quinolone specified in JIS K 8279 to the mixture
solution of 35 mL of nitric acid and 100 mL of water.
e) Quimosiac solution: Add 60 g of citric acid monohydrate specified in JIS K 8283 tothe mixture solution of 85 mL nitric acid and 150 mL of water to dissolve. Add
gradually total volume of the sodium molybdate solution to mix. Add gradually the total
volume of the quinoline solution while mixing the solution. After leaving at rest
overnight, filter the total volume with Type 3 filter paper. Add acetone specified in JIS
K 8962, and further add water to make 1,000 mL.
f) Catalyst(1): Mix potassium sulfate specified in JIS K 8962 and copper (II) sulfate
pentahydrate(2) specified in JIS K 8983 in the ratio of 9 to 1.
Note (1) A tablet is commercially available.
(2) Crush into powder as appropriate.
(3)
a)
b)
c)

Apparatus and instruments: Apparatus and instruments are as follows:
Water bath: Water bath that can be adjusted to 60 ºC – 65 ºC.
Drying apparatus: A drying apparatus that can be adjusted to 220 ºC±5 ºC.
Crucible type glass filter: Put a crucible type glass filter 1G4 specified in JIS R 3503
into a drying apparatus, let it stand to cool in a desiccator after heating at 220 ºC ± 5 ºC
and measure the mass to the order of 1 mg.
d) Digestion flask: Kjeldahl flask(4) Test procedures(4.1) Kjeldahl method: Conduct digestion as shown below:a) Weigh 0.5 g - 5 g of an analytical sample to the order of 1 mg, and put it in a 300-mL
digestion flask.
b) Add 5 g - 10 g of catalyst, and further add 20 mL - 40 mL of sulfuric acid to shake tomix, and heat gently.
c) After bubbles cease to form, heat until white smoke of sulfuric acid evolves.
d) Ignite temperature until organic matters are completely digested (3).
e) After standing to cool, add a small amount of water, mix well by shaking, and transfer
to a 250-mL - 500-mL volumetric flask with water.
f) After standing to cool, add water up to the marked line.
g) Filter with Type 3 filter paper to make the sample solution.
88

Note (3) After the change in the color of the solution ends, heat additionally for no lessthan 2 hours.
Comment 1 The procedures in (4.1) are the same as in (4.1.1) in 4.2.1.a. In addition, thesample solution prepared in (4.1.2) in 4.2.1.a and (4.1) a) - h) in 4.9.1.a canalso be used.
(4.2) Measurement: Conduct measurement as shown below.
a) Transfer a predetermined volume (the equivalents of 10 mg - 30 mg as P2O5 and no
more than 5 mL as sulfuric acid) of sample solution to a 300-mL tall beaker.
b) Add 5 mL of nitric acid and add water to make about 80 mL.
c) Cover with a watch glass and wash the watch glass and inside the tall beaker with water
after boiling for about 3 minutes, and then add water to make about 100 mL.
d) Immediately, add 50mL of quimosiac solution, heat for about 15 minutes whilesometimes mixing in a water bath at 60 ºC - 65 ºC to produce the precipitate ofquinolyum molybdate.
e) After standing to cool down to room temperature while sometimes mixing, filter under
reduced pressure with a crucible type glass filter, wash the tall beaker 3 times withwater and transfer the whole precipitate into a crucible type glass filter, and further wash
7 - 8 times with water.
f) Transfer the precipitate together with the crucible type glass filter into a drying
apparatus and heat at 220 ºC ± 5 ºC for about 30 minutes.
g) As soon as heating is complete, move it into a desiccator and let it stand to cool.
h) After standing to cool, remove the crucible type glass filter from the desiccator andmeasure the mass to the order of 1 mg.
i) Calculate total phosphate (T-P2O5) in the analytical sample by the following formula.
Total phosphate (T-P2O5) (% (mass fraction)) in an analytical sample= A × 0.03207 × (V1/V2) × (1/B) × 100
A: Mass (g) of the precipitate in h)
B: Mass of an analytical sample
V1: Predetermined volume (mL) of the sample solution
V2: Volume (mL) of the sample solution transferred in a)
Reference
1) Masayoshi KOSHINO: Second Revision of The Methods of Analysis of Fertilizers(Details), p.98 - 106, Yokendo, Tokyo (1988)

89

(5)

Flow sheet for total phosphate: The flow sheet for total phosphate in fertilizers isshown below:
0.5 g- 5 g
Weigh to the order of 1 mg into a 300-mL digestion flask.
analytical sample← 5 g - 10 g catalyst← 20 mL - 40 mL sulfuric acid
Heating
GentlyHeating

After bubbles cease to form,
ignite until organic matters are completely digested

Standing to coolRoom temperature← Small mount of waterTransfer
250-mL - 500-mL volumetric flask, water← Water (up to the marked line)
Filtration
Type 3 filter paper
Aliquot(predetermined
300-mL tall beakervolume)← 5 mL nitrate acid
← Water (to reach about 100 mL)
Cover with a watch glass for 3 minutes
Boiling
Wash a watch glass and inside the tall beaker with water← Water (to be about 100 mL)← Quimosiac solution 50 mL
Produce precipitate
60 ºC - 65 ºC, for 15 minutes, sometimes mixing
Standing to coolRoom temperature

Filtration
under reducedpressure

Crucible type glass filtering apparatus 1G4, 3 times with water

Washing

Wash 7 - 8 times with water

Drying220 ºC ± 5 ºC for about 30 minutes

Standing to cool

Desiccator

Measurement

Measure the mass to the order of 1 mg

Figure The flow sheet for total phosphate in fertilizers

904.2.2 Soluble phosphoric acid
4.2.2.a Ammonium vanadomolybdate absorptiometric analysis(1) Summary
This test method is applicable to fertilizers that do not contain the matter not colored by thehydrolysis with nitrate acids such as phosphonic acid.
Extract by adding water to an analytical sample, then extract by adding ammonium citric acidsolution, and combine them to make ammonia alkaline ammonium citrate soluble phosphoricacid (soluble phosphoric acid (S-P2O5)) and further heat after adding nitric acid (1+1),hydrolyze water-soluble phosphoric acid to orthophosphate ion and measure the absorbanceof phosphovanadomolybdate salt formed by the reaction with ammonium vanadate (V),hexaammonium heptamolybdate and nitric acid to obtain soluble phosphoric acid (S-P2O5).
(2) Reagents: Reagents are as shown below:a) Nitric acid: A JIS Guaranteed Reagent (HNO3 60 % (mass fraction)) specified in JIS K
8541 or a reagent of equivalent quality.
b) Ammonia solution: A JIS Guaranteed Reagent (NH3 28 % (mass fraction)) specified in
JIS K 8085 or a reagent of equivalent quality.
c) Petermans citrate solution: Add 173 g of citric acid monohydrate specified in JIS K
8283 in water to dissolve and add gradually ammonia solution equivalent to 42 g ofnitrogen while cooling. After standing to cool, add water to make 1,000 mL.
Additionally, check that the specific gravity of the solution is 1.082 - 1.083 (15 ºC) andthe nitrogen content per 1 mL is 42 mg.
d) Coloring reagent solution(1) (2): Dissolve 1.12 g of ammonium vanadate (V)(3) specified
in JIS K 8747 in water, add 150 mL of nitric acid, then add 50 g of hexaammonium
heptamolybdate tetrahydrate(4) specified in JIS K 8905 while dissolving in water, andfurther add water to make 1,000 mL.(5)e) Phosphate standard solution (P2O5 10 mg/mL)(1): Heat potassium dihydrogen
phosphate specified in JIS K 9007 at 105 ºC ± 2 ºC for about 2 hours, let it stand to coolin a desiccator, and weigh 19.17 g to a weighing dish. Dissolve with a small amount ofwater, transfer to a 1,000-mL volumetric flask, add 2 mL - 3 mL of nitric acid, and add
water up to the marked line.
f) Phosphate standard solution (P2O5 0.5 mg/mL)(1): Transfer 50 mL of phosphatestandard solution (P2O5 10 mg/mL) to a 1,000-mL volumetric flask, add 2 mL - 3 mL ofnitric acid, and add water up to the marked line.
Note (1) This is an example of preparation; prepare an amount as appropriate.
(2) This corresponds to reagent “b” solution in the Official Methods of Analysis of
Fertilizers (1992).
(3) This corresponds to ammonium metavanadate in the Official Methods ofAnalysis of Fertilizers (1992).
(4) This corresponds to ammonium molybdate in the Official Methods of Analysis
of Fertilizers (1992).
(5) Store in an amber bottle. However the reagent solution cannot tolerate long termpreservation.
Comment 1 The coloring reagent solution in d) can also be prepared by the following
method.
Dissolve 2.24 g of ammonium vanadate (V) (3) specified in JIS K 8747 inwater, add 300 mL of nitric acid, and add water to make 1,000 mL. Separately,
add 100 g of hexaammonium heptamolybdate tetrahydrate (4) specified in JIS91

K 8905 while dissolving in water, and further add water to make 1,000 mL. In
the case of usage, mix equal volumes of the two solutions.
(3)
a)
b)
c)

Instruments: Instruments are as follows:
Water bath: Water bath that can be adjusted to the test temperature ± 25 ºC.
Hot plate: A hot plate whose surface temperature can be adjusted up to 250 ºC.
Spectrophotometer: A spectrophotometer specified in JIS K 0115.

(4) Test procedures(4.1) Extraction: Conduct extraction as shown below.
a) Weigh 2.5 g of an analytical sample to the order of 1 mg, and put it in a small mortar.
b) Add about 20 mL - 25 mL of water, grind well and filter the supernatant with Type 6
filter paper into a 250-mL volumetric flask.
c) Further, after repeating procedure in b) 3 times, transfer non-dissolved matter in thesmall mortar onto a filter paper and wash with water until the filtrate becomes about 200mL.
d) Add a small amount of nitric acid to the filtrate, and further add water up to the marked
line to make the sample solution (1).
e) Transfer the non-dissolved matter on the filter paper together with the filter paper toanother 250- mL volumetric flask(6), add 100 mL of Petermans citrate solution andstopple, then shake to mix until the filter paper breaks down.
f) Heat the volumetric flask in e) in water bath at 65 ºC ± 2 ºC for 1 hour while shaking tomix every 15 minutes.
g) After standing to cool, add water up to the marked line.
h) Filter with Type 6 filter paper to make the sample solution (2).
Note (6) It is recommended to use a 250-mL short-neck volumetric flask.
Comment 2 The procedure in (4.1) is the same as the procedure in (4.1) of 4.2.2.b.
Comment 3 When the determination is affected by the coloring of the sample solution ofd) and h), transfer the predetermined volume (equivalent volume) (7) of thesample solution (1) and the sample solution (2) to a 100-mL volumetric flask,
add a few drops of hydrochloric acid (1+1) to acidify, then add no more than
0.1 g of active carbon. After leaving at rest for a little while, add water up to
the marked line and filter. The filtrate is to be the mixture solution for the
sample solution of (4.2) a). Additionally, as phosphorus contained in active
carbon has the possibility to elute and affects the determination value, a blanktest is required.
(4.2) Coloring: Conduct coloring as follows:a) Transfer a predetermined amount (the equivalents of 0.5 mg - 6 mg as P2O5 and no morethan the equivalents of 2 mL of Petermans citrate solution)(7) of sample solution (1) andsample solution (2) to a 100-mL tall beaker.
b) Add the solution to make Petermans citrate solution equivalent to 2 mL.
c) Add 4 mL of nitric acid (1+1) (8), and heat to boil (9).
d) After standing to cool, transfer it to a 100- mL volumetric flask with water (10).
e) Add 20 mL of coloring reagent solution, and further add water up to the marked line,
and then leave at rest for about 30 minutes (8).
Comment 4 In the procedure in a), a 100-mL volumetric flask can be used instead of a
100-mL tall beaker. However the volumetric flask used should be92

distinguished as a flask to be used for phosphate coloring operation andshould not be used for other purposes. Additionally, “transfer to a 100-mL
volumetric flask with water” in d) is replaced by “add a proper amount ofwater (11)”.
Note (7) The transferred volume of sample solution (1) and sample solution (2) should beequivalent.
(8) When the solution is muddled by adding nitric acid (1+1), conduct
centrifugation after the procedure in e).
(9) When it does not contain non-orthophosphate, the boiling operation is notnecessary.
(10) The volume of solution after transferring should be up to about 60 mL.
(11) If no water is added, precipitate is produced in some cases when coloring
reagent solution is added.
(4.3)

Measurement: Conduct measurement according to JIS K 0115 and as shown below.
Specific measurement procedures are according to the operation method of thespectrophotometer used in measurement.
a) Measurement conditions for the spectrophotometer: Set up the measurement
conditions for the spectrophotometer considering the following:
Detection wavelength: 420 nmb) Calibration curve preparation
1) Transfer 1 mL - 12 mL of phosphate standard solution (P2O5 0.5 mg/mL) to 100-mL
volumetric flasks step-by-step.
2) Add 2 mL of Petermans citrate solution, 4 mL of nitric acid (1+1) and a properamount of water(11), and conduct the same procedure as (4.2) e) to make the P2O5 0.5
mg/100 mL - 6 mg/100 mL phosphate standard solutions for the calibration curve
preparation.
3) Conduct the same procedures as 2) for another 100-mL volumetric flask to make theblank test solution for the calibration curve preparation.
4) Measure absorbance at a wavelength of 420 nm of the phosphate standard solutions
for the calibration curve preparation using the blank test solution for the calibration
curve preparation as the control (12).
5) Prepare the calibration curve of the phosphate concentration and absorbance of thephosphate standard solutions for the calibration curve preparation.
c) Sample measurement
1) Regarding the solution of (4.2) e), measure the absorbance by the same procedure asb) 4) (12).
2) Obtain the phosphate (P2O5) content from the calibration curve, and calculate soluble
phosphoric acid (S-P2O5) in the analytical sample.
Note (12) Measure within 2 hours after adding the coloring reagent solution.
Comment 5 Table 1 shows results from a simultaneous analysis with the same samples(proficiency testing and external quality control testing) hosted by the Japan
Fertilizer Quality Assurance Committee analyzed by the robust method.

93

Table 1 Results and analysis results from a simultaneous analysis with the same samples
for soluble phosphoric acid hosted by the Japan Fertilizer Quality Assurance Committee 1)
5)
RSDrob
Number of Median Μ2) NIQR4)
Year
Samplelaboratories
(%)
(%)3)
(%)3)
High analysis
144
10.88
0.11
1.0
compound fertilizer
Ordinary compound
2009
124
6.37
0.12
1.9
fertilizer
High analysis
2011
113
17.44
0.22
1.3
compound fertilizer
1) Proficiency testing and external quality control testing2) Median (M) agrees with the mean in normal distribution.
3) Mass fraction4) Robust standard deviation (NIQR) agrees with standard deviation in normal distribution.
5) RSDrob stands for relative standard deviation obtained from the robust method
2006by the formula shown below:
RSDrob = (NIQR/M) × 100

Reference
1) Masayoshi KOSHINO: Second Revision of The Methods of Analysis of Fertilizers(Details), p.108 - 114, Yokendo, Tokyo (1988)

94

(5)

Flow sheet for soluble phosphoric acid: The flow sheet for soluble phosphoric acid in
fertilizers is shown below:
2.5 g analytical sample
Repeat 3 times

Weigh to the order of 1 mg to a small mortar← About 20 mL - 25 mL water
Grinding

Supernatant filtration＜Residue＞

Type 6 filter paper, 250-mL volumetric flask

Transfer
Transfer residue to a filter paper← Washing with water, until filtrate reaches about 200 mL＜Residue＞＜Filtrate＞← A small amount of nitric acid
← Water (up to the marked line)
Sample solution (1)Transfer
Together with filter paper, 250-mL volumetric flask← 100 ml of Petermans citrate solution
Shaking to mix
Stopple and shake to mix until a filter paler breaks down
Heating

At 65 ºC ± 2 ºC for 1 hour while shaking to mix every 15 minutes.

Standing to cool
← Water (up to the marked line)
Filtration
Type 6 filter paper
Sample solution (2)Aliquot
Transfer the same volume of sample solution (1)(predetermined volume)and sample solution (2) to 100-mL volumetric flasks← Petermans citrate solution, until it reaches the equivalent of 2 mL
← 4 mL of nitric acid (1+1)
Heating
Boiling← A proper amount of water← 20 mL of coloring reagent solution← Water (up to the marked line)
Leaving at rest
For about 30 minutes
Measurement

Spectrophotometer (420nm)

Figure The flow sheet for soluble phosphoric acid in fertilizers

954.2.2.b Quinoline gravimetric analysis(1) Summary
This test method is applicable to fertilizers containing no phosphonic acid, etc.
It is suitable for the fertilizers containing a relatively a high content of phosphate.
Extract by adding water to an analytical sample, then extract by adding ammonium citric acidsolution and combine them to make ammonia alkaline ammonium citrate soluble phosphoricacid (soluble phosphoric acid (S-P2O5)) and measure the mass of quinolium molybdate
formed by the reaction with quinoline, molybdic acid and nitric acid to obtain soluble
phosphoric acid (S-P2O5).
(2) Reagents: Reagents are as shown below:a) Nitric acid: A JIS Guaranteed Reagent (HNO3 60 % (mass fraction)) specified in JIS K
8541 or a reagent of equivalent quality.
b) Ammonia solution: A JIS Guaranteed Reagent (NH3 28 % (mass fraction)) specified in
JIS K 8085 or a reagent of equivalent quality.
c) Petermans citrate solution: Dissolve 173 g of citric acid monohydrate specified in JIS
K 8283 by adding to water and add gradually ammonia solution equivalent to 42 g ofnitrogen while cooling. After standing to cool, add water to make 1,000 mL.
Additionally, check that the specific gravity of the solution is 1.082 -1.083 (15 ºC) andthe volume of nitrogen per 1 mL is 42 mg.
d) Sodium molybdate solution: Dissolve 70 g of sodium molybdate dihydrate in 150 mL
of water.
e) Quinoline solution: Add 5 mL of quinoline specified in JIS K 8279 to the mixture
solution of 35 mL of nitric acid and 100 mL of water.
f) Quimosiac solution: Add 60 g of citric acid monohydrate specified in JIS K 8283 tothe mixture solution of 85 mL nitric acid and 150 mL of water to dissolve. Add
gradually total volume of the sodium molybdate solution to mix them. Add gradually
the total volume of the quinoline solution while mixing the solution. After leaving at
rest overnight, filter the total volume with Type 3 filter paper. Add 280 mL of acetone
specified in JIS K 8962, and further add water to make 1,000 mL.
(3) Apparatus and instruments: Apparatus and instruments are as follows:a) Water bath: A water bath that can be adjusted to the test temperature ± 2 ºC.
b) Drying apparatus: A drying apparatus that can be adjusted to 220 ºC ± 5 ºC.
c) Crucible type glass filter: Put a crucible type glass filter 1G4 specified in JIS R 3503
into a drying apparatus, stand to cool in a desiccator after heating at 220 ºC ± 5 ºC andmeasure the mass to the order of 1 mg.
(4) Test procedures(4.1) Extraction: Conduct extraction as shown below.
a) Weigh 2.5 g of an analytical sample to the order of 1 mg, and put it in a small mortar.
b) Add about 20 mL - 25 mL of water, grind well and filter the supernatant with Type 6
filter paper into a 250-mL volumetric flask.
c) Further, after repeating 3 times the procedure in b), transfer non-dissolved matter in thesmall mortar on a filter paper and wash with water until the filtrate becomes about 200mL.
d) Add a small amount of nitric acid to the filtrate, and further add water up to the marked
line to make the sample solution (1).
e) Transfer the non-dissolved matter on the filter paper together with the filter paper toanother 250- mL (1) volumetric flask, and add 100 mL of Petermans citrate solution andstopple. Then shake to mix until the filter paper breaks down.
96

f)Heat the volumetric flask in e) in a water bath at 65 ºC ± 2 ºC for 1 hour while shaking
to mix every 15 minutes.
g) After standing to cool, add water up to the marked line.
h) Filter with Type 6 filter paper to make the sample solution (2).
Note (1) It is recommended to use a short-neck volumetric flask.
Comment 1 The procedure in(4.1) is the same as the procedure in (4.1) of 4.2.2.a.
(4.2) Measurement: Conduct measurement as shown below.
a) Transfer a predetermined volume (the equivalents of 10 mg - 30 mg as P2O5 and no
more than the equivalents of 8 mL of Petermans citrate solution) (2) of sample solution(1) and sample solution (2) to a 300-mL tall beaker.
b) Add 5 mL of nitric acid and add water to make about 80 mL.
c) Cover with a watch glass. After boiling for about 3 minutes, wash the watch glass andthe inside of the tall beaker with water and add water to make about 100 mL.
d) Immediately, add 50 mL of quimosiac solution, heat for about 15 minutes whilesometimes mixing in a water bath at 60 ºC - 65 ºC and produce the precipitate ofquinolyum molybdate.
e) After standing to cool down to room temperature while sometimes mixing, filter under
reduced pressure with a crucible type glass filter, wash the tall beaker 3 times withwater and transfer the whole precipitate into a crucible type glass filter, further wash 7 8 times with water.
f) Transfer the precipitate together with the crucible type glass filter into a drying
apparatus and heat at 220 ºC ± 5 ºC for about 30 minutes.
g) As soon as heating is complete, move it into a desiccator and let it stand to cool.
h) After standing to cool, remove the crucible type glass filter from the desiccator andmeasure the mass to the order of 1 mg.
i) Calculate soluble phosphoric acid (S-P2O5) by the following formula.
Soluble phosphoric acid (% (mass fraction)) in an analytical sample= A × 0.03207 × (V1/V2) × (1/B) × 100
A: Mass (g) of the precipitate in h)
B: Mass of an analytical sample (2.5 g)
V1: Predetermined volume (250 mL) of the sample solution
V2: Volume (mL) of the sample solution transferred in a)
Note (2) The transferred volume of sample solution (1) and the sample solution (2)should be equivalent.
Reference
1) Masayoshi KOSHINO: Second Revision of The Methods of Analysis of Fertilizers(Details), p.98 - 106, Yokendo, Tokyo (1988)

97

(5) Flow sheet for soluble phosphoric acid: The flow sheet for soluble phosphoric acidin fertilizers is shown below:
2.5 g analytical sample
Repeat 3 times

Weigh to the order of 1 mg to a small mortar← About 20 mL - 25 mL water
Grinding

Supernatant filtration＜Residue＞

Type 6 filter paper, 250-mL volumetric flask

Transfer
Transfer residue to a filter paper← Wash with water, until filtrate reaches about 200 mL＜Residue＞＜Filtrate＞← A small amount of nitric acid
← Water (up to the marked line)
Sample solution (1)Transfer
Together with filter paper, 250-mL volumetric flask← 100 ml Petermans citrate solution
Shaking to mix
Stopple and shake to mix until a filter paler breaks down
Heating

At 65 ºC ± 2 ºC for 1 hour while shaking to mix every 15 minutes.

Standing to cool
← Water (up to the marked line)
Filtration
Type 6 filter paper
Sample solution (2)Aliquot
Transfer the same volume of sample solution (1) and(predetermined volume)
sample solution (2) to 300-mL volumetric flasks← 5 mL nitrate acid
← Water (to reach about 100 mL)
Cover with a watch glass for 3 minutes
Boiling
Wash a watch glass and inside a tall beaker with water← Water (to make about 100 mL)← 50 mL of quimosiac
Forming precipitate
60 ºC - 65 ºC, for 15 minutes, sometimes mixing
Standing to cool
Filtration
under reduced pressure

Room temperature
Crucible type glass filtering apparatus 1G4, 3 times with water

Washing

Wash 7 - 8 times with water

Drying220 ºC ± 5 ºC for about 30 minutes

Standing to cool

Desiccator

Measurement

Measure the mass to the order of 1 mg

Figure The flow sheet for soluble phosphoric acid in fertilizers

984.2.3

Citrate soluble phosphoric acid

4.2.3.a Ammonium vanadomolybdate absorptiometric analysis(1) Summary
This test method is applicable to the fertilizers that do not contain the matter not colored by
the hydrolysis with nitrate acids such as phosphonic acid.
Extract by adding citric acid solution to an analytical sample, and hydrolyze citrate soluble
phosphoric acid (C-P2O5) to orthophosphate ion and measure the absorbance ofphosphovanadomolybdate salt formed by the reaction with ammonium vanadate (V),hexaammonium heptamolybdate and nitric acid to obtain citrate soluble phosphoric acid(C-P2O5).
(2) Reagents: Reagents are as shown below:a) Nitric acid: A JIS Guaranteed Reagent (HNO3 60 % (mass fraction)) specified in JIS K
8541 or a reagent of equivalent quality.
b) Citric acid solution (1): Dissolve 20 g of citric acid monohydrate specified in JIS K
8283 in water to make 1,000 mL.
c) Coloring reagent solution(1) (2): Dissolve 1.12 g of ammonium vanadate (V)(3) specified
in JIS K 8747 in water, add 150 mL of nitric acid, then add 50 g of hexaammonium
heptamolybdate tetrahydrate(4) specified in JIS K 8905 while dissolving in water, andfurther add water to make 1,000 mL(5).
d) Phosphate standard solution (P2O5 10 mg/mL)(1): Heat potassium dihydrogen
phosphate specified in JIS K 9007 at 105 ºC ± 2 ºC for about 2 hours, let it stand to coolin a desiccator, and weigh 19.17 g to a weighing dish. Dissolve with a small amount ofwater, transfer to a 1,000-mL volumetric flask, add 2 mL - 3 mL of nitric acid, and add
water up to the marked line.
e) Phosphate standard solution (P2O5 0.5 mg/mL)(1): Transfer 50 mL of phosphatestandard solution (P2O5 10 mg/mL) to a 1,000-mL volumetric flask, add 2 mL - 3 mL ofnitric acid, and add water up to the marked line.
Note (1) This is an example of preparation; prepare an amount as appropriate.
(2) This corresponds to reagent “b” solution in the Official Methods of Analysis of
Fertilizers (1992).
(3) This corresponds to ammonium metavanadate in the Official Methods ofAnalysis of Fertilizers (1992).
(4) This corresponds to ammonium molybdate in the Official Methods of Analysis
of Fertilizers (1992).
(5) Store in an amber bottle. However the reagent solution does not tolerate longterm preservation.
Comment 1 The coloring reagent solution in c) can also be prepared by the following
method.
Dissolve 2.24 g of ammonium vanadate (V) (3) specified in JIS K 8747 inwater, add 300 mL of nitric acid, and add water to make 1,000 mL. Separately,
dissolve 100 g of hexaammonium heptamolybdate tetrahydrate (4) specified in
JIS K 8905 in water, and further add water to make 1,000 mL. Mix equalvolume of two solutions in the case of usage.
(3)

Instruments: Instruments are as follows:99a) Constant-temperature rotary shaker: A constant-temperature rotary shaker that canrotate a 250-mL volumetric flask, set up in a thermostat adjustable to 30 ºC ± 1 ºC,
upside down at 30 - 40 revolutions/min.
b) Hot plate: A hot plate whose surface temperature can be adjusted up to 250 ºC.
c) Spectrophotometer: A spectrophotometer specified in JIS K 0115.
(4) Test procedures(4.1) Extraction: Conduct extraction as shown below.
a) Weigh 1 g of an analytical sample to the order of 1 mg, and put it in a 250-mL
volumetric flask.
b) Add 150 mL of citric acid solution heated up to about 30 ºC, and shake to mix at 30 - 40
revolutions/min (30 ºC ± 1 ºC) for 1 hour.
c) After standing to cool, add water up to the marked line.
d) Filter with Type 3 filter paper to make the sample solution.
Comment 2 The procedure in (4.1) is the same as the procedure in (4.1) of 4.2.3.b, 4.3.2.a,4.3.2.b, 4.6.2.a, 4.7.2.a and 4.8.1.a.
Comment 3 When the determination is affected by the coloring of the sample solution ofd), transfer a predetermined volume of the sample solution to a 100-mL
volumetric flask, add a few drops of hydrochloric acid (1+1) to make itacidity, and then add no more than 0.1 g of active carbon. After leaving at rest
for a little while, add water up to the marked line and filter. The filtrate isprepared as the sample solution of (4.2) a). Additionally, as phosphorus
contained in active carbon has the possibility to elute and affects thedetermination value, a blank test is required.
Comment 4 For a by-product phosphate fertilizer or a fertilizer containing a by-productphosphate, if pH of the sample solution in d) is neutral or basic, prepare asample solution anew by replacing “1 g of an analytical sample” in theprocedure in a) with “0.5 g of an analytical sample”.
(4.2) Coloring: Conduct coloring as follows:a) Transfer a predetermined amount (the equivalents of 0.5 mg - 6 mg as P2O5 and no morethan the equivalents of 17 mL of the citric acid solution) of the sample solution to a
100-mL tall beaker.
b) Add the citric acid solution to make the equivalents of 17 mL of the citric acid solution(20 mg/mL).
c) Add 4 mL of nitric acid (1+1) (6), and heat to boil (7).
d) After standing to cool, transfer to a 100- mL volumetric flask with water (8).
e) Add 20 mL of coloring reagent solution, and further add water up to the marked line,
and then leave at rest for about 30 minutes.
Comment 5 In the procedure in a), a 100-mL volumetric flask can be used instead of a
100-mL tall beaker. However the volumetric flask used should be
distinguished as a flask to be used for phosphate coloring operation andshould not be used for other purposes. Additionally, “transfer to a 100-mL
volumetric flask with water” in d)” is replaced by “add a proper amount ofwater (9)”.
Note (6) When the solution is muddled by adding nitric acid (1+1), conduct
centrifugation after the procedure in e).
100(7) When it does not contain non-orthophosphate, the boil operation is notnecessary.
(8) The volume of solution after transferring should be up to about 60 mL.
(9) If no water is added, precipitate is produced in some cases when a coloring
reagent solution is added.
(4.3) Measurement: Conduct measurement according to JIS K 0115 and as shown below.
Specific measurement procedures are according to the operation method of thespectrophotometer used in measurement.
a) Measurement conditions for the spectrophotometer: Set up the measurement
conditions for the spectrophotometer considering the following:
Detection wavelength: 420 nmb) Calibration curve preparation
1) Transfer 1 mL - 12 mL of phosphate standard solution (P2O5 0.5 mg/mL) to 100-mL
volumetric flasks step-by-step.
2) Add 17 mL of citric acid solution, then add 4 mL of nitric acid (1+1), further add aproper amount of water(9) and conduct the same procedure as (4.2) e) to make theP2O5 0.5 mg/100 mL - 6 mg/100 mL phosphate standard solution for the calibration
curve preparation.
3) Conduct the same procedures as 2) for another 100-mL volumetric flask to make theblank test solution for the calibration curve preparation.
4) Measure absorbance at a wavelength of 420 nm of the phosphate standard solutions
for the calibration curve preparation using the blank test solution for the calibration
curve preparation as the control (10).
5) Prepare the calibration curve of the phosphate concentration and absorbance of thephosphate standard solutions for the calibration curve preparation.
c) Sample measurement
1) Regarding the solution in (4.2) e), measure the absorbance by the same procedure asb) 4) (10).
2) Obtain the phosphate (P2O5) content from the calibration curve, and calculate citratesoluble phosphoric acid (C-P2O5) in the analytical sample.
Note (10) Measure within 2 hours after adding the coloring reagent solution.
Comment 6 Table 1 shows results from a simultaneous analysis with the same samples(proficiency testing and external quality control testing) hosted by the Japan
Fertilizer Quality Assurance Committee analyzed by the robust method.

101

Table 1

Results and analysis results from simultaneous analysis with the same samples
1)
for nitrate nitrogen hosted by the Japan Fertilizer Quality Assurance Committee
2)

Year Sample

Number of Median Μ
3)laboratories
(%)

4)

5)

NIQR

RSDrob

3)

(%)

(%)

2007 Organic compound fertilizer
143
9.81
0.13
1.3
2008 High analysis compound fertilizer
144
15.82
0.13
0.8
2010 High analysis compound fertilizer
140
14.59
0.18
1.2
2013 High analysis compound fertilizer
128
20.60
0.19
0.9
1) Proficiency testing and external quality control testing2) Median (M) agrees with the mean in normal distribution.
3) Mass fraction4) Robust standard deviation (NIQR) agrees with standard deviation in normal distribution.
5) RSDrob stands for relative standard deviation obtained from the robust methodby the formula shown below:
RSDrob = (NIQR/M) × 100

Reference
1) Masayoshi KOSHINO: Second Revision of The Methods of Analysis of Fertilizers(Details), p.108 - 114, Yokendo, Tokyo (1988)(5)

Flow sheet for citrate soluble phosphoric acid: The flow sheet for citrate soluble
phosphoric acid in fertilizers is shown below:
1 g analytical sample
Weigh to the order of 1 mg to a 250-mL volumetric flask← 150 mL of citric acid solution [about 30 ºC]
Shaking to mix
Constant-temperature rotary shaker (30 -40 revolutions/min)
30 ºC ± 1 ºC, for 1 hourStanding to cool
← Water (up to the marked line)
Filtration
Type 3 filter paper
Aliquot
100-mL volumetric flask(predetermined volume)← Citric acid solution, until it reaches the equivalents of 17 mL← 4 mL of nitric acid(1+1)
Heating
Boiling← A proper amount of water← 20 mL of coloring reagent solution← Water (up to the marked line)
Leaving at rest
For about 30 minutes
Measurement

Spectrophotometer (420nm)

Figure The flow sheet for citrate soluble phosphoric acid in fertilizers

1024.2.3.b Quinoline gravimetric analysis(1) Summary
This test method is applicable to fertilizers containing no phosphonic acid, etc. It is suitable
for the fertilizers containing relatively a high content of phosphate.
Extract by adding citric acid solution to an analytical sample, and measure the mass ofquinonium phosphomolybdate formed by the reaction of citrate soluble phosphoric acid(C-P2O5) with quinoline, molybdic acid and nitric acid to obtain citrate soluble phosphoricacid (C-P2O5).
(2) Reagents: Reagents are as shown below:a) Nitric acid: A JIS Guaranteed Reagent (HNO3 60 % (mass fraction)) specified in JIS K
8541 or a reagent of equivalent quality.
b) Citric acid solution (1): Dissolve citric acid monohydrate specified in JIS K 8283 inwater to make 1,000 mL.
c) Sodium molybdate solution: Dissolve 70 g of sodium molybdate dihydrate in 150 mL
of water.
d) Quinoline solution: Add 5 mL of quinoline specified in JIS K 8279 to the mixture
solution of 35 mL of nitric acid and 100 mL of water.
e) Quimosiac solution: Add 60 g of citric acid monohydrate specified in JIS K 8283 tothe mixture solution of 85 mL of nitric acid and 150 mL of water to dissolve. Add
gradually total volume of the sodium molybdate solution to mix. Add gradually the total
volume of the quinoline solution while mixing the solution. After leaving at rest
overnight, filter the total volume with Type 3 filter paper. Add 280 mL of acetone
specified in JIS K 8962, and further add water to make 1,000 mL.
Note (1) This is an example of preparation; prepare an amount as appropriate.
(3) Instruments: Instruments are as follows:a) Constant-temperature rotary shaker: A constant-temperature rotary shaker that canrotate a 250-mL volumetric flask, set up in a thermostat adjustable to 30 ºC ± 1 ºC,
upside down at 30 - 40 revolutions/min.
b) Water bath: Water bath that can be adjusted to 60 ºC - 65 ºC.
c) Drying apparatus: A drying apparatus that can be adjusted to 220 ºC ± 5 ºC.
d) Crucible type glass filter: Put a crucible type glass filter 1G4 specified in JIS R 3503
into a drying apparatus, stand to cool in a desiccator after heating at 220 ºC ± 5 ºC andmeasure the mass to the order of 1 mg.
(4) Test procedures(4.1) Extraction:
Conduct extraction as shown below.
a) Weigh 1 g of an analytical sample to the order of 1 mg, and transfer to a 250-mL
volumetric flask.
b) Add 150 mL of citric acid solution heated up to about 30 ºC, and shake to mix at 30-40
revolutions/min (30 ºC ± 1 ºC) for 1 hour.
c) After standing to cool, add water up to the marked line.
d) Filter with Type 3 filter paper to make the sample solution.
Comment 1 The procedure in (4.1) is the same as the procedure in (4.1) of 4.2.3.a.
Comment 2 For a by-product phosphate fertilizer or a fertilizer containing a by-productphosphate, if pH of the sample solution of d) is neutral or basic, prepare asample solution anew by replacing “1 g of an analytical sample” in theprocedure in a) with “0.5 g of an analytical sample”.
103(4.2) Measurement: Conduct measurement as shown below.
a) Transfer a predetermined volume (the equivalents of 10 mg - 30 mg as P2O5) of sample
solution to a 300-mL tall beaker.
b) Add 5 mL of nitric acid and add water to make about 80 mL.
c) Cover with a watch glass. After boiling for about 3 minutes, wash the watch glass andthe inside the tall beaker with water and add water to make about 100 mL.
d) Immediately, add 50 mL of quimosiac solution, heat for about 15 minutes whilesometimes mixing in a water bath at 60 ºC - 65 ºC and produce the precipitate ofquinolyum molybdate.
e) After standing to cool down to room temperature while sometimes mixing, filter under
reduced pressure with a crucible type glass filter, wash the tall beaker 3 times withwater and transfer the whole precipitate into a crucible type glass filter, further wash 7 8 times with water.
f) Transfer the precipitate together with the crucible type glass filter into a drying
apparatus and heat at 220 ºC ± 5 ºC for about 30 minutes.
g) As soon as heating is complete, move into a desiccator and let it stand to cool.
h) After standing to cool, remove the crucible type glass filter from the desiccator andmeasure the mass to the order of 1 mg.
i) Calculate citrate soluble phosphoric acid (C-P2O5) by the following formula.
Citrate soluble phosphoric acid (C-P2O5) (% (mass fraction)) in an analytical sample= A × 0.03207 × (V1/V2) × (1/B) × 100
A: Mass (g) of the precipitate in h)
B: Mass of an analytical sample (1 g)
V1: Predetermined volume (250 mL) of the sample solution
V2: Volume (mL) of the sample solution transferred in a)
Reference
1) Masayoshi KOSHINO: Second Revision of The Methods of Analysis of Fertilizers(Details), p.98 - 106, Yokendo, Tokyo (1988)

104

(5)

Flow sheet for citrate soluble phosphoric acid: The flow sheet for citrate soluble
phosphoric acid in fertilizers is shown below:
1 g analytical sample
Weigh to the order of 1 mg to a 250-mL volumetric flask← 150 mL of citric acid solution [about 30 ºC]
Shaking to mix
Constant-temperature rotary shaker (30 - 40 revolutions/min)
30 ºC ± 1 ºC, for 1 hourStanding to cool
← Water (up to the marked line)
Filtration
Type 3 filter paper
Aliquot
300-mL tall beaker(predetermined volume)← 5 mL nitric acid
← Water (to reach about 100 mL)
Cover with a watch glass for 3 minutes
Boiling
Wash a watch glass and the inside the tall beaker with water← Water (to be about 100 mL)← 50mL of quimosiac solution
Forming precipitation
60 ºC - 65 ºC, for 15 minutes, sometimes mixing
Standing to cool
Filtration
under reduced pressure

Room temperature
Crucible type glass filtering apparatus 1G4, 3 times with water

Washing

Wash 7 - 8 times with water

Drying220 ºC ± 5 ºC for about 30 minutes

Standing to cool

Desiccator

Measurement

Measure the mass to the order of 1 mg

Figure The flow sheet for citrate soluble phosphoric acid in fertilizers

1054.2.4 Water-soluble phosphoric acid
4.2.4.a Ammonium vanadomolybdate absorptiometric analysis(1) Summary
This test method is applicable to the fertilizers that do not contain matters not colored byhydrolysis with nitrate acids such as phosphonic acid.
Extract by adding water to an analytical sample, add nitric acid (1+1) to heat, and hydrolyze
water-soluble phosphoric acid to orthophosphate ion, then measure the absorbance ofphosphovanadomolybdate salt formed by the reaction with ammonium vanadate (V),hexaammonium heptamolybdate and nitric acid to obtain water-soluble phosphoric acid
(W-P2O5).
(2) Reagents: Reagents are as shown below:a) Nitric acid: A JIS Guaranteed Reagent (HNO3 60 % (mass fraction)) specified in JIS K
8541 or a reagent of equivalent quality.
b) Ammonia solution: A JIS Guaranteed Reagent (NH3 28 % (mass fraction)) specified in
JIS K 8085 or a reagent of equivalent quality.
c) Coloring reagent solution(1) (2): Dissolve 1.12 g of ammonium vanadate (V)(3) specified
in JIS K 8747 in water, add 250 mL of nitric acid, then add 27 g of hexaammonium
heptamolybdate tetrahydrate(4) specified in JIS K 8905 while dissolving in water, andfurther add water to make 1,000 mL(5).
d) Phenolphthalein solution (1 g/100 mL): Dissolve 1 g of phenolphthalein specified in
JIS K 8799 in 100 mL of ethanol (95) specified in JIS K 8102.
e) Phosphate standard solution (P2O5 10 mg/mL)(1): Heat potassium dihydrogen
phosphate specified in JIS K 9007 at 105 ºC ± 2 ºC for about 2 hours, let it stand to coolin a desiccator, and then weigh 19.17 g to a weighing dish. Dissolve with a small
amount of water, transfer to a 1,000-mL volumetric flask, add 2 mL - 3 mL of nitric acid,and add water up to the marked line.
f) Phosphate standard solution (P2O5 0.5 mg/mL)(1): Transfer 50 mL of phosphatestandard solution (P2O5 10 mg/mL) to a 1,000-mL volumetric flask, add 2 mL - 3 mL ofnitric acid, and add water up to the marked line.
Note (1) This is an example of preparation; prepare an amount as appropriate.
(2) This corresponds to reagent “a” solution in the Official Methods of Analysis of
Fertilizers (1992).
(3) This corresponds to ammonium metavanadate in the Official Methods ofAnalysis of Fertilizers (1992).
(4) This corresponds to ammonium molybdate in the Official Methods of Analysis
of Fertilizers (1992).
(5) Store in an amber bottle.
(3) Instruments: Instruments are as follows:a) Rotary shaker: A rotary shaker that can rotate a 500-mL volumetric flask upside down
at 30 - 40 revolutions/min.
b) Hot plate: A hot plate whose surface temperature can be adjusted up to 250 ºC.
c) Spectrophotometer: A spectrophotometer specified in JIS K 0115.
(4) Test procedures(4.1) Extraction: Conduct extraction as shown below.
a) Weigh 5 g of an analytical sample to the order of 1 mg, and transfer to a 500-mL
volumetric flask.
106b) Add about 400 mL of water, and shake to mix at 30 - 40 revolutions /min for about 30
minutes.
c) Add water up to the marked line.
d) Filter with Type 3 filter paper to make the sample solution.
Comment 1 In the procedure in a), it is also allowed to weigh 2.5 g of an analyticalsample to the order of 1 mg, and put it to a 250-mL volumetric flask.
Comment 2 The procedure in (4.1) is the same as the procedure in (4.1) of 4.2.4.b, 4.2.4.cand 4.7.3.a, the procedure in (4.1.1) of 4.1.2.b, and the procedure in (4.1.2) of
4.3.3.a, 4.3.3.b and 4.3.3.c. In addition, the solution is also usable as the
sample solution of 4.9.2.a, 4.10.2.a, 4.13.1.a and 4.14.1.a.
Comment 3 When the determination is affected by the coloring of the sample solution ofd), transfer the predetermined volume of the sample solution to a 100-mL
volumetric flask, add a few drops of hydrochloric acid (1+1) to make it acidic,
then add no more than 0.1 g of active carbon. After leaving at rest for a little
while, add water up to the marked line and filter. The filtrate is prepared as
the sample solution of (4.2) a). Additionally, as phosphorus contained in
active carbon has the possibility to elute and affects the determination value, ablank test is required.
(4.2) Coloring: Conduct coloring as follows:a) Transfer a predetermined amount (the equivalents of 0.5 mg - 6 mg as P2O5) of thesample solution to a 100-mL tall beaker.
b) Add 4 mL of nitric acid (1+1) (6), and heat to boil (7).
c) After standing to cool, add 1 - 2 drop(s) of phenolphthalein solution (1 g/100 mL), andneutralize by adding ammonia solution (1+1) until the color of the solution becomeslight red-purple.
d) Add nitric acid (1+10) until the light red-purple color of the solution disappears to makeit slightly acidic. Transfer with water to a 100-mL volumetric flask.
e) Add 20 mL of coloring reagent solution, and further add water up to the marked line,
and then leave at rest for about 30 minutes (6).
Comment 4 In the procedure in a), a volumetric flask can be used instead of a 100 mL tallbeaker. However the volumetric flask used should be distinguished as a flask
to be used for phosphoric coloring operation and should not be used for theother purposes. Additionally, “transfer to a 100 mL volumetric flask withwater (8)” in d) is replaced by “add a proper amount of water (9)”.
Note (6) When the solution is muddled by adding nitric acid (1+1), conduct
centrifugation after the procedure in e).
(7) When it does not contain non-orthophosphate, the procedure in b) is notnecessary.
(8) The volume of solution after transferring should be up to about 60 mL.
(9) If no water is added, precipitate is produced in some cases when coloring
reagent solution is added.
(4.3) Measurement: Conduct measurement according to JIS K 0115 and as shown below.
Specific measurement procedures are according to the operation method of thespectrophotometer used in measurement.
a) Measurement conditions for the spectrophotometer: Set up the measurement
conditions for the spectrophotometer considering the following:
Detection wavelength: 420 nm
107b) Calibration curve preparation
1) Transfer 1 mL - 12 mL of phosphate standard solution (P2O5 0.5 mg/mL) to 100-mL
volumetric flasks step-by-step.
2) Add a proper amount of water (9) and conduct the same procedure as (4.2) e) to makethe P2O5 0.5 mg/100 mL - 6 mg/100 mL phosphate standard solution for thecalibration curve preparation.
3) Conduct the same procedures as 2) for another 100-mL volumetric flask to make theblank test solution for the calibration curve preparation.
4) Measure the absorbance at a wavelength of 420 nm of the phosphate standard
solutions for the calibration curve preparation using the blank test solution for thecalibration curve preparation as the control (10).
5) Prepare the calibration curve of the phosphate concentration and absorbance of thephosphate standard solutions for the calibration curve preparation.
c) Sample measurement
1) Regarding the solution in (4.2) e), measure absorbance by the same procedure as b)4) (10).
2) Obtain the phosphate (P2O5) content from the calibration curve, and calculate
water-soluble phosphoric acid (W-P2O5) in the analytical sample.
Note (10) Measure within 6 hours after adding the coloring reagent solution in theprocedure in (4.2) e).
Comment 5 Table 1 shows results from a simultaneous analysis with the same samples(proficiency testing and external quality control testing) hosted by the Japan
Fertilizer Quality Assurance Committee analyzed by the robust method.

108

Table 1

Results and analysis results from simultaneous analysis with the same samples
for nitrate nitrogen hosted by the Japan Fertilizer Quality Assurance Committee 1)

Year
2006
2007
2008
2009
2010
2011
2012

SampleHigh analysis
compound fertilizer
Organic compound
fertilizer
High analysis
compound fertilizer
Ordinary compound
fertilizer
High analysis
compound fertilizer
High analysis
compound fertilizer
Fluid mixed fertilizer

2)

4)

5)

Number oflaboratories

Median Μ(%)(%)

RSDrob
(%)

157

9.02

0.12

1.3

143

7.02

0.23

3.3

146

9.16

0.24

2.7

142

4.57

0.08

1.7

143

11.56

0.52

4.5

132

14.41

0.19

1.3

128

2.88

0.06

2.1

3)

NIQR

3)

High analysis
133
12.08
0.19
0.9
compound fertilizer
1) Proficiency testing and external quality control testing2) Median (M) agrees with the mean in normal distribution.
3) Mass fraction4) Robust standard deviation (NIQR) agrees with standard deviation in normal distribution.
5) RSDrob stands for relative standard deviation obtained from the robust methodby the formula shown below:
RSDrob = (NIQR/M) × 100
2013

Reference
1) Masayoshi KOSHINO: Second Revision of The Methods of Analysis of Fertilizers(Details), p.108 - 114, Yokendo, Tokyo (1988)

109

(5)

Flow sheet for water-soluble phosphoric acid: The flow sheet for water-soluble
phosphoric acid in fertilizers is shown below:
5 g analytical sample
Weigh to the order of 1 mg into a 500-mL volumetric flask← Water, about 400 mL
Shaking to mix
Rotary shaker (30 - 40 revolutions/min) for 30 minutes← Water (up to the marked line)
Filtration
Type 3 filter paper←Water (up to the marked line)
Aliquot
100-mL volumetric flask(predetermined volume)← 4 mL of nitric acid(1+1)
Heating
BoilingStanding cool
← 1-2 drop(s) of phenolphthalein solution (1 g/100 mL)← Ammonia solution (1+1) [neutralization]← Nitric acid (1+10) [slightly acidic]← A proper amount of water← 20 mL of coloring reagent solution← Water (up to the marked line)
Leaving at rest
For about 30 minutes
Measurement

Spectrophotometer (420 nm)

Figure The flow sheet for water-soluble phosphoric acid in fertilizers

1104.2.4.b Ammonium vanadomolybdate absorptiometric analysis (Fluid mixed fertilizers
containing phosphonic acid or phosphite)(1) Summary
This test method is applicable to the fertilizers containing fluid mixed fertilizers containing
phosphonic acid or phosphite
Extract by adding water to an analytical sample, add hydrochloric acid - sulfuric acid to heat,and oxygenate phosphonic acid ion to orthophosphate ion, and then measure the absorbanceof phosphovanadomolybdate salt formed by the reaction with ammonium vanadate (V),hexaammonium heptamolybdate and nitric acid to obtain water-soluble phosphoric acid
(W-P2O5).
(2) Reagents: Reagents are as shown below:a) Hydrochloric acid: A JIS Guaranteed Reagent specified in JIS K 8180 or a reagent ofequivalent quality.
b) Nitric acid: A JIS Guaranteed Reagent (HNO3 60 % (mass fraction)) specified in JIS K
8541 or a reagent of equivalent quality.
c) Ammonia solution: A JIS Guaranteed Reagent (NH3 28 % (mass fraction)) specified in
JIS K 8085 or a reagent of equivalent quality.
d) Coloring reagent solution(1) (2): Dissolve 1.12 g of ammonium vanadate (V)(3) specified
in JIS K 8747 in water, add 250 mL of nitric acid, then add 27 g of hexaammonium
heptamolybdate tetrahydrate(4) specified in JIS K 8905 while dissolving in water, andfurther add water to make 1,000 mL(5).
e) Phenolphthalein solution (1 g/100 mL): Dissolve 1 g of phenolphthalein specified in
JIS K 8799 in 100 mL of ethanol (95) specified in JIS K 8102.
f) Phosphate standard solution (P2O5 10 mg/mL)(1): Heat potassium dihydrogen
phosphate specified in JIS K 9007 at 105 ºC ± 2 ºC for about 2 hours, let it stand to coolin a desiccator, and weigh 19.17 g to a weighing dish. Dissolve with a small amount ofwater, transfer to a 1,000-mL volumetric flask, add 2 mL - 3 mL of nitric acid, and add
water up to the marked line.
g) Phosphate standard solution (P2O5 0.5 mg/mL)(1): Transfer 50 mL of phosphatestandard solution (P2O5 10 mg/mL) to a 1,000-mL volumetric flask, add 2 mL - 3 mL ofnitric acid, and add water up to the marked line.
Note (1) This is an example of preparation; prepare an amount as appropriate.
(2) This corresponds to reagent “a” solution in the Official Methods of Analysis of
Fertilizers (1992).
(3) This corresponds to ammonium metavanadate in the Official Methods ofAnalysis of Fertilizers (1992).
(4) This corresponds to ammonium molybdate in the Official Methods of Analysis
of Fertilizers (1992).
(5) Store in an amber bottle.
(3) Instruments: Instruments are as follows:a) Rotary shaker: A rotary shaker that can rotate a 500-mL volumetric flask upside
down at 30 - 40 revolutions/min.
b) Hot plate or sand bath: A hot plate whose surface temperature can be adjusted up to
250 ºC. Adjust the amounts of gas and silica sand of a sand bath so that the sand bathtemperature can be set to 250 ºC.
c) Spectrophotometer: A spectrophotometer specified in JIS K 0115.
(4)

Test procedures111

(4.1) Extraction: Conduct extraction as shown below.
a) Weigh 5 g of an analytical sample to the order of 1 mg, and transfer to a 500-mL
volumetric flask.
b) Add about 400 mL of water, and shake to mix at 30 - 40 revolutions /min for about 30
minutes.
c) Add water up to the marked line.
d) Filter with Type 3 filter paper to make the sample solution.
Comment 1
Comment 2

In the procedure in a), it is also allowed to weigh 2.5 g of an analyticalsample to the order of 1 mg, and put it into a 250-mL volumetric flask.
The procedure in (4.1) is the same as the procedure in (4.1) of 4.2.4.a.

(4.2) Coloring: Conduct coloring as shown below:a) Transfer a predetermined amount (the equivalents of 0.5 mg - 6 mg as P2O5) of thesample solution to a 100-mL - 200-mL tall beaker.
b) Add 3 mL of hydrochloric acid and 1 mL of nitric acid.
c) Cover the tall beaker with a watch glass, heat on a hot plate or sand bath at 200 ºC - 250ºC and condense (7) until the solution volume becomes 1 mL - 3 mL (6).
d) After standing to cool, transfer to a 100-mL volumetric flask with water (8).
e) Add 1 - 2 drop(s) of phenolphthalein solution (1 g/100 mL), and neutralize by adding
ammonia solution (1+1) until the color of the solution becomes light red-purple.
f) Add nitric acid (1+10) until the light red-purple color of the solution disappears to makeit slightly acidic.
g) Add 20 mL of coloring reagent solution, and further add water up to the marked line,
and then leave at rest for about 30 minutes.
Note (6) It is recommended to transfer 1 mL - 3 mL of water to a 100-mL - 200-mL tallbeaker in advance and confirm the volume.
(7) Care should be taken not to evaporate completely. When it is evaporated
completely, the determined value becomes lower than usual in some cases.
(8) The volume of solution after transferring should be up to about 60 mL.
(4.3) Measurement: Conduct measurement according to JIS K 0115 and as shown below.
Specific measurement procedures are according to the operation method of thespectrophotometer used in measurement.
a) Measurement conditions for the spectrophotometer: Set up the measurement
conditions for the spectrophotometer considering the following:
Detection wavelength: 420 nmb) Calibration curve preparation
1) Transfer 1mL - 12 mL of phosphate standard solution (P2O5 0.5 mg/mL) to 100-mL
volumetric flasks step-by-step.
2) Add a proper amount of water (9) and conduct the same procedure as (4.2) g) to makethe P2O5 0.5 mg/100 mL - 6 mg/100 mL phosphate standard solution for thecalibration curve preparation.
3) Conduct the same procedures as 2) for another 100-mL volumetric flask to make theblank test solution for the calibration curve preparation.
4) Measure absorbance at a wavelength of 420 nm of the phosphate standard solutions
for the calibration curve preparation using the blank test solution for the calibration
curve preparation as the control (10).
5) Prepare the calibration curve of the phosphate concentration and absorbance of thephosphate standard solutions for the calibration curve preparation.
112c) Sample measurement
1) Regarding the solution in (4.2) g), measure the absorbance by the same procedure asb) 4) (10).
2) Obtain the phosphate (P2O5) content from the calibration curve, and calculate
water-soluble phosphoric acid (W-P2O5) in the analytical sample.
Note (9) If no water is added, precipitate is produced in some cases when coloring
reagent solution is added.
(10) Measure within 6 hours after adding the coloring reagent solution in theprocedure in (4.2) g).
Comment 3 As the result of a recovery testing using 7 kinds of fluid mixed fertilizers (7samples) containing the equivalents of 0.2 % - 50 %(mass fraction) as
water-soluble phosphoric acid, the average rate of recovery is 100.8 % 102.5 % and its repeatability is 0.00 % - 0.04 % as standard deviation and0.07 - 1.69 % as relative standard deviation.
Additionally, Table 1 shows results and analysis results from a collaborative
study for test method validation. In this case, the minimum quantitation of thetesting method is about 0.01 % (mass fraction).
Table 1 Results and analysis results from a collaborative study
for the test method validation of water-soluble phosphoric acid
Sample name

Number of1)laboratories

Mean

2)

sr4)

RSDr5)sR6)

RSDR7)

(%)3)

(%)3)

(%)

(%)3)

(%)

0.59

1.8

0.30

1.7

0.31

3.8

0.33

2.8

0.47

2.0

Fluid mixed
12
33.56
0.25
0.7
fertilizers 1
Fluid mixed
12
17.93
0.08
0.5
fertilizers 2
Fluid mixed
12
7.99
0.12
1.5
fertilizers 3
Fluid mixed
11
11.93
0.13
1.1
fertilizers 4
Fluid mixed
11
24.1
0.08
0.3
fertilizers 51) Number of laboratories used in analysis
2) Mean (n= number of laboratories x number of samples (2))3) Mass fraction
4) Repeatability standard deviation
5) Repeatability relative standard deviation
6) Reproducibility standard deviation
7) Reproducibility relative standard deviation

References
1) Toshiaki HIROI, Masakazu SAIKI and Kimie KATO: Development and Validation ofSpectrophotometry for Determination of Water-Soluble Phosphoric acid in Liquid
Mixed Fertilizer Containing Phosphonic acid, Research Report of Fertilizer, Vol.1 2008.
(25 - 33)
2) Toshiaki HIROI, Masakazu SAIKI and Kimie KATO: Determination of Water-Soluble
Phosphoric Acid in Liquid Mixed Fertilizer Containing Phosphonic Acid by
113

Spectrophotometry: A Collaborative Study, Research Report of Fertilizer, Vol.1 2008.
(34 - 40)
(5)

Flow sheet for water-soluble phosphoric acid of the fluid mixed fertilizers
containing phosphonic acid, etc.: The flow sheet for water-soluble phosphoric acid of
the fluid mixed fertilizers containing phosphonic acid, etc. is shown below:
5 g analytical sample
Weigh to the order of 1 mg to a 500-mL volumetric flask← Water, about 400 mL
Shaking to mix
Rotary shaker (30 - 40 revolutions/min) for 30 minutes← Water (up to the marked line)
Filtration
Type 3 filter paper← Water (up to the marked line)
Aliquot
100-mL - 200-mL tall beaker(predetermined volume)← 3 mL of hydrochloric acid← 1 mL of nitric acid
Cover the tall beaker with a watch glass, heat on a hot plate or sand bath
Heating
at 200 ºC - 250 ºC, condense until the solution volume reaches 1 mL – 3 mL
Standing to coolTransfer
100-mL volumetric flask, water← 1-2 drop(s) of phenolphthalein solution (1 g/100 mL)← Ammonia solution (1+1) [neutralization]← Nitric acid (1+10) [slightly acidic]← 20 mL of coloring reagent solution← Water (up to the marked line)
Leaving at rest
For about 30 minutes
Measurement

Spectrophotometer (420 nm)

Figure The flow sheet for water-soluble phosphate acidin fluid mixed fertilizers containing phosphonic acid, etc.

114

4.2.4.c Quinoline gravimetric analysis(1) Summary
This test method is applicable to fertilizers containing no phosphonic acid, etc.
It is suitable for the fertilizers containing relatively a high content of phosphate.
Extract by adding water to an analytical sample, and measure the mass of quinonium
phosphomolybdate formed by the reaction with quinoline, molybdic acid and nitric acid toobtain water-soluble phosphoric acid (W-P2O5).
(2) Reagents: Reagents are as shown below:a) Nitric acid: A JIS Guaranteed Reagent (HNO3 60 % (mass fraction)) specified in JIS K
8541 or a reagent of equivalent quality.
b) Sodium molybdate solution: Dissolve 70 g of sodium molybdate dihydrate in 150 mL
of water.
c) Quinoline solution: Add 5 mL of quinoline specified in JIS K 8279 to the mixture
solution of 35 mL of nitric acid and 100 mL of water.
d) Quimosiac solution: Add 60 g of citric acid monohydrate specified in JIS K 8283 tothe mixture solution of 85 mL nitric acid and 150 mL of water to dissolve. Add
gradually total volume of the sodium molybdate solution to mix them. Add gradually
the total volume of the quinoline solution while mixing the solution. After leaving at
rest overnight, filter the total volume with Type 3 filter paper. Add 280 mL of acetone
specified in JIS K 8962, and further add water to make 1,000 mL.
(3) Instruments: Instruments are as follows:a) Rotary shaker: A rotary shaker that can rotate a 500-mL volumetric flask upside down
at 30 - 40 revolutions/min.
b) Water bath: Water bath that can be adjusted to the test temperature ± 2 ºC.
c) Drying apparatus: A drying apparatus that can be adjusted to 220 ºC ± 5 ºC.
d) Crucible type glass filter: Put a crucible type glass filter 1G4 specified in JIS R 3503
into a drying apparatus, let it stand to cool in a desiccator after heating at 220 ºC ± 5 ºC
and measure the mass to the order of 1 mg.
(4) Test procedures(4.1) Extraction: Conduct extraction as shown below.
a) Weigh 1 g of an analytical sample to the order of 1 mg, and put to a 500-mL volumetricflask.
b) Add about 400 mL of water, and shake to mix at 30 - 40 revolutions/min for 30 minutes.
c) Add water up to the marked line.
d) Filter with Type 3 filter paper to make the sample solution.
Comment 1 In the procedure in a), it is allowed to weigh 2.5 g of an analytical sample tothe order of 1 mg, and put it into a 250-mL volumetric flask.
Comment 2 The procedure in (4.1) is the same procedure in (4.1) of 4.2.4.a.
(4.2) Measurement: Conduct measurement as shown below.
a) Transfer a predetermined volume (the equivalents of 10 mg - 30 mg as P2O5 and no
more than 20 mL as the total solution volume) of sample solution to a 300-mL tallbeaker.
b) Add 5 mL of nitric acid and add water to make about 80 mL.
c) Cover with a watch glass. After boiling for about 3 minutes, wash the watch glass andthe inside the tall beaker with water and add water to make about 100 mL.
115d) Immediately, add 50 mL of quimosiac solution, heat for about 15 minutes whilesometimes mixing in a water bath at 60 ºC - 65 ºC and produce precipitate of quinolyum
molybdate.
e) After standing to cool down to room temperature while sometimes mixing, filter under
reduced pressure with a crucible type glass filter, wash the tall beaker 3 times withwater and transfer the whole precipitate into a crucible type glass filter, further wash 7 8 times with water.
f) Transfer the precipitate together with a crucible type glass filter into a drying apparatus
and heat at 220 ºC ± 5 ºC for about 30 minutes.
g) As soon as heating is complete, move it into a desiccator and let it stand to cool.
h) After standing to cool, remove the crucible type glass filter from the desiccator andmeasure the mass to the order of 1 mg.
i) Calculate water-soluble phosphoric acid (W-P2O5) by the following formula.
Water-soluble phosphoric acid (W-P2O5) (% (mass fraction)) in an analytical sample= A × 0.03207 × (V1/V2) × (1/B) × 100
A: Mass (g) of the precipitate in h)
B: Mass of an analytical sample (5 g)
V1: Predetermined volume (500 mL) of the sample solution
V2: Volume (mL) of the sample solution transferred in a)
Reference
1) Masayoshi KOSHINO: Second Revision of The Methods of Analysis of Fertilizers(Details), p.98 - 114, Yokendo, Tokyo (1988)

116

(5)

Flow sheet for water-soluble phosphoric acid: The flow sheet for water-soluble
phosphoric acid in fertilizers is shown below:
5 g analytical sample
Weigh to the order of 1 mg to a 500-mL volumetric flask← About 400 mL of water
Shaking to mix
Rotary shaker (30 - 40 revolutions/min) for 30 minutes← Water (up to the marked line)
Filtration
Type 3 filter paper
Aliquot
300-mL tall beaker(predetermined volume)← 5 mL nitrate acid
← Water (to make about 80 mL)
Cover with a watch glass for 3 minutes
Boiling
Wash a watch glass and the inside the tall beaker with water← Water (to make about 100 mL)← 50 mL quimosiac solution
Forming precipitate
60 ºC - 65 ºC, for 15 minutes, some times mix
Standing to cool
Filtration
under reduced pressure

Room temperature

Crucible type glass filtering apparatus 1G4, 3 times with water

Washing

Wash 7 - 8 times with water

Drying220 ºC ± 5 ºC for about 30 minutes

Standing to cool

Desiccator

Measurement

Measure the mass to the order of 1 mg

Figure The flow sheet for water-soluble phosphoric acid in fertilizers

117

4.3

Potassium

4.3.1 Total potassium
4.3.1.a Flame atomic absorption spectrometry or flame photometry(1) Summary
This test method is applicable to fertilizers containing organic matters.
Pretreat an analytical sample with incineration and hydrochloric acid to convert total
potassium into potassium ion, add interference suppressor solution, and then spray in anacetylene-air flame, and measure the atomic absorption with potassium at a wavelength of
766.5 nm or 769.9 nm to quantify total potassium. Or, determine the intensity of the emission
line at a wavelength of 766.5 nm or 769.9 nm produced in flame to quantify total potassium.
(2) Reagents: Reagents are as shown below:a) Hydrochloric acid: A JIS Guaranteed Reagent specified in JIS K 8180 or a reagent ofequivalent quality.
b) Interference suppressor solution: Weigh 12.5 g of calcium carbonate specified in JIS
K 8617 into a 2,000-mL beaker, add a small amount of water, gradually add 105 mL ofhydrochloric acid, and heat for a little while. After standing to cool, add water to make
1,000 mL.
c) Potassium standard solution (K2O 1 mg/mL)(1): Heat potassium chloride specified in
JIS K 8121 at 110 ºC ± 2 ºC for about 2 hours, let it stand to cool in a desiccator, andweigh 1.583 g into a weighing dish. Dissolve in a small amount of water, transfer to a
1,000-mL volumetric flask, and add water up to the marked line.
d) Potassium standard solution (K2O 5 µg/mL - 50 µg/mL) for the calibration curve
preparation (1): Transfer 2.5 mL - 25 mL of potassium standard solution (K2O 1
mg/mL) to 500-mL volumetric flasks step-by-step, add about 50 mL of interference
suppressor solution (2), and add water up to the marked line.
e) Blank test solution for the calibration curve preparation (1): Transfer about 50 mL ofinterference suppressor solution to a 500-mL volumetric flask (2), and add water up to themarked line.
Note (1) This is an example of preparation; prepare an amount as appropriate.
(2) Add interference suppressor solution that is 1/10 volume of the volume to beprepared.
Comment 1 Instead of potassium standard solution (K2O 1 mg/mL) in (2) c), potassium
standard solution (K 1 mg/mL) for atomic absorption traceable to a specified
reference material (national measurement standard) according to Article 134of the Measurement Act can also be used. Additionally, it is recommended tomultiple by the conversion factor (1.2046) to use as potassium standard
solution (K2O 1.2046 mg/mL).
(3) Instruments: Instruments are as shown below:a) Analytical instrument: An atomic absorption spectrometer or a flame photometer asshown below:aa) Flame atomic absorption spectrometer: An atomic absorption spectrometer specified
in JIS K 0121.
1) Light source: A potassium hollow cathode lamp
2) Gas: Gas for heating by flame(i) Fuel gas: acetylene(ii) Auxiliary gas: Air sufficiently free of dust and moisture.
118ab) Flame photometer:
1) Gas: Gas for heating by flame(i) Fuel gas: acetylene(ii) Auxiliary gas: Air sufficiently free of dust and moisture.
b) Electric furnace: An electric furnace that can be adjusted to 550 ºC ± 5 ºC.
c) Hot plate or sand bath: A hot plate whose surface temperature can be adjusted up to
250 ºC. Adjust the amounts of gas and silica sand of a sand bath so that the sand bathtemperature can be set to 250 ºC.
(4) Test procedures(4.1) Extraction: Conduct extraction as follows:a) Weigh 5 g of an analytical sample to the order of 1 mg, and put it in a 200-mL - 300-mLtall beaker.
b) Put the tall beaker in an electric furnace, and heat gently to char(3).
c) Ignite at 550 ºC ± 5 ºC for no less than 4 hours to incinerate.
d) After standing to cool, moisten the residue with a small amount of water, add gradually
about 10 mL of hydrochloric acid, and further add water to make about 20 mL.
e) Cover the tall beaker with a watch glass, and heat on a hot plate or a sand bath to boil
for about 5 minutes.
f) After standing to cool, transfer to a 100-mL - 500-mL volumetric flask.
g) Add water up to the marked line.
h) Filter with Type 3 filter paper to make the sample solution.
Note (3) Example of the charring procedure: Heat at about 250 ºC until smoke is nolonger formed.
Comment 2 The procedures in (4.1) are the same as in (4.1.2) in 4.2.1.a.
Comment 3 The sample solution prepared in (4.1) a) - h) in 4.9.1.a can also be used.
(4.2) Measurement: Conduct measurement according to JIS K 0121 and as shown below.
Specific measurement procedures are according to the operation method of the atomic
absorption spectrometer or flame photometer used in measurement.
a) Measurement conditions for the atomic absorption spectrometer or flame
photometer: Set up the measurement conditions for the atomic absorption spectrometeror flame photometer considering the following:
Analytical line wavelength: 766.5 nm or 769.9 nmb) Calibration curve preparation
1) Spray the potassium standard solutions for the calibration curve preparation and theblank test solution for the calibration curve preparation into a flame, and read theindicated value at a wavelength of 766.5 nm or 769.9 nm.
2) Prepare a curve for the relationship between the potassium concentration and theindicated value of the potassium standard solutions for the calibration curve
preparation and the blank test solution for the calibration curve preparation.
c)
Sample measurement
1) Transfer a predetermined amount of the sample solution (the equivalents of 0.5 mg - 5
mg as K2O) to a 100-mL volumetric flask.
2) Add about 10 mL of interference suppressor solution (2), and add water up to themarked line.
3) Conduct similarly as in b) 1) to read the indicated value.
4) Obtain the potassium content from the calibration curve, and calculate total potassiumin the analytical sample.
119Comment 4 Replicate testing was conducted using composted sludge fertilizer (2samples), human waste sludge fertilizer (2 samples), compost (1 sample),designated blended fertilizer containing organic fertilizer (1 sample) andcompound fertilizer containing organic fertilizer (1 sample); as a result, themean was in the range of 0.163 % - 12.40 % (mass fraction), and the standard
deviation and relative standard deviation were 0.001 % - 0.03 % (massfraction) and 0.1 % - 4.4 %, respectively.
References
1) Masayoshi KOSHINO: Second Revision of The Methods of Analysis of Fertilizers(Details), p.132 - 138, Yokendo, Tokyo (1988)
2) Kimie KATO, Masayuki YOSHIMOTO and Yuji SHIRAI: Systematization of
Determination Methods of Major Components in Sludge Fertilizer, Compost andOrganic Fertilizer, Research Report of Fertilizer, Vol.3 2010. (107 - 116)

120

(5)

Flow sheet for total potassium: The flow sheet for total potassium in fertilizers isshown below:
5 g analytical sample
Charring
Incineration

Weigh to the order of 1 mg into a 200-mL - 300-mL tall beaker.
Heat gently
550 ºC ± 5 ºC, no less than 4 hours

Standing to coolRoom temperature← A small amount of water, moisten the residue← About 10 mL of hydrochloric acid
← Water (up to about 20 mL)
Heating
Cover with a watch glass, and boil for 5 minutes.
Standing to coolTransfer

Room temperature
250-mL - 500-mL volumetric flask, water← Water (up to the marked line)
Filtration

Type 3 filter paper

Aliquot(predetermined
100-mL volumetric flask, wateramount)← About 10 mL interference suppressor solution← Water (up to the marked line)
Measurement
Atomic absorption spectrometer or flame photometer
Figure Flow sheet for total potassium in fertilizers

1214.3.1.b Sodium tetraphenylborate gravimetric analysis(1) Summary
This test method is applicable to fertilizers containing organic matters. It is suitable for
fertilizers containing relatively a high content of potassium.
Pretreat an analytical sample by incineration and hydrochloric acid to convert total potassium
into potassium ion, mask co-existing ammonium and other salts with formaldehyde andethylenediamine tetraacetate, and measure the mass of potassium tetraphenylborate formed by
the reaction with tetraphenylborate to obtain total potassium (T-K2O).
(2) Reagents: Reagents are as shown below:a) Hydrochloric acid: A JIS Guaranteed Reagent specified in JIS K 8180 or a reagent ofequivalent quality.
b) Formaldehyde solution: A JIS Guaranteed Reagent specified in JIS K 8872 or areagent of equivalent quality.
c) Sodium hydroxide solution (200 g/L) (1): Dissolve 200 g of sodium hydroxide
specified in JIS K 8576 in water to make 1,000 mL.
d) Aluminum chloride solution (1): Dissolve 12 g of aluminum chloride (III) hexahydrate
specified in JIS K 8114 in water to make 100 mL.
e) Tetraphenylborate solution (1): Transfer 6.1 g of sodium tetraphenylborate specified in
JIS K 9521to a 250-mL volumetric flask, dissolve by adding about 200 mL of water andadd 10 mL of aluminum chloride solution. Add methyl red solution (0.1 g/100 mL) as
an indicator, and neutralize with sodium hydroxide solution (200 g/L) until the color of
the solution changes to yellow, and then add water up to the marked line. Filter withType 3 filter paper and add 0.5 mL of sodium hydroxide solution (200 g/L) to the total
filtrate. Filter with Type 3 filter paper in the case of usage.
f) Tetraphenylborate washing solution (1): Dilute 40 mL of tetraphenylborate solution
with water to make 1,000mL.
g) Ethylenediaminetetraacetate - Sodium hydroxide solution (1): Dissolve 10 g ofethylenediaminetetraacetic acid disodium dihydrogen dihydrate specified in JIS K 8107
and 8 g of sodium hydroxide specified in JIS K 8576 in a proper amount of water. Add
6 mL - 10 mL of tetraphenylborate solution while mixing according to the potassium
content coexisting as impurity after standing to cool, and then add water to make 100
mL. After leaving at rest for about 30 minutes while sometimes mixing, filter with Type
3 filter paper.
h) Methyl red solution (0.1 g/100 mL): Dissolve 0.10 g of methyl red specified in JIS K
8896 in 100 mL of ethanol (95) specified in JIS K 8102.
Note (1) This is an example of preparation; prepare an amount as appropriate.
(3)
a)
b)
c)

Instruments: Instruments are as shown below:
Electric furnace: An electric furnace that can be adjusted to 550 ºC ± 5 ºC.
Drying apparatus: A drying apparatus that can be adjusted to 120 ºC ± 2 ºC.
Crucible type glass filter: Put a crucible type glass filter 1G4 specified in JIS R 3503
into a drying apparatus, stand to cool in a desiccator after heating at 120 ºC ± 2 ºC andmeasure the mass to the order of 1 mg.
d) Hot plate or sand bath: A hot plate whose surface temperature can be adjusted up to
250 ºC. Adjust the amounts of gas and silica sand of a sand bath so that the sand bathtemperature can be set to 250 ºC.

(4)

Test procedures122

(4.1) Extraction: Conduct extraction as follows:a) Weigh 5 g of an analytical sample to the order of 1 mg, and put it in a 200- mL 300-mL tall beaker.
b) Put the tall beaker in an electric furnace, and heat gently to char(2).
c) Ignite at 550 ºC ± 5 ºC for no less than 4 hours to incinerate.
d) After standing to cool, moisten the residue with a small amount of water, add gradually
about 10 mL of hydrochloric acid, and further add water to make 20 mL.
e) Cover the tall beaker with a watch glass, and heat on a hot plate or a sand bath to boil
for about 5 minutes.
f) After standing to cool, transfer to a 100-mL - 500-mL volumetric flask.
g) Add water up to the marked line.
h) Filter with Type 3 filter paper to make the sample solution.
Note (2) Example of the charring procedure: Heat at about 250 ºC until smoke is nolonger formed.
Comment 1

The procedure in (4.1) is the same as (4.1.2) in 4.2.1.a. In addition, thesample solution prepared in (4.1) a) - h) in 4.9.1.a can also be used.

(4.2) Measurement: Conduct measurement as shown below.
a) Transfer a predetermined volume (the equivalents of 15 mg - 30 mg as K2O) of sample
solution to a 100-mL tall beaker.
b) Add water to the solution to reach 50 mL when the procedure in e) is complete.
c) Add hydrochloric acid (1+9) , so that the hydrochloric acid becomes equivalent to 0.2mL.
d) Add 5 mL of formaldehyde solution, and then add 5 mL of ethylenediamine tetraacetate- sodium hydroxide solution.
e) Add necessary volume (3) of tetraphenylborate solution at the rate of one or two drop(s)per second while mixing, and further add 4 mL of the same solution in the same manner.
f) Leave at rest for about 30 minutes while sometimes mixing to form the precipitate ofpotassium tetraphenylborate.
g) Filter supernatant with a crucible type glass filter, wash the tall beaker 5 times with 5
mL of tetraphenylborate washing solution and transfer the whole precipitate to the
crucible type glass filter and further wash 2 times with 2 ml of water.
h) Transfer the precipitate together with the crucible type glass filter into a drying
apparatus and heat at 120 ºC ± 2 ºC for 1 hour.
i) After heating, move it quickly into a desiccator and let it stand to cool.
j) After standing to cool, remove the crucible type glass filter from the desiccator andmeasure the mass to the order of 1 mg.
k) Calculate total potassium (T-K2O) by the following formula.
Total potassium (T-K2O) (% (mass fraction)) in an analytical sample= A × 0.1314 × (V1/V2) /W × 100
A: Mass (g) of the precipitate
V1: Predetermined volume (mL) of the sample solution in (4.1) g)
V2: Volume (mL) of the sample solution transferred in (4.2) a)
B: Mass of an analytical sample (g)Note (3) About 3 ml of tetraphenylborate solution per 10 mg of K2O is required to form
the precipitate of potassium tetraphenylborate
123

Reference
1) Masayoshi KOSHINO: Second Revision of The Methods of Analysis of Fertilizers(Details), p.122 - 128, Yokendo, Tokyo (1988)(5)

Flow sheet for total potassium: The flow sheet for total potassium in fertilizers isshown below:
5 g analytical sample
Charring
Incineration

Weigh to the order of 1 mg into a 200-mL - 300-mL tall beaker.
Heat gently
550 ºC ± 5 ºC, no less than 4 hours

Standing to coolRoom temperature← A small amount of water, moisten the residue← About 10 mL of hydrochloric acid
← Water (up to about 20 mL)
Heating
Cover with a watch glass, and boil for 5 minutes.
Standing to coolRoom temperature

Transfer
250-mL - 500-mL volumetric flask, water← Water (up to the marked line)
Filtration
Type 3 filter paper
Aliquot
100-mL tall beaker(predetermined amount)← Water (add tetraphenylborate solution to reach 50 mL)← Hydrochloric acid (1+9) (the equivalents of 0.2 mL of hydrochloric acid)← 5 mL of formaldehyde solution← 5 mL of ethylenediamine tetraacetate- sodium hydroxide solution← Tetraphenylborate solution (necessary volume + 4 mL)
Forming precipitate
For 30 minutes, sometimes mixing
Transfer

Crucible type glass filtering apparatus 1G4,
5 times with 5 mL of tetraphenylborate washing solution

Washing

Wash 2 times with 2 mL of water

Drying120 ºC ± 5 ºC for 1 hour.

Standing to cool

Desiccator

Measurement

Measure the mass to the order of 1 mg

Figure Flow sheet for total potassium in fertilizers

1244.3.2 Citrate soluble potassium
4.3.2.a Flame atomic absorption spectrometry or flame photometry(1) Summary
This test method is applicable to fertilizers containing potassium silicate fertilizers, etc.
Extract by adding citric acid solution to an analytical sample, add interference suppressor
solution, and then spray in an acetylene-air flame, and measure the atomic absorption withpotassium at a wavelength of 766.5 nm or 769.9 nm to quantify citrate soluble potassium(C-K2O). Or, measure the intensity of the emission line at a wavelength of 766.5 nm or 769.9
nm produced in flame to quantify citrate soluble potassium (C-K2O).
(2) Reagents: Reagents are as shown below:a) Hydrochloric acid: A JIS Guaranteed Reagent specified in JIS K 8180 or a reagent ofequivalent quality.
b) Citric acid solution (1): Dissolve 20 g of citric acid monohydrate specified in JIS K
8283 in water to make 1,000 mL.
c) Interference suppressor solution: Weigh 12.5 g of calcium carbonate specified in JIS
K 8617 into a 2,000-mL beaker, add a small amount of water, gradually add 105 mL ofhydrochloric acid, and heat for a little while. After standing to cool, add water to make
1,000 mL.
d) Potassium standard solution (K2O 1 mg/mL)(1): Heat potassium chloride specified in
JIS K 8121 at 110 ºC ± 2 ºC for about 2 hours, let it stand to cool in a desiccator, andweigh 1.583 g into a weighing dish. Dissolve with a small amount of water, transfer to a
1,000-mL volumetric flask, and add water up to the marked line.
e) Potassium standard solution (K2O 5µg/mL - 50 µg/mL) for the calibration curve
preparation (1): Transfer 2.5 mL - 25 mL of potassium standard solution (K2O 1
mg/mL) to 500-mL volumetric flasks step-by-step, add about 50 mL of interference
suppressor solution (2), and add water up to the marked line.
f) Blank test solution for the calibration curve preparation (1): Transfer about 50 mL ofinterference suppressor solution to a 500-mL volumetric flask (2), and add water up to themarked line.
Note (1) This is an example of preparation; prepare an amount as appropriate.
(2) Add interference suppressor solution that is 1/10 volume of the volume to beprepared.
Comment 1 Instead of potassium standard solution (K2O 1 mg/mL) in (2) d), potassium
standard solution (K 1 mg/mL) for atomic absorption traceable to a specified
reference material (national measurement standard) according to Article 134of the Measurement Act can also be used. Additionally, it is recommended tomultiple by the conversion factor (1.2046) to use as potassium standard
solution (K2O 1.2046 mg/mL).
(3) Instruments: Instruments are as follows:a) Constant-temperature rotary shaker: A constant-temperature rotary shaker that canrotate a 250-mL volumetric flask, set up in a thermostat adjustable to 30 ºC ± 1 ºC,
upside down at 30 - 40 revolutions/min.
b) Analysis instrument: An atomic absorption spectrometer or a flame photometer asshown below:ba)Flame atomic absorption spectrometer: An atomic absorption spectrometer
specified in JIS K 0121.
125

1) Light source: A potassium hollow cathode lamp
2) Gas: Gas for heating by flame(i) Fuel gas: acetylene(ii) Auxiliary gas: Air sufficiently free of dust and moisture.
bb)
Flame photometer:
1) Gas: Gas for heating by flame(i) Fuel gas: acetylene(ii) Auxiliary gas: Air sufficiently free of dust and moisture.
(4) Test procedures(4.1) Extraction: Conduct extraction as shown below.
a) Weigh 1 g of an analytical sample to the order of 1 mg, and transfer to a 250-mL
volumetric flask.
b) Add 150 mL of citric acid solution heated up to about 30 ºC, and shake to mix at 30 - 40
revolutions/min (30 ºC ± 1 ºC) for 1 hour.
c) After standing to cool, add water up to the marked line.
d) Filter with Type 3 filter paper to make the sample solution.
Comment 2 The procedure in (4.1) is the same as the procedure in (4.1) of 4.2.3.a.
(4.2) Measurement: Conduct measurement according to JIS K 0121 and as shown below.
Specific measurement procedures are according to the operation method of the atomic
absorption spectrometer or flame photometer used in measurement.
a) Measurement conditions for the atomic absorption spectrometer or flame
photometer: Set up the measurement conditions for the atomic absorption spectrometeror flame photometer considering the following:
Analytical line wavelength: 766.5 nm or 769.9 nmb) Calibration curve preparation
1) Spray the potassium standard solutions for the calibration curve preparation and theblank test solution for the calibration curve preparation into a flame, and read theindicated value at a wavelength of 766.5 nm or 769.9 nm.
2) Prepare a curve for the relationship between the potassium concentration and theindicated value of the potassium standard solutions for the calibration curve
preparation and the blank test solution for the calibration curve preparation.
c) Sample measurement
1) Transfer a predetermined amount of the sample solution (the equivalents of 0.5 mg 5 mg as K2O) to a 100-mL volumetric flask.
2) Add about 10 mL of interference suppressor solution (2), and add water up to themarked line.
3) Conduct similarly as in b) 1) to read the indicated value.
4) Obtain the potassium content from the calibration curve, and calculate citrate soluble
potassium (C-K2O) in the analytical sample.
Comment 3 Table 1 shows results from a simultaneous analysis with the same samples(proficiency testing and external quality control testing) hosted by the Japan
Fertilizer Quality Assurance Committee analyzed by the robust method.

126

Table 1

Results and analysis results from simultaneous analysis with the same samples
1)
for citrate soluble potassium hosted by the Japan Fertilizer Quality Assurance Committee
Measurement
Number of Median Μ2) NIQR4) RSDrob5)
Year
Samplemethodlaboratories
(%)
(%)3)
(%)3)
High analysis
Atomic absorption
2011
50
10.35
0.15
1.4
compound fertilizer spectrometry
Flame photometry

1)
2)
3)
4)
5)

68
10.45
0.22
2.1
Proficiency testing and external quality control testingMedian (M) agrees with the mean in normal distribution.
Mass fraction
Robust standard deviation (NIQR) agrees with standard deviation in normal distribution.
RSDrob stands for relative standard deviation obtained from the robust methodby the formula shown below:
RSDrob = (NIQR/M) × 100

Reference
1) Masayoshi KOSHINO: Second Revision of The Methods of Analysis of Fertilizers(Details), p.136 - 138, Yokendo, Tokyo (1988)(5)

Flow sheet for citrate soluble potassium: The flow sheet for citrate soluble potassiumin fertilizers is shown below:
1 g analytical sample
Weigh to the order of 1 mg into a 250-mL volumetric flask.
← 150 mL citrate solution [about 30 ºC]
Constant temperature rotary shaker (30 - 40 revolutions/min)Shaking to mix
30 ºC ± 1 ºC, 1 hourStanding to cool
← Water (up to the marked line)
Filtration
Type 3 filter paper
Aliquot
100-mL volumetric flask(predetermined amount)← About 10 mL interference suppressor solution← Water (up to the marked line)
Measurement
Atomic absorption spectrometer or flame photometer
Figure Flow sheet for citrate soluble potassium in fertilizers

1274.3.2.b Sodium tetraphenylborate gravimetric analysis(1) Summary
This test method is applicable to fertilizers containing potassium silicate fertilizer, etc.
Extract by adding citric acid solution to an analytical sample, mask co-existing ammonium
and other salts with formaldehyde and ethylenediamine tetraacetate and measure the mass of
citrate soluble potassium (C-K2O) and the mass of potassium tetraphenylborate formed by thereaction with tetraphenylborate to obtain citrate soluble potassium (C-K2O).
(2) Reagents: Reagents are as shown below:a) Citric acid solution (1): Dissolve 20 g of citric acid monohydrate specified in JIS K
8283 in water to make 1,000 mL.
b) Formaldehyde solution: A JIS Guaranteed Reagent specified in JIS K 8872 or areagent of equivalent quality.
c) Sodium hydroxide solution (200 g/L) (1): Dissolve 200 g of sodium hydroxide
specified in JIS K 8576 in water to make 1,000 mL.
d) Aluminum chloride solution (1): Dissolve 12 g of aluminum chloride (III) hexahydrate
specified in JIS K 8114 in water to make 100 mL.
e) Tetraphenylborate solution (1): Transfer 6.1 g of Sodium tetraphenylborate specified in
JIS K 9521 to a 250-mL volumetric flask, dissolve by adding about 200 mL of waterand add 10 mL of aluminum chloride solution. Add methyl red solution (0.1 g/100 mL)
as an indicator, and neutralize with sodium hydroxide solution (200 g/L) until the colorof the solution changes to yellow, and then add water up to the marked line. Filter withType 3 filter paper and add sodium hydroxide solution (200 g/L) to the total filtrate.
Filter with Type 3 filter paper in the case of usage.
f) Tetraphenylborate washing solution (1): Dilute 40 mL of tetraphenylborate solution
with water to make 1,000mL.
g) Ethylenediamine tetraacetate- Sodium hydroxide solution (1): Dissolve 10 g ofethylenediaminetetraacetic acid dihydrogen disodium dihydrate specified in JIS K 8107
and 8 g of sodium hydroxide specified in JIS K 8576 in a proper amount of water, add 6
mL - 10 mL of tetraphenylborate solution while mixing according to the volume ofpotassium coexisting as impurity after standing to cool and then add water to make 100
mL. After leaving at rest for about 30 minutes while sometimes mixing, filter with Type
3 filter paper.
h) Methyl red solution (0.1 g/100 mL): Dissolve 0.10 g of methyl red specified in JIS K
8896 in 100 mL of ethanol (95) specified in JIS K 8102.
Note (1) This is an example of preparation; prepare an amount as appropriate.
(3) Apparatus and instruments: Apparatus and instruments are as follows:a) Constant-temperature rotary shaker: A constant-temperature rotary shaker that canrotate a 250-mL volumetric flask, set up in a thermostat adjustable to 30 ºC ± 1 ºC,
upside down at 30 - 40 revolutions/min.
b) Drying apparatus: A drying apparatus that can be adjusted to 120 ºC ± 2 ºC.
c) Crucibles type glass filter: Put a crucibles type glass filter 1G4 specified in JIS R 3503
into a drying apparatus, stand to cool in a desiccator after heating at 120 ºC ± 2 ºC andmeasure the mass to the order of 1 mg.
(4) Test procedures(4.1) Extraction: Conduct extraction as shown below.
a) Weigh 1 g of an analytical sample to the order of 1 mg, and transfer to a 250-mL
volumetric flask.
128b) Add 150 mL of citric acid solution heated up to about 30 ºC, and shake to mix at 30-40
revolutions/min (30 ºC ± 1 ºC) for 1 hour.
c) After standing to cool, add water up to the marked line.
d) Filter with Type 3 filter paper to make the sample solution.
Comment 1 The procedure (4.1) is the same as the procedure (4.1) of 4.2.3.a.
(4.2) Measurement: Conduct measurement as shown below.
a) Transfer 20 mL of sample solution to a 100-mL tall beaker.
b) Add water to the solution to reach 50 mL when the procedure in d) is complete.
c) Add 5 mL of formaldehyde solution, and then add 5 mL of ethylenediamine
tetraacetate- sodium hydroxide solution.
d) Add necessary volume (2) of tetraphenylborate solution at the rate of one or two drop(s)per second while mixing, and further add 4 mL of the same solution in the same manner.
e) Leave at rest for about 30 minutes while sometimes mixing to form the precipitate ofpotassium tetraphenylborate.
f) Filter supernatant with a crucible type glass filter, wash the vessel 5 times with 5 mL oftetraphenylborate washing solution and transfer the whole precipitate to the crucible
type glass filter and further wash 2 times with 2 ml of water.
g) Transfer the precipitate together with the crucible type glass filter into a drying
apparatus and heat at 120 ºC ± 2 ºC for 1 hour.
h) After heating, move it quickly into a desiccator and let it stand to cool.
i) After standing to cool, remove the crucible type glass filter from the desiccator andmeasure the mass to the order of 1 mg.
j) Calculate citrate soluble potassium (C-K2O) by the following formula in the analyticalsample.
Citrate soluble potassium (C-K2O) (% (mass fraction)) in an analytical sample= A × 0.1314 × (V1/V2) /W × 100
A: Mass (g) of the precipitate
V1: Predetermined volume (mL) of the sample solution in (4.1) c)
V2: Transferred volume (mL) of the sample solution in (4.2) a)
B: Mass of an analytical sample (g)Note (2) About 3 ml of tetraphenylborate solution per 10 mg of K2O is required to form
the precipitate of potassium tetraphenylborate
Reference
1) Masayoshi KOSHINO: Second Revision of The Methods of Analysis of Fertilizers(Details), p.122 - 128, Yokendo, Tokyo (1988)

129

(5)

Flow sheet for the testing method: The flow sheet for citrate soluble potassium in
fertilizers is shown below:
1 g analytical sample
Weigh to the order of 1 mg into a 250-mL volumetric flask.
← 150 mL citrate solution [about 30 ºC]
Constant temperature rotary shaker (30 - 40 revolutions/min)Shaking to mix
30 ºC ± 1 ºC, 1 hourStanding to cool
← Water (up to the marked line)
Filtration
Type 3 filter paper
Aliquot(predetermined amount)

100-mL tall beaker← Water (add tetraphenylborate solution to reach 50 mL)← 5 mL of formaldehyde solution← 5 mL of ethylenediamine tetraacetate - sodium hydroxide solution← Tetraphenylborate solution (the equivalents of potassium + 4 mL)
Forming precipitate

For 30 minutes, sometimes mixing

Transfer

Crucible type glass filtering apparatus 1G4,
5 times with 5 mL of tetraphenylborate washing solution

Washing

Wash 2 times with 2 mL of water

Drying120 ºC ± 5 ºC for 1 hour.

Standing to cool

Desiccator

Measurement

Measure the mass to the order of 1 mg

Figure Flow sheet for citrate soluble potassium in fertilizers

1304.3.2.c Sodium tetraphenylborate volumetric analysis(1) Summary
This test method is applicable to fertilizers containing potassium silicate fertilizer, etc. but notorganic matters.
Extract by adding citric acid solution to an analytical sample, mask co-existing ammonium
and other salts with formaldehyde, and make potassium ion and tetraphenylborate react witheach other. Conduct precipitate titration for unconsumed tetraphenylborate with benzaikonium
chloride to obtain citrate soluble potassium (C-K2O).
(2) Reagents: Reagents are as shown below:a) Citric acid solution (1): Dissolve 20 g of citric acid monohydrate specified in JIS K
8283 in water to make 1,000 mL.
b) Formaldehyde solution: A JIS Guaranteed Reagent specified in JIS K 8872 or areagent of equivalent quality.
c) Sodium hydroxide solution (120 g/L) (1): Dissolve 30 g of sodium hydroxide specified
in JIS K 8576 in water to make 250 mL.
d) Tetraphenylborate solution (1): Transfer 12.2 g of sodium tetraphenylborate to a
1,000-mL volumetric flask, dissolve by adding about 800 mL of water and add 3 mL of
sodium hydroxide (120 g/L) to the total filtrate, and further add water up to the marked
line. Filter with Type 3 filter paper in the case of usage.
e) Benzaikonium chloride solution (3.3 g/500 mL) (1): Dissolve 3.3 g of benzaikonium
chloride in 500 mL of water.
f) Methyl red solution (0.1 g/100 mL): Dissolve 0.10 g of methyl red specified in JIS K
8896 in 100 mL of ethanol (95) specified in JIS K 8102.
g) Titan Yellow solution (0.04 g/100mL): Dissolve 0.04 g of Titan Yellow in 100 mL ofwater in the case of usage.
h) Potassium standard solution (K2O 2 mg/mL)(1): Heat potassium chloride specified in
JIS K 8121 at 110 ºC ± 2 ºC for about 2 hours, let it stand to cool in a desiccator, andthen weigh 3.166 g into a weighing dish. Dissolve in a small amount of water, transfer
to a 1,000-mL volumetric flask, and add water up to the marked line.
Note (1) This is an example of preparation; prepare an amount as appropriate.
(3) Instruments: Instruments are as follows:a) Constant-temperature rotary shaker: A constant-temperature rotary shaker that canrotate a 250-mL volumetric flask, set up in a thermostat adjustable to 30 ºC ± 1 ºC,
upside down at 30 - 40 revolutions/min.
(4) Test procedures(4.1) Extraction: Conduct extraction as shown below.
a) Weigh 1 g of an analytical sample to the order of 1 mg, and transfer to a 250-mL
volumetric flask.
b) Add 150 mL of citric acid solution heated up to about 30 ºC, and shake to mix at 30 - 40
revolutions/min (30 ºC ± 1 ºC) for 1 hour.
c) After standing to cool, add water up to the marked line.
d) Filter with Type 3 filter paper to make the sample solution.
Comment 1 The procedure (4.1) is the same as the procedure (4.1) of 4.2.3.a.
(4.2) Precipitate formation: Form precipitate as shown below.
131a) Transfer 5 mL - 15 mL (no more than the equivalents of 30 mg as K2O) of the extract toa 100-mL volumetric flask.
b) Add water to the solution to make about 30 mL.
c) Add about 5 mL of formaldehyde solution and add 5 mL of sodium hydroxide solution(120 g/L).
d) Add one or two drop (s) of tetraphenylborate solution per second while shaking to mix.
e) Add water up to the marked line and leave at rest for about 10 minutes.
f) Filter with Type 3 filter paper to make the sample solution.
(4.3) Measurement: Conduct measurement as shown below.
a) Calibration curve preparation
1) Transfer 1 mL - 15 mL of potassium standard solution (K2O 2 mg/mL) to 100-mL
volumetric flasks step-by-step.
2) Conduct the same procedures as (4.2 b) - f) to make K2O 2 mg/100 mL - 30 mg/100
mL of the potassium standard solutions for the calibration curve preparation.
3) Conduct the same procedure as 2) to make the blank test solution for the calibration
curve preparation in another 100-mL volumetric flask.
4) Transfer 40 mL of the potassium standard solutions for a calibration curve
preparation and the blank test solution for a calibration curve preparation to an
Erlenmeyer flask respectively.
5) Add a few drops of Titan Yellow solution.
6) Titrate with benzaikonium chloride solution (3.3 g/500 mL) until the color of thesolution changes to light red (2).
7) Prepare a curve for the relationship between the potassium concentration and thevolume of the benzaikonium chloride solution (3.3 g/500 mL) required for thetitration of the potassium standard solutions for the calibration curve preparation andthe blank test solution for the calibration curve preparation.
b) Sample measurement
1) Transfer 40 mL of the sample solution of (4.2) f) to a 100-mL volumetric flask.
2) Conduct similarly as in a) 5) - 6) to obtain the volume of the benzaikonium chloride
solution (3.3 g/500 mL) required for the titration.
3) Obtain the potassium content from the calibration curve, and calculate citrate soluble
potassium (C-K2O) in the analytical sample.
Note (2) If the solution temperature is no more than 20 ºC, the reaction does not advancein some cases. Therefore it is recommended to heat the solution up to about 30ºC.
Reference
1) Masayoshi KOSHINO: Second Revision of The Methods of Analysis of Fertilizers(Details), p.128 - 132, Yokendo, Tokyo (1988)

132

(5)

Flow sheet for citrate soluble potassium: The flow sheet for citrate soluble potassiumin fertilizers is shown below:
1 g analytical sample
Weigh to the order of 1 mg into a 250-mL volumetric flask.
← 150 mL citrate solution [about 30 ºC]
Constant temperature rotary shaker (30 - 40 revolutions/min)Shaking to mix
30 ºC ± 1 ºC, 1 hourStanding to cool
← Water (up to the marked line)
Filtration
Type 3 filter paper
Aliquot
100-mL volumetric flask(5 mL - 15 mL)← Water (the volume to reach about 30 mL)← About 5 mL of formaldehyde solution← 5 mL of sodium hydroxide solution (120 g/L)
← 25 mL tetraphenylborate solution(one or two drop (s) per second while shaking to mix)
← Water (up to the marked line)
Leaving at rest
10 minutes
Filtration
Type 3 filter paper←A few drops of Titan Yellow solution
Aliquot
100-mL Erlenmeyer flask(40 mL)← A few drops of titan yellow solution
Benzaikonium chloride solution (3.3 g/500 mL)
Titration(until the solution changes to light red)
Figure Flow sheet for citrate soluble potassium in fertilizers

1334.3.3 Water-soluble potassium
4.3.3.a Flame atomic absorption spectrometry or flame photometry(1) Summary
This test method is applicable to fertilizers containing potassium salts.
Extract by adding water to an analytical sample, add interference suppressor solution, andthen spray in an acetylene-air flame, and measure the atomic absorption with potassium at awavelength of 766.5 nm or 769.9 nm to quantify water-soluble potassium (W-K2O). Or,
determine the intensity of the emission line at a wavelength of 766.5 nm or 769.9 nm
produced in flame to quantify water-soluble potassium (W-K2O).
(2) Reagents: Reagents are as shown below:a) Hydrochloric acid: A JIS Guaranteed Reagent specified in JIS K 8180 or a reagent ofequivalent quality.
b) Interference suppressor solution: Weigh 12.5 g of calcium carbonate specified in JIS
K 8617 into a 2,000-mL beaker, add a small amount of water, gradually add 105 mL ofhydrochloric acid, and heat for a little while. After standing to cool, add water to make
1,000 mL.
c) Potassium standard solution (K2O 1 mg/mL)(1): Heat potassium chloride specified in
JIS K 8121 at 110 ºC ± 2 ºC for about 2 hours, let it stand to cool in a desiccator, andthen weigh 1.583 g into a weighing dish. Dissolve with a small amount of water,transfer to a 1,000-mL volumetric flask, and add water up to the marked line.
d) Potassium standard solution (K2O 5 µg/mL - 50 µg/mL) for the calibration curve
preparation (1): Transfer 2.5 mL - 25 mL of potassium standard solution (K2O 1
mg/mL) to 500-mL volumetric flasks step-by-step, add about 50 mL of interference
suppressor solution (2), and add water up to the marked line.
e) Blank test solution for the calibration curve preparation (1): Transfer about 50 mL ofinterference suppressor solution to a 500-mL volumetric flask (2), and add water up to themarked line.
Note (1) This is an example of preparation; prepare an amount as appropriate.
(2) Add interference suppressor solution that is 1/10 volume of the volume to beprepared.
Comment 1 Instead of potassium standard solution (K2O 1 mg/mL) in (2) c), potassium
standard solution (K 1 mg/mL) for atomic absorption traceable to a specified
reference material (national measurement standard) according to Article 134of the Measurement Act can also be used. Additionally, it is recommended tomultiply by the conversion factor (1.2046) to use as potassium standard
solution (K2O 1.2046 mg/mL).
(3) Instruments: Instruments are as follows:a) Rotary shaker: A rotary shaker that can rotate a 500-mL volumetric flask upside down
at 30 - 40 revolutions/min.
b) Analytical instrument: An atomic absorption spectrometer or a flame photometer asshown below:ba) Flame atomic absorption spectrometer: An atomic absorption spectrometer specified
in JIS K 0121.
1) Light source: A potassium hollow cathode lamp
2) Gas: Gas for heating by flame(i) Fuel gas: acetylene134

(ii) Auxiliary gas: Air sufficiently free of dust and moisture.
bb) Flame photometer:
1) Gas: Gas for heating by flame(i) Fuel gas: acetylene(ii) Auxiliary gas: Air sufficiently free of dust and moisture.
c) Hot plate: A hot plate whose surface temperature can be adjusted up to 250 ºC.
(4) Test procedures(4.1) Extraction: Conduct extraction as shown below.
(4.1.1) Mixed fertilizers containing potassium salts and magnesium potassium sulfatea) Weigh 2.5 g of an analytical sample to the order of 1 mg, and transfer to a 300-mL tallbeaker.
b) Add about 200 mL of water, and cover with a watch glass and heat on a hot plate to boil
for about 15 minutes.
c) After standing to cool, transfer to a 250-mL volumetric flask with water.
d) Add water up to the marked line.
e) Filter with Type 3 filter paper to make the sample solution.
Comment 2 In the procedure in a), a 250-mL volumetric flask can be used instead of a
300-mL tall beaker. However the volumetric flask used should be
distinguished as an extraction flask and should not be used for the otherpurposes. Additionally, “cover with a watch glass” in b) is replaced by “placea long-stem funnel”, and “transfer to a 250-mL volumetric flask with water”
in the procedure in c) is skipped.
Comment 3 The procedure in (4.1.1) is the same as (4.1.1) of 4.3.3.b and 4.3.3.c, and(4.1) of 4.8.2.a.
(4.1.2) Mixed fertilizers containing no magnesium potassium sulfatea) Weigh 5 g of an analytical sample to the order of 1 mg, and transfer to a 500-mL
volumetric flask.
b) Add about 400 mL of water, and shake to mix at 30 - 40 revolutions/min for about 30
minutes.
c) Add water up to the marked line.
d) Filter with Type 3 filter paper to make the sample solution.
Comment 4 In the procedure in a), it is also allowed to weigh 2.5 g of an analyticalsample to the order of 1 mg, and put it into a 250-mL volumetric flask
Comment 5 The procedure in (4.1.2) is the same as the procedure in (4.1) of 4.2.4.a.
(4.2) Measurement: Conduct measurement according to JIS K 0121 and as shown below.
Specific measurement procedures are according to the operation method of the atomic
absorption spectrometer or flame photometer used in measurement.
a) Measurement conditions for the atomic absorption spectrometer or flame
photometer: Set up the measurement conditions for the atomic absorption spectrometeror flame photometer considering the following:
Analytical line wavelength: 766.5 nm or 769.9 nmb) Calibration curve preparation
1) Spray the potassium standard solutions for a calibration curve preparation and theblank test solution for a calibration curve preparation into a flame, and read theindicated value at a wavelength of 766.5 nm or 769.9 nm.
1352) Prepare a curve for the relationship between the potassium concentration and theindicated value of the potassium standard solutions for the calibration curve
preparation and the blank test solution for the calibration curve preparation.
c) Sample measurement
1) Transfer a predetermined amount of the sample solution (the equivalents of 0.5 mg 5 mg as K2O) to a 100-mL volumetric flask.
2) Add about 10 mL of interference suppressor solution (2), and add water up to themarked line.
3) Conduct similarly as in b) 1) to read the indicated value.
4) Obtain the potassium content from the calibration curve, and calculate thewater-soluble potassium (W-K2O) in the analytical sample.
Comment 6 Table 1 shows results from a simultaneous analysis with the same samples(proficiency testing and external quality control testing) hosted by the Japan
Fertilizer Quality Assurance Committee analyzed by the robust method.
Table 1Year

Results and analysis results from simultaneous analysis with the same samples
1)
for water-soluble potassium hosted by the Japan Fertilizer Quality Assurance Committee
Sample2008

High analysis
compound fertilizer
Organic
compound fertilizer
High analysis
compound fertilizer

2009

Ordinary
compound fertilizer

2006
2007

2010

2011

High analysis
compound fertilizer
High analysis
compound fertilizer

2012

Fluid mixed fertilizer

2013

High analysis
compound fertilizer

Measurement
methodAtomic absorption
spectrometry 6)
Flame photometry
Atomic absorption
spectrometry
Flame photometry
Atomic absorption
spectrometry
Flame photometry
Atomic absorption
spectrometry
Flame photometry
Atomic absorption
spectrometry
Flame photometry
Atomic absorption
spectrometry
Flame photometry

1)
2)
3)
4)
5)

2)

Number of Median Μ
3)laboratories
(%)

4)

NIQR
(%)

3)

156

12.38

0.22

1.8

145

8.43

0.15

1.8

75

11.38

0.13

1.1

57

11.42

0.16

1.4

78

8.36

0.13

1.6

54

8.35

0.09

1.1

84

14.7

0.21

1.4

49

14.72

0.12

0.8

51

10.35

0.13

1.3

75

10.45

0.17

1.7

47

2.44

0.04

1.8

75

2.42

0.06

2.6

57

11.72

0.93

3.3

65

11.81

0.33

2.8

Proficiency testing and external quality control testingMedian (M) agrees with the mean in normal distribution.
Mass fraction
Robust standard deviation (NIQR) agrees with standard deviation in normal distribution.
RSDrob stands for relative standard deviation obtained from the robust methodby the formula shown below:
RSDrob = (NIQR/M) × 100
6) Analyzed separately according to the measurement methods from 2008

136

RSDrob(%)5)

Reference
1) Masayoshi KOSHINO: Second Revision of The Methods of Analysis of Fertilizers(Details), p.136 - 138, Yokendo, Tokyo (1988)(5)

Flow sheet for water-soluble potassium: The flow sheet for water-soluble potassiumin fertilizers is shown below:
2.5 g analytical sample
Weigh to the order of 1 mg into a 300-mL tall beaker(potassium salts, etc.)← About 200 mL water
Heating
Cover with a watch dish, boil for 15 minutes
Standing to coolTransfer
Water, 250-mL volumetric flask← Water (up to the marked line)
5 g analytical sample
Weigh to the order of 1 mg into a 500-mL volumetric flask(compound fertilizer)← About 400 mL water
Rotary shaker (30 - 40 revolutions/min)Shaking to mix
For 30 minutes← Water (up to the marked line)
Filtration

Type 3 filter paper

Aliquot
100-mL volumetric flask(predetermined volume)← About 10 mL of interference suppressor solution← Water (up to the marked line)
Atomic absorption spectrometer or flame photometer
Measurement
Figure Flow sheet for water-soluble potassium in fertilizers

1374.3.3.b Sodium tetraphenylborate gravimetric analysis(1) Summary
This test method is applicable to fertilizers containing potassium salts.
Extract by adding water to an analytical sample, mask co-existing ammonium and other salts
with formaldehyde and ethylenediamine tetraacetate and measure the mass of potassium
tetraphenylborate formed by the reaction with tetraphenylborate to obtain water-soluble
potassium (W-K2O).
(2) Reagents: Reagents are as shown below:a) Hydrochloric acid: A JIS Guaranteed Reagent specified in JIS K 8180 or a reagent ofequivalent quality.
b) Formaldehyde solution: A JIS Guaranteed Reagent specified in JIS K 8872 or areagent of equivalent quality.
c) Sodium hydroxide solution (200 g/l) (1): Dissolve 200 g of sodium hydroxide specified
in JIS K 8576 in water to make 1,000 mL.
d) Aluminum chloride solution (1): Dissolve 12 g of aluminum chloride (III) hexahydrate
specified in JIS K 8114 in water to make 100 mL.
e) Tetraphenylborate solution (1): Transfer 6.1 g of Sodium tetraphenylborate to a
250-mL volumetric flask, dissolve by adding about 200 mL of water and add 10 mL ofaluminum chloride solution. Add methyl red solution (0.1 g/100 mL) as an indicator,and neutralize with sodium hydroxide solution (200 g/L) until the color of the solutionchanges to yellow, and then add water up to the marked line. Filter with Type 3 filter
paper and add sodium hydroxide solution (200 g/L) to the total filtrate. Filter with Type
3 filter paper in the case of usage.
f) Tetraphenylborate washing solution (1): Dilute 40 mL of tetraphenylborate solution
with water to make 1,000 mL.
g) Ethylenediamine tetraacetate-sodium hydroxide solution (1): Dissolve 10 g ofethylenediaminetetraacetic acid dihydrogen disodium dihydrate specified in JIS K 8107
and 8 g of sodium hydroxide specified in JIS K 8576 in a proper amount of water, add 6
mL - 10 mL of tetraphenylborate solution while mixing according to potassium content
coexisting as impurity after standing to cool and then add water to make 100 mL. Afterleaving at rest for about 30 minutes while sometimes mixing, filter with Type 3 filter
paper.
h) Methyl red solution (0.1 g/100 mL): Dissolve 0.10 g of methyl red specified in JIS K
8896 in 100 mL of ethanol (95) specified in JIS K 8102.
Note (1) This is an example of preparation; prepare an amount as appropriate.
(3) Apparatus and instruments: Apparatus and instruments are as follows:a) Rotary shaker: A rotary shaker that can rotate a 500-mL volumetric flask upside down
at 30- 40 revolutions/min.
b) Drying apparatus: A drying apparatus that can be adjusted to 120 ºC ± 2 ºC.
c) Crucible type glass filter: Put a crucible type glass filter 1G4 specified in JIS R 3503
into a drying apparatus, stand to cool in a desiccator after heating at 120 ºC ± 2 ºC andmeasure the mass to the order of 1 mg.
c) Hot plate: A hot plate whose surface temperature can be adjusted up to 250 ºC.
(4) Test procedures(4.1) Extraction: Conduct extraction as shown below.
(4.1.1) Mixed fertilizers containing potassium salts and magnesium potassium sulfate
138a) Weigh 2.5 g of an analytical sample to the order of 1 mg, and transfer to a 300-mL tallbeaker.
b) Add about 200 mL of water, and cover with a watch glass and heat on a hot plate to boil
for about 15 minutes.
c) After standing to cool, transfer to a 250-mL volumetric flask with water.
d) Add water up to the marked line.
e) Filter with Type 3 filter paper to make the sample solution.
Comment 1 In the procedure in a), a 250-mLvolumetric flask can be used instead of a
300-mL tall beaker. However the volumetric flask used should be
distinguished as an extraction flask and should not be used for the otherpurposes. Additionally, “cover with a watch glass” in b) is replaced by “placea long-stem funnel”, and “transfer to a 250-mL volumetric flask with water”
in the procedure in c) is skipped.
Comment 2 The procedure in (4.1.1) is the same as (4.1.1) of 4.3.3.a(4.1.2) Mixed fertilizers containing no magnesium potassium sulfatea) Weigh 5 g of an analytical sample to the order of 1 mg, and transfer to a 500-mL
volumetric flask.
b) Add about 400 mL of water, and shake to mix at 30-40 revolutions/min for about 30
minutes.
c) Add water up to the marked line.
d) Filter with Type 3 filter paper to make the sample solution.
Comment 3 In the procedure in a), it is also allowed to weigh 2.5 g of an analyticalsample to the order of 1 mg, and put it to a 250-mL volumetric flask
Comment 4 The procedure in (4.1.2) is the same as the procedure in (4.1) of 4.2.4.a.
(4.2) Measurement: Conduct measurement as shown below.
a) Transfer a predetermined volume (the equivalents of 15 mg - 30 mg as K2O) of sample
solution to a 100-mL tall beaker.
b) Add water to the solution to reach 50 mL when the procedure in e) is complete.
c) Add 2 mL of hydrochloric acid (1+9).
d) Add 5 mL of formaldehyde solution, and then add 5 mL of ethylenediamine
tetraacetate-sodium hydroxide solution.
e) Add necessary volume (2) of tetraphenylborate solution at the rate of one or two drop(s)per second while mixing, and further add 4 mL of the same solution in the same manner.
f) Leave at rest for about 30 minutes while sometimes mixing to form the precipitate ofpotassium tetraphenylborate.
g) Filter supernatant with a crucible type glass filter, wash the vessel 5 times with 5 mL oftetraphenylborate washing solution and transfer the whole precipitate to the crucible
type glass filter and further wash 2 times with 2 mL of water.
h) Transfer the precipitate together with the crucible type glass filter into a drying
apparatus adjusted to 120 ºC ± 2 ºC and heat for 1 hour.
i) After heating, move it quickly into a desiccator and let it stand to cool.
j) After standing to cool, remove a ground-in stoppered weighing bottle from thedesiccator and measure the mass to the order of 1 mg.
k) Calculate water-soluble potassium (W-K2O) in the analytical sample by the following
formula.
Water-soluble potassium (W-K2O) (% (mass fraction)) in an analytical sample
139

= A × 0.1314 × (V1/V2)/W × 100
A: Mass (g) of precipitate
V1: Predetermined volume (mL) of the sample solution in (4.1.1) d) or (4.1.2) c)
V2: Volume (mL) of the sample solution transferred in (4.2) a)
W: Mass of an analytical sample (g)Note (2) About 3 mL of tetraphenylborate solution per 10 mg of K2O is required to form
the precipitate of potassium tetraphenylborate
Reference
1) Masayoshi KOSHINO: Second Revision of The Methods of Analysis of Fertilizers(Details), p.122 - 128, Yokendo, Tokyo (1988)

140

(5)

Flow sheet for water-soluble potassium: The flow sheet for water-soluble potassiumin fertilizers is shown below:
2.5 g analytical sample
Weigh to the order of 1 mg into a 300-mL tall beaker(potassium salts, etc.)← About 200 mL water
Heating
Cover with a watch glass, boil for 15 minutes
Standing to coolTransfer
Water, 250-mL volumetric flask← Water (up to the marked line)
5 g analytical sample
Weigh to the order of 1 mg into a 500-mL volumetric flask(compound fertilizer)← About 400 mL of water
Rotary shaker (30 - 40 revolutions/min)Shaking
For 30 minutes← Water (up to the marked line)
Filtration

Type 3 filter paper

Aliquot
100-mL tall beaker(predetermined← Water (add tetraphenylborate solution to reach 50 mL)← 2 mL of hydrochloric acid (1+9)← 5 mL of formaldehyde solution← 5 mL of ethylenediamine tetraacetate-sodium hydroxide solution← Tetraphenylborate solution (the equivalents of potassium + 4 mL)
Forming precipitate
For 30 minutes, sometimes mixing
Transfer

Crucible type glass filtering apparatus 1G4,
5 times with 5 mL of tetraphenylborate washing solution

Washing

Wash 2 times with 2 mL of water

Drying120 ºC ± 2 ºC for 1 hour.

Standing to cool

Desiccator

Measurement

Measure the mass to the order of 1 mg

FigureFlow sheet for water-soluble potassium in fertilizers

1414.3.3.c Sodium tetraphenylborate volumetric analysis(1) Summary
This test method is applicable to fertilizers containing potassium salt but not organic matters.
Extract by adding water to an analytical sample, mask co-existing ammonium and other salts
with formaldehyde, and make potassium ion and tetraphenylborate react with each other.
Conduct precipitate titration for unconsumed tetraphenylborate with benzaikonium chloride
to obtain water-soluble potassium (W-K2O).
(2) Reagents: Reagents are as shown below:a) Formaldehyde solution: A JIS Guaranteed Reagent specified in JIS K 8872 or areagent of equivalent quality.
b) Sodium hydroxide solution (120 g/L) (1): Dissolve 30 g of sodium hydroxide specified
in JIS K 8576 in water to make 250 mL.
c) Tetraphenylborate solution (1): Transfer 12.2 g of Sodium tetraphenylborate to a
1,000-mL volumetric flask, dissolve by adding about 800 mL of water and add 3 mL of
sodium hydroxide (120g/L) to total filtrate, and further add water up to the marked line.
Filter with Type 3 filter paper in the case of usage.
d) Benzaikonium chloride solution (3.3 g/500 mL) (1): Dissolve 3.3 g of benzaikonium
chloride in 500 mL of water.
e) Methyl red solution (0.1 g/100 mL): Dissolve 0.10 g of methyl red specified in JIS K
8896 in 100 mL of ethanol (95) specified in JIS K 8102.
f) Titan Yellow solution (0.04 g/100 mL): Dissolve 0.04 g of Titan Yellow in 100 mL ofwater in the case of usage.
g) Potassium standard solution (K2O 2 mg/mL)(1): Heat potassium chloride specified in
JIS K 8121 at 110 ºC ± 2 ºC for about 2 hours, let it stand to cool in a desiccator, andweigh 3.166 g into a weighing dish. Dissolve with a small amount of water, transfer to a
1,000-mL volumetric flask, and add water up to the marked line.
Note (1) This is an example of preparation; prepare an amount as appropriate.
(3) Apparatus and instruments: Apparatus and instruments are as follows:a) Rotary shaker: A rotary shaker that can rotate a 500-mL volumetric flask upside down
at 30- 40 revolutions/min.
b) Hot plate: A hot plate whose surface temperature can be adjusted up to 250 ºC.
(4) Test procedures(4.1) Extraction: Conduct extraction as shown below.
(4.1.1) Mixed fertilizers containing potassium salts and magnesium potassium sulfatea) Weigh 2.5 g of an analytical sample to the order of 1 mg, and transfer to a 300-mL tallbeaker.
b) Add about 200 mL of water, and cover with a watch glass and heat on a hot plate to boil
for about 15 minutes.
c) After standing to cool, transfer to a 250-mL volumetric flask with water.
d) Add water up to the marked line.
e) Filter with Type 3 filter paper to make the extract.
Comment 1 In the procedure in a), a 250-mL volumetric flask can be used instead of a
300-mL tall beaker. However the volumetric flask used should be
distinguished as an extraction flask and should not be used for the otherpurposes. Additionally, “cover with a watch glass” in b) is replaced by “place142

a long-stem funnel”, and “transfer to a 250-mL volumetric flask with water”
in the procedure in c) is skipped.
Comment 2 The procedure of (4.1.1) is the same as (4.1.1) of 4.3.3.a(4.1.2) Mixed fertilizers containing no magnesium potassium sulfatea) Weigh 5 g of an analytical sample to the order of 1 mg, and transfer to a 500-mL
volumetric flask.
b) Add about 400 mL of water, and shake to mix at 30 - 40 revolutions/min for about 30
minutes.
c) Add water up to the marked line.
d) Filter with Type 3 filter paper to make the sample solution.
Comment 3 In the procedure of a), it is also allowed to weigh 2.5 g of an analyticalsample to the order of 1 mg, and put it into a 250-mL volumetric flask
Comment 4 The procedure in (4.1.2) is the same as the procedure (4.1) of 4.2.4.a.
(4.2) Precipitate formation: Form precipitate as shown below.
a) Transfer 5 mL - 15 mL (no more than the equivalents of 30 mg as K2O) of the extract toa 100-mL volumetric flask.
b) Add water to the solution to make about 30 mL.
c) Add about 5 mL of formaldehyde solution and add 5 mL of sodium hydroxide (120g/L).
d) Add 25 mL of tetraphenylborate solution at the rate of one or two drop(s) per second
while shaking to mix.
e) Add water up to the marked line and leave at rest for about 10 minutes.
f) Filter with Type 3 filter paper to make the sample solution.
(4.3) Measurement: Conduct measurement as shown below.
a) Calibration curve preparation
1) Transfer 1 mL - 15 mL of potassium solution (K2O 2 mg/mL) to 100-mL volumetricflasks step-by-step.
2) Conduct the same procedures as (4.2 b) - f) to make K2O 2 mg/100 mL - 30 mg/100
mL of the potassium standard solutions for the calibration curve preparation.
3) Conduct the same procedure as 2) to make the blank test solution for the calibration
curve preparation in another 100-mL volumetric flask.
4) Transfer 40 mL of the potassium standard solutions for the calibration curve
preparation and the blank test solution for the calibration curve preparation to an
Erlenmeyer flask respectively.
5) Add a few drops of Titan Yellow solution.
6) Titrate with benzaikonium chloride solution (3.3 g/500 mL) until the color of thesolution changes to light red (2).
7) Prepare a curve for the relationship between the potassium concentration and thevolume of the benzaikonium chloride solution (3.3 g/500 mL) required for thetitration of the potassium standard solutions for the calibration curve preparation andthe blank test solution for the calibration curve preparation.
b) Sample measurement
1) Transfer 40 mL of the sample solution of (4.2) f) to a 100-mL volumetric flask.
2) Conduct similarly as in a) 5) - 6) to obtain the volume of the benzaikonium chloride
solution (3.3 g/500 mL) required for the titration.

1433) Obtain the potassium content from the calibration curve, and calculate water-soluble
potassium (W-K2O) in the analytical sample.
Note (2) If the solution temperature is no more than 20 ºC, the reaction does not advancein some cases. Therefore it is recommended to heat the solution up to about 30ºC.
Reference
1) Masayoshi KOSHINO: Second Revision of The Methods of Analysis of Fertilizers(Details), p.128 - 132, Yokendo, Tokyo (1988)
5) Flow sheet for water-soluble potassium t: The flow sheet for water-soluble potassiumin fertilizers is shown below:
2.5 g analytical sample
Weigh to the order of 1 mg into a 300-mL tall beaker(potassium salts, etc.)← About 200 mL of water
Heating
Cover with a watch glass, boil for about 15 minutes
Standing to coolTransfer
Water, 250-mL volumetric flask← Water (up to the marked line)
5 g analytical sample
Weigh to the order of 1 mg into a 500-mL volumetric flask(compound fertilizer)← Water (up to the marked line)
Rotary shaker (30 - 40 revolutions/min)Shaking to mix
For 30 minutes← Water (up to the marked line)
Filtration

Type 3 filter paper

Aliquot
100-mL volumetric flask(5 mL to 15 mL)← Water (the liquid volume to reach about 30 mL)← About 5mL of formaldehyde solution← 5 mL of sodium hydroxide solution (120 g/L)
← 25 mL tetraphenylborate solution(one or two drop (s) per second while shaking to mix)
← Water (up to the marked line)
Leaving at rest
10 minutes
Filtration

Type 3 filter paper

Aliquot
100-mL Erlenmeyer flask(40 mL)← A few drops of Titan Yellow solution
Benzaikonium chloride solution (3.3 g/500 mL)
Titration(until the solution changes to light red)
FigureFlow sheet for water soluble potassium in fertilizers

144

4.4

Silicic acid

4.4.1 Soluble silicic acid
4.4.1.a Potassium fluoride method(1) Summary
This test method is applicable to fertilizers containing no silica gel fertilizers.
Extract by adding hydrochloric acid (1+23) to an analytical sample, add hydrochloric acid,potassium chloride and potassium fluoride solution and cool in a refrigerator, and then filter
after forming precipitate as potassium silicofluoride. Put the precipitate in water and heat, andmeasure by neutralization titration to obtain the hydrochloric acid (1+23) soluble silicic acid(soluble silicic acid (S-SiO2)) in an analytical sample.
(2) Reagents: Reagents are as shown below:a) 0.1 mol/L - 0.2 mol/L sodium hydroxide solution(1): Transfer about 30 mL of water toa polyethylene bottle, dissolve about 35 g of sodium hydroxide specified in JIS K 8576
by adding in small portions while cooling, seal tightly and leave at rest for 4 - 5 days.
Transfer 5.5 mL - 11 mL of the supernatant to a ground-in stoppered storage container,
and add 1,000 mL of water containing no carbonic acid.
Standardization: Dry sulfamic acid reference material for volumetric analysis
specified in JIS K 8005 by leaving at rest in a desiccator at no more than 2 kPa for about
48 hours, then transfer about 2.5 g to a weighing dish, and measure the mass to the orderof 0.1 mg. Dissolve with a small amount of water, transfer to a 250-mL volumetric flask,
and add water up to the marked line (1). Transfer a predetermined amount of the solution
to a 200-mL - 300-mL Erlenmeyer flask, add a few drops of bromothymol blue solution(0.1 mg/100 mL) as an indicator, and titrate with 0.1 mol/L - 0.2 mol/L sodiumhydroxide solution until the color of the solution becomes green. Calculate the factor of
a 0.1 mol/L - 0.2 mol/L sodium hydroxide solution by the following formula:
Factor of 0.1 mol/L - 0.2 mol/L sodium hydroxide solution (f)= (W × A × 0.01/97.095) × (V1/V2) × (1,000/V3) × (1/C)
W: Mass (g) of sulfamic acid sampled
A: Purity (%) of sulfamic acidV1: Volume (mL) of sulfamic acid solution transferred
V2: Constant volume (250 mL) of sulfamic acid solution
V3: Volume (mL) of 0.1 mol/L - 0.2 mol/L sodium hydroxide solution needed fortitration
C: Set concentration (mol/L) of 0.1 mol/L - 0.2 mol/L sodium hydroxide solutionb) Hydrochloric acid: A JIS Guaranteed Reagent specified in JIS K 8180 or a reagent ofequivalent quality.
c) Potassium chloride: A JIS Guaranteed Reagent specified in JIS K 8121 or a reagent ofequivalent quality.
d) Potassium chloride solution (1): Add 250 mL of ethanol specified in JIS K 8101
to 750 mL of water to mix, and add 150 mL of potassium chloride to dissolve.
Add a few drops of methyl red solution (0.1 g/100 mL) as an indicator and drophydrochloric acid until the color of the solution becomes red to make it acidic. Afterleaving at rest for 1 day, neutralize with the 0.1 mol/L - 0.2 mol/L sodium hydroxide
solution.
e) Potassium fluoride solution (1): Dissolve 58 g of potassium fluoride specified in JIS K
8815 in 1,000 mL of water (2).
145

f)

Methyl red solution (0.1 g/100 mL): Dissolve 0.10 g of methyl red specified in JIS K
8896 in 100 mL of ethanol (95) specified in JIS K 8102.
g) Phenolphthalein solution (1 g/100 mL): Dissolve 1 g of phenolphthalein specified in
JIS K 8799 in 100 mL of ethanol (95) specified in JIS K 8102.
Note (1) This is an example of preparation; prepare an amount as appropriate.
(2) Store in a container made of polyethylene, etc. that contains no silicon.
(3) Apparatus and instruments: Apparatus and instruments are as follows:a) Constant-temperature rotary shaker: A constant-temperature rotary shaker that canrotate a 250-mL volumetric flask, set up in a thermostat adjustable to 30 ºC ± 1 ºC,
upside down at 30 - 40 revolutions/min.
b) Hot plate: A hot plate whose surface temperature can be adjusted up to 250 ºC.
(4) Test procedures(4.1) Extraction: Conduct extraction as shown below.
a) Weigh 1 g of an analytical sample to the order of 1 mg, and transfer to a 250-mL
volumetric flask.
b) Add 150 mL of hydrochloric acid heated up to about 30 ºC, and shake to mix at 30 - 40
revolutions/min (30 ºC ± 1 ºC) for 1 hour.
c) After standing to cool, add water up to the marked line.
d) Filter with Type 3 filter paper to make the sample solution.
Comment 1 The procedure in (4.1) is the same as the procedure in (4.1) of 4.4.1.d.
(4.2) Measurement: Conduct measurement as shown below.
a) Transfer a predetermined volume (the equivalents of 20 mg - 50 mg as SiO2 and no
more than 25 mL of liquid volume) to a 200-mL beaker made of polyethylene.
b) Add about 10 mL of hydrochloric acid and about 15 mL of potassium fluoride solution,and further add about 2 g of potassium chloride to dissolve, and then cool in arefrigerator for 30 minutes or more (3) to form the precipitate of potassium fluoride.
c) Filter with a Gooch crucible (4) made of polyethylene topped with Type 6 filter paper,and wash the container 3 times with potassium chloride solution, then move the whole
precipitate into the crucible, and further wash 6 - 7 times with a small amount ofpotassium chloride solution (5).
d) Move the precipitate on the filter together with the filter paper into a 300-mL tall beakerwith water, and further add water to make about 200 mL and heat it up to 70 ºC - 80 ºC
on a hot plate.
e) Add a few drops of phenolphthalein solution (1 g/100 mL) to the sample solution as anindicator and titrate with the 0.1 mol/L - 0.2 mol/L sodium hydroxide solution until thecolor of the solution becomes light red.
f) Calculate soluble silicic acid (S-SiO2) by the following formula.
Soluble silicic acid (S-SiO2) (% (mass fraction)) in an analytical sample= VS × C × f × (V4/V5) × (15.021/W) × (100/1,000)
VS: Volume of the 0.1 mol/L - 0.2 mol/L sodium hydroxide solution required fortitration
C: Set concentration (mol/L) of 0.1 mol/L - 0.2 mol/L sodium hydroxide solution
f: Factor of 0.1 mol/L - 0.2 mol/L sodium hydroxide solution
V4: Predetermined volume (mL) of the extract in (4.1) c)146

V5: Volume (mL) of the extract transferred in (4.2) a)
W: Mass (g) of an analytical sample
Note (3) To be no more than 10 ºC(4) Filter paper pulp can be stuffed to restrain precipitate from outflowing.
(5) Until the filtrate becomes neutral.
Comment 2 Table 1 shows results from a simultaneous analysis with the same samples(proficiency testing and external quality control testing) hosted by the Japan
Fertilizer Quality Assurance Committee analyzed by the robust method.
Table 1Results and analysis results from simultaneous analysis with the same samples
1)
for soluble silicic acid hosted by the Japan Fertilizer Quality Assurance Committee
2)

Year

SampleNumber of Median Μ
3)laboratories
(%)

4)

5)

NIQR

RSDrob

(%)

3)

(%)

2008

Slag silicate fertilizer

55

33.34

0.47

1.4

2009

Slag silicate fertilizer

59

32.67

0.59

1.8

2010

Slag silicate fertilizer

52

33.50

0.59

1.8

2011

Slag silicate fertilizer

46

30.69

0.76

2.5

2012

Slag silicate fertilizer

46

35.96

0.37

1.0

2013 Slag silicate fertilizer
46
35.14
0.55
1.6
1) Proficiency testing and external quality control testing2) Median (M) agrees with the mean in normal distribution.
3) Mass fraction4) Robust standard deviation (NIQR) agrees with standard deviation in normal distribution.
5) RSDrob stands for relative standard deviation obtained from the robust methodby the formula shown below:
RSDrob = (NIQR/M) × 100

Reference
1) Masayoshi KOSHINO: Second Revision of The Methods of Analysis of Fertilizers(Details), p.144 - 146, Yokendo, Tokyo (1988)

147

5) Flow sheet for soluble silicic acid: The flow sheet for soluble silicic acid in fertilizers isshown below:
1 g analytical sample
Weigh to the order of 1 mg to a 250-mL volumetric flask.
← About 150 mL hydrochloric acid (1+23) [about 30 ºC]
Rotary shaker (30 - 40 revolutions/min)Shaking to mix
30 ºC ± 1 ºC, 1 hourStanding to coolRoom temperature← Water (up to the marked line)
Filtration
Type 3 filter paper
Aliquot(predetermined amount)

200-mL beaker made of polyethylene← About 10 mL hydrochloric acid
← About 15mL potassium fluoride solution← 2 g of potassium chloride
Cooling
For 30 minutes or more in a refrigerator
Filtration

Gooch crucible made of polyethylene
Type 6 filter paper

Washing

Wash 6 - 7 times with potassium chloride solution

Transfer
300-mL tall beaker, water← Water (until the liquid volume reaches about 200 mL)
Heating
70 ºC - 80 ºC← A few drops of phenolphthalein solution (1 g/100 mL)
0.1 mol/L - 0.2 mol/L sodium hydroxide solution
Titration(Until the solution becomes light red)
Figure Flow sheet for soluble silicic acid in fertilizers

148

4.4.1.b Potassium fluoride method (Silica gel fertilizers, etc.)(1) Summary
This test method is applicable to silica gel fertilizers and silica hydrogel fertilizers.
Extract by adding sodium hydroxide (20 g/L) to an analytical sample, add hydrochloric acid,potassium chloride and a potassium fluoride solution and cool in a refrigerator. After forming
the precipitate as potassium silicofluoride, put the precipitate in water and heat , then measureby neutralization titration to obtain the sodium hydroxide (20 g/L) soluble silicic acid (soluble
silicic acid (S-SiO2)) in the analytical sample.
(2) Reagents: Reagents are as shown below:a) 0.1 mol/L - 0.2 mol/L sodium hydroxide solution(1): Transfer about 30 mL of water toa polyethylene bottle, dissolve about 35 g of sodium hydroxide specified in JIS K 8576
by adding in small portions while cooling, seal tightly and leave at rest for 4 - 5 days.
Transfer 5.5 mL - 11 mL of the supernatant to a ground-in stoppered storage container,
and add 1,000 mL of water containing no carbonic acid.
Standardization: Dry sulfamic acid reference material for volumetric analysis
specified in JIS K 8005 by leaving at rest in a desiccator at no more than 2 kPa for about
48 hours, then transfer about 2.5 g to a weighing dish, and measure the mass to the orderof 0.1 mg. Dissolve with a small amount of water, transfer to a 250-mL volumetric flask,
and add water up to the marked line (1). Transfer a predetermined amount of the solution
to a 200-mL - 300-mL Erlenmeyer flask, add a few drops of bromothymol blue solution(0.1 mg/100 mL) as an indicator, and titrate with a 0.1 mol/L - 0.2 mol/L sodiumhydroxide solution until the color of the solution becomes green. Calculate the factor of
0.1 mol/L - 0.2 mol/L sodium hydroxide solution by the following formula:
Factor of 0.1 mol/L - 0.2 mol/L sodium hydroxide solution (f1)
= (W1 × A × 0.01/97.095) × (V1/V2) × (1,000/V3) × (1/C1)
W1: Mass (g) of sulfamic acid sampled
A: Purity (%) of sulfamic acidV1: Volume (mL) of sulfamic acid solution transferred
V2: Constant volume (250 mL) of sulfamic acid solution
V3: Volume (mL) of 0.1 mol/L - 0.2 mol/L sodium hydroxide solution needed fortitration
C1: Set concentration (mol/L) of 0.1 mol/L - 0.2 mol/L sodium hydroxide solutionb) Sodium hydroxide: A JIS Guaranteed Reagent specified in JIS K 8576 or a reagent ofequivalent quality.
c) Hydrochloric acid: A JIS Guaranteed Reagent specified in JIS K 8180 or a reagent ofequivalent quality.
d) Potassium chloride: A JIS Guaranteed Reagent specified in JIS K 8121 or a reagent ofequivalent quality.
e) Potassium chloride solution (1): Add 250 mL of ethanol specified in JIS K 8101
to 750 mL of water to mix, and add 150 mL of potassium chloride to dissolve.
Add a few drops of methyl red solution (0.1 g/100 mL) as an indicator and drophydrochloric acid until the color of the solution becomes red to make it acidic, and after
leaving at rest for 1 day, neutralize with a 0.1 mol/L - 0.2 mol/L sodium hydroxide
solution.
f) Potassium fluoride solution (1): Dissolve 58 g of potassium fluoride specified in JIS K
8815 in 1,000 mL of water (2).
149

g) Methyl red solution (0.1 g/100 mL): Dissolve 0.10 g of methyl red specified in JIS K
8896 in 100 mL of ethanol (95) specified in JIS K 8102.
h) Phenolphthalein solution (1 g/100 mL): Dissolve 1 g of phenolphthalein specified in
JIS K 8799 in 100 mL of ethanol (95) specified in JIS K 8102.
Note (1) This is an example of preparation; prepare an amount as appropriate.
(2) Store in a container made of polyethylene, etc. that contains no silicon.
(3) Apparatus and instruments: Apparatus and instruments are as follows:a) Constant-temperature bath: A constant-temperature bath adjustable up to 60 ºC ± 2ºC.
b) Hot plate: A hot plate whose surface temperature can be adjusted up to 250 ºC.
c) Volumetric flask made of polymer: A volumetric flask that is made of polyethylene,
etc. of a quality of material which prevents silicic acid from eluting in the extract
procedure in (4.1).
(4) Test procedures(4.1) Extraction: Conduct extraction as shown below.
a) Weigh 1 g of an analytical sample to the order of 1 mg, and transfer to a 250-mL
volumetric flask made of polymer.
b) Add about 150 mL of sodium hydroxide solution heated up to about 65 ºC, and heat for
1 hour while shaking to mix at every 10 minutes in a water bath at 65 ºC ± 2 ºC.
c) After standing to cool, add water up to the marked line.
d) Filter with Type 3 filter paper to make the sample solution.
(4.2) Measurement: Conduct measurement as shown below.
a) Transfer a predetermined volume (the equivalents of 20 mg - 50 mg as SiO2 and no
more than 25 mL of liquid volume) to a 200-mL beaker made of polyethylene.
b) Add about 10 mL of hydrochloric acid and about 15 mL of potassium fluoride solution,and further add about 2 g of potassium chloride to dissolve, and then cool in arefrigerator for about 30 minutes (3) to form the precipitate of potassium fluoride.
c) Filter with a Gooch crucible (4) made of polyethylene topped with Type 6 filter paper,and wash the container 3 times with potassium chloride solution, then move the whole
precipitate into the crucible, and further wash 6 - 7 times with a small amount ofpotassium chloride solution (5).
d) Move the precipitate on the filter together with the filter paper into a 300-mL tall beakerwith water, and further add water to make about 200 mL and heat up to 70 ºC - 80 ºC on
a hot plate.
e) Add a few drops of phenolphthalein solution (1 g/100 mL) to the sample solution as anindicator and titrate with the 0.1 mol/L - 0.2 mol/L sodium hydroxide solution until thecolor of the solution becomes light red.
f) Calculate soluble silicic acid (S-SiO2) in the analytical sample by the following formula.
Soluble silicic acid (S-SiO2) (% (mass fraction)) in an analytical sample= VS × C × f × (V4/V5) × (15.021/W) × (100/1,000)
VS: Volume of 0.1 mol/L - 0.2 mol/L sodium hydroxide required for titration
C: Set concentration (mol/L) of 0.1 mol/L - 0.2 mol/L sodium hydroxide solution
f: factor of 0.1 mol/L - 0.2 mol/L sodium hydroxide solution
V4: Predetermined volume (mL) of the extract in (4.1) c)
V5: Volume (mL) of the extract transferred in (4.2) a)150

W: Mass (g) of an analytical sample
Note (3) To be no more than 10 ºC(4) Filter paper pulp can be stuffed to restrain precipitate from outflowing.
(5) Until the filtrate becomes neutral.
Comment 1 Replicate testing was conducted using silica gel fertilizer (8 samples); as aresult, the mean was in the range of 66.81 % - 91.31 % (mass fraction), andthe standard deviation and relative standard deviation were 0.27 % - 0.86 %(mass fraction) and 0.3 % - 1.0 %, respectively.
Additionally, table 1 shows results and analysis results from a collaborative
study for test method validation.
.
Table 1 Results and analysis results from a collaborative study
for the test method validation of soluble silicic acid in silica gel fertilizers
4)

RSDr

3)

(%)

Silica gel fertilizers 1
8
79.37
0.23
0.3
Silica gel fertilizers 2
8
84.68
0.42
0.5
Silica gel fertilizers 3
8
89.58
0.4
0.4
Silica gel fertilizers 4
8
84.44
0.37
0.4
Silica gel fertilizers 5
8
85.77
0.46
0.5
1) Number of laboratories used in analysis
2) Mean (n= number of laboratories × number of samples (2))3) Mass fraction
4) Repeatability standard deviation
5) Repeatability relative standard deviation
6) Reproducibility standard deviation
7) Reproducibility relative standard deviation

Sample name

Number of

2)

sr

3)

(%)

Mean

5)

6)

RSDR

(%)

3)

(%)

0.55
0.85
0.51
0.77
0.59

0.7
1
0.6
0.9
0.7

sR

7)

1)

laboratories(%)

References
1) Takeshi HASHIMOTO, Akira SHIMIZU and Kaori OKADA: Validation of a Method of
Potassium Fluoride for Determination of Sodium Hydroxide-Soluble Silicic Acid in
Silica Gel Fertilizer, Research Report of Fertilizer, Vol.3, 2010. (19 - 24)
2) Akira SHIMIZU, Shin ABE and Jun ITO: Determination of Solubility Silicic Acid in Silica gel Fertilizer
and Silica gel-including Fertilizer by Potassium Fluoride Method: A Collaborative Study, Research
Report of Fertilizer, Vol.5, 2012. (31 - 40)

1515) Flow sheet for soluble silicic acid: The flow sheet for soluble silicic acid in silica gel
fertilizers, etc. is shown below:
1g
Weigh to the order of 1 mg
analytical sample
into a 250-mL volumetric flask made of polymer.
← About 150 mL sodium hydroxide (20 g/L) [about 65 ºC]
65 ºC ± 2 ºC, 1 hour, shaking to mix every 10 minutes
HeatingStanding to coolRoom temperature← Water (up to the marked line)
Filtration
Type 3 filter paper
Aliquot(predetermined amount)

200-mL beaker made of polyethylene← About 10 mL of hydrochloric acid
← About 15 mL of potassium fluoride solution← 2 g of potassium chloride
Standing to cool
For 30 minutes in a refrigerator
Filtration

Gooch crucible made of polyethylene
Type 6 filter paper

Washing

Wash 6 - 7 times with potassium chloride solution

Transfer
300-mL tall beaker, water← Water (until the liquid volume reaches about 200 mL)
Heating
70 ºC - 80 ºC← A few drops of phenolphthalein solution (1 g/100 mL)
0.1 mol/L - 0.2 mol/L sodium hydroxide solution
Titration(Until the solution becomes light red)
FigureFlow sheet for soluble silicic acid in silica gel fertilizers

1524.4.1.c Potassium fluoride method (Fertilizers containing silica gel fertilizers)(1) Summary
This test method is applicable to fertilizers containing silica gel fertilizers.
Mix the equivalent volumes of the extract which is extracted by adding hydrochloric acid
(1+23) to an analytical sample and the liquid by extracting non-dissolved matter on a filter
paper with sodium hydroxide (20 g/L), and add hydrochloric acid, potassium fluoride solution
and potassium chloride. Cool it in a refrigerator, and form precipitate as potassium
silicofluoride. Then transfer water in the precipitate, heat and measure by neutralization
titration to obtain soluble silicic acid (S-SiO2) in an analytical sample.
(2) Reagents: Reagents are as shown below:a) 0.1 mol/L - 0.2 mol/L sodium hydroxide solution(1): Transfer about 30 mL of water toa polyethylene bottle, dissolve about 35 g of sodium hydroxide specified in JIS K 8576
by adding in small portions while cooling, seal tightly and leave at rest for 4 - 5 days.
Transfer 5.5 mL - 11 mL of the supernatant to a ground-in stoppered storage container,
and add 1,000 mL of water containing no carbonic acid.
Standardization: Dry sulfamic acid reference material for volumetric analysis
specified in JIS K 8005 by leaving at rest in a desiccator at no more than 2 kPa for about
48 hours, then transfer about 2.5 g to a weighing dish, and measure the mass to the orderof 0.1 mg. Dissolve with a small amount of water, transfer to a 250-mL volumetric flask,
and add water up to the marked line (1). Transfer a predetermined amount of the solution
to a 200-mL - 300-mL Erlenmeyer flask, add a few drops of bromothymol blue solution(0.1 mg/100 mL) as an indicator, and titrate with 0.1 mol/L - 0.2 mol/L sodiumhydroxide solution until the color of the solution becomes green. Calculate the factor of
a 0.1 mol/L - 0.2 mol/L sodium hydroxide solution by the following formula:
Factor of 0.1 mol/L - 0.2 mol/L sodium hydroxide solution (f)= (W × A × 0.01/97.095) × (V1/V2) × (1,000/V3) × (1/C)
W: Mass (g) of sulfamic acid sampled
A: Purity (%) of sulfamic acidV1: Volume (mL) of sulfamic acid solution transferred
V2: Constant volume (250 mL) of sulfamic acid solution
V3: Volume (mL) of 0.1 mol/L - 0.2 mol/L sodium hydroxide solution needed fortitration
C: Estimated concentration (mol/L) of 0.1 mol/L - 0.2 mol/L sodium hydroxide
solutionb) Sodium hydroxide: A JIS Guaranteed Reagent specified in JIS K 8576 or a reagent ofequivalent quality.
c) Hydrochloric acid: A JIS Guaranteed Reagent specified in JIS K 8180 or a reagent ofequivalent quality.
d) Potassium chloride: A JIS Guaranteed Reagent specified in JIS K 8121 or a reagent ofequivalent quality.
e) Potassium chloride solution (1): Add 250 mL of ethanol specified in JIS K 8101
to 750 mL of water to mix, and add 150 mL of potassium chloride to dissolve.
Add a few drops of methyl red solution (0.1 g/100 mL) as an indicator and drophydrochloric acid until the color of the solution becomes red to make it acidic. Afterleaving at rest for 1 day, neutralize with the 0.1 mol/L - 0.2 mol/L sodium hydroxide
solution.
153

Potassium fluoride solution (1): Dissolve 58 g of potassium fluoride specified in JIS K
8815 in 1,000 mL of water (2).
g) Methyl red solution (0.1 g/100 mL): Dissolve 0.10 g of methyl red specified in JIS K
8896 in 100 mL of ethanol (95) specified in JIS K 8102.
h) Phenolphthalein solution (1 g/100 mL): Dissolve 1 g of phenolphthalein specified in
JIS K 8799 in 100 mL of ethanol (95) specified in JIS K 8102.
f)

Note (1) This is an example of preparation; prepare an amount as appropriate.
(2) Store in a container made of polyethylene, etc. that contains no silicon.
(3) Instruments: Instruments are as follows:a) Constant-temperature bath: A constant-temperature bath adjustable up to 80 ºC which
can keep test temperature ± 2 ºC and set a 250-mL volumetric flask made ofpolyethylene that contains no silicon in it.
b) Hot plate: A hot plate whose surface temperature can be adjusted up to 250 ºC.
c) Volumetric flask made of polymer: A volumetric flask that is made of polyethylene,
etc. of a quality of material which prevents silicic acid from eluting in the extract
procedure in (4.1).
(4) Test procedures(4.1) Extraction: Conduct extraction as shown below.
a) Weigh 1 g of an analytical sample to the order of 1 mg, and transfer to a 300-mL tallbeaker.
b) Add 150 mL of hydrochloric acid (1+23) warmed up to about 30 ºC, and warm it in awater bath at 30 ºC ± 2 ºC while stirring every 10minutes with a glass rod for 60
minutes.
c) After standing to cool, filter with Type 6 filter paper to a 250-mL volumetric flask as an
acceptor. Wash the tall beaker with water, then move the whole residue on the filter
paper and add water up to the marked line to make the sample solution (1).
d) Transfer the non-dissolved matter on the filter paper together with the filter paper to a
250- mL volumetric flask made of polyethylene.
e) Add about 150 mL of sodium hydroxide solution (20 g/L) warmed up to about 65 ºC,
and heat for 60 minutes while shaking to mix at every 10 minutes in a water bath at 65ºC ± 2 ºC.
f) After standing to cool, add water up to the marked line and filter with Type 3 filter paper
to make the sample solution (2).
(4.2) Measurement: Conduct measurement as shown below.
a) Transfer a predetermined volume (the equivalents of 20 mg - 50 mg as SiO2) (3) of thesample solution (1) and (2) to a 200-mL beaker made of polyethylene.
b) Add about 10 mL of hydrochloric acid and about 15 mL of potassium fluoride solution,and further add about 2 g of potassium chloride to dissolve, and then cool in arefrigerator for about 30 minutes (4) to form the precipitate of potassium fluoride.
c) Filter with a Gooch crucible (5) made of polyethylene topped with Type 6 filter paper,and wash the container 3 times with potassium chloride solution, then move the whole
precipitate into the crucible, and further wash 6 - 7 times with a small amount ofpotassium chloride solution (6).
d) Move the precipitate on the filter together with the filter paper into a 300-mL tall beakerwith water, and further add water to make about 200 mL of solution.
e) Heat up to 70 ºC - 80 ºC on a hot plate. Then, add a few drops of phenolphthaleinsolution (1 g/100 mL) to the sample solution as an indicator and titrate with the 0.1
154mol/L - 0.2 mol/L sodium hydroxide solution until the color of the solution becomeslight red.
f) Calculate soluble silicic acid (S-SiO2) in the analytical sample by the following formula.
Soluble silicic acid (S-SiO2) (%) in an analytical sample= VS × C × f × (V1/V2) × (15.021/W) × (100/1000)
VS: Volume of 0.1 mol/L - 0.2 mol/L sodium hydroxide required for titration
C: Estimated concentration (mol/L) of sodium hydroxide solution (0.1 mol/L - 0.2
mol/L)
f: factor of 0.1 mol/L - 0.2 mol/L sodium hydroxide solution
V1: Predetermined volume (250 mL) of the sample solution in (4.1) c)
V2: Volume (mL) of the sample solution transferred in (4.2) a)
W: Mass (g) of an analytical sample
Note (3) The transferred volumes of the sample solution (1) and (2) should be equal.
(4) To be no more than 10 ºC(5) Filter paper pulp can be stuffed to restrain precipitate from outflowing.
(6) Until the filtrate becomes neutral.

Comment 1 Replicate testing was conducted using fertilizers (13 samples) including asilica gel fertilizer; as a result, the mean was in the range of 12.85 % 42.49 % (mass fraction), and the standard deviation and repeatability relative
standard deviation were 0.04 % - 0.29 % (mass fraction) and 0.2 % - 1.2 %,respectively.
Additionally, table 1 shows results and analysis results from a collaborative
study for test method validation.

155

Table 1 Results and analysis results from a collaborative study for the test method validationof soluble silicic acid in fertilizers including a silica gel fertilizer
4)
5)
6)
7)
Number of Mean2)
sr
RSDr
sR
RSDR
laboratorie
Sample name
3)
3)
3)
(%)
(%)
(%)
(%)
(%)
s1)
Mixed phosphate
8.00
24.99
0.16
0.6
0.33
1.3
fertilizer 1
Mixed phosphate
fertilizer 2

8.00

34.50

0.26

0.7

0.48

1.4

Compoundfertilizers 1

8.00

30.30

0.13

0.4

0.60

2.0

Compoundfertilizers 2

8.00

33.34

0.13

0.4

0.47

1.4

Compound
8.00
15.76
0.11
0.7
0.21
fertilizers 3
1) Number of laboratories used in analysis
2) Total mean (n= number of laboratories × number of replication(2))3) Mass fraction
4) Repeatability standard deviation
5) Repeatability relative standard deviation
6) Reproducibility standard deviation
7) Reproducibility relative standard deviation

1.3

References
1) Akira SHIMIZU, Jun ITO and Shin ABE: Method Validation of Potassium Fluoride Method
for Determination of Acid-Soluble and Base-Soluble Silicic Acid in Fertilizer containing
Silica gel, Research Report of Fertilizer, Vol.4, 2011. (1 - 8)2) Akira SHIMIZU: Method Validation of Potassium Fluoride Method for Determinationof Acid-Soluble and Base-Soluble Silicic Acid in Fertilizer containing Silica Gel,
Research Report of Fertilizer, Vol.6, 2013. (1 - 8)

1565) Flow sheet for soluble silicic acid: The flow sheet for soluble silicic acid in
fertilizers including silica gel fertilizers is shown below:

1g
Weigh to the order of 1 mg
analytical sample
into a 300-mL tall beaker← About 150 mL hydrochloric acid (1+23) [30 ºC]
60 minutes in water bath at 30 ºC ± 2 ºC
Warming(Stir at every 10 minutes)
Filtration(supernatant)<Residue>Type 6 filter paper, a 250-mL volumetric flask

Transfer
Move the residue to a filter paper← Wash with water<Filtrate>← Wash (up to the marked line)
Sample solution (1)Transfer

250-mL volumetric flask made of polyethylene,
together wiyh a filter paper← Add about 150 mL of sodium hydroxide solution (20 g/L) [65ºC] to stopple.
Then, shake to mix until the filter paper completely breaks down
Heating
65 ºC ± 2 ºC, while shaking to mix
at every 10 minutes for 60 minutes
Standing to cool
← Water (up to the marked line)
Filtration
Type 3 filter paper
Sample solution (2)Aliquot

Transfer the equal volume of the sample (1) and (2) to(predetermined amount) a 200-mL beaker made of polyethylene← About 10 mL of hydrochloric acid
← About 15 mL of potassium fluoride solution← About 2 g of potassium chloride
Cooling
For 30 minutes in a refrigerator
Filtration

Gooch crucible made of polyethylene
Type 6 filter paper

Washing

Wash 6 - 7 times with potassium chloride solution

Transfer
300-mL tall beaker, water← Water (until the liquid volume reaches about 200 mL)
Heating
70 ºC - 80 ºC← A few drops of phenolphthalein solution (1 g/100 mL)
0.1 mol/L - 0.2 mol/L sodium hydroxide solution
Titration(Until the color of solution becomes light red)
FigureFlow sheet for soluble silicic acid in fertilizers

157

4.4.1.d Perchloric acid method(1) Summary
Extract by adding hydrochloric acid (1+23) to an analytical sample, add perchloric acid andheat, and then measure the formed silicic acid anhydride to obtain hydrochloric acid (1+23)
soluble silicic acid (soluble silicic acid (S-SiO2)) in the analytical sample.
(2) Reagents: Reagents are as shown below:a) Hydrochloric acid: A JIS Guaranteed Reagent specified in JIS K 8180 or a reagent ofequivalent quality.
b) Perchloric acid: A JIS Guaranteed Reagent specified in JIS K 8223 or a reagent ofequivalent quality.
(3) Apparatus and instruments: Apparatus and instruments are as follows:a) Constant-temperature rotary shaker: A constant-temperature rotary shaker that canrotate a 250-mL volumetric flask, set up in a thermostat adjustable to 30 ºC ± 1 ºC,
upside down at 30 - 40 revolutions/min.
b) Hot plate: A hot plate whose surface temperature can be adjusted up to 250 ºC.
c) Electric funnel: An electric funnel that can be adjusted to 1,000 ºC - 1,100 ºC.
d) Crucible: After heating a chemical analysis porcelain crucible specified in JIS R 1301
in an electric funnel at 1,000 ºC - 1,100 ºC, let it stand to cool in a desiccator andmeasure the mass to the order of 1 mg.
(4) Test procedures(4.1) Extraction:a) Weigh 1 g of an analytical sample to the order of 1 mg, and transfer to a 250-mL
volumetric flask.
b) Add 150 mL of hydrochloric acid heated up to about 30 ºC, and shake to mix at 30 - 40
revolutions/min (30 ºC ± 1 ºC) for 1 hour.
c) After standing to cool, add water up to the marked line.
d) Filter with Type 3 filter paper to make the sample solution.
Comment 1 The procedure in (4.1) is the same as the procedure in (4.1) of 4.4.1.a.
(4.2) Measurement:a) Transfer a predetermined volume to a 100-mL tall beaker.
b) Add about 10 mL of perchloric acid and heat.
c) When white smoke from the perchloric acid starts evolving, cover with a watch glass,then heat for 15 - 20 minutes to form precipitate of silicone dioxide.
d) After standing to cool, add about 50 mL of hydrochloric acid (1+4) and cover with awatch glass and heat at 70 ºC - 80 ºC on a hot plate for several minutes.
e) Immediately after heating, filter with Type 5-C filter paper, wash the container withheated hydrochloric acid (1+10) and move the whole precipitate to a filter paper.
f) Wash the precipitate and the filter paper 2 times with heated hydrochloric acid (1+10),
and further wash several times with hot water (1).
g) Put the precipitate together with the filter paper into the crucible.
h) Put the crucible into a drying apparatus and dry at about 120 ºC for 1 hour.
i) After standing to cool, put the crucible into an electric funnel and heat gently to char.
j) Ignite at 1,000 ºC - 1,100 ºC for 1 hour.
k) After ignition, move the crucible to a desiccator and let it stand to cool.
l) After standing to cool, remove the crucible from the desiccator and measure the mass tothe order of 1 mg.
158m) Calculate soluble silicic acid (S-SiO2) in an analytical sample by the following formula.
Soluble silicic acid (S-SiO2) (% (mass fraction)) in an analytical sample= A × (V1/V2)/B × 100
A: Mass (g) of precipitate
B: Mass (g) of an analytical sample
V1: Predetermined volume (mL) of the sample solution in (4.1) c)
V2: Volume (mL) of the sample solution transferred in (4.2) a)
Note (1) Wash until no reaction by chloride appears in the filtrate.
Comment 2 Table 1 shows results from a simultaneous analysis with the same samples(proficiency testing and external quality control testing) hosted by the Japan
Fertilizer Quality Assurance Committee analyzed by the robust method.
Table 1 Results and analysis results from simultaneous analysis with the same samples
for soluble silicic acid hosted by the Japan Fertilizer Quality Assurance Committee1)
5)
2)
4)RSDrob
Number of Median Μ
NIQR
Year
Sample3)3)laboratories
(%)
(%)
(%)
Slag silicate
13
33.58
0.42
1.2
fertilizer
Slag silicate
2010
12
33.72
0.30
0.9
fertilizer
Slag silicate
2012
13
36.09
0.63
1.7
fertilizer
Slag silicate
2013
11
35.50
0.70
2.0
fertilizer
1) Proficiency testing and external quality control testing2) Median (M) agrees with the mean in normal distribution.
3) Mass fraction4) Robust standard deviation (NIQR) agrees with standard deviation in normal distribution.
5) RSDrob stands for relative standard deviation obtained from the robust methodby the formula shown below:
RSDrob = (NIQR/M) × 100
2008

Reference
1) Masayoshi KOSHINO: Second Revision of The Methods of Analysis of Fertilizers(Details), p.143 - 144, Yokendo, Tokyo (1988)

159

5) Flow sheet for soluble silicic acid: The flow sheet for soluble silicic acid in fertilizers isshown below:
1 g analytical sample
Weigh to the order of 1 mg into a 250-mL volumetric flask.
← About 150 mL of hydrochloric acid (1+23) [about 30 ºC]
Rotary shaker (30 - 40 revolutions/min)Shaking to mix
30 ºC ± 1 ºC, 1 hourStanding to coolRoom temperature← Water (up to the marked line)
Filtration
Type 3 filter paper
Aliquot(predetermined amount)

100-mL tall beaker←About 10 mL of perchloric acid
When white smoke of perchloric acid starts evolving,
Heating
cover with a watch glass, heat for 15 - 20 minutes
Standing to cool
←About 50 mL of hydrochloric acid (1+4)
Cover with a watch glass,
Heatingheat at 70 ºC - 80 ºC for several minutes
Filtration

Type 5-C filter paper←Wash 2 times with about 10 mL of hydrochloric acid (1+10)←Wash several times with hot water
DryingDrying apparatus, about 120 ºC, 1 hourStanding to coolCharring
Incineration

Heat gently in an electric furnace
1,000 ºC - 1,100 ºC for no less than 1 hourStanding to cool

Desiccator

Measurement

Measure the mass to the order of 1 mg

FigureFlow sheet for soluble silicic acid in fertilizers

160

4.4.2 Water-soluble silicic acid
4.4.2.a Potassium fluoride method(1) Summary
This test method is applicable to liquid potassium silicate fertilizers.
Extract by adding water to an analytical sample, add hydrochloric acid, potassium chloride
and potassium fluoride solution and cool in a refrigerator, and then filter after forming
precipitate as potassium silicofluoride. Put the precipitate in water and heat, and measure by
neutralization titration to obtain the water- soluble silicic acid (W-SiO2) in an analyticalsample.
(2) Reagents: Reagents are as shown below:a) 0.1 mol/L - 0.2 mol/L sodium hydroxide solution(1): Transfer about 30 mL of water toa polyethylene bottle, dissolve about 35 g of sodium hydroxide specified in JIS K 8576
by adding in small portions while cooling, seal tightly and leave at rest for 4 - 5 days.
Transfer 5.5 mL - 11 mL of the supernatant to a ground-in stoppered storage container,
and add 1,000 mL of water containing no carbonic acid.
Standardization: Dry sulfamic acid reference material for volumetric analysis
specified in JIS K 8005 by leaving at rest in a desiccator at no more than 2 kPa for about
48 hours, then transfer about 2.5 g to a weighing dish, and measure the mass to the orderof 0.1 mg. Dissolve with a small amount of water, transfer to a 250-mL volumetric flask,
and add water up to the marked line (1). Transfer a predetermined amount of the solution
to a 200-mL - 300-mL Erlenmeyer flask, add a few drops of bromothymol blue solution(0.1 mg/100 mL) as an indicator, and titrate with 0.1 mol/L - 0.2 mol/L sodiumhydroxide solution until the color of the solution becomes green. Calculate the factor of
a 0.1 mol/L - 0.2 mol/L sodium hydroxide solution by the following formula:
Factor of 0.1 mol/L - 0.2 mol/L sodium hydroxide solution (f)= (W × A × 0.01/97.095) × (V1/V2) × (1,000/V3) × (1/C)
W: Mass (g) of sulfamic acid sampled
A: Purity (%) of sulfamic acidV1: Volume (mL) of sulfamic acid solution transferred
V2: Constant volume (250 mL) of sulfamic acid solution
V3: Volume (mL) of 0.1 mol/L - 0.2 mol/L sodium hydroxide solution needed fortitration
C: Set concentration (mol/L) of 0.1 mol/L - 0.2 mol/L sodium hydroxide solutionb) Hydrochloric acid: A JIS Guaranteed Reagent specified in JIS K 8180 or a reagent ofequivalent quality.
c) Potassium chloride: A JIS Guaranteed Reagent specified in JIS K 8121 or a reagent ofequivalent quality.
d) Potassium chloride solution (1): Add 250 mL of ethanol specified in JIS K 8101
to 750 mL of water to mix, and add 150 mL of potassium chloride to dis solve.
Add a few drops of methyl red solution (0.1 g/100 mL) as an indicator and drophydrochloric acid until the color of the solution becomes red to make it acidic. Afterleaving at rest for 1 day, neutralize with the 0.1 mol/L - 0.2 mol/L sodium hydroxide
solution.
e) Potassium fluoride solution (1): Dissolve 58 g of potassium fluoride specified in JIS K
8815 in 1,000 mL of water (2).
f) Methyl red solution (0.1 g/100 mL): Dissolve 0.10 g of methyl red specified in JIS K
8896 in 100 mL of ethanol (95) specified in JIS K 8102.
161g) Phenolphthalein solution (1 g/100 mL): Dissolve 1 g of phenolphthalein specified in
JIS K 8799 in 100 mL of ethanol (95) specified in JIS K 8102.
Note (1) This is an example of preparation; prepare an amount as appropriate.
(2) Store in a container made of polyethylene, etc. that contains no silicon.
(3) Apparatus and instruments: Apparatus and instruments are as follows:a) Constant-temperature rotary shaker: A constant-temperature rotary shaker that canrotate a 500-mL volumetric flask upside down at 30 - 40 revolutions/min.
b) Hot plate: A hot plate whose surface temperature can be adjusted up to 250 ºC.
(4) Test procedures(4.1) Extraction: Conduct extraction as shown below.
a) Weigh 5 g of an analytical sample to the order of 1 mg, and transfer to a 500-mL
volumetric flask.
b) Add 400 mL of water, and shake to mix at 30 - 40 revolutions/min for about 30 minutes.
c) Add water up to the marked line.
d) Filter with Type 3 filter paper to make the sample solution.
Comment 1 In the procedure of a), it is also allowed to weigh 2.5 g of an analyticalsample to the order of 1 mg, and put it into a 250-mL volumetric flask
Comment 2 The procedure in (4.1) is the same as the procedure in (4.1.2) of 4.3.3.a,4.3.3.b and 4.3.3.c.
(4.2) Measurement: Conduct measurement as shown below.
a) Transfer a predetermined volume (the equivalents of 20 mg - 50 mg as SiO2 and no
more than 25 mL of liquid volume) to a 200-mL beaker made of polyethylene.
b) Add about 10 mL of hydrochloric acid and about 15 mL of potassium fluoride solution,and further add about 2 g of potassium chloride to dissolve, and then cool in arefrigerator for 30 minutes or more (3) to form the precipitate of potassium fluoride.
c) Filter with a Gooch crucible (4) made of polyethylene topped with Type 6 filter paper,and wash the container 3 times with potassium chloride solution, then move the whole
precipitate into the crucible, and further wash 6 - 7 times with a small amount ofpotassium chloride solution (5).
d) Move the precipitate on the filter together with the filter paper into a 300-mL tall beakerwith water, and further add water to make about 200 mL and heat it up to 70 ºC - 80 ºC
on a hot plate.
e) Add a few drops of phenolphthalein solution (1 g/100 mL) to the sample solution as anindicator and titrate with the 0.1 mol/L - 0.2 mol/L sodium hydroxide solution until thecolor of the solution becomes light red.
f) Calculate soluble silicic acid (S-SiO2) by the following formula.
Soluble silicic acid (S-SiO2) (% (mass fraction)) in an analytical sample= VS × C × f × (V4/V5) × (15.021/W) × (100/1,000)
VS: Volume of the 0.1 mol/L - 0.2 mol/L sodium hydroxide solution required fortitration
C: Set concentration (mol/L) of 0.1 mol/L - 0.2 mol/L sodium hydroxide solution
f: Factor of 0.1 mol/L - 0.2 mol/L sodium hydroxide solution
V4: Predetermined volume (mL) of the extract in (4.1) c)
V5: Volume (mL) of the extract transferred in (4.2) a)162

W: Mass (g) of an analytical sample
Note (3) To be no more than 10 ºC(4) Filter paper pulp can be stuffed to restrain precipitate from outflowing.
(5) Until the filtrate becomes neutral.
Reference
1) Masayoshi KOSHINO: Second Revision of The Methods of Analysis of Fertilizers(Details), p.144 - 146, Yokendo, Tokyo (1988)
5) Flow sheet for soluble silicic acid: The flow sheet for soluble silicic acid in fertilizers isshown below:
5 g analytical sample
Weigh to the order of 1 mg to a 500-mL volumetric flask.
← About 400 mL of water
Rotary shaker (30 - 40 revolutions/min)Shaking to mix
30 minutes← Water (up to the marked line)
Filtration
Type 3 filter paper
Aliquot(predetermined amount)

200-mL beaker made of polyethylene← About 10 mL hydrochloric acid
← About 15mL potassium fluoride solution← 2 g of potassium chloride
Cooling
For 30 minutes or more in a refrigerator
Filtration

Gooch crucible made of polyethylene
Type 6 filter paper

Washing

Wash 6 - 7 times with potassium chloride solution

Transfer
300-mL tall beaker, water← Water (until the liquid volume reaches about 200 mL)
Heating
70 ºC - 80 ºC← A few drops of phenolphthalein solution (1 g/100 mL)
0.1 mol/L - 0.2 mol/L sodium hydroxide solution
Titration(Until the solution becomes light red)
Figure Flow sheet for soluble silicic acid in fertilizers

163

4.5

Lime, calcium and alkalinity

4.5.1 Total lime
4.5.1.a Flame atomic absorption spectrometry(1) Summary
This test method is applicable to fertilizers containing organic matters.
Pretreat an analytical sample with incineration and hydrochloric acid, add an interference
suppressor solution, and then spray in an acetylene-air flame, and measure the atomic
absorption with calcium at a wavelength of 422.7 nm to quantify total lime.
(2) Reagents: Reagents are as shown below:a) Hydrochloric acid: A JIS Guaranteed Reagent specified in JIS K 8180 or a reagent ofequivalent quality.
b) Interference suppressor solution: Weigh 60.9 g - 152.1 g of strontium chloride
hexahydrate specified in JIS K 8132(1) into a 2,000-mL beaker, add a small amount ofwater, gradually add 420 mL of hydrochloric acid to dissolve, and further add water tomake 1,000 mL.
c) Calcium standard solution (CaO 1 mg/mL)(2): Heat calcium carbonate specified in
JIS K 8617 at 110 ºC ± 2 ºC for about 2 hours, let it stand to cool in a desiccator, andweigh 1.785 g into a weighing dish. Transfer to a 1,000-mL volumetric flask with asmall amount of water, add 20 mL of hydrochloric acid (1+3) to dissolve, and add water
up to the marked line.
d) Calcium standard solution (CaO 5 µg/mL - 50 µg/mL) for the calibration curve
preparation(2): Transfer 2.5 mL - 25 mL of a calcium standard solution (CaO 1 mg/mL)
to 500-mL volumetric flasks step-by-step, add about 50 mL of interference suppressor
solution(3), and add water up to the marked line(4).
e) Blank test solution for the calibration curve preparation (2): Transfer about 50 mL of
an interference suppressor solution to a 500-mL volumetric flask (3), and add water up to
the marked line (4).
Note (1) 29 g of lanthanum oxide (atomic absorption analysis grade or equivalents) can
also be used.
(2) This is an example of preparation; prepare an amount as appropriate.
(3) Add an interference suppressor solution that is 1/10 volume of the volume to beprepared.
(4) For storage, use a sealable container made of materials which are not likely todissolve calcium, such as borosilicate glass-1 specified in JIS R 3503 or Teflon.
Comment 1 Instead of a calcium standard solution (CaO 1 mg/mL) in (2) c), a calcium
standard solution (Ca 1 mg/mL) for atomic absorption traceable to a specified
reference material (national measurement standard) according to Article 134of the Measurement Act can also be used. Additionally, it is recommended tomultiply by the conversion factor to use as a calcium standard solution (CaO
1.3992 mg/mL).
(3) Instruments: Instruments are as shown below:a) Flame atomic absorption spectrometer: An atomic absorption spectrometer specified
in JIS K 0121.
1) Light source: A calcium hollow cathode lamp
2) Gas: Gas for heating by flame(i) Fuel gas: acetylene(ii) Auxiliary gas: Air sufficiently free of dust and moisture.
164b) Electric furnace: An electric furnace that can be adjusted to 550 ºC ± 5 ºC.
c) Hot plate or sand bath: A hot plate whose surface temperature can be adjusted up to
250 ºC. Adjust the amounts of gas and silica sand of a sand bath so that the sand bathtemperature can be set to 250 ºC.
(4) Test procedures(4.1) Extraction: Conduct extraction as follows:a) Weigh about 5 g of an analytical sample to the order of 1 mg, and put it in a 200-mL300-mL tall beaker.
b) Put the tall beaker in an electric furnace, and heat gently to char(5).
c) Ignite (at 550 ºC ± 5 ºC) for no less than 4 hours to incinerate.
d) After standing to cool, moisten the residue with a small amount of water, add gradually
about 10 mL of hydrochloric acid, and further add water to make about 20 mL.
e) Cover the tall beaker with a watch glass, and heat on a hot plate or a sand bath to boil
for about 5 minutes.
f) After standing to cool, transfer the solution to a 250-mL - 500-mL volumetric flask withwater.
g) Add water up to the marked line.
h) Filter with Type 3 filter paper to make the sample solution.
Note (5) Example of the charring procedure: Heat at about 250 ºC until smoke is nolonger formed.
Comment 2 The procedures in (4.1) are the same as in (4.1.2) in 4.2.1.a.
Comment 3 The sample solution prepared in (4.1) a) - h) in 4.9.1.a can also be used.
(4.2) Measurement: Conduct measurement according to JIS K 0121 and as shown below.
Specific measurement procedures are according to the operation method of the atomic
absorption spectrometer used in measurement.
a) Measurement conditions for the atomic absorption spectrometer: Set up themeasurement conditions for the atomic absorption spectrometer considering thefollowing:
Analytical line wavelength: 422.7 nmb) Calibration curve preparation
1) Spray the calcium standard solutions for the calibration curve preparation and theblank test solution for the calibration curve preparation into a flame, and read theindicated value at a wavelength of 422.7 nm.
2) Prepare a curve for the relationship between the calcium concentration and theindicated value of the calcium standard solutions for the calibration curve
preparation and the blank test solution for the calibration curve preparation.
c) Sample measurement
1) Transfer a predetermined amount of the sample solution (the equivalents of 0.5 mg- 5 mg as CaO) to a 100-mL volumetric flask.
2) Add about 10 mL of an interference suppressor solution (3), and add water up to themarked line.
3) Conduct similarly as in b) 1) to read the indicated value.
4) Obtain the calcium content from the calibration curve, and calculate total lime in
the analytical sample.
Comment 4

Replicate testing was conducted using composted sludge fertilizer (2samples), human waste sludge fertilizer (2 samples) and compost (1 sample),165

designated blended fertilizer containing organic fertilizer (1 sample) andcompound fertilizer containing organic fertilizer (1 sample); as a result, themean was in the range of 1.38 % - 2.44% (mass fraction), and the standard
deviation and relative standard deviation were 0.004 % - 0.02% (massfraction) and 0.2 % - 1.5%, respectively.
References
1) Masayoshi KOSHINO: Second Revision of The Methods of Analysis of Fertilizers(Details), p.156 – 158, Yokendo, Tokyo (1988)
2) Kimie KATO, Masayuki YOSHIMOTO and Yuji SHIRAI: Systematization of
Determination Methods of Major Components in Sludge Fertilizer, Compost andOrganic Fertilizer, Research Report of Fertilizer, Vol.3, 2010. (107 - 116)

(5)

Flow sheet for total lime: The flow sheet for total lime in fertilizers is shown below:

5 g analytical sample
Charring
Incineration

Weigh to the order of 1 mg into a 200-mL- 300-mL tall beaker.
Heat gently
Heat at 550 ºC ±5 ºC, no less than 4 hours

Standing to coolRoom temperature← Small amount of water, moisten the residue← About 10 mL of hydrochloric acid
← Water (up to about 20 mL)
Heating
Cover with a watch glass, and boil for 5 minutes.
Standing to coolRoom temperature

Transfer

250-mL - 500-mL volumetric flask, water

Filtration

Type 3 filter paper

Aliquot(predetermined amount)

100-mL volumetric flask←About 10 mL interference suppressor solution← Water (up to the marked line)
Measurement
Atomic absorption spectrometer
Figure Flow sheet for total lime in fertilizers.

1664.5.2 Soluble lime
4.5.2.a Flame atomic absorption spectrometry(1) Summary
This test method is applicable to fertilizers that guarantee alkalinity.
Add hydrochloric acid (1+23) to an analytical sample, boil to extract, and add an interference
suppressor solution, and then spray in an acetylene-air flame, and measure the atomic
absorption with calcium at a wavelength of 422.7 nm to quantify hydrochloric acid (1+23)
soluble lime (soluble lime (S-CaO)).
(2) Reagents: Reagents are as shown below:a) Hydrochloric acid: A JIS Guaranteed Reagent specified in JIS K 8180 or a reagent ofequivalent quality.
b) Interference suppressor solution(2): Weigh 60.9g - 152.1g of strontium chloride
hexahydrate specified in JIS K 8132(1) into a 2,000-mL beaker, add a small amount ofwater, add gradually 420 mL of hydrochloric acid to dissolve, and further add water tomake 1,000 mL.
c) Calcium standard solution (CaO 1 mg/mL)(2): Put calcium carbonate specified in JIS
K 8617 in a drying apparatus, heat at 110 ºC ± 2 ºC for about 2 hours, let it stand to coolin a desiccator, and weigh 1.785 g into a weighing dish. Transfer to a 1,000-mL
volumetric flask with a small amount of water, add about 20 mL of hydrochloric acid(1+3) to dissolve, and add water up to the marked line.
d) Calcium standard solution (CaO 5 µg/mL - 50 µg/mL) for the calibration curve
preparation(2): Transfer 2.5 mL - 25 mL of a calcium standard solution (CaO 1 mg/mL)
to 500-mL volumetric flasks step-by-step, add about 50 mL of an interference
suppressor solution(3), and add water up to the marked line(4).
e) Blank test solution for the calibration curve preparation (2): Transfer about 50 mL ofinterference suppressor solution used in the procedure in d) to a 500-mL volumetricflask (3), and add water up to the marked line (4).
Note (1) 29 g of lanthanum oxide (atomic absorption analysis grade or equivalents) can
also be used.
(2) This is an example of preparation; prepare an amount as appropriate.
(3) Add an interference suppressor solution that is 1/10 volume of the volume to beprepared.
(4) For storage, use a sealable container made of materials which are not like todissolve calcium, such as borosilicate glass-1 specified in JIS R 3503 or Teflon.
Comment 1 Instead of a calcium standard solution (CaO 1 mg/mL) in (2) c), a calcium
standard solution (Ca 1 mg/mL) for atomic absorption traceable to a specified
reference material (national measurement standard) according to Article 134of the Measurement Act can also be used. Additionally, it is recommended tomultiply by the conversion factor to use as the calcium standard solution(CaO 1.3992 mg/mL).
(3) Instruments: Instruments are as shown below:a) Flame atomic absorption spectrometer: An atomic absorption spectrometer specified
in JIS K 0121.
1) Light source: A calcium hollow cathode lamp
2) Gas: Gas for heating by flame(i) Fuel gas: acetylene
167

(ii) Auxiliary gas: Air sufficiently free of dust and moisture.
b) Hot plate: A hot plate whose surface temperature can be adjusted up to 250 ºC.
(4) Test procedures(4.1) Extraction: Conduct extraction as follows:a) Weigh about 2 g of an analytical sample to the order of 1 mg, and put it in a 500-mL tallbeaker.
b) Add about 20 mL of hydrochloric acid (1+23), cover with a watch glass, and boil on a
hot plate for about 5 minutes (5).
c) After standing to cool, transfer to a 250-mL - 500-mL volumetric flask with water.
d) Add water up to the marked line.
e) Filter with Type 3 filter paper to make the sample solution.
Note (5) Be aware that an analytical sample should not solidify in the bottom of a beaker.
Comment 2

In the case of a by-product magnesia fertilizer or a fertilizer containing a
by-product magnesia, if the pH of the sample solution of d) is neutral or basic,
prepare a sample solution anew by replacing “2 g of an analytical sample” inthe procedure in a) with “1 g - 1.5 g of an analytical sample”.
Comment 3 In the procedure in a), a 500-mL volumetric flask can be used instead of a
500-mL tall beaker. However the volumetric flask used should be
distinguished as an extraction flask and should not be used for the otherpurposes. In addition, “cover with a watch glass” in b) is replaced by “place along-stem funnel”, and “transfer to a 250-mL - 500 mL volumetric flask withwater” in the procedure in c) is skipped.
Comment 4 The procedure in (4.1) is the same as in (4.1) in 4.6.1.a and 4.7.1.a(4.2) Measurement: Conduct measurement according to JIS K 0121 and as shown below.
Specific measurement procedures are according to the operation method of the atomic
absorption spectrometer used in measurement.
a) Measurement conditions for the atomic absorption spectrometer: Set up themeasurement conditions for the atomic absorption spectrometer considering thefollowing:
Analytical line wavelength: 422.7 nmb) Calibration curve preparation
1) Spray the calcium standard solutions for the calibration curve preparation and theblank test solution for the calibration curve preparation into a flame, and read theindicated value at a wavelength of 422.7 nm.
2) Prepare a curve for the relationship between the calcium concentration and theindicated value of the calcium standard solutions for the calibration curve
preparation and the blank test solution for the calibration curve preparation.
c) Sample measurement
1) Transfer a predetermined amount of the sample solution (the equivalents of 0.5 mg- 5 mg as CaO) to a 100-mL volumetric flask.
2) Add about 10 mL of interference suppressor solution (3), and add water up to themarked line.
3) Conduct similarly as in b) 1) to read the indicated value.
4) Obtain the calcium content from the calibration curve, and calculate soluble lime
(S-CaO) in the analytical sample.

168

Comment 5 Table 1 shows results from a simultaneous analysis with the same samples(proficiency testing and external quality control testing) hosted by the Japan
Fertilizer Quality Assurance Committee analyzed by the robust method.
Table 1 Results and analysis results from simultaneous analysis with the same samples
for soluble lime hosted by the Japan Fertilizer Quality Assurance Committee1)
2)

Year

SampleNumber of Median Μ
3)laboratories
(%)

4)

NIQR

3)

(%)
0.90
0.78
0.79
0.60
0.65

RSDrob5)(%)

2009 Slag silicate fertilizer
58
32.68
2.8
2010 Slag silicate fertilizer
60
41.64
1.9
2011 Slag silicate fertilizer
55
40.78
1.9
2012 Slag silicate fertilizer
59
40.53
1.5
2013 Slag silicate fertilizer
59
30.06
2.2
1) Proficiency testing and external quality control testing2) Median (M) agrees with the mean in normal distribution.
3) Mass fraction4) Robust standard deviation (NIQR) agrees with standard deviation in normal distribution.
5) RSDrob stands for relative standard deviation obtained from the robust methodby the formula shown below:
RSDrob = (NIQR/M) × 100

Reference
1) Masayoshi KOSHINO: Second Revision of The Methods of Analysis of Fertilizers(Details), p.167 - 169, Yokendo, Tokyo (1988)(5) Flow sheet for soluble lime: The flow sheet for soluble lime in fertilizers is shown
below:
2 g analytical sample
Weigh to the order of 1 mg into a 500-mL tall beaker.
← About 200 mL of hydrochloric acid (1+23)
Heating
Cover with a watch glass, and boil for 5 minutes.
Standing to coolRoom temperature

Transfer
Water, 250-mL - 500-mL volumetric flask← Water (up to the marked line)
Filtration
Type 3 filter paper
Aliquot
100-mL volumetric flask(predetermined amount)←About 10 mL interference suppressor solution← Water (up to the marked line)
Measurement
Atomic absorption spectrometer
Figure Flow sheet for soluble lime in fertilizers.

1694.5.3 Water-soluble calcium
4.5.3.a Flame atomic absorption spectrometry(1) Summary
This test method is applicable to fertilizers that indicate calcium content as a response
modifier.
Extract by adding water to an analytical sample, and add an interference suppressor solution,and then spray in an acetylene-air flame, and measure the atomic absorption with calcium at awavelength of 422.7 nm to quantify water-soluble calcium (W-Ca).
(2) Reagents: Reagents are as shown below:a) Hydrochloric acid: A JIS Guaranteed Reagent specified in JIS K 8180 or a reagent ofequivalent quality.
b) Interference suppressor solution (2): Weigh 60.9 g-152.1 g of strontium chloride
hexahydrate (1) specified in JIS K 8132 into a 2,000-mL beaker, add a small amount ofwater, add gradually 420 mL of hydrochloric acid to dissolve, and further add water tomake 1,000 mL.
c) Calcium standard solution (CaO 1 mg/mL)(2): Put calcium carbonate specified in JIS
K 8617 in a drying apparatus, heat at 110 ºC ± 2 ºC for about 2 hours, let it stand to coolin a desiccator, and weigh 1.785 g into a weighing dish. Transfer to a 1,000-mL
volumetric flask with a small amount of water, add 20 mL of hydrochloric acid (1+3) todissolve, and add water up to the marked line.
d) Calcium standard solution (CaO 5 µg/mL - 50 µg/mL) for the calibration curve
preparation(2): Transfer 2.5 mL - 25 mL of a calcium standard solution (CaO 1 mg/mL)
to 500-mL volumetric flasks step-by-step, add about 50 mL of an interference
suppressor solution(3), and add water up to the marked line(4).
e) Blank test solution for the calibration curve preparation (2): Transfer about 50 mL ofinterference suppressor solution used in the procedure in d) to a 500-mL volumetricflask (3), and add water up to the marked line (4).
Note (1) 29 g of lanthanum oxide (atomic absorption analysis grade or equivalents) can
also be used.
(2) This is an example of preparation; prepare an amount as appropriate.
(3) Add an interference suppressor solution that is 1/10 volume of the volume to beprepared.
(4) For storage, use a sealable container made of materials which are not like todissolve calcium, such as borosilicate glass-1 specified in JIS R 3503 or Teflon.
Comment 1 Instead of a calcium standard solution (CaO 1 mg/mL) in (2) c), a calcium
standard solution (Ca 1 mg/mL) for atomic absorption traceable to a specified
reference material (national measurement standard) according to Article 134of the Measurement Act can also be used. Additionally, it is recommended tomultiply by the conversion factor to use as the calcium standard solution(CaO 1.3992 mg/mL).
3)
Instruments: Instruments are as shown below:a) Rotary shaker: A rotary shaker that can rotate a 500-mL volumetric flask upside down
at 30 - 40 revolutions/min.
b) Flame atomic absorption spectrometer: An atomic absorption spectrometer specified
in JIS K 0121.
1) Light source: A calcium hollow cathode lamp
1702) Gas: Gas for heating by flame(i) Fuel gas: acetylene(ii) Auxiliary gas: Air sufficiently free of dust and moisture.
(4) Test procedures(4.1) Extraction: Conduct extraction as follows:a) Weigh 1.00 g of an analytical sample, and put it in a 500-mL volumetric flask.
b) Add about 400 mL of water, and shake to mix at 30 - 40 revolutions/min for about 30
minutes.
c) Add water up to the marked line.
d) Filter with Type 3 filter paper to make the sample solution.
(4.2) Measurement: Conduct measurement according to JIS K 0121 and as shown below.
Specific measurement procedures are according to the operation method of the atomic
absorption spectrometer used in measurement.
a) Measurement conditions for the atomic absorption spectrometer: Set up themeasurement conditions for the atomic absorption spectrometer considering thefollowing:
Analytical line wavelength: 422.7 nmb) Calibration curve preparation
1) Spray the calcium standard solutions for the calibration curve preparation and theblank test solution for the calibration curve preparation into a flame, and read theindicated value at a wavelength of 422.7 nm.
2) Prepare a curve for the relationship between the calcium concentration and theindicated value of the calcium standard solutions for the calibration curve
preparation and the blank test solution for the calibration curve preparation.
c) Sample measurement
1) Transfer a predetermined amount of the sample solution (the equivalents of 0.5 mg- 5 mg as CaO) to a 100-mL volumetric flask.
2) Add about 10 mL of interference suppressor solution (3), and add water up to themarked line.
3) Conduct similarly as in b) 1) to read the indicated value.
4) Obtain the calcium content from the calibration curve, and calculate water-soluble
calcium (W-Ca) in the analytical sample by the following formula.
Water-soluble calcium (W-Ca) (% (mass fraction)) in an analytical sample= A × 0.7147
A: Content volume of W-CaO (% (mass fraction))

171

(5)

Flow sheet for water-soluble calcium: The flow sheet for water-soluble calcium in
fertilizers is shown below:
1.00 g analytical sample
500-mL volumetric flask← About 400 mL of water
Shaking to mix
Rotary shaker (30 - 40 revolutions/min), for 30 minutes← Water (up to the marked line)
Filtration
Type 3 filter paper
Aliquot
100-mL volumetric flask(predetermined amount)← About 10 mL interference suppressor solution← Water (up to the marked line)
Measurement
Atomic absorption spectrometer (422.7 nm)
Figure Flow sheet for water-soluble calcium in fertilizers.

1724.5.4 Alkalinity
4.5.4.a Ethylenediamine tetraacetate method(1) Summary
This test method is applicable to fertilizers that guarantee alkalinity.
Add hydrochloric acid (1+23) to an analytical sample, boil to extract, and mask with
2,2’,2”-nitrilotriethanol and a potassium cyanide solution, add a 0.01 mol/L ethylenediamine
tetraacetate standard solution, and titrate with a 0.01 mol/L magnesium standard solution to
obtain alkalinity (AL). Or after masking, titrate with an ethylenediamine tetraacetate standard
solution to obtain alkalinity (AL).
(2) Reagents: Reagents are as shown below:a) Hydrochloric acid: A JIS Guaranteed Reagent specified in JIS K 8180 or a reagent ofequivalent quality.
b) Sodium hydroxide: A JIS Guaranteed Reagent specified in JIS K 8576 or a reagent ofequivalent quality.
c) Ascorbic acid: A JIS Guaranteed Reagent specified in JIS K 9502 or a reagent ofequivalent quality.
d) 2,2’,2”-nitrilotriethanol (1): A JIS Guaranteed Reagent specified in JIS K 8663 or areagent of equivalent quality.
e) Acetone: A JIS Guaranteed Reagent specified in JIS K 8034 or a reagent of equivalentquality.
f) Ammonia solution: A JIS Guaranteed Reagent (NH3 28 % (mass fraction)) specified in
JIS K 8085 or a reagent of equivalent quality.
g) 0.01 mol/L ethylenediamine tetraacetate standard solution: Dissolve 3.72 g ofethylenediaminetetraacetic acid disodium dihydrate in water to make 1,000 mL.
Standardization: Wash zinc reference material for volumetric analysis specified in JIS
K 8005 with hydrochloric acid (1+3), water, ethanol (99.5) specified in JIS K 8101, anddiethyl ethel specified in JIS K 8103 successively, and immediately leave at rest in a
desiccator for about 12 hours under no more than 2 kPa to dry, and then weigh about
0.65 mg to the order of 0.1 mg, transfer it to a 1,000-mL volumetric flask, add about 10
mL of hydrochloric acid to dissolve and then add water up to the marked line. Transfer
25 mL of the solution to a 200-mL - 300-mL Erlenmeyer flask, add about 15 mL ofwater and about 5 mL of ammonium chloride buffer solution, and titrate with 0.01
mol/L ethylenediamine tetraacetate standard solution, while adding Erio-chrome BlackT solution as an indicator, until the color of the solution becomes blue. Calculate thefactor of 0.01 mol/L ethylenediamine tetraacetate standard solution by the following
formula.
Factor of 0.01 mol/L ethylenediamine tetraacetate standard solution (f1)
= W × (A/100) × (1/65.409) × (V1/V2) × (1,000/V3) × (1/C1)= W × A × (1/65.409) × (0.25/V3)
W: Mass (g) of d zinc sampled
A: Purity (% (mass fraction)) of zinc
V1: Volume (mL) of zinc solution transferred
V2: Constant volume (25 mL) of zinc solution
V3: Volume (mL) of 0.01 mol/L ethylenediamine tetraacetate standard solution
needed for titration
C1: Set concentration (0.01 mol/L) of 0.01 mol/L ethylenediamine tetraacetatestandard solution
173h) 0.01 mol/L magnesium standard solution: Put 0.24 g of magnesium specified in JIS K
8875 into a 1,000-mL beaker, add about 10 mL of hydrochloric acid to dissolve, add aproper amount of water, and while adding methyl red solution (0.1 g/100 mL) as anindicator, neutralize with an ammonia solution (1+3) until the color of the solution
becomes achromatic, and then add water to make 1,000 mL.
Standardization: Transfer 25 mL of 0.01 mol/L magnesium standard solution to a
200-mL - 300-mL Erlenmeyer flask, add about 15 mL of water and about 5 mL ofammonium chloride buffer solution, and while adding an Erio-chrome Black T solution
as an indicator, titrate with 0.01 mol/L ethylenediamine tetraacetate standard solution
until the color of solution becomes blue. Calculate the factor of 0.01 mol/L magnesium
standard solution by the following formula.
Factor of 0.01 mol/L magnesium standard solution (f1)
= (C1 × f1 ×V4) × (1/V5) × (1/C2)= (f1 × V4) × (1/V5)
C1: Set concentration (0.01 mol/L) of 0.01 mol/L ethylenediamine tetraacetatestandard solution
C2: Set concentration (0.01 mol/L) of 0.01 mol/L magnesium standard solution
f1: Factor of 0.01 mol/L ethylenediamine tetraacetate standard solution
V4: Volume (mL) of 0.01 mol/L ethylenediamine tetraacetate standard solution
needed for titration
V5: Volume (mL) of 0.01 mol/L magnesium standard solution transferred.
i) Ammonium chloride solution: Dissolve 70 g of ammonium chloride specified in JIS K
8116 and 570 mL of ammonia solution in water to make 1,000 mL.
j) 2-aminoethanol solution: Add 400 mL of water to 150 mL of 2-aminoethanol specified
in JIS K 8109, add gradually hydrochloric acid to a pH 10.6.
k) Potassium cyanide solution: Dissolve 100 g of potassium cyanide specified in JIS K
8443 in water to make 1,000 mL.
l) Erio-chrome Black T solution: Dissolve 0.5 g of Erio-chrome Black T specified in JIS
K 8736 and 4.5 g of hydroxyl-ammonium chloride in methanol - water (95+5) to make100 mL.
m) Methyl red solution (0.1 g/100 mL): Dissolve 0.10 g of methyl red specified in JIS K
8896 in 100 mL of ethanol (95) specified in JIS K 8102.
n) Methanol: A JIS Guaranteed Reagent specified in JIS K 8891 or a reagent of equivalentquality.
Note (1) The reagent corresponds to triethanolamine in the Official Methods of Analysis
of Fertilizers (1992).
3)
Instruments: Instruments are as shown below:a) Hot plate: A hot plate whose surface temperature can be adjusted up to 250 ºC.
(4) Test procedures(4.1) Extraction: Conduct extraction as follows:a) Weigh about 2 g of an analytical sample to the order of 1 mg, and put it in a 500-mL tallbeaker.
b) Add about 200 mL of hydrochloric acid (1+23), cover with a watch glass, and boil on a
hot plate for about 5 minutes (2).
174c) After standing to cool, move it to a 250-mL - 500-mL volumetric flask with water.
d) Add water up to the marked line.
e) Filter with Type 3 filter paper to make the sample solution.
Note (2) Be aware that an analytical sample should not solidify in the bottom of a beaker.
Comment 1

In the case of a by-product magnesia fertilizer or a fertilizer containing a
by-product magnesia, if the pH of the sample solution of d) is neutral or basic,
prepare a sample solution anew by replacing “2 g of an analytical sample” inthe procedure in a) with “1 g - 1.5 g of an analytical sample”.
Comment 2 In the procedure in a), a 500-mL volumetric flask can be used instead of a
500-mL tall beaker. However the volumetric flask used should be
distinguished as an extraction flask and should not be used for the otherpurposes. In addition, “cover with a watch glass” in b) is replaced by “place along-stem funnel”, and “transfer to a 250-mL - 500-mL volumetric flask withwater” in the procedure in c) is skipped.
Comment 3 The procedure in (4.1) is the same as in (4.1) in 4.5.2.a.
(4.2) Measurement: Conduct measurement as shown below. Two examples of titration are
shown as follows.
(4.2.1) Measurement (A): Titration with a magnesium standard solution (0.01 mol/L)a) Transfer a predetermined volume (the equivalents of 5 mg - 20 mg as CaO + MgO) of
sample solution to a 200-mL - 300-mL Erlenmeyer flask.
b) Add a proper amount of water, add a drop of methyl red solution as an indicator anddrop sodium hydroxide (5 g/100 mL) to neutralize until the color of the solution
becomes yellow.
c) Add 0.1 g of ascorbic acid, 1 ml - 10 mL of 2,2’,2”-nitrilotriethanol - water (1+3) and 1
mL - 10 mL of potassium cyanide solution.
d) Add a predetermined volume of 0.01 mol/L ethylenediamine tetraacetate (3).
e) Add 20 mL of ammonium chloride solution or 2-aminoethanol solution.
f) Add several drops of Erio-chrome Black T solution, and titrate with 0.01 mol/L
magnesium standard solution until the color of solution becomes red.
g) Calculate the alkalinity (AL) content in an analytical sample by the following formula.
Alkalinity (AL) (% (mass fraction)) in an analytical sample= ((C1 × f1 × V6/1,000) – (C2 × f2 × V7/1,000)) × 56.077 × (V8/V9) × (1/W) × 100= ((f1 × V1) – (f2 × V2)) × 56.077 × (V3/V4) × (1/2,000)
C1: Set concentration (0.01 mol/L) of 0.01 mol/L ethylenediamine tetraacetatestandard solution
C2: Set concentration (0.01 mol/L) of 0.01 mol/L magnesium standard solution
f 1: Factor of 0.01 mol/L ethylenediamine tetraacetate standard solution
f 2: Factor of 0.01 mol/L magnesium standard solution
V6: Additive volume (mL) of 0.01 mol/L ethylenediamine tetraacetate standard
solution
V7: Volume (mL) of 0.01 mol/L magnesium standard solution needed for titration
V8: Predetermined volume (mL) of the sample solution in (4.1) d)
V9: Transferred volume (mL) of the sample solution subjected to titration in (4.2)a).
W: Mass (2 g) of sample a solution
175

Note (3) Add excess volume since 1.8 mL of ethylenediamine tetraacetate standard
solution (0.01 mol/L) is required for 1 mg of CaO.
(4.2.2) Measurement (B): Titration with an ethylenediamine tetraacetate standard solution(0.01 mol/L)a) Transfer a predetermined volume (the equivalents of 5 mg - 20 mg as CaO + MgO) of
sample solution to a 200-mL - 300-mL Erlenmeyer flask.
b) Add a proper amount of water, add a drop of methyl red solution as an indicator anddrop sodium hydroxide (5 g/100 mL) to neutralize until the color of the solution
becomes yellow.
c) Add 0.1 g of ascorbic acid, 1 ml - 10 mL of 2,2’,2”-nitrilotriethanol - water (1+3) and 1
mL - 10 mL of potassium cyanide solution.
d) Add 20 mL of ammonium chloride solution or 2-aminoethanol solution.
e) Add several drops of an Erio-chrome Black T solution, and titrate with 0.01 mol/L
ethylenediamine tetraacetate standard solution until the color of solution becomesblue-green.
f) Calculate the alkalinity (AL) in an analytical sample by the following formula.
Alkalinity (AL) (% (mass fraction)) in an analytical sample= (C1 × f1 × V10/1,000) × 56.077 × (V11/V12) × (1/W) × 100= (f1 × V10) × 56.077 × (V11/V12) × (1/2,000)
C1: Set concentration (0.01 mol/L) of 0.01 mol/L ethylenediamine tetraacetatestandard solution
f1: Factor of 0.01 mol/L ethylenediamine tetraacetate standard solution
V10: Volume (mL) of 0.01 mol/L ethylenediamine tetraacetate standard solution
needed for titration
V11: Predetermined volume (mL) of the sample solution in (4.1) d)
V12: Transferred volume (mL) of the sample solution subjected to titration in (4.2)a)
W: Mass (2 g) of a sample solution
Comment4

Care should be fully taken in the case of using potassium cyanide and its
solution in accordance with the Safety Data Sheet (SDS). In addition, observe
laws and ordinances concerned such as the Poisonous and Deleterious
Substance Control Law.
Criteria of the abolition in the Poisonous and Deleterious Substance Control
Law (for reference):
Add sodium hydroxide solution to make it alkalinity more than pH 11, andfurther add oxidizer (sodium hypochlorite, bleaching powder) solution to
conduct oxidative degradation processes. After dissolving CN ingredient,
neutralize with sulfuric acid, and discard it after diluting it with a large
amount of water. Take enough time to dissolve the CN ingredient withalkalinity.

Comment 5 Table 1 shows results from a simultaneous analysis with the same samples(proficiency testing and external quality control testing) hosted by the Japan
Fertilizer Quality Assurance Committee analyzed by the robust method.

176

Table 1 Results and analysis results from simultaneous analysis with the same samples
for alkalinity hosted by the Japan Fertilizer Quality Assurance Committee
2)

Year
2008
2009
2010
2011
2012

1)
2)
3)
4)
5)

SampleSlag silicate fertilizer
Slag silicate fertilizer
Slag silicate fertilizer
Slag silicate fertilizer
Slag silicate fertilizer

Number of Median Μ
5)laboratories
(%)
19
51.06
21
39.69
20
49.16
16
49.44
17
49.76

3)

NIQR

RSDrob

5)

(%)
0.8
3.2
1.3
1.2
0.7

(%)
0.42
1.25
0.65
0.59
0.35

1)

4)

Proficiency testing and external quality control testingMedian (M) agrees with the mean in normal distribution.
Mass fraction
Robust standard deviation (NIQR) agrees with standard deviation in normal distribution.
RSDrob stands for relative standard deviation obtained from the robust methodby the formula shown below:
RSDrob = (NIQR/M) × 100

Reference
1) Masayoshi KOSHINO: Second Revision of The Methods of Analysis of Fertilizers(Details), p.162 - 164, Yokendo, Tokyo (1988)(5)

Flow sheet for alkalinity: The flow sheets for alkalinity in fertilizers are shown below:
2 g analytical sample
Weigh to the order of 1 mg to a 500-mL tall beaker← About 200 mL of hydrochloric acid (1+23)
Boiling
Cover with a watch glass for 5 minutes
Cooling
Transfer
250-mL - 500-mL volumetric flask← Water (up to the marked line)
Filtration
Type 3 filter paper
Sample solution
Aliquot
200-mL - 300-mL Erlenmeyer Flask(predetermined amount)← Water (proper amount)← A drop of methyl-red solution
Sodium hydroxide (5 g/100 mL)
Neutralization(until the solution becomes yellow )← 0.1 g ascorbic acid← 1 mL - 10 mL of 2,2’,2”-nitrilotriethanol - solution (1+3)← 1 mL - 10 mL potassium cyanide solution← Predetermined volume of 0.01 mol/L ethylenediamine tetraacetate standard solution← 20 mL of ammonium chloride solution or 2-aminoethanol solution← Several drops of Erio-chrome Black T solution
0.01 mol/L magnesium standard solution
Titration(until the solution becomes red)
Figure Flow sheet for alkalinity in fertilizers (A).
177

2 g analytical sample
Weigh to the order of 1 mg into a 500-mL tall beaker← About 200 mL of hydrochloric acid (1+23)
Boiling
Cover with a watch glass for 5 minutes
CoolingType 3 filter paper

Transfer
250-mL - 500-mL volumetric flask← Water (up to the marked line)
Filtration
Type 3 filter paper
Sample solution
Aliquot
200-mL - 300-mL Erlenmeyer Flask(predetermined amount)← Water (proper amount)← A drop of methyl-red solution
Sodium hydroxide (5 g/100 mL)
Neutralization(until the solution becomes yellow )← 0.1 g of ascorbic acid← 1 mL - 10 mL of 2,2’,2”-nitrilotriethanol - solution (1+3)← 1 mL - 10 mL of potassium cyanide solution← 20 mL of ammonium chloride solution or 2-aminoethanol solution← Several drops of Erio-chrome Black T solution
0.01 mol/L ethylenediamine tetraacetate standard solution
Titration(until the solution becomes blue-green)
Figure Flow sheet for alkalinity in fertilizers (B).

178

4.5.4.b Calculation from soluble lime and soluble magnesia(1) Summary
This test method is applicable to fertilizers that guarantee alkalinity (AL).
Multiply the soluble magnesia (S-MgO) obtained in 4.6.1 by the factor (1.3934) and add tothe soluble lime (S-CaO) obtained in 4.5.2 to calculate alkalinity (AL).
(2) Calculation of alkalinitya) Calculate the alkalinity (AL) in a teat sample by the following formula.
Alkalinity (AL) (% (mass fraction)) in a test sample= (S-CaO) + 1.3914 × (S-MgO)
S-CaO: Soluble lime (% (mass fraction)) (1) obtained in 4.5.2 in an analytical sample
S-MgO: Soluble magnesia (% (mass fraction)) (1) obtained in 4.6.1 in an analyticalsampleNote (1) S-CaO and S-MgO use raw data without rounding numerical value
Comment 1 Table 1 shows results from a simultaneous analysis with the same samples(proficiency testing and external quality control testing) hosted by the Japan
Fertilizer Quality Assurance Committee analyzed by the robust method.
Table 1 Results and analysis results from simultaneous analysis with the same samples
for alkalinity hosted by the Japan Fertilizer Quality Assurance Committee1)
Year

SampleNumber of Median Μ2)
3)laboratories(%)

NIQR4)
3)

(%)
0.75
0.90
0.89
0.62
0.72
0.86

RSDrob5)(%)

2008 Slag silicate fertilizer
62
50.81
1.5
2009 Slag silicate fertilizer
59
38.82
2.3
2010 Slag silicate fertilizer
62
49.27
1.8
2011 Slag silicate fertilizer
56
49.48
1.3
2012 Slag silicate fertilizer
56
50.07
1.4
2013 Slag silicate fertilizer
59
36.56
2.4
1) Proficiency testing and external quality control testing2) Median (M) agrees with the mean in normal distribution.
3) Mass fraction4) Robust standard deviation (NIQR) agrees with standard deviation in normal distribution.
5) RSDrob stands for relative standard deviation obtained from the robust methodby the formula shown below:
RSDrob = (NIQR/M) × 100

179

4.6

Magnesia

4.6.1

Soluble magnesia

4.6.1.a Flame atomic absorption spectrometry(1) Summary
This test method is applicable to fertilizers containing by-product magnesia fertilizers andfertilizers that guarantee alkalinity.
Add hydrochloric acid (1+23) to an analytical sample, boil to extract and add an interference
suppressor solution, and then spray in an acetylene-air flame, and measure the atomic
absorption with magnesium at a wavelength of 285.2 nm to quantify hydrochloric acid (1+23)
soluble magnesia (soluble magnesia (S-MgO)).
(2) Reagents: Reagents are as shown below:a) Hydrochloric acid: A JIS Guaranteed Reagent specified in JIS K 8180 or a reagent ofequivalent quality.
b) Interference suppressor solution (1): Weigh 60.9 g - 152.1g of strontium chloride
hexahydrate (2) specified in JIS K 8132 into a 2,000-mL beaker, add a small amount ofwater, add gradually 420 mL of hydrochloric acid to dissolve, and further add water tomake 1,000 mL.
c) Magnesium standard solution (MgO 1 mg/mL) (1): Weigh 0.603 g of guaranteed
magnesium (powder) specified in JIS K 8876 into a weighing dish. Transfer to a
1,000-mL volumetric flask with a small amount of water, add about 10 mL ofhydrochloric acid to dissolve, and add water up to the marked line.
d) Magnesium standard solution (MgO 0.1 mg/mL): Transfer 10 mL of magnesium
standard solution (MgO 1 mg/mL) to a 100-mL volumetric flask and add water up to themarked line.
e) Magnesium standard solution (MgO 1 µg/mL-10 µg/mL) for the calibration curve
preparation (1): Transfer 2.5 mL-25 mL of magnesium standard solution (MgO 0.1
mg/mL) to 250-mL volumetric flasks step-by-step, add about 25 mL of interference
suppressor solution (3), and add water up to the marked line.
f) Blank test solution for the calibration curve preparation (1): Transfer about 25 mL ofinterference suppressor solution used in the procedure e) to a 250-mL volumetric flask(3), and add water up to the marked line.
Note (1) This is an example of preparation; prepare an amount as appropriate.
(2) 29 g of lanthanum oxide (atomic absorption analysis grade or equivalents) can
also be used.
(3) Add an interference suppressor solution that is 1/10 volume of the volume to beprepared.
Comment 1 Instead of a magnesium standard solution (MgO 1 mg/mL) in (2) c), a
magnesium standard solution (Mg 1 mg/mL) for atomic absorption traceable
to a specified reference material (national measurement standard) according
to Article 134 of the Measurement Act can also be used. Additionally, it isrecommended to multiply by the conversion factor to use as the magnesium
standard solution (MgO 1.658 mg/mL).
3)
Instruments: Instruments are as shown below:a) Flame atomic absorption spectrometer: An atomic absorption spectrometer specified
in JIS K 0121.
1) Light source: A magnesium hollow cathode lamp1802) Gas: Gas for heating by flame(i) Fuel gas: acetylene(ii) Auxiliary gas: Air sufficiently free of dust and moisture.
b) Hot plate: A hot plate whose surface temperature can be adjusted up to 250 ºC.
(4) Test procedures(4.1) Extraction: Conduct extraction as follows:a) Weigh about 2 g of an analytical sample to the order of 1 mg, and put it in a 500-mL tallbeaker.
b) Add about 200 mL of hydrochloric acid (1+23), cover with a watch glass, and boil on a
hot plate for about 5 minutes (4).
c) After standing to cool, transfer to a 250-mL - 500-mL volumetric flask with water.
d) Add water up to the marked line.
e) Filter with Type 3 filter paper to make the sample solution.
Note (4) Be aware that an analytical sample should not solidify in the bottom of a beaker.
Comment 2

In the case of a by-product magnesia fertilizer or a fertilizer containing a
by-product magnesia, if the pH of the sample solution of d) is neutral or basic,
prepare a sample solution anew by replacing “2 g of an analytical sample” inthe procedure in a) with “1 g - 1.5 g of an analytical sample”.
Comment 3 In the procedure in a), a 500-mL volumetric flask can be used instead of a
500-mL tall beaker. However the volumetric flask used should be
distinguished as an extraction flask and should not be used for the otherpurposes. In addition, “cover with a watch glass” in b) is replaced by “place along-stem funnel”, and “transfer to a 250-mL - 500 mL volumetric flask withwater” in the procedure in c) is skipped.
Comment 4 The procedure in (4.1) is the same as in (4.1) in 4.5.2.a.
(4.2) Measurement: Conduct measurement according to JIS K 0121 and as shown below.
Specific measurement procedures are according to the operation method of the atomic
absorption spectrometer used in measurement.
a) Measurement conditions for the atomic absorption spectrometer: Set up themeasurement conditions for the atomic absorption spectrometer considering thefollowing:
Analytical line wavelength: 285.2 nmb) Calibration curve preparation
1) Spray the magnesium standard solutions for the calibration curve preparation andthe blank test solution for the calibration curve preparation into a flame, and readthe indicated value at a wavelength of 285.2 nm.
2) Prepare a curve for the relationship between the magnesium concentration and theindicated value of the magnesium standard solutions for the calibration curve
preparation and the blank test solution for the calibration curve preparation.
c) Sample measurement
1) Transfer a predetermined amount of the sample solution (the equivalents of 0.1 mg- 1 mg as MgO) to a 100-mL volumetric flask.
2) Add about 10 mL of interference suppressor solution (3), and add water up to themarked line.
3) Conduct similarly as in b) 1) to read the indicated value.
4) Obtain the magnesium content from the calibration curve, and calculate the soluble
magnesia (S-MgO) in the analytical sample.
181

Comment 5 Table 1 shows results from a simultaneous analysis with the same samples(proficiency testing and external quality control testing) hosted by the Japan
Fertilizer Quality Assurance Committee analyzed by the robust method.
Table 1 Results and analysis results from simultaneous analysis with the same samples
for soluble magnesia hosted by the Japan Fertilizer Quality Assurance Committee
2)

Year

SampleNumber of Median Μ
3)laboratories
(%)

4)

5)

NIQR

RSDrob

3)

(%)

(%)
0.10
0.08
0.12
0.14
0.15

1)

2009 Slag silicate fertilizer
58
4.48
2.3
2010 Slag silicate fertilizer
60
5.47
1.4
2011 Slag silicate fertilizer
55
6.24
1.9
2012 Slag silicate fertilizer
56
6.86
2.0
2013 Slag silicate fertilizer
59
4.62
3.2
1) Proficiency testing and external quality control testing2) Median (M) agrees with the mean in normal distribution.
3) Mass fraction4) Robust standard deviation (NIQR) agrees with standard deviation in normal distribution.
5) RSDrob stands for relative standard deviation obtained from the robust methodby the formula shown below:
RSDrob = (NIQR/M) × 100

Reference
1) Masayoshi KOSHINO: Second Revision of The Methods of Analysis of Fertilizers(Details), p.167 - 169, Yokendo, Tokyo (1988)(5)

Flow sheet for soluble magnesia: The flow sheet for soluble magnesia in fertilizers isshown below:
2 g analytical sample
Weigh to the order of 1 mg into a 500-mL tall beaker.
← About 200 mL of hydrochloric acid (1+23)
Heating
Cover with a watch glass, and boil for 5 minutes.
Standing to coolTransfer
Water, 250-mL - 500-mL volumetric flask← Water (up to the marked line)
Filtration
Type 3 filter paper
Aliquot
100-mL volumetric flask(predetermined amount)← About 10 mL of interference suppressor solution← Water (up to the marked line)
Measurement
Atomic absorption spectrometer
Figure Flow sheet for soluble magnesia in fertilizers.

1824.6.2

Citrate soluble magnesia

4.6.2.a Flame atomic absorption spectrometry(1) Summary
This test method is applicable to fertilizers containing magnesia hydroxide fertilizers, etc.
Extract by adding a citric acid solution to an analytical sample and add an interference
suppressor solution, and then spray in an acetylene-air flame, and measure the atomic
absorption with magnesium at a wavelength of 285.2 nm to quantify citrate soluble magnesia(C-MgO).
(2) Reagents: Reagents are as shown below:a) Hydrochloric acid: A JIS Guaranteed Reagent specified in JIS K 8180 or a reagent ofequivalent quality.
b) Citric acid solution (1): Dissolve 20 g of citric acid monohydrate specified in JIS K
8283 in water to make 1,000 mL.
c) Interference suppressor solution (1): Weigh 60.9 g - 152.1 g of strontium chloride
hexahydrate (2) specified in JIS K 8132 into a 2,000-mL beaker, add a small amount ofwater, add gradually 420 mL of hydrochloric acid to dissolve, and further add water tomake 1,000 mL.
d) Magnesium standard solution (MgO 1 mg/mL) (1): Weigh 0.603 g of the guaranteed
magnesium (powder) specified in JIS K 8876 into a weighing dish. Transfer to a
1,000-mL volumetric flask with a small amount of water, add about 10 mL ofhydrochloric acid to dissolve, and add water up to the marked line.
e) Magnesium standard solution (MgO 0.1 mg/mL): Transfer 10 mL of magnesium
standard solution (MgO 1 mg/mL) to a 100-mL volumetric flask and add water up to themarked line.
f) Magnesium standard solution (MgO 1 µg/mL - 10 µg/mL) for the calibration curve
preparation (1): Transfer 2.5 mL - 25 mL of magnesium standard solution (MgO 0.1
mg/mL) to 250-mL volumetric flasks step-by-step, add about 25 mL of interference
suppressor solution (3), and add water up to the marked line.
g) Blank test solution for the calibration curve preparation (1): Transfer about 25 mL ofinterference suppressor solution used in the procedure in f) to a 250-mL volumetricflask (3), and add water up to the marked line.
Note (1) This is an example of preparation; prepare an amount as appropriate.
(2) 29 g of lanthanum oxide (atomic absorption analysis grade or equivalents) can
also be used.
(3) Add an interference suppressor solution that is 1/10 volume of the volume to beprepared.
Comment 1 Instead of a magnesium standard solution (MgO 0.1 mg/mL) in (2) e), a
magnesium standard solution (Mg 0.1 mg/mL) for atomic absorption
traceable to a specified reference material (national measurement standard)
according to Article 134 of the Measurement Act can also be used.
Additionally, it is recommended to multiply by the conversion factor to use asa magnesium standard solution (MgO 0.1658 mg/mL).
(3) Instruments: Instruments are as follows:a) Constant-temperature rotary shaker: A constant-temperature rotary shaker that canrotate a 250-mL volumetric flask, set up in a thermostat adjustable to 30 ºC±1 ºC, upside
down at 30 - 40 revolutions/min.
183b) Flame atomic absorption spectrometer: An atomic absorption spectrometer specified
in JIS K 0121.
1) Light source: A magnesium hollow cathode lamp
2) Gas: Gas for heating by flame(i) Fuel gas: acetylene(ii) Auxiliary gas: Air sufficiently free of dust and moisture.
(4) Test procedures(4.1) Extraction: Conduct extraction as shown below.
a) Weigh 1 g of an analytical sample to the order of 1 mg, and transfer to a 250-mL
volumetric flask.
b) Add 150 mL of citric acid solution heated up to about 30 ºC, and shake to mix at 30 - 40
revolutions/min (30 ºC ± 1 ºC) for 1 hour.
c) After standing to cool, add water up to the marked line.
d) Filter with Type 3 filter paper to make the sample solution.
Comment 2

In the case of a by-product magnesia fertilizer or a fertilizer containing a
by-product magnesia, if the pH of the sample solution of d) is neutral or basic,
prepare a sample solution anew by replacing “1 g of an analytical sample” inthe procedure in a) with “0.5 g of an analytical sample”.
Comment 3 In some slag silicate fertilizers, the variation of measurement value of citratemagnesia (C-MgO) may be observed according to the time variation ofheating state after adding citric acid solution. Therefore, in the case of slag
silicate fertilizers, it is necessary to conduct the procedures of c) – d) as
quickly as possible after confirming the time of shaking to mix in theprocedure in b).
Comment 4 The procedure in (4.1) is the same as the procedure in (4.1) in 4.2.3.a.
(4.2) Measurement: Conduct measurement according to JIS K 0121 and as shown below.
Specific measurement procedures are according to the operation method of the atomic
absorption spectrometer used in measurement.
a) Measurement conditions for the atomic absorption spectrometer: Set up themeasurement conditions for the atomic absorption spectrometer considering thefollowing:
Analytical line wavelength: 285.2 nmb) Calibration curve preparation
1) Spray the magnesium standard solutions for the calibration curve preparation andthe blank test solution for the calibration curve preparation into a flame, and readthe indicated value at a wavelength of 285.2 nm.
2) Prepare a curve for the relationship between the magnesium concentration and theindicated value of the magnesium standard solutions for the calibration curve
preparation and the blank test solution for the calibration curve preparation.
c) Sample measurement
1) Transfer a predetermined amount of the sample solution (the equivalents of 0.1 mg- 1 mg as MgO) to a 100-mL volumetric flask.
2) Add about 10 mL of interference suppressor solution (3), and add water up to themarked line.
3) Conduct similarly as in b) 1) to read the indicated value.
4) Obtain the magnesium content from the calibration curve, and calculate the citratesoluble magnesia (C-MgO) in the analytical sample.
184

Comment 5 Table 1 shows results from a simultaneous analysis with the same samples(proficiency testing and external quality control testing) hosted by the Japan
Fertilizer Quality Assurance Committee analyzed by the robust method.
Table 1 Results and analysis results from simultaneous analysis with the same samples
for soluble magnesia hosted by the Japan Fertilizer Quality Assurance Committee1)
5)
2)
4)RSDrob
Number of Median Μ
NIQR
Year
Samplelaboratories
(%)
(%)3)
(%)3)
2006
2007
2008
2009

Slag silicate fertilizer
Organic compound fertilizer
Slag silicate fertilizer
Organic compound fertilizer
Slag silicate fertilizer
Slag silicate fertilizer

78
137
86
127
76
75

6.18
3.41
3.13
4.62
5.83
2.31

0.13
0.07
0.21
0.11
0.16
0.17

2.0
2.2
6.6
2.5
2.8
7.5

2010

High analysis compound fertilizer
123
3.11
0.07
2.2
Slag silicate fertilizer
76
5.42
0.12
2.1
2011 High analysis compound fertilizer
116
2.48
0.06
2.4
Slag silicate fertilizer
69
3.33
0.20
6.0
2012 Slag silicate fertilizer
69
6.59
0.18
2.7
2013 High analysis compound fertilizer
116
6.17
0.13
2.3
Slag silicate fertilizer
68
3.75
0.11
3.1
1) Proficiency testing and external quality control testing2) Median (M) agrees with the mean in normal distribution.
3) Mass fraction4) Robust standard deviation (NIQR) agrees with standard deviation in normal distribution.
5) RSDrob stands for relative standard deviation obtained from the robust methodby the formula shown below:
RSDrob = (NIQR/M) × 100

Reference
1) Masayoshi KOSHINO: Second Revision of The Methods of Analysis of Fertilizers(Details), p.167 - 169, Yokendo, Tokyo (1988)

185

(5)

Flow sheet for citrate soluble magnesia: The flow sheet for citrate soluble magnesia
in fertilizers is shown below:
1 g analytical sample

Weigh to the order of 1 mg into a 250-mL tall beaker.

← About 150 mL of citrate solution [about 30 ºC]
Rotary shaker (30 - 40 revolutions/min),Shaking to mix
30 ºC ± 1 ºC, for 1 hourStanding to cool
← Water (up to the marked line)
Filtration
Type 3 filter paper
Aliquot
100-mL volumetric flask(predetermined amount)← About 10 mL of interference suppressor solution← Water (up to the marked line)
Measurement
Atomic absorption spectrometer
Figure Flow sheet for citrate soluble magnesia in fertilizers.

1864.6.3

Water-soluble magnesia

4.6.3.a Flame atomic absorption spectrometry(1) Summary
This test method is applicable to fertilizers containing magnesia sulfate fertilizers, etc.
Add water to an analytical sample and boil to extract. Add an interference suppressor solution,then spray in an acetylene-air flame and measure the atomic absorption with magnesium at awavelength of 285.2 nm to quantify water-soluble magnesia (W-MgO).
(2) Reagents: Reagents are as shown below:a) Hydrochloric acid: A JIS Guaranteed Reagent specified in JIS K 8180 or a reagent ofequivalent quality.
b) Interference suppressor solution (1): Weigh 60.9 g - 152.1 g of strontium chloride
hexahydrate (2) specified in JIS K 8132 into a 2,000-mL beaker, add a small amount ofwater, gradually add 420 mL of hydrochloric acid to dissolve, and further add water tomake 1,000 mL.
c) Magnesium standard solution (MgO 1 mg/mL) (1): Weigh 0.603 g of guaranteed
magnesium (powder) specified in JIS K 8876 into a weighing dish. Transfer to a
1,000-mL volumetric flask with a small amount of water, add about 10 mL ofhydrochloric acid to dissolve, and add water up to the marked line.
d) Magnesium standard solution (MgO 1 mg/mL): Transfer 10 mL of magnesium
standard solution (MgO 1 mg/mL) to a 100-mL volumetric flask and add water up to themarked line.
e) Magnesium standard solution (MgO 1 µg/mL-10 µg/mL) for the calibration curve
preparation (1): Transfer 2.5 mL - 25 mL of magnesium standard solution (MgO 0.1
mg/mL) to 250-mL volumetric flasks step-by-step, add about 25 mL of interference
suppressor solution (3), and add water up to the marked line.
f) Blank test solution for the calibration curve preparation (1): Transfer about 25 mL ofinterference suppressor solution used in the procedure in e) to a 250-mL volumetricflask (3), and add water up to the marked line.
Note (1) This is an example of preparation; prepare an amount as appropriate.
(2) 29 g of lanthanum oxide (atomic absorption analysis grade or equivalents) can
also be used.
(3) Add an interference suppressor solution that is 1/10 volume of the volume to beprepared.
Comment 1 Instead of a magnesium standard solution (MgO 1 mg/mL) in (2) c), a
magnesium standard solution (Mg 1 mg/mL) for atomic absorption traceable
to a specified reference material (national measurement standard) according
to Article 134 of the Measurement Act can also be used. Additionally, it isrecommended to multiply by the conversion factor to use as a magnesium
standard solution (MgO 1.658 mg/mL).
(3) Apparatus and instruments: Apparatus and instruments are as shown below:a) Flame atomic absorption spectrometer: An atomic absorption spectrometer specified
in JIS K 0121.
1) Light source: A magnesium hollow cathode lamp
2) Gas: Gas for heating by flame(i) Fuel gas: acetylene(ii) Auxiliary gas: Air sufficiently free of dust and moisture.
187b) Extraction flask (4): A 500-mL volumetric flask made of borosilicate glassc) Hot plate: A hot plate whose surface temperature can be adjusted up to 250 ºC.
Note (4) The volumetric flask used for extraction should be distinguished as an extraction
flask and should not be used for the other purposes.
(4) Test procedures(4.1) Extraction: Conduct extraction as follows:a) Weigh 1 g of an analytical sample to the order of 1 mg, and put it in an extraction flask.
b) Add about 400 mL of water, and place a long-stem funnel to boil on a hot plate forabout 30 minutes.
c) After standing to cool, add water up to the marked line.
e) Filter with Type 3 filter paper to make the sample solution.
(4.2) Measurement: Conduct measurement according to JIS K 0121 and as shown below.
Specific measurement procedures are according to the operation method of the atomic
absorption spectrometer used in measurement.
a) Measurement conditions for the atomic absorption spectrometer: Set up themeasurement conditions for the atomic absorption spectrometer considering thefollowing:
Analytical line wavelength: 285.2 nmb) Calibration curve preparation
1) Spray the magnesium standard solutions for the calibration curve preparation andthe blank test solution for the calibration curve preparation into a flame, and readthe indicated value at a wavelength of 285.2 nm.
2) Prepare a curve for the relationship between the magnesium concentration and theindicated value of the magnesium standard solutions for the calibration curve
preparation and the blank test solution for the calibration curve preparation.
c) Sample measurement
1) Transfer a predetermined amount of the sample solution (the equivalents of 0.1 mg- 1 mg as MgO) to a 100-mL volumetric flask.
2) Add about 10 mL of interference suppressor solution (3), and add water up to themarked line.
3) Conduct similarly as in b) 1) to read the indicated value.
4) Obtain the magnesium content from the calibration curve, and calculate thewater-soluble magnesia (W-MgO) in the analytical sample.
Comment 2 Table 1 shows results from a simultaneous analysis with the same samples(proficiency testing and external quality control testing) hosted by the Japan
Fertilizer Quality Assurance Committee analyzed by the robust method.

188

Table 1 Results and analysis results from simultaneous analysis with the same samples
1)
for water-soluble magnesia hosted by the Japan Fertilizer Quality Assurance Committee
Year
2011
2012
2013
1)
2)
3)
4)
5)

SampleHigh analysis
compound fertilizer
Fluid mixed
fertilizer
High analysis
compound fertilizer

Number of Median Μ2)laboratories(%)3)

NIQR4)

RSDrob5)

(%)3)

(%)

110

1.94

0.08

4.1

104

1.69

0.04

2.6

109

3.80

0.63

16.6

Proficiency testing and external quality control testingMedian (M) agrees with the mean in normal distribution.
Mass fraction
Robust standard deviation (NIQR) agrees with standard deviation in normal distribution.
RSDrob stands for relative standard deviation obtained from the robust methodby the formula shown below:
RSDrob = (NIQR/M) × 100

Reference
1) Masayoshi KOSHINO: Second Revision of The Methods of Analysis of Fertilizers(Details), p.167 - 169, Yokendo, Tokyo (1988)(5)

Flow sheet for water-soluble magnesia: The flow sheet for water-soluble magnesia
in fertilizers is shown below:
1g analytical sample
Weigh to the order of 1 mg into an extraction flask.
← About 400 mL of water
Heating
Place a long-stem funnel, boil for about 30 minutes
Standing to cool
← Water (up to the marked line)
Filtration
Type 3 filter paper
Aliquot
100-mL volumetric flask(predetermined←About 10 mL of interference suppressor solution← Water (up to the marked line)
Measurement
Atomic absorption spectrometer
Figure Flow sheet for water-soluble magnesia in fertilizers.

189

4.7

Manganese

4.7.1

Soluble manganese

4.7.1.a Flame atomic absorption spectrometry(1) Summary
This test method is applicable to fertilizers containing manganese carbonate fertilizers.
Add hydrochloric acid (1+23) to an analytical sample, boil to extract and add an interference
suppressor solution, and then spray into an acetylene-air flame, and measure the atomic
absorption with manganese at a wavelength of 279.5 nm to quantify the hydrochloric acid
(1+23) soluble manganese (soluble manganese (S-MnO)).
(2) Reagents: Reagents are as shown below:a) Hydrochloric acid: A JIS Guaranteed Reagent specified in JIS K 8180 or a reagent ofequivalent quality.
b) Interference suppressor solution (1): Weigh 60.9 g - 152.1 g of strontium chloride
hexahydrate (2) specified in JIS K 8132 into a 2,000-mL beaker, add a small amount ofwater, gradually add 420 mL of hydrochloric acid to dissolve, and further add water tomake 1,000 mL.
c) Manganese standard solution (MnO 1 mg/mL) (1): Weigh 0.775 g of manganese
powder (purity no less than 99 % (mass fraction)) into a weighing dish. Transfer to a
1,000-mL volumetric flask with a small amount of water, add about 10 mL ofhydrochloric acid to dissolve, and add water up to the marked line.
d) Manganese standard solution (MnO 0.1 mg/mL): Transfer 10 mL of manganesestandard solution (MnO 1 mg/mL) to a 100-mL volumetric flask and add water up to themarked line.
e) Manganese standard solution (MnO 1 µg/mL - 10 µg/mL) for the calibration curve
preparation (1): Transfer 2.5 mL - 25 mL of manganese standard solution (MnO 0.1
mg/mL) to 250-mL volumetric flasks step-by-step, add about 25 mL of interference
suppressor solution (3), and add water up to the marked line.
f) Blank test solution for the calibration curve preparation (1): Transfer about 25 mL ofinterference suppressor solution used in the procedure in e) to a 250-mL volumetricflask (3), and add water up to the marked line.
Note (1) This is an example of preparation; prepare an amount as appropriate.
(2) 29 g of lanthanum oxide (a reagent of atomic absorption analysis grade orequivalents) can also be used.
(3) Add an interference suppressor solution that is 1/10 volume of the volume to beprepared.
Comment 1 Instead of a manganese standard solution (MnO 0.1 mg/mL) in (2) d), a
manganese standard solution (Mn 0.1 mg/mL) for atomic absorption traceable
to a specified reference material (national measurement standard) according
to Article 134 of the Measurement Act can also be used. Additionally, it isrecommended to multiple by the conversion factor to use as a manganesestandard solution (MnO 0.1291 mg/mL).
(3) Instruments: Instruments are as shown below:a) Flame atomic absorption spectrometer: An atomic absorption spectrometer specified
in JIS K 0121.
1) Light source: A manganese hollow cathode lamp
2) Gas: Gas for heating by flame
190

(i) Fuel gas: acetylene(ii) Auxiliary gas: Air sufficiently free of dust and moisture.
b) Hot plate: A hot plate whose surface temperature can be adjusted up to 250 ºC.
(4) Test procedures(4.1) Extraction: Conduct extraction as follows:a) Weigh about 2 g of an analytical sample to the order of 1 mg, and put it in a 500-mL tallbeaker.
b) Add about 200 mL of hydrochloric acid (1+23), cover with a watch glass, and boil on a
hot plate for about 5 minutes (4).
c) After standing to cool, transfer to a 250-mL - 500-mL volumetric flask with water.
d) Add water up to the marked line.
e) Filter with Type 3 filter paper to make the sample solution.
Note (4) Be aware that an analytical sample should not solidify in the bottom of a beaker.
Comment 2 In the procedure in a), a 500-mL volumetric flask can be used instead of a
500-mL tall beaker. However the volumetric flask used should be
distinguished as an extraction flask and should not be used for the otherpurposes. In addition, “cover with a watch glass” in b) is replaced by “place along-stem funnel”, and “transfer to a 250-mL - 500-mL volumetric flask withwater” in the procedure in c) is skipped.
Comment 3 The procedure in (4.1) is the same as in (4.1) in 4.5.2.a.
(4.2) Measurement: Conduct measurement according to JIS K 0121 and as shown below.
Specific measurement procedures are according to the operation method of the atomic
absorption spectrometer used in measurement.
a) Measurement conditions for the atomic absorption spectrometer: Set up themeasurement conditions for the atomic absorption spectrometer considering thefollowing:
Analytical line wavelength: 279.5 nmb) Calibration curve preparation
1) Spray the manganese standard solutions for the calibration curve preparation andthe blank test solution for the calibration curve preparation into a flame, and readthe indicated value at a wavelength of 279.5 nm.
2) Prepare a curve for the relationship between the manganese concentration and theindicated value of the manganese standard solutions for the calibration curve
preparation and the blank test solution for the calibration curve preparation.
c) Sample measurement
1) Transfer a predetermined amount of the sample solution (the equivalents of 0.1 mg- 1 mg as MnO) to a 100-mL volumetric flask.
2) Add about 10 mL of interference suppressor solution (3), and add water up to themarked line.
3) Conduct similarly as in b) 1) to read the indicated value.
4) Obtain the manganese content from the calibration curve, and calculate the soluble
manganese (S-MnO) in the analytical sample.
Reference
1) Masayoshi KOSHINO: Second Revision of The Methods of Analysis of Fertilizers(Details), p.176 - 177, Yokendo, Tokyo (1988)
191

(5)

Flow sheet for soluble manganese: The flow sheet for soluble manganese infertilizers is shown below:
2 g analytical sample
Weigh to the order of 1 mg into a 500-mL tall beaker.
← About 200 mL of hydrochloric acid (1+23)
Heating
Cover with a watch glass, and boil for about 5 minutes.
Standing to coolTransfer
Water, 250-mL - 500-mL volumetric flask← Water (up to the marked line)
Filtration
Type 3 filter paper
Aliquot(predetermined amount)

100-mL volumetric flask← About 10 mL of interference suppressor solution← Water (up to the marked line)
Measurement
Atomic absorption spectrometer
Figure Flow sheet for soluble manganese in fertilizers.

192

4.7.2

Citrate soluble manganese

4.7.2.a Flame atomic absorption spectrometry(1) Summary
This test method is applicable to fertilizers containing manganese carbonate fertilizers.
Extract by adding citric acid solution to an analytical sample and add an interference
suppressor solution, and then spray into an acetylene-air flame, and measure the atomic
absorption with manganese at a wavelength of 279.5 nm to quantify citrate soluble manganese(citrate soluble manganese(C-MnO)).
(2) Reagents: Reagents are as shown below:a) Hydrochloric acid: A JIS Guaranteed Reagent specified in JIS K 8180 or a reagent ofequivalent quality.
b) Citric acid solution (1): Dissolve 20 g of citric acid monohydrate specified in JIS K
8283 in water to make 1,000 mL.
c) Interference suppressor solution (1): Weigh 60.9 g - 152.1 g of strontium chloride
hexahydrate (2) specified in JIS K 8132 into a 2,000-mL beaker, add a small amount ofwater, add gradually 420 mL of hydrochloric acid to dissolve, and further add water tomake 1,000 mL.
d) Manganese standard solution (MnO 1 mg/mL) (1): Weigh 0.775 g of manganese
powder (purity no less than 99 % (mass fraction)) into a weighing dish. Transfer to a
1,000-mL volumetric flask with a small amount of water, add about 10 mL ofhydrochloric acid to dissolve, and add water up to the marked line.
e) Manganese standard solution (MnO 0.1 mg/mL): Transfer 10 mL of manganesestandard solution (MnO 1 mg/mL) to a 100-mL volumetric flask and add water up to themarked line.
f) Manganese standard solution (MnO 1 µg/mL - 10 µg/mL) for the calibration curve
preparation (1): Transfer 2.5 mL - 25 mL of manganese standard solution (MnO 0.1
mg/mL) to 250-mL volumetric flasks step-by-step, add about 25 mL of interference
suppressor solution (3), and add water up to the marked line.
g) Blank test solution for the calibration curve preparation (1): Transfer about 25 mL ofinterference suppressor solution used in the procedure in f) to a 250-mL volumetricflask (3), and add water up to the marked line.
Note (1) This is an example of preparation; prepare an amount as appropriate.
(2) 29 g of lanthanum oxide (atomic absorption analysis grade or equivalents) can
also be used.
(3) Add an interference suppressor solution that is 1/10 volume of the volume to beprepared.
Comment 1 Instead of a manganese standard solution (MnO 0.1 mg/mL) in (2) e), a
manganese standard solution (Mn 0.1 mg/mL) for atomic absorption traceable
to a specified reference material (national measurement standard) according
to Article 134 of the Measurement Act can also be used. Additionally, it isrecommended to multiply by the conversion factor to use as a manganesestandard solution (MnO 0.1291 mg/mL).
(3) Instruments: Instruments are as follows:a) Constant-temperature rotary shaker: A constant-temperature rotary shaker that canrotate a 250-mL volumetric flask, set up in a thermostat adjustable to 30 ºC ± 1 ºC,
upside down at 30 - 40 revolutions/min.
193b) Flame atomic absorption spectrometer: An atomic absorption spectrometer specified
in JIS K 0121.
1) Light source: A manganese hollow cathode lamp
2) Gas: Gas for heating by flame(i) Fuel gas: acetylene(ii) Auxiliary gas: Air sufficiently free of dust and moisture.
(4) Test procedures(4.1) Extraction: Conduct extraction as shown below.
a) Weigh 1 g of an analytical sample to the order of 1 mg, and transfer to a 250-mL
volumetric flask.
b) Add 150 mL of citric acid solution heated up to about 30 ºC, and shake to mix at 30 - 40
revolutions/min (30 ºC ± 1 ºC) for 1 hour.
c) After standing to cool, add water up to the marked line.
d) Filter with Type 3 filter paper to make the sample solution.
Comment 2 The procedure in (4.1) is the same as the procedure (4.1) in 4.2.3.a.
(4.2) Measurement: Conduct measurement according to JIS K 0121 and as shown below.
Specific measurement procedures are according to the operation method of the atomic
absorption spectrometer used in measurement.
a) Measurement conditions for the atomic absorption spectrometer: Set up themeasurement conditions for the atomic absorption spectrometer considering thefollowing:
Analytical line wavelength: 279.5 nmb) Calibration curve preparation
1) Spray the manganese standard solutions for the calibration curve preparation andthe blank test solution for the calibration curve preparation into a flame, and readthe indicated value at a wavelength of 279.5 nm.
2) Prepare a curve for the relationship between the manganese concentration and theindicated value of the manganese standard solutions for the calibration curve
preparation and the blank test solution for the calibration curve preparation.
c) Sample measurement
1) Transfer a predetermined amount of the sample solution (the equivalents of 0.1 mg- 1 mg as MnO) to a 100-mL volumetric flask.
2) Add about 10 mL of interference suppressor solution (3), and add water up to themarked line.
3) Conduct similarly as in b) 1) to read the indicated value.
4) Obtain the manganese content from the calibration curve, and citrate soluble
manganese (C-MnO) in the analytical sample.
Comment 3 Table 1 shows results from a simultaneous analysis with the same samples(proficiency testing and external quality control testing) hosted by the Japan
Fertilizer Quality Assurance Committee analyzed by the robust method.

194

Table 1 Results and analysis results from simultaneous analysis with the same samples
1)
for citrate soluble manganese hosted by the Japan Fertilizer Quality Assurance Committee
5)2)
4)
Number of Median Μ
RSDrobNIQR
Year
Sample3)3)laboratories
(%)
(%)
(%)
High analysis
2009
110
0.547
0.00173.0
compound fertilizer
High analysis
102
0.513
0.013
2.5
compound fertilizer
1) Proficiency testing and external quality control testing2) Median (M) agrees with the mean in normal distribution.
3) Mass fraction4) Robust standard deviation (NIQR) agrees with standard deviation in normal distribution.
5) RSDrob stands for relative standard deviation obtained from the robust methodby the formula shown below:
RSDrob = (NIQR/M) × 100
2013

Reference
1) Masayoshi KOSHINO: Second Revision of The Methods of Analysis of Fertilizers(Details), p.176 - 177, Yokendo, Tokyo (1988)(5)

Flow sheet for citrate soluble manganese: The flow sheet for citrate soluble
manganese in fertilizers is shown below:
1g analytical sample
Weigh to the order of 1 mg into a 250-mL tall beaker.
← About 150 mL of citrate solution [about 30 ºC]
Shaking to mix

Constant temperature rotary shaker (30 - 40 revolutions/min),
30 ºC ± 1 ºC, for 1 hourStanding to cool
← Water (up to the marked line)
Filtration
Type 3 filter paper
Aliquot
100-mL volumetric flask(predetermined amount)← About 10 mL of interference suppressor solution← Water (up to the marked line)
Measurement
Atomic absorption spectrometer
Figure Flow sheet for citrate soluble manganese in fertilizers.

1954.7.3

Water-soluble manganese

4.7.3.a Flame atomic absorption spectrometry(1) Summary
This test method is applicable to fertilizers containing manganese sulfate fertilizers.
Extract by adding water to an analytical sample and add an interference suppressor solution,and then spray in an acetylene-air flame, and measure the atomic absorption with manganese
at a wavelength of 279.5 nm to quantify water-soluble manganese (W-MnO).
(2) Reagents: Reagents are as shown below:a) Hydrochloric acid: A JIS Guaranteed Reagent specified in JIS K 8180 or a reagent ofequivalent quality.
b) Interference suppressor solution (1): Weigh 60.9 g - 152.1 g of strontium chloride
hexahydrate (2) specified in JIS K 8132 into a 2,000-mL beaker, add a small amount ofwater, add gradually 420 mL of hydrochloric acid to dissolve, and further add water tomake 1,000 mL.
c) Manganese standard solution (MnO 1 mg/mL) (1): Weigh 0.775 g of manganese
powder (purity no less than 99 % (mass fraction)) into a weighing dish. Transfer to a
1,000-mL volumetric flask with a small amount of water, add about 10 mL ofhydrochloric acid to dissolve, and add water up to the marked line.
d) Manganese standard solution (MnO 0.1 mg/mL): Transfer 10 mL of manganesestandard solution (MnO 1 mg/mL) to a 100-mL volumetric flask and add water up to themarked line.
e) Manganese standard solution (MnO 1 µg/mL - 10 µg/mL) for the calibration curve
preparation (1): Transfer 2.5 mL - 25 mL of manganese standard solution (MnO 0.1
mg/mL) to 250-mL volumetric flasks step-by-step, add about 25 mL of interference
suppressor solution (3), and add water up to the marked line.
f) Blank test solution for the calibration curve preparation (1): Transfer about 25 mL ofinterference suppressor solution used in the procedure e) to a 250-mL volumetric flask(3), and add water up to the marked line.
Note (1) This is an example of preparation; prepare an amount as appropriate.
(2) 29 g of lanthanum oxide (atomic absorption analysis grade or equivalents) can
also be used.
(3) Add an interference suppressor solution that is 1/10 volume of the volume to beprepared.
Comment 1 Instead of a manganese standard solution (MnO 0.1 mg/mL) in (2) d), a
manganese standard solution (Mn 0.1 mg/mL) for atomic absorption traceable
to a specified reference material (national measurement standard) according
to Article 134 of the Measurement Act can also be used. Additionally, it isrecommended to multiply by the conversion factor to use as manganesestandard solution (MnO 0.1291 mg/mL).
(3) Instruments: Instruments are as follows:a) Rotary shaker: A rotary shaker that can rotate upside down a 500-mL volumetric flask
at 30 - 40 revolutions/min.
b) Flame atomic absorption spectrometer: An atomic absorption spectrometer specified
in JIS K 0121.
1) Light source: A manganese hollow cathode lamp
2) Gas: Gas for heating by flame196

(i) Fuel gas: acetylene(ii) Auxiliary gas: Air sufficiently free of dust and moisture.
(4) Test procedures(4.1) Extraction: Conduct extraction as shown below.
a) Weigh 5 g of an analytical sample to the order of 1 mg, and transfer to a 500-mL
volumetric flask.
b) Add about 400 mL of water, and shake to mix at 30 - 40 revolutions/min for about 30
minutes.
c) Add water up to the marked line.
d) Filter with Type 3 filter paper to make the sample solution.
Comment 2 In the procedure in a), it is also allowed to weigh 2.5 g of the analyticalsample to the order of 1 mg and transfer to a 250-mL volumetric flask.
Comment 3 The procedure in (4.1) is the same as the procedure in (4.1) in 4.2.4.a(4.2) Measurement: Conduct measurement according to JIS K 0121 and as shown below.
Specific measurement procedures are according to the operation method of the atomic
absorption spectrometer used in measurement.
a) Measurement conditions for the atomic absorption spectrometer: Set up themeasurement conditions for the atomic absorption spectrometer considering thefollowing:
Analytical line wavelength: 279.5 nmb) Calibration curve preparation
1) Spray the manganese standard solutions for the calibration curve preparation andthe blank test solution for the calibration curve preparation into a flame, and readthe indicated value at a wavelength of 279.5 nm.
2) Prepare a curve for the relationship between the manganese concentration and theindicated value of the manganese standard solutions for the calibration curve
preparation and the blank test solution for the calibration curve preparation.
c) Sample measurement
1) Transfer a predetermined amount of the sample solution (the equivalents of 0.1 mg- 1 mg as MnO) to a 100-mL volumetric flask.
2) Add about 10 mL of interference suppressor solution (3), and add water up to themarked line.
3) Conduct similarly as in b) 1) to read the indicated value.
4) Obtain the manganese content from the calibration curve, and water-soluble
manganese (W-MnO) in the analytical sample.
Comment 4 Table 1 shows results from a simultaneous analysis with the same samples(proficiency testing and external quality control testing) hosted by the Japan
Fertilizer Quality Assurance Committee analyzed by the robust method.

197

Table 1 Results and analysis results from simultaneous analysis with the same samples
for water-soluble manganese hosted by the Japan Fertilizer Quality Assurance Committee 1)
Year

SampleNumber oflaboratories

2)

4)Median Μ

NIQR

(%)3)

(%)3)

5)

RSDrob(%)
Fluid mixed
2012
99
1.23
0.03
2.7
fertilizer
1) Proficiency testing and external quality control testing2) Median (M) agrees with the mean in normal distribution.
3) Mass fraction4) Robust standard deviation (NIQR) agrees with standard deviation in normal distribution.
5) RSDrob stands for relative standard deviation obtained from the robust methodby the formula shown below:
RSDrob = (NIQR/M) × 100

Reference
1) Masayoshi KOSHINO: Second Revision of The Methods of Analysis of Fertilizers(Details), p.176 - 177, Yokendo, Tokyo (1988)(5)

Flow sheet for water-soluble manganese: The flow sheet for water-soluble
manganese in fertilizers is shown below:
5 g analytical sample
Weigh up to 1 mg into a 500-mL volumetric flask.
← Water, About 400 mL
Shaking to mix
Rotary shaker (30 - 40 revolutions/min), for 30 minutes← Water (up to the marked line)
Filtration
Type 3 filter paper
Aliquot
100-mL volumetric flask(predetermined amount)← About 10 mL of interference suppressor solution← Water (up to the marked line)
Measurement
Atomic absorption spectrometer
Figure Flow sheet for water-soluble manganese in fertilizers.

198

4.8

Boron

4.8.1

Citrate soluble boron

4.8.1.a Azomethine-H method(1) Summary
This test method is applicable to fertilizers containing borate fertilizers, etc.
Extract by adding citric acid solution to an analytical sample, mask co-existing copper, ironand other salts with ethylenediamine tetraacetate and measure the absorbance withazomethine-H borate formed by the reaction with azomethine-H to obtain citrate solubleboron (C-B2O3).
(2) Reagents: Reagents are as shown below:a) Citric acid solution (1): Dissolve 20 g of citric acid monohydrate specified in JIS K
8283 in water to make 1,000 mL.
b) Ethylenediamine tetraacetate solution (1): Dissolve 37.2 g ofethylenediaminetetraacetic acid disodium dihydrate specified in JIS K 8107 in water tomake 1,000 mL.
c) Ammonium acetate solution (1): Dissolve 250 g of ammonium acetate specified in JIS
K 8359 in water to make 500 mL and adjust pH with sulfuric acid (1+4) to pH 5.2 ± 0.1.
d) Azomethine-H solution: Add water to 0.6 g of azomethine-H and 2 g of L (+) –ascorbic acid specified in JIS K 9502, and heat up to 35 ºC - 40 ºC to dissolve and add
water after cooling to make 100 mL.
e) Boron standard solution (B2O3 2.5 mg/mL) (1): After leaving boric acid specified in
JIS K 8863 at rest in a desiccator for about 24 hours to dry, weigh 4.441 g to a weighing
dish. Dissolve with a small amount of water, transfer to a 1,000-mL volumetric flask
and add water up to the marked line.
f) Boron standard solution (B2O3 0.05 mg/mL): Dilute with water exactly 50 times thepredetermined volume of boron standard solution (B2O3 2.5 mg/mL).
Note (1) This is an example of preparation; prepare an amount as appropriate.
(3) Instruments: Instruments are as follows:a) Constant-temperature rotary shaker: A constant-temperature rotary shaker that canrotate a 250-mL volumetric flask, set up in a thermostat adjustable to 30 ºC ± 1 ºC,
upside down at 30 - 40 revolutions/min.
b) Spectrophotometer: A spectrophotometer specified in JIS K 0115
(4) Test procedures(4.1) Extraction: Conduct extraction as shown below.
a) Weigh 1 g of an analytical sample to the order of 1 mg, and transfer to a 250-mL
volumetric flask.
b) Add 150 mL of citric acid solution heated up to about 30 ºC, and shake to mix at 30 - 40
revolutions/min (30 ºC ± 1 ºC) for 1 hour.
c) After standing to cool, add water up to the marked line.
d) Filter with Type 3 filter paper to make the sample solution.
Comment 1

The procedure in (4.1) is the same as the procedure in (4.1) in 4.2.3.a.

(4.2) Coloring: Conduct coloring as shown below.

199a) Transfer a predetermined volume (the equivalents of 0.05 mg -1 mg of B2O3, and no
more than the equivalents of 15 mL of citric acid solution) of an analytical sample to a
100-mL volumetric flask.
b) Add the solution to make the citric acid solution equivalent to 15 mL.
c) Add 25 mL of ethylenediamine tetraacetate solution, and add 10 mL of ammonium
acetate solution and then 10 mL of azomethine-H solution successively, and further add
water up to the marked line, then leave at rest for about 2 hours.
(4.3) Measurement: Conduct measurement according to JIS K 0115 and as shown below.
Specific measurement procedures are according to the operation method of thespectrophotometer used in measurement.
a) Measurement conditions for the spectrophotometer: Set up the measurement
conditions for the spectrophotometer considering the following:
Detection wavelength: 415 nmb) Calibration curve preparation
1) Transfer 1mL - 20 mL of boron standard solution (B2O3 0.05 mg/mL) to 100-mL
volumetric flasks step-by-step.
2) Add 15 mL of citric acid solution and conduct the same procedure as (4.2) c) to makethe B2O3 0.05 mg/100 mL - 1 mg/100 mL boron standard solution for the calibration
curve preparation.
3) Conduct the same procedures as 2) for another 100-mL volumetric flask to make theblank test solution for the calibration curve preparation.
4) Measure absorbance at a wavelength of 415 nm of the boron standard solutions forthe calibration curve preparation using the blank test solution for the calibration
curve preparation as the control.
5) Prepare a curve for the relationship between the boron concentration and absorbanceof the boron standard solutions for the calibration curve preparation.
c) Sample measurement
1) Regarding the solution in (4.2) c), measure the absorbance by the same procedure asb) 4).
2) Obtain the boron (B2O3) content from the calibration curve, and calculate the citratesoluble boron (C-B2O3) in the analytical sample.
Comment 2 Table 1 shows results from a simultaneous analysis with the same samples(proficiency testing and external quality control testing) hosted by the Japan
Fertilizer Quality Assurance Committee analyzed by the robust method.

200

Table 1 Results and analysis results from simultaneous analysis with the same samples
for citrate soluble boron hosted by the Japan Fertilizer Quality Assurance Committee
2)

Year
2009
2014

SampleHigh analysis
compound fertilizer
High analysis
compound fertilizer

Number of Median Μ
3)laboratories
(%)

4)

NIQR
(%)

3)

RSDrob
(%)

110

0.280

0.015

5.3

95

0.243

0.014

5.6

1)

5)

1) Proficiency testing and external quality control testing2) Median (M) agrees with the mean in normal distribution.
3) Mass fraction4) Robust standard deviation (NIQR) agrees with standard deviation in normal distribution.
5) RSDrob stands for relative standard deviation obtained from the robust methodby the formula shown below:
RSDrob = (NIQR/M) × 100

Reference
1) Masayoshi KOSHINO: Second Revision of The Methods of Analysis of Fertilizers(Details), p.184 - 187, Yokendo, Tokyo (1988)(5)

Flow sheet for citrate soluble boron: The flow sheet for citrate soluble boron in
fertilizers is shown below:
1 g analytical sample
Weigh to the order of 1 mg into a 250-mL volumetric flask← 150 mL of citric acid solution [about 30 ºC]
Shaking to mix

Constant temperature rotary shaker (30 - 40 revolutions/min),
30 ºC ± 1 ºC, for 1 hourStanding to cool
← Water (up to the marked line)
Filtration
Type 3 filter paper
Aliquot
100-mL volumetric flask(predetermined amount)← Citric acid solution, until the volume reaches
to the equivalents of 15 mL← 25 mL of ethylenediamine tetraacetate solution← 10 mL of ammonium acetate solution← 10 mL of azomethine-H solution← Water (up to the marked line)
Leaving at rest
For about 2 hours
Measurement

Spectrophotometer (415 nm)

Figure Flow sheet for citrate soluble boron in fertilizers.

201

4.8.2

Water-soluble boron

4.8.2.a Azomethine-H method(1) Summary
This test method is applicable to fertilizers containing borate fertilizers, etc.
Extract by adding water to an analytical sample, boil to extract, and mask co-existing copper,
iron and other salts with ethylenediamine tetraacetate and measure the absorbance withazomethine-H borate formed by the reaction with azomethine-H to obtain water-soluble boron(W-B2O3).
(2) Reagents: Reagents are as shown below:a) Ethylenediamine tetraacetate solution (1): Dissolve 37.2 g ofethylenediaminetetraacetic acid disodium dihydrate specified in JIS K 8107 in water tomake 1,000 mL.
b) Ammonium acetate solution (1): Dissolve 250 g of ammonium acetate specified in JIS
K 8359 in water to make 500 mL and adjust pH with sulfuric acid (1+4) to pH 5.2 ± 0.1.
c) Azomethine-H solution (1): Add water to 0.6 g of azomethine-H and 2 g of L (+) –ascorbic acid specified in JIS K 9502, and heat up to 35 ºC - 40 ºC to dissolve and add
water after cooling to make100 mL.
d) Boron standard solution (B2O3 2.5 mg/mL) (1): After leaving boric acid specified in
JIS K 8863 at rest in a desiccator for about 24 hours to dry, weigh 4.441 g to a weighing
dish. Dissolve with a small amount of water, transfer to a 1,000-mL volumetric flask
and add water up to the marked line.
e) Boron standard solution (B2O3 0.05 mg/mL): Dilute with water precisely 50 times thepredetermined volume of boron standard solution (B2O3 2.5 mg/mL).
Note (1) This is an example of preparation; prepare an amount as appropriate.
(3) Instruments: Instruments are as follows:a) Spectrophotometer: A spectrophotometer specified in JIS K 0115b) Hot plate: A hot plate whose surface temperature can be adjusted up to 250 ºC.
(4) Test procedures(4.1) Extraction: Conduct extraction as follows:a) Weigh about 2.5 g of an analytical sample to the order of 1 mg, and put it in a 300-mLtall beaker.
b) Add about 200 mL of water, cover with a watch glass, and boil on a hot plate for about
15 minutes.
c) After standing to cool, transfer to a 250-mL volumetric flask with water.
d) Add water up to the marked line.
e) Filter with Type 3 filter paper to make the sample solution.
Comment 1 In the procedure in a), a 250-mLvolumetric flask can be used instead of a
300-mL tall beaker. However the volumetric flask used should be
distinguished as an extraction flask and should not be used for the otherpurposes. In addition, “cover with a watch glass” in b) is replaced by “place along-stem funnel”, and “transfer to a 250-mL volumetric flask with water” inthe procedure in c) is skipped.
Comment 2 The procedure in (4.1) is the same as in (4.1) in 4.3.3.a.
(4.2) Coloring: Conduct coloring as shown below.
202a) Transfer a predetermined volume (the equivalents of 0.05 mg -1 mg as B2O3) of ananalytical sample to a 100-mL volumetric flask.
b) Add 25 mL of ethylenediamine tetraacetate solution, and add 10 mL of ammonium
acetate solution and 10 mL of azomethine-H solution successively, and further add
water up to the marked line, then leave at rest for about 2 hours.
Comment 3 Water-soluble boron can be measured simultaneously with citrate solubleboron by adding 15 mL of citric acid solution before the procedure in (4.2) b).
(4.3) Measurement: Conduct measurement according to JIS K 0115 and as shown below.
Specific measurement procedures are according to the operation method of thespectrophotometer used in measurement.
a) Measurement conditions for the spectrophotometer: Set up the measurement
conditions for the spectrophotometer considering the following:
Detection wavelength: 415 nmb) Calibration curve preparation
1) Transfer 1mL - 20 mL of boron standard solution (B2O3 0.05 mg/mL) to 100-mL
volumetric flasks step-by-step.
2) Conduct the same procedure as (4.2) b) to make the B2O3 0.05 mg/100 mL - 1
mg/100 mL boron standard solution for the calibration curve preparation.
3) Conduct the same procedures as 2) for another a 100-mL volumetric flask to makethe blank test solution for the calibration curve preparation.
4) Measure the absorbance at a wavelength of 415 nm of the boron standard solutions
for the calibration curve preparation using the blank test solution for the calibration
curve preparation as the control .
5) Prepare a curve for the relationship between the boron concentration and absorbanceof the boron standard solutions for the calibration curve preparation.
c) Sample measurement
1) Regarding the solution in (4.2) b), measure the absorbance by the same procedure asb) 4).
2) Obtain the boron (B2O3) content from the calibration curve, and calculate thewater-soluble boron (W-B2O3) in the analytical sample.
Comment 4 Water-soluble boron can be measured simultaneously with citrate solubleboron by adding 15 mL of citric acid solution before the procedure in (4.3) b)
2).
Comment 5 Table 1 shows results from a simultaneous analysis with the same samples(proficiency testing and external quality control testing) hosted by the Japan
Fertilizer Quality Assurance Committee analyzed by the robust method.

203

Table 1 Results and analysis results from simultaneous analysis with the same samples
1)
for water-soluble boron hosted by the Japan Fertilizer Quality Assurance Committee
2)

Year
2006
2008
2010
2012

1)
2)
3)
4)
5)

SampleHigh analysis
compound fertilizer
High analysis
compound fertilizer
High analysis
compound fertilizer
Fluid mixed
fertilizer

Number of Median Μ
3)laboratories
(%)

4)

NIQR
(%)

3)

RSDrob
(%)

132

0.40

0.02

4.2

113

0.26

0.01

2.9

109

0.291

0.009

3.1

92

0.240

0.008

3.2

5)

Proficiency testing and external quality control testingMedian (M) agrees with the mean in normal distribution.
Mass fraction
Robust standard deviation (NIQR) agrees with standard deviation in normal distribution.
RSDrob stands for relative standard deviation obtained from the robust methodby the formula shown below:
RSDrob = (NIQR/M) × 100

Reference
1) Masayoshi KOSHINO: Second Revision of The Methods of Analysis of Fertilizers(Details), p.184 - 187, Yokendo, Tokyo (1988)(5)

Flow sheet for water-soluble boron: The flow sheet for water-soluble boron in
fertilizers is shown below:
2.5 g analytical sample
Weigh to the order of 1 mg into a 300-mL tall beaker.
← About 200 mL of Water
Heating
Cover with a watch glass, boil for 15 minutes
Standing to coolTransfer
Water, 250-mL volumetric flask← Water (up to the marked line)
Filtration
Type 3 filter paper
Aliquot
100-mL volumetric flask(predetermined amount)← 25 mL of ethylenediamine tetraacetate solution← 10 mL of ammonium acetate solution← 10 mL of azomethine-H solution← Water (up to the marked line)
Leaving at rest
For about 2 hours
Measurement

Spectrophotometer (415 nm)

Figure Flow sheet for water-soluble boron in fertilizers.

204

4.9
4.9.1

Zinc
Total zinc

4.9.1.a Flame atomic absorption spectrometry(1) Summary
This test method is applicable to fertilizers.
Pretreat an analytical sample with incineration and nitric acid-hydrochloric acid (1+3), andthen spray in an acetylene-air flame, and measure the atomic absorption with zinc at awavelength of 213.9 nm to quantify total zinc.
(2) Reagents, etc.: Reagents and water are as shown below:a) Water: Water of A3 specified in JIS K 0557.
b) Nitric acid: A reagent of harmful metal analysis grade, microanalysis grade orequivalents.
c) Hydrochloric acid: A reagent of harmful metal analysis grade, microanalysis grade orequivalents.
d) Zinc standard solution (0.1 mg/mL): Zinc standard solution (0.1 mg/mL) for atomic
absorption traceable to a specified reference material (national measurement standard)
according to Article 134 of the Measurement Act.
e) Zinc standard solutions (0.5 µg - 5 µg/mL) for the calibration curve preparation(1):Transfer 2.5 mL - 25 mL of zinc standard solution (0.1 mg/mL) to 500-mL volumetricflasks step-by-step, and add hydrochloric acid (1+23) to the marked line.
f) Blank test solution for the calibration curve preparation (1): Hydrochloric acid
(1+23) used in the procedure in e).
Note (1) This is an example of preparation; prepare an amount as appropriate.
(3) Instruments: Instruments are as shown below:a) Flame atomic absorption spectrometer: An atomic absorption spectrometer specified
in JIS K 0121 with the background correction (2) function.
1) Light source: A zinc hollow cathode lamp (when the continuous source method as
the background correction method is used, the light source is a deuterium lamp.)
2) Gas: Gas for heating by flame(i) Fuel gas: acetylene(ii) Auxiliary gas: Air sufficiently free of dust and moisture.
b) Electric furnace: An electric furnace that can be adjusted to 450 ºC ± 5 ºC.
c) Hot plate or sand bath: A hot plate whose surface temperature can be adjusted up to
250 ºC. Adjust the amounts of gas and silica sand of a sand bath so that the sand bathtemperature can be set to 250 ºC.
Note (2) There are the continuous source method, the Zeeman method, the non-resonancespectrum method, and the self-reversal method, etc.
(4) Test procedures(4.1) Extraction: Conduct extraction as follows:a) Weigh 5.00 g of an analytical sample, and put it in a 200-mL - 300-mL tall beaker.
b) Put the tall beaker in an electric furnace, and heat gently to char(3).
c) Ignite at 450 ºC ± 5 ºC to incinerate (4).
d) After standing to cool, moisten the residue with a small amount of water, and add about
10 mL of nitric acid and about 30 mL of hydrochloric acid.
e) Cover the tall beaker with a watch glass, and heat on a hot plate or a sand bath to digest.
205

Slightly move the watch glass (5), and continue heating on the hot plate or sand bath toconcentrate until nearly dried up.
g) After standing to cool, add 25 mL - 50 mL of hydrochloric acid (1+5)(6) to the digest,cover the tall beaker with the watch glass, and heat quietly to dissolve.
h) After standing to cool, transfer to a 100-mL - 200-mL volumetric flask with water, add
water up to the marked line, and filter with Type 3 filter paper to make the sample
solution.
i) As a blank test, conduct the procedures in b) - h) using another tall beaker to prepare theblank test solution.
f)Note (3) Example of the charring procedure: Heat at about 250 ºC until smoke is nolonger formed.
(4) Example of ignition period: 8 - 16 hours(5) The watch glass can be removed.
(6) Add hydrochloric acid (1+5) so that the hydrochloric acid concentration of thesample solution will be hydrochloric acid (1+23). For example, when a 100-mL
volumetric flask is used in the procedure in h), about 25 mL of hydrochloric acid(1+5) should be added.
Comment 1 Do not conduct the procedures in (4.1) b) - c) in the case of fertilizers notcontaining organic matters.
Comment 2 The procedures in (4.1) are the same as in (4.1) in 4.9.1.b, 4.10.1.a, 4.10.1.b,
5.3.a, 5.3.b, 5.4.a, 5.4.b, 5.5.a, 5.5.d, 5.5.a and 5.6.b. Additionally, it also canbe used as the sample solution in 4.2.1.a, 4.2.1.b, 4.3.1.a, 4.3.1.b, and 4.5.1.a.
Comment 3 The sample solution prepared in (4.1.2) in 4.2.1.a can also be used.
(4.2) Measurement: Conduct measurement according to JIS K 0121 and as shown below.
Specific measurement procedures are according to the operation method of the atomic
absorption spectrometer used in measurement.
a) Measurement conditions for the atomic absorption spectrometer: Set up themeasurement conditions for the atomic absorption spectrometer considering thefollowing:
Analytical line wavelength: 213.9 nmb) Calibration curve preparation
1) Spray the zinc standard solutions for the calibration curve preparation and the blanktest solution for the calibration curve preparation into a flame, and read theindicated value at a wavelength of 213.9 nm.
2) Prepare a curve for the relationship between the zinc concentration and theindicated value of the zinc standard solutions for the calibration curve preparationand the blank test solution for the calibration curve preparation.
c) Sample measurement
1) Subject the sample solution (7) to the same procedure as in b) 1) to read the indicatedvalue.
2) Subject the blank test solution to the same procedure as in b) 1) to read theindicated value, and correct the indicated value obtained for the sample solution.
3) Obtain the zinc content from the calibration curve, and calculate the zincconcentration in the analytical sample.
Note (7) If there is a possibility that the zinc concentration in the sample solution will
exceed the maximum limit of the calibration curve, dilute a predeterminedamount with hydrochloric acid (1+23).
206

Comment 4 The zinc concentration in the analytical sample can also be corrected bysubjecting the blank test solution to the same procedures as in 1) and 3) toobtain the zinc content in the blank test solution.
Comment 5 Replicate testing was conducted using human waste sludge fertilizer, mixed
sludge fertilizer and composted sludge fertilizer (3 samples); as a result,repeatability in the range of 510 mg/kg - 1,100 mg/kg total zinc (meandetermined value) was 0.6 % - 1.9 % relative standard deviation.
References
1) Masayoshi KOSHINO: Second Revision of The Methods of Analysis of Fertilizers(Details), p.193 - 194, Yokendo, Tokyo (1988)
2) Kimie KATO, Masayuki YOSHIMOTO and Yuji SHIRAI: Systematization of
Determination Methods of Major Components in Sludge Fertilizer, Compost andOrganic Fertilizer, Research Report of Fertilizer, Vol.3, 2010. (107-116)(5)

Flow sheet for total zinc: Flow sheet for total zinc in fertilizers is shown below:
5.00 ganalytical sample
Charring
Incineration

200-mL - 300-mL tall beaker.
Heat gently
Ignite at 450 ºC ± 5 ºC

Standing to coolRoom temperature← A small amount of water← About 10 mL of nitric acid
← About 30 mL of hydrochloric acid
Heating
Cover with a watch glass, and digest
Heating

Slightly move a watch glass to remove acid

Standing to coolRoom temperature← 25 mL - 50 mL of hydrochloric acid (1+5)
Heating
Cover with a watch glass, and dissolve
Standing to coolRoom temperature

Transfer
100-mL - 200-mL volumetric flask, water← Water (up to the marked line)
Filtration
Type 3 filter paper
Measurement

Atomic absorption spectrometer (213.9 nm)

Figure Flow sheet for total zinc in fertilizers

207

4.9.1.b ICP Atomic Emission Spectrometry <Reference method>(1) Summary
The test method is applicable to sludge fertilizers, etc.
Pretreat an analytical sample with incineration, nitric acid - hydrochloric acid (1+3), introduce
it to ICP Atomic Emission Spectrometer (“ICP-AES”) and measure the emission with zinc at
a wavelength of 206.191 nm to quantify zinc.
(2) Reagents, etc.: Reagents and water are as shown below:a) Water: Water of A3 specified in JIS K 0557.
b) Nitric acid: A reagent of harmful metal analysis grade, microanalysis grade orequivalents.
c) Hydrochloric acid: A reagent of harmful metal analysis grade, microanalysis grade orequivalents.
d) Zinc standard solution (0.1 mg/mL): A zinc standard solution (0.1 mg/mL) for atomic
absorption spectrometry traceable to a specified reference material (national
measurement standard) according to Article 134 of the Measurement Act.
e) Zinc standard solutions (2.5 µg/mL) (1):Dilute a predetermined amount of zinc standard solution (0.1 mg/mL) with hydrochloric
acid (1+23) to prepare a zinc standard solution (25 µg/mL).
Note (1) This is an example of preparation; prepare an amount as appropriate.
(3) Instruments: Instruments are as shown below:a) ICP Atomic Emission Spectrometer: An atomic emission spectrometer specified in
JIS K 0116.
1) Gas: Argon gas specified in JIS K 1105 of no less than 99.5 % (volume fraction) inpurityb) Electric furnace: An electric furnace that can keep the test temperature at  5 ºCc) Hot plate or sand bath: A hot plate whose surface temperature can be adjusted up to
250 ºC. Adjust the amounts of gas and silica sand of a sand bath so that the sand bathtemperature can be set to 250 ºC.
(4) Test procedures(4.1) Extraction: Conduct extraction as follows:a) Weigh 5.00 g of an analytical sample, and put it in a 200-mL - 300-mL tall beaker.
b) Put the tall beaker in an electric furnace, and heat gently to char (2).
c) Ignite at 450 ºC ± 5 ºC to incinerate (3).
d) After standing to cool, moisten the residue with a small amount of water, and add about
10 mL of nitric acid and about 30 mL of hydrochloric acid.
e) Cover the tall beaker with a watch glass, and heat on a hot plate or a sand bath to digest.
f) Slightly move the watch glass (4), and continue heating on the hot plate or sand bath toconcentrate until nearly dried up.
g) After standing to cool, add 25 mL - 50 mL of hydrochloric acid (1+5) (5) to the digest,cover the tall beaker with the watch glass, and heat quietly to dissolve.
h) After standing to cool, transfer to a 100-mL - 200-mL volumetric flask with water, add
water up to the marked line, and filter with Type 3 filter paper to make the sample
solution.
i) As a blank test, conduct the procedures in b) - h) using another tall beaker to prepare theblank test solution.

208

Note (2) Example of the charring procedure: Heat at about 250 ºC until smoke is nolonger formed.
(3) Example of ignition period: 8 - 16 hours(4) The watch glass can be removed.
(5) Add hydrochloric acid (1+5) so that the hydrochloric acid concentration of thesample solution will be hydrochloric acid (1+23). For example, when a 100-mL
volumetric flask is used in the procedure in h), about 25 mL of hydrochloric acid(1+5) should be added.
Comment 1 Do not conduct the procedures in (4.1) b) - c) in the case of fertilizers notcontaining organic matters.
Comment 2 The procedures in (4.1) are the same as in (4.1) in 4.9.1.a.
(4.2) Measurement: Conduct measurement (Standard Addition Method) according to JIS K
0116 and as shown below. Specific measurement procedures are according to theoperation method of the ICP Atomic Emission Spectrometry used in measurement.
a) Measurement conditions for the ICP Atomic Emission Spectrometer: Set up themeasurement conditions for the ICP Atomic Emission Spectrometer considering thefollowing:
Analytical line wavelength: 206.191 nmb) Calibration curve preparation and sample measurement.
1) Put 5mL of sample solution to three 10-mL volumetric flasks respectively.
2) Add 2mL and 4 mL (0.25 μg/mL) of zinc standard solution to volumetric flasks of1) above, then add hydrochloric acid (1+23) up to the marked line to make thesample solution of Standard Addition Method.
3) Add hydrochloric acid (1+23) to the marked line of the remaining volumetric flaskof 1) above to make the sample solution without standard solution.
4) Spray the sample solution of Standard Addition Method and the sample solution
without standard solution into the induction plasma, and read the indicated value ata wavelength of 206.191 nm.
5) Transfer 5 mL of blank test solution to a 10-mL volumetric flask, conduct the sameprocedures as in 3) - 4) to read the indicated value, and correct the indicated value
obtained from the respective sample solutions.
6) Prepare a curve for the relationship between the added zinc concentration and thecorrected indicated value of the sample solution for Standard Addition Method andthe sample solution without standard solution.
7) Obtain the zinc content from the intercept of the calibration curve to calculate theconcentration of zinc in the analytical sample.
Comment 3 The zinc concentration in the analytical sample can also be corrected bysubjecting the blank test solution to the same procedures as in 1) - 4) and 6) 7) to obtain the zinc content in the blank test solution.
Comment 4 Simultaneous measurement of multiple elements by ICP-AES is available. In
this case, mix a predetermined amount of copper standard solution (0.1mg/mL), zinc standard solution (0.1 mg/mL), cadmium standard solution (0.1mg/mL), nickel standard solution (0.1 mg/mL), chromium standard solution(0.1 mg/mL) and lead standard solution (0.1 mg/mL), dilute withhydrochloric acid (1 +23) to prepare mixture standard solution (Cu 25μg/mL,
Zn 25 μg/mL, Cd 0.25μg/mL, Ni 2.5 μg/mL, Cr 2.5 μg/mL and Pb 2.5
μg/mL) (1). Use the mixture solution instead of zinc standard solution (25μg/mL) in (4.2) b) 2). After that, conduct the same procedures as in (4.2) b) to209

calculate the concentration of respective elements in the analytical sample.
Note that the wavelength for measurement of respective elements is Cu
324.754 nm, Zn 206.191 nm, Cd 228.802 nm, Ni 231.604 nm, Cr 205.552 nm
and Pb 220.351 nm.
In addition, the respective element concentrations for respective standard
addition samples are shown in the below Table.
Table The additive amount in mixture standard solution
and the additive concentration of respective element in respective sample solution
Additive amount
Cd
Pb
Ni
Cr
Cu
(mL) in mixture(mg/L) (mg/L) (mg/L) (mg/L) (mg/L)
standard solution
Sample solution without
0
0
0
0
0
0
standard solution
Sample solution 1 of
2
0.05
0.5
0.5
0.5
5
Standard Addition Method
Sample solution 2 of
4
0.1
1
1
1
10
Standard Addition Method

Zn(mg/L)
05
10Comment 5 For each one sample of sewage sludge fertilizer, human waste sludge fertilizer,
industrial sludge fertilizer, mixture sludge fertilizer, calcined sludge fertilizer
and composted sludge fertilizer, the repeatability obtained from triplicatesmeasurement is 0.1 % - 2.3 % relative standard deviation.
Additionally, the minimum limit of quantification of the test method is about
8 mg/kg.
Reference
1) Masahiro ECHI, Tomoe INOUE, Megumi TABUCHI and Tetuya NOMURA:
Simultaneous Determination of Cadmium, Lead, Nickel, Chromium, Copper and Zincin Sludge Fertilizer using Inductively Coupled Plasma-Atomic Emission Spectrometry(ICP-AES), Research Report of Fertilizer, Vol.4, 2011. (36 - 48)

210

(5)

Flow sheet for zinc: The flow sheet for zinc in fertilizers is shown below:
5.00 ganalytical sample
Charring
Incineration

200-mL - 300-mL tall beaker.
Heat gently
Ignite at 450 ºC ± 5 ºC

Standing to coolRoom temperature← A small amount of water← About 10 mL of nitric acid
← About 30 mL of hydrochloric acid
Heating
Cover with a watch glass, and digest for 30 minutes
Heating

Slightly move a watch glass to remove acid

Standing to coolRoom temperature← 25 mL - 50 mL of hydrochloric acid (1+5)
Heating
Cover with a watch glass, and dissolve
Standing to coolRoom temperature

Transfer
100-mL - 200-mL volumetric flask, water← Water (up to the marked line)
Filtration
Type 3 filter paper
Aliquot 5 mL
10-mL volumetric flask, 3 flasks← 0, 2 and 4 mL of zinc standard solution (25 μg / mL)← Hydrochloric acid (1+23) ( up to the marked line)
Measurement
ICP Atomic Emission Spectrometer (206.191nm)
Figure Flow sheet for zinc in fertilizers

2114.9.2

Water-soluble zinc

4.9.2.a Flame atomic absorption spectrometry(1) Summary
This test method is applicable to fertilizers that indicate zinc content as a response modifier.
Extract by adding water to an analytical sample, spray in an acetylene-air flame and measure
the atomic absorption with zinc at a wavelength of 213.9 nm to quantify water-soluble zinc.
(2) Reagents, etc.: Reagents and water are as shown below:a) Water: Water of A3 specified in JIS K 0557.
b) Hydrochloric acid: A JIS Guaranteed Reagent specified in JIS K 8180 or a reagent ofequivalent quality.
c) Zinc standard solution (0.1 mg/mL): A zinc standard solution (0.1 mg/mL) for atomic
absorption traceable to a specified reference material (national measurement standard)
according to Article 134 of the Measurement Act.
d) Zinc standard solutions (0.5 µg - 5 µg/mL) for the calibration curve preparation (1):Transfer 2.5 mL - 25 mL of zinc standard solution (0.1 mg/mL) to 500-mL volumetricflasks step-by-step, and add hydrochloric acid (1+23) to the marked line.
e) Blank test solution for the calibration curve preparation (1): Hydrochloric acid
(1+23) used in the procedure in d).
Note (1) This is an example of preparation; prepare an amount as appropriate.
(3) Instruments: Instruments are as follows:a) Rotary shaker: A rotary shaker that can rotate upside down a 500-mL volumetric flask
at 30 - 40 revolutions/min.
b) Flame atomic absorption spectrometer: An atomic absorption spectrometer specified
in JIS K 0121 with the background correction (2) function.
1) Light source: A zinc hollow cathode lamp (when the continuous source method as
the background correction method is used, the light source is a deuterium lamp.)
2) Gas: Gas for heating by flame(i) Fuel gas: acetylene(ii) Auxiliary gas: Air sufficiently free of dust and moisture.
Note (2) There are the continuous source method, the Zeeman method, the non-resonancespectrum method, and the self-reversal method, etc.
(4) Test procedures(4.1) Extraction: Conduct extraction as shown below.
a) Weigh 5.00 g of an analytical sample and transfer to a 500-mL volumetric flask.
b) Add about 400 mL of water, and shake to mix at 30 - 40 revolutions/min for about 30
minutes.
c) Add water up to the marked line.
d) Filter with Type 3 filter paper to make the sample solution.
Comment 1 In the procedure in a), it is also allowed to weigh 2.50 g of the analyticalsample and transfer to a 250-mL volumetric flask.
Comment 2 The procedure in (4.1) is the same as the procedure in (4.1) in 4.10.2.a,4.13.1.a and 4.14.1.a.
Comment 3 The sample solution prepared in (4.1) in 4.2.4.a can also be used.
212

(4.2) Measurement: Conduct measurement according to JIS K 0121 and as shown below.
Specific measurement procedures are according to the operation method of the atomic
absorption spectrometer used in measurement.
a) Measurement conditions for the atomic absorption spectrometer: Set up themeasurement conditions for the atomic absorption spectrometer considering thefollowing:
Analytical line wavelength: 213.9 nmb) Calibration curve preparation
1) Spray the zinc standard solutions for the calibration curve preparation and the blanktest solution for the calibration curve preparation into a flame, and read theindicated value at a wavelength of 213.9 nm.
2) Prepare a curve for the relationship between the zinc concentration and theindicated value of the zinc standard solutions for the calibration curve preparationand the blank test solution for the calibration curve preparation.
c) Sample measurement
1) Transfer a predetermined amount of the sample solution (the equivalents of 0.05 mg- 0.5 mg as Zn) to a 100-mL volumetric flask.
2) Add about 25 mL of hydrochloric acid (1+5), and add water up to the marked line.
3) Conduct similarly as in b) 1) to read the indicated value.
4) Obtain the zinc content from the calibration curve, and calculate the water-soluble
zinc (W-Zn) in the analytical sample.
Comment 4 Table 1 shows results from a simultaneous analysis with the same samples(proficiency testing and external quality control testing) hosted by the Japan
Fertilizer Quality Assurance Committee analyzed by the robust method.
Table 1 Results and analysis results from simultaneous analysis with the same samples
1)
for water-soluble zinc hosted by the Japan Fertilizer Quality Assurance Committee
5)2)
4)RSDrob
NIQR
Number of Median ΜYear
Sample3)3)laboratories
(%)
(%)
(%)
Fluid mixed
75
0.05860.0023
3.7
fertilizer
1) Proficiency testing and external quality control testing2) Median (M) agrees with the mean in normal distribution.
3) Mass fraction4) Robust standard deviation (NIQR) agrees with standard deviation in normal distribution.
5) RSDrob stands for relative standard deviation obtained from the robust methodby the formula shown below:
RSDrob = (NIQR/M) × 100
2012

Reference
1) Masayoshi KOSHINO: Second Revision of The Methods of Analysis of Fertilizers(Details), p.192 - 194, Yokendo, Tokyo (1988)

213

(5)

Flow sheet for water-soluble zinc: The flow sheet for water-soluble zinc in fertilizers
is shown below:
5.00 g analytical sample
500-mL volumetric flask.
← About 400 mL of water,
Shaking to mix
Rotary shaker (30 - 40 revolutions/minutes), for 30 minutes← Water (up to the marked line)
Filtration
Type 3 filter paper
Aliquot
100-mL volumetric flasks(predetermined amount)← 25 mL of hydrochloric acid (1+5)← Water (up to the marked line)
Measurement
Atomic absorption spectrometer (213.9 nm)
Figure Flow sheet for water-soluble zinc in fertilizers.

2144.10 Copper
4.10.1 Total copper
4.10.1.a Flame atomic absorption spectrometry(1) Summary
This test method is applicable to fertilizers.
Pretreat an analytical sample with incineration and nitric acid-hydrochloric acid (1+3), andthen spray in an acetylene-air flame, and measure the atomic absorption with copper at awavelength of 324.8 nm to quantify total copper.
(2) Reagents, etc.: Reagents and water are as shown below:a) Water: Water of A3 specified in JIS K 0557.
b) Nitric acid: A reagent of harmful metal analysis grade, microanalysis grade orequivalents.
c) Hydrochloric acid: A reagent of harmful metal analysis grade, microanalysis grade orequivalents.
d) Copper standard solution (0.1 mg/mL): A copper standard solution (Cu 0.1 mg/mL)
for atomic absorption traceable to a specified reference material (national measurement
standard) according to Article 134 of the Measurement Act.
e) Copper standard solutions (0.5 µg - 5 µg/mL) for the calibration curve
preparation(1): Transfer 2.5 mL - 25 mL of copper standard solution (0.1 mg/mL) to
500-mL volumetric flasks step-by-step, and add hydrochloric acid (1+23) up to themarked line.
f) Blank test solution for the calibration curve preparation (1): Hydrochloric acid
(1+23) used in the procedure in e).
Note (1) This is an example of preparation; prepare an amount as appropriate.
(3) Instruments: Instruments are as shown below:a) Flame atomic absorption spectrometer: An atomic absorption spectrometer specified
in JIS K 0121 with the background correction (2) function.
1) Light source: A copper hollow cathode lamp (when the continuous source method
as the background correction method is used, the light source is a deuterium lamp.)
2) Gas: Gas for heating by flame(i) Fuel gas: acetylene(ii) Auxiliary gas: Air sufficiently free of dust and moisture.
b) Electric furnace: An electric furnace that can be adjusted to 450 ºC ± 5 ºC.
c) Hot plate or sand bath: A hot plate whose surface temperature can be adjusted up to
250 ºC. Adjust the amounts of gas and silica sand of a sand bath so that the sand bathtemperature can be set to 250 ºC.
Note (2) There are the continuous source method, the Zeeman method, the non-resonancespectrum method, and the self-reversal method, etc.
(4) Test procedures(4.1) Extraction: Conduct extraction as follows:a) Weigh 5.00 g of an analytical sample, and put it in a 200-mL - 300-mL tall beaker.
b) Put the tall beaker in an electric furnace, and heat gently to char(3).
c) Ignite at 450 ºC ± 5 ºC to incinerate (4).
d) After standing to cool, moisten the residue with a small amount of water, and add about
10 mL of nitric acid and about 30 mL of hydrochloric acid.
e) Cover the tall beaker with a watch glass, and heat on a hot plate or a sand bath to digest.
215

Slightly move the watch glass (5), and continue heating on the hot plate or sand bath toconcentrate until nearly dried up.
g) After standing to cool, add 25 mL - 50 mL of hydrochloric acid (1+5)(6) to the digest,cover the tall beaker with the watch glass, and heat quietly to dissolve.
h) After standing to cool, transfer the solution to a 100-mL - 200-mL volumetric flask withwater, add water up to the marked line, and filter with Type 3 filter paper to make thesample solution.
i) As a blank test, conduct the procedures in b) - h) using another tall beaker to prepare theblank test solution.
f)Note (3) Example of the charring procedure: Heat at about 250 ºC until smoke is nolonger formed.
(4) Example of ignition period: 8 - 16 hours(5) The watch glass can be removed.
(6) Add hydrochloric acid (1+5) so that the hydrochloric acid concentration of thesample solution will be hydrochloric acid (1+23). For example, when a 100-mL
volumetric flask is used in the procedure in h), about 25 mL of hydrochloric acid(1+5) should be added.
Comment 1 Do not conduct the procedures in (4.1) b) - c) in the case of fertilizers notcontaining organic matters.
Comment 2 The procedures in (4.1) are the same as in (4.1) in 4.9.1.a.
Comment 3 The sample solution prepared in (4.1.2) in 4.2.1.a can also be used.
(4.2) Measurement: Conduct measurement according to JIS K 0121 and as shown below.
Specific measurement procedures are according to the operation method of the atomic
absorption spectrometer used in measurement.
a) Measurement conditions for the atomic absorption spectrometer: Set up themeasurement conditions for the atomic absorption spectrometer considering thefollowing:
Analytical line wavelength: 324.8 nmb) Calibration curve preparation
1) Spray the copper standard solutions for the calibration curve preparation and theblank test solution for the calibration curve preparation into a flame, and read theindicated value at a wavelength of 324.8 nm.
2) Prepare a curve for the relationship between the copper concentration and theindicated value of the copper standard solutions for the calibration curve
preparation and the blank test solution for the calibration curve preparation.
c) Sample measurement
1) Subject the sample solution (7) to the same procedure as in b) 1) to read theindicated value.
2) Subject the blank test solution to the same procedure as in b) 1) to read theindicated value, and correct the indicated value obtained for the sample solution.
3) Obtain the copper content from the calibration curve, and calculate the total copperin the analytical sample.
Note (7) If there is a possibility that the copper concentration in the sample solution will
exceed the maximum limit of the calibration curve, dilute a predeterminedamount with hydrochloric acid (1+23).

216

Comment 4 The copper concentration in the analytical sample can also be corrected bysubjecting the blank test solution to the same procedures as in 1) and 3) toobtain the copper content in the blank test solution.
Comment 5 Replicate testing was conducted using human waste sludge fertilizer (2samples), calcined sludge fertilizer (2 samples) and composted sludge
fertilizer (2 samples); as a result, repeatability in the range of 210 mg/kg - 830
mg/kg total copper (mean determined value) was 0.6 % - 3.7 % relative
standard deviation.
References
1) Masayoshi KOSHINO: Second Revision of The Methods of Analysis of Fertilizers(Details), p.254 - 255, Yokendo, Tokyo (1988)
2) Kimie KATO, Masayuki YOSHIMOTO and Yuji SHIRAI: Systematization of
Determination Methods of Major Components in Sludge Fertilizer, Compost andOrganic Fertilizer, Research Report of Fertilizer, Vol.3, 2010. (107 - 116)

217

(5)

Flow sheet for total copper: The flow sheet for total copper in fertilizers is shown
below:
5.00 ganalytical sample
Charring
Incineration

200-mL - 300-mL tall beaker.
Heat gently
Ignite at 450 ºC ± 5 ºC

Standing to coolRoom temperature← A small amount of water← About 10 mL of nitric acid
← About 30 mL of hydrochloric acid
Heating
Cover with a watch glass, and digest
Heating

Slightly move a watch glass to remove acid

Standing to coolRoom temperature← 25 mL - 50 mL of hydrochloric acid (1+5)
Heating
Cover with a watch glass, and dissolve
Standing to coolRoom temperature

Transfer
100-mL - 200-mL volumetric flask, water← Water (up to the marked line)
Filtration
Type 3 filter paper
Measurement

Atomic absorption spectrometer (324.8nm)

Figure Flow sheet for total copper in sludge fertilizers

2184.10.1.b ICP Atomic Emission Spectrometry <Reference method>(1) Summary
The test method is applicable to sludge fertilizers, etc.
Pretreat an analytical sample with incineration, nitric acid - hydrochloric acid (1+3), introduce
it to ICP Atomic Emission Spectrometry (“ICP-AES”) and measure the emission with copperat a wavelength of 324.754 nm to quantify copper.
(2) Reagents, etc.: Reagents and water are as shown below:a) Water: Water of A3 specified in JIS K 0557.
b) Nitric acid: A reagent of harmful metal analysis grade, microanalysis grade orequivalents.
c) Hydrochloric acid: A reagent of harmful metal analysis grade, microanalysis grade orequivalents.
d) Copper standard solution (0.1 mg/mL): Copper standard solution (0.1 mg/mL) for
atomic absorption spectrometry traceable to a specified reference material (national
measurement standard) according to Article 134 of the Measurement Act.
e) Copper standard solutions (2.5 µg/ mL) (1):Dilute a predetermined amount of copper standard solution (0.1 mg/mL) withhydrochloric acid (1+23) to prepare copper standard solution (2.5 µg/mL).
Note (1) This is an example of preparation; prepare an amount as appropriate.
(3) Instruments: Instruments are as shown below:a) ICP Atomic Emission Spectrometer: An atomic emission spectrometer specified in
JIS K 0116.
1) Gas: Argon gas specified in JIS K 1105 of no less than 99.5 % (volume fraction) inpurityb) Electric furnace: An electric furnace that can keep the test temperature at  5 ºCc) Hot plate or sand bath: A hot plate whose surface temperature can be adjusted up to
250 ºC. Adjust the amounts of gas and silica sand of a sand bath so that the sand bathtemperature can be set to 250 ºC.
(4) Test procedures(4.1) Extraction: Conduct extraction as follows:a) Weigh 5.00 g of an analytical sample, and put it in a 200-mL - 300-mL tall beaker.
b) Put the tall beaker in an electric furnace, and heat gently to char(2).
c) Ignite at 450 ºC ± 5 ºC to incinerate (3).
d) After standing to cool, moisten the residue with a small amount of water, and add about
10 mL of nitric acid and about 30 mL of hydrochloric acid.
e) Cover the tall beaker with a watch glass, and heat on a hot plate or a sand bath to digest.
f) Slightly move the watch glass (4), and continue heating on the hot plate or sand bath toconcentrate until nearly dried up.
g) After standing to cool, add 25 mL - 50 mL of hydrochloric acid (1+5) (5) to the digest,cover the tall beaker with the watch glass, and heat quietly to dissolve.
h) After standing to cool, transfer to a 100-mL - 200-mL volumetric flask with water, add
water up to the marked line, and filter with Type 3 filter paper to make the sample
solution.
i) As a blank test, conduct the procedures in b) - h) using another tall beaker to prepare theblank test solution.

219

Note (2) Example of the charring procedure: Heat at about 250 ºC until smoke is nolonger formed.
(3) Example of ignition period: 8 - 16 hours(4) The watch glass can be removed.
(5) Add hydrochloric acid (1+5) so that the hydrochloric acid concentration of thesample solution will be hydrochloric acid (1+23). For example, when a 100-mL
volumetric flask is used in the procedure in h), about 25 mL of hydrochloric acid(1+5) should be added.
Comment 1 Do not conduct the procedures in (4.1) b) - c) in the case of fertilizers notcontaining organic matters.
Comment 2 The procedures in (4.1) are the same as in (4.1) in 4.9.1.a.
(4.2) Measurement: Conduct measurement (Standard Addition Method) according to JIS K
0116 and as shown below. Specific measurement procedures are according to theoperation method of the ICP Atomic Emission Spectrometer used in measurement.
a) Measurement conditions for the ICP Atomic Emission Spectrometer: Set up themeasurement conditions for the ICP Atomic Emission Spectrometer considering thefollowing:
Analytical line wavelength: 324.754 nmb) Calibration curve preparation and sample measurement.
1) Put 5mL of sample solution to three 10-mL volumetric flasks respectively.
2) Add 2mL and 4 mL (2.5 μg/mL) of copper standard solution to volumetric flasks of1) above, then add hydrochloric acid (1+23) to the marked line to make the sample
solution of Standard Addition Method.
3) Add hydrochloric acid (1+23) to the marked line of the remaining volumetric flaskof 1) above to make the sample solution without standard solution.
4) Spray the sample solution of Standard Addition Method and the sample solution
without standard solution into the induction plasma, and read the indicated value ata wavelength of 324.754 nm.
5) Transfer 5mL of the blank test solution to a 10-mL volumetric flask, conduct thesame procedures as in 3) - 4) to read the indicated value, and correct the indicatedvalue obtained from the respective sample solutions.
6) Prepare a curve for the relationship between the added copper concentration and thecorrected indicated value of the sample solution for Standard Addition Method andthe sample solution without standard solution.
7) Obtain the copper content from the intercept of the calibration curve to calculate theconcentration of copper in the analytical sample.
Comment 3 The copper concentration in the analytical sample can also be corrected bysubjecting the blank test solution to the same procedures as in 1) - 4) and 6) 7) to obtain the copper content in the blank test solution.
Comment 4 Simultaneous measurement of multiple elements by ICP-AES is available. In
this case, mix a predetermined amount of copper standard solution (0.1mg/mL), zinc standard solution (0.1 mg/mL), cadmium standard solution (0.1mg/mL), nickel standard solution (0.1 mg/mL), chromium standard solution(0.1 mg/mL) and lead standard solution (0.1 mg/mL), dilute withhydrochloric acid (1 +23) to prepare mixture standard solution (Cu 25μg/mL,
Zn 25 μg/mL, Cd 0.25μg/mL, Ni 2.5 μg/mL, Cr 2.5 μg/mL and Pb 2.5
μg/mL) (1) Use the mixture solution instead of copper standard solution (25μg/mL) in (4.2) b) 2). After that, conduct the same procedures as in (4.2) b) to220

calculate the concentration of respective elements in the analytical sample.
Note that the wavelength for measurement of respective elements is Cu
324.754 nm, Zn 206.191 nm, Cd 228.802 nm, Ni 231.604 nm, Cr 205.552 nm
and Pb 220.351 nm.
In addition, the respective element concentrations for respective standard
addition samples are shown in the below Table.
Table The additive amount in mixture standard solution
and the additive concentration of respective elements in respective sample solutions
Additive amount
Cd
Pb
Ni
Cr
Cu
Zn
(mL) in mixture(mg/L) (mg/L) (mg/L) (mg/L) (mg/L) (mg/L)
standard solution
Sample solution without
standard solution

0

0

0

0

0

0

0

Sample solution 1 ofStandard Addition Method

2

0.05

0.5

0.5

0.5

5

5

Sample solution 2 ofStandard Addition Method

4

0.1

1

1

1

10

10

Comment 5 For each one sample of sewage sludge fertilizer, human waste sludge fertilizer,
industrial sludge fertilizer, mixture sludge fertilizer, calcined sludge fertilizer
and composted sludge fertilizer, the repeatability obtained from triplicatesmeasurement is 0.6 % - 1.8 % relative standard deviation. Additionally, theminimum limit of quantification of the test method is about 3 mg/kg.
Reference
1) Masahiro ECHI, Tomoe INOUE, Megumi TABUCHI and Tetuya NOMURA:
Simultaneous Determination of Cadmium, Lead, Nickel, Chromium, Copper and Zincin Sludge Fertilizer using Inductively Coupled Plasma-Atomic Emission Spectrometry(ICP-AES), Research Report of Fertilizer, Vol.4, 2011. (36 - 48)

221

(5)

Flow sheet for copper: The flow sheet for copper in fertilizers is shown below:
5.00 ganalytical sample
Charring
Incineration

200-mL - 300-mL tall beaker.

Heat gently
Ignite at 450 ºC ± 5 ºC

Standing to coolRoom temperature← A small amount of water← About 10 mL of nitric acid
← About 30 mL of hydrochloric acid
Heating
Cover with a watch glass, and digest for 30 minutes
Heating

Slightly move a watch glass to remove acid

Standing to coolRoom temperature← 25 mL - 50 mL of hydrochloric acid (1+5)
Heating
Cover with a watch glass, and dissolve
Standing to coolRoom temperature

Transfer
100-mL - 200-mL volumetric flask, water← Water (up to the marked line)
Filtration
Type 3 filter paper
Aliquot 5 mL
10-mL volumetric flask, 3 flasks← 0, 2 and 4 mL of copper standard solution (25 μg / mL)← Hydrochloric acid (1+23) ( up to the marked line)
Measurement
ICP Atomic Emission Spectrometer (324.754 nm)
Figure Flow sheet for copper in fertilizers

222

4.10.2 Water-soluble copper
4.10.2.a Flame atomic absorption spectrometry(1) Summary
This test method is applicable to fertilizers that indicate copper content as a response modifier.
Extract by adding water to an analytical sample, spray in an acetylene-air flame and measure
the atomic absorption with copper at a wavelength of 324.8 nm to quantify water-soluble
copper (W-Cu).
(2) Reagents, etc.: Reagents and water are as shown below:a) Water: Water of A3 specified in JIS K 0557.
b) Hydrochloric acid: A JIS Guaranteed Reagent specified in JIS K 8180 or a reagent ofequivalent quality.
c) Copper standard solution (0.1 mg/mL): A copper standard solution (0.1 mg/mL) for
atomic absorption traceable to a specified reference material (national measurement
standard) according to Article 134 of the Measurement Act.
d) Copper standard solutions (0.5 µg - 5 µg/mL) for the calibration curve preparation(1): Transfer 2.5 mL - 25 mL of copper standard solution (0.1 mg/mL) to 500-mL
volumetric flasks step-by-step, and add hydrochloric acid (1+23) up to the marked line.
e) Blank test solution for the calibration curve preparation (1): Hydrochloric acid
(1+23) used in the procedure in d).
Note (1) This is an example of preparation; prepare an amount as appropriate.
(3) Instruments: Instruments are as follows:a) Rotary shaker: A rotary shaker that can rotate upside down a 500-mL volumetric flask
at 30 - 40 revolutions/min.
b) Flame atomic absorption spectrometer: An atomic absorption spectrometer specified
in JIS K 0121 with the background correction (2) function.
1) Light source: A copper hollow cathode lamp (when the continuous source method
as the background correction method is used, the light source is a deuterium lamp.)
2) Gas: Gas for heating by flame(i) Fuel gas: acetylene(ii) Auxiliary gas: Air sufficiently free of dust and moisture.
Note (2) There are the continuous source method, the Zeeman method, the non-resonancespectrum method, and the self-reversal method, etc.
(4) Test procedures(4.1) Extraction: Conduct extraction as shown below.
a) Weigh 5.00 g of an analytical sample and transfer to a 500-mL volumetric flask.
b) Add about 400 mL of water, and shake to mix at 30 - 40 revolutions/min for about 30
minutes.
c) Add water up to the marked line.
d) Filter with Type 3 filter paper to make the sample solution.
Comment 1 In the procedure in a), it is also allowed to weigh 2.50 g of the analyticalsample and put it into a 250-mL volumetric flask.
Comment 2 The procedure in (4.1) is the same as the procedure in (4.1) in 4.9.2.a.

223

(4.2) Measurement: Conduct measurement according to JIS K 0121 and as shown below.
Specific measurement procedures are according to the operation method of the atomic
absorption spectrometer used in measurement.
a) Measurement conditions for the atomic absorption spectrometer: Set up themeasurement conditions for the atomic absorption spectrometer considering thefollowing:
Analytical line wavelength: 324.8 nmb) Calibration curve preparation
1) Spray the copper standard solutions for the calibration curve preparation and theblank test solution for the calibration curve preparation into a flame, and read theindicated value at a wavelength of 324.8 nm.
2) Prepare a curve for the relationship between the copper concentration and theindicated value of the copper standard solutions for the calibration curve
preparation and the blank test solution for the calibration curve preparation.
c) Sample measurement
1) Transfer a predetermined amount of the sample solution (the equivalents of 0.05 mg- 0.5 mg as Cu) to a 100-mL volumetric flask.
2) Add about 25 mL of hydrochloric acid, and add water up to the marked line.
3) Conduct similarly as in b) 1) to read the indicated value.
4) Obtain the copper content from the calibration curve, and calculate thewater-soluble copper (W-Cu).
Comment 3 Table 1 shows results from a simultaneous analysis with the same samples(proficiency testing and external quality control testing) hosted by the Japan
Fertilizer Quality Assurance Committee analyzed by the robust method.
Table 1 Results and analysis results from simultaneous analysis with the same samples
for water-soluble cupper hosted by Japan Ferttilizer Quality Assuarance Committee 1)
Number of Median Μ2) NIQR4) RSDrob5)
Year
Sample3)3)laboratories
(%)
(%)
(%)
2012
Fluid mixed fertilizer
76
0.05460.00142.5
1) Proficiency testing and external quality control testing2) Median (M) agrees with the mean in normal distribution.
3) Mass fraction4) Robust standard deviation (NIQR) agrees with standard deviation in normal distribution.
5) RSDrob stands for relative standard deviation obtained from the robust methodby the formula shown below:
RSDrob = (NIQR/M) × 100

Reference
1) Masayoshi KOSHINO: Second Revision of The Methods of Analysis of Fertilizers(Details), p.254 - 255, Yokendo, Tokyo (1988)

224

(5)

Flow sheet for water-soluble copper: The flow sheet for water-soluble copper in
fertilizers is shown below:
5.00 g analytical sample
500-mL volumetric flask.
← Water, about 400 mL
Shaking to mix
Rotary shaker (30 - 40 revolutions/min), for 30 minutes← Water (up to the marked line)
Filtration
Type 3 filter paper
Aliquot
100-mL volumetric flask(predetermined amount)← 25 mL of hydrochloric acid (1+5)← Water (up to the marked line)
Measurement
Atomic absorption spectrometer (324.8 nm)
Figure Flow sheet for water-soluble cupper in fertilizers.

225

4.11 Organic carbon and carbon-nitrogen ratio
4.11.1 Organic carbon
4.11.1.a Dichromate oxidation(1) Summary
This test method is applicable to sludge fertilizers and compost, etc.
Add a potassium dichromate-sulfuric acid solution to an analytical sample and heat to oxidizeorganic carbon with potassium dichromate. Quantify unconsumed potassium dichromate byoxidation-reduction titration to obtain organic carbon (O-C). This test method is also referred
to as the Method of Tyulin.
(2) Reagents: Reagents are as shown below:a) Sulfuric acid: A JIS Guaranteed Reagent specified in JIS K 8951 or a reagent ofequivalent quality.
b) 0.2 mol/L ammonium iron (II) sulfate solution (1): Weigh 80 g of ammonium iron (II)sulfate hexahydrate specified in JIS K 8979 into a 2,000-mL beaker, and add 1,000 mLof sulfuric acid (1+50) to dissolve.
Standardization: Grind potassium dichromate reference material for volumetricanalysis specified in JIS K 8005 in an agate mortar to powder, heat at 150 ºC ± 2 ºC for
1 hour, let it stand to cool in a desiccator, and then transfer about 1 g to a weighing dish,and weigh the mass to the order of 0.1 mg. Dissolve in a small amount of water, transfer
to a 100-mL volumetric flask, and add water up to the marked line to make thepotassium dichromate standard solution(1) (2). On each day to use 0.2 mol/L ammonium
iron (II) sulfate solution, transfer 10 mL of the potassium dichromate standard solution
to a 100-mL Erlenmeyer flask, add about 5 mL of sulfuric acid (1+2), and then conduct
the procedures in (4.2) b) - c), and calculate the factor of 0.2 mol/L ammonium iron (II)sulfate solution by the following formula:
Factor (f) of 0.2 mol/L ammonium iron (II) sulfate solution= W × (A/100) × (6/294.18) × (V1/V2) × (1,000/V3) /C= (W × A/V3) × (30/294.18)
W:
A:
V1:
V2:
V3:
C:

Mass (g) of potassium dichromate weighed
Purity (% (mass fraction)) of potassium dichromate
Volume (10 mL) of potassium dichromate solution transferred
Constant volume (100 mL) of potassium dichromate solution
Volume (mL) of 0.2 mol/L ammonium iron (II) sulfate solution needed fortitration
Set concentration (0.2 mol/L) of 0.2 mol/L ammonium iron (II) sulfate
solutionc) Potassium dichromate-sulfuric acid solution (1): Weigh 40 g of potassium dichromate
specified in JIS K 8517 to a 3,000-mL beaker. Add 1,000 mL of water to dissolve, andfurther add gradually 1,000 mL of sulfuric acid while cooling and mixing.
d) N-Phenylanthranilic acid solution: Dissolve 0.2 g of N-phenylanthranilic acid of noless than 98 % (mass fraction) in purity and 0.2 g of sodium carbonate specified in JIS
K 8625 in a small amount of water, and add water to make 100 mL.
Note (1) This is an example of preparation; prepare an amount as appropriate.

226

(2) This corresponds to the standard potassium dichromate solution (0.2 M (1/6
K2Cr2O7) solution) in 7.1 B 1) in the Official Methods of Analysis of Fertilizers(1992).
(3) Apparatus and instruments: Apparatus and instruments are as follows:a) Hot plate: A hot plate whose surface temperature can be adjusted up to 250 ºC.
b) Sample digestion flask (3): A 100-mL borosilicate glass volumetric flask 100 mL (180mm total height, 13 mm mouth diameter)
Note (3) Distinguish the volumetric flask used in digestion as a sample digestion flask
and do not use it for any other purposes.
(4) Test procedures(4.1) Dichromate oxidation: Conduct oxidation as follows:a) Weigh 0.05 g of an analytical sample to the order of 0.1 mg (4), and transfer to a sample
digestion flask.
b) Add 25 mL of potassium dichromate-sulfuric acid solution.
c) Heat on a hot plate at 200 ºC until organic matters are completely digested.
d) After standing to cool, fill up with water to 100 mL to make the sample solution.
e) As a blank test, conduct the procedures in b) and d) using another sample digestion
flask to prepare the blank test solution.
Note (4) Up to about 28 mg as organic carbon (O-C).
(5) Heat for no less than 1 hour after boiling(4.2) Measurement: Conduct measurement as shown below.
a) Transfer 20 mL of the sample solution to a 100-mL Erlenmeyer flask.
b) Add 0.2 mol/L ammonium iron (II) sulfate solution drop-by-drop until the brown colorof dichromate ion almost disappears from the sample solution.
c) Add about 0.25 mL of N-phenylanthranilic acid solution(6), and titrate with 0.2 mol/Lammonium iron (II) sulfate solution until the color of the solution changes from dark
red-purple to blue-green.
d) Transfer 20 mL of the blank test solution to a 100-mL Erlenmeyer flask, and conduct theprocedures in b) - c) to titrate.
e) Calculate the organic carbon (O-C) in the analytical sample by the following formula:
Organic carbon (% (mass fraction)) in the analytical sample= (V4 - V5) × C × f × (12.011/4)/W × (100/1,000) × (V6/V7)= (V4 - V5) × f × (12.011/40)/W
V4:
V5:
C:
f:
V6:
V7:

Volume (mL) of 0.2 mol/L ammonium iron (II) sulfate solution needed forthe titration of the blank test solution
Volume (mL) of 0.2 mol/L ammonium iron (II) sulfate solution needed forthe titration of the sample solution
Set concentration (0.2 mol/L) of 0.2 mol/L ammonium iron (II) sulfate
solution
Factor of 0.2 mol/L ammonium iron (II) sulfate solution
Predetermined volume (100 mL) of the sample solution and the blank test
solution in (4.1) d)
Transferred volume (20 mL) of the sample solution and the blank test
solution subjected to titration in (4.2) a) and (4.2) d)
227

W:

Mass (g) of an analytical sample

Note (6) About 5 drops with a 1-mL - 2-mL Komagome pipette. Add the same volume tothe sample solution and the blank test solution.
Comment 1 Sample an analytical sample from a test sample prepared in 2.2.3 Grinding(3) Procedure (3.1) b) by grinding with a mill until it completely passes
through a sieve of 500 µm aperture.
Comment 2 Replicate testing was conducted using composted sludge fertilizer (2samples), human waste sludge fertilizer (1 sample), industrial sludge fertilizer(2 samples), compost (3 samples) and animal excreta (1 sample); as a result,the mean was in the range of 14.2 % - 50.7 % (mass fraction), and thestandard deviation and relative standard deviation were 0.04 % - 0.6 % (massfraction) and 0.1 % - 2.6 %, respectively.
Additionally, the minimum limit of quantification of this test method is about
1.5 % (mass fraction).
Reference
1) Yuji SHIRAI, Yuko SEKINE, and Toshiaki HIROI: Validation of Determination Method
for Organic Form Carbon in Sludge Fertilizer and Compost, Research Report of
Fertilizer, Vol.3, 2010. (117 - 122)
(5)

Flow sheet for organic carbon: The flow sheet for organic carbon in sludge fertilizers
and compost, etc. is shown below:
0.05 gWeigh to the order of 0.1 mg into a sample digestion flask.
analytical sample(Up to about 28 mg as organic carbon.)← 25 mL potassium dichromate-sulfuric acid solution
Heating
Boil for about 1 hour, 200 ºC
Standing to coolRoom temperature← Water (fill up to100 mL)
100-mL Erlenmeyer flask
20 mL aliquot
Addingdrop by drop

0.2 mol/L Ammonium iron (II) sulfate solution(until the brown color of dichromate ion

almost disappears from the solution)← About 0.25 mL N-phenylanthranilic acid solution
Titration

0.2 mol/L ammonium iron (II) sulfate solution(until the solution becomes blue-green)

Figure Flow sheet for organic carbon in sludge fertilizers and compost, etc.

228

4.11.1.b Combustion method(1) Summary
This test method is applicable to compost and sludge fertilizers.
Drop hydrochloric acid (1+3) to an analytical sample and evaporate inorganic carbon as
carbon dioxide, then thermally decompose carbon compounds using a total nitrogen-total
carbon analyzer by the combustion method to measure carbon dioxide gas with a thermal
conductivity detector.
(2) Reagents: Reagents are as shown below:a) Sea sand: Particle diameter 425 µm – 850 µmb) Hydrochloric acid: A JIS Guaranteed Reagent specified in JIS K 8180 or a reagent ofequivalent quality.
Comment 1 Sea sand (particle diameter 425 µm – 850 µm) is commercially sold by Wako
Pure Chemical Industries, Ltd. and YONETAMA YAKUHIN KOGYO Co.,Ltd.
(3) Instruments: Instruments are as shown below:a) Total nitrogen-total carbon analyzer by the combustion method: A total
nitrogen-total carbon analyzer configured on the basis of the principle of the combustion
method (modified Dumas’ method).
1) Turn on the total nitrogen-total carbon analyzer by the combustion method (1), andadjust so that stable indicated values can be obtained.
(i) Combustion gas: Oxygen having purity no less than 99.99 % (volume percentage)(ii) Carrier gas: Helium having purity no less than 99.99 % (volume percentage)b) Hot plate: A hot plate whose surface temperature can be adjusted up to 250 ºC.
c) Drying apparatus: A drying apparatus that can adjust test temperature to ± 2 ºC.
Note (1) The setup of the program and the parameters of the analyzer are according to thespecification and the operation method of the total nitrogen-total carbon analyzer
by the combustion method used.
(4)

Test procedures: Conduct measurement as shown below. However, confirm that there
is no difference from the measured value of organic carbon obtained in advanceaccording to 4.11.1.a by using an analytical sample.

(4.1) Hydrochloric acid treatmenta) Weigh 0.05 g of an analytical sample to the order of 0.1 mg, and transfer to a container
for combustion.
b) Cover the analytical sample with about 0.2 g of sea sand and moisten the analyticalsample by dropping a few drops of water.
c) After dropping 0.5 mL - 0.7 mL of hydrochloric acid (1+3) little by little (2), drop about
0.3 mL of water (3) (4).
d) Heat a container for combustion on a hot plate at 100 ºC for 90 minutes to make it dryup.
e) Put the container for combustion into a drying apparatus and heat for 30 minutes (5).
f) After heating, let it stand to cool to make a test sample.
Note (2) The additive amount of hydrochloric acid (1+3) is merely a target. It is enoughto make the whole analytical sample come into contact with hydrochloric acid.
Let it stand for a short time in the case of producing bubbles.
229

(3) Shake calmly the container for combustion to make the analytical sample
completely come into contact with hydrochloric acid.
(4) Let it stand for a short time in the case of producing bubbles when droppinghydrochloric acid (1+3).
(5) Remove hydrochloric acid completely
Comment 2 Sample an analytical sample from a test sample prepared in 2.2.3 Grinding(3) Procedure (3.1) b) by grinding with a mill until it completely passes
through a sieve of 500 µm aperture.
Comment 3 When it is confirmed that, for example, the volatilization of hydrogenchloride is not detected by using a test paper, etc. and hydrochloric iscompletely removed in the procedure d), the procedure e) can be skipped.
(4.2) Measurement: Specific measurement procedures are according to the operation methodof a total nitrogen-total carbon analyzer by the combustion method.
a) Measurement conditions for the total nitrogen-total carbon analyzer by the
combustion method: Set up the total nitrogen-total carbon analyzer considering thefollowing:
Combustion temperature: No less than 870 ºCb) Calibration curve preparation
1) Turn on the total nitrogen-total carbon analyzer by the combustion method (1), andadjust so that stable indicated values can be obtained.
2) Weigh a predetermined amount of the standard for calibration curves (6) to the orderof 0.1 mg into a combustion vessel.
3) Insert the combustion vessel into the total nitrogen-total carbon analyzer by the
combustion method, and read the indicated value.
4) Conduct the procedure in 3) for another combustion vessel for a blank test, and readthe indicated value.
5) Prepare a curve for the relationship between the carbon content and the indicatedvalue of the standard for calibration curves and the blank test for calibration curves.
c) Sample measurement
1) Insert the combustion vessel containing the test sample to the total nitrogen-total
carbon analyzer by the combustion method, and read the indicated value.
2) Obtain the carbon content from the calibration curve, and calculate organic carbon in
the analytical sample.
Note (6) Standard for calibration curves: DL-Aspartic acid (purity no less than 99 %(mass fraction)), EDTA (purity no less than 99 % (mass fraction)), hippuric acid(purity no less than 98 % (mass fraction)) or other reagents having equivalent
purity recommended by the total nitrogen-total carbon analyzer by the
combustion method used.
Comment 4 Replicate testing was conducted using sewage sludge fertilizer (1 sample),human waste sludge fertilizer (1 samples), industrial sludge fertilizer (1sample), calcined sludge fertilizer (1 sample) and composted sludge fertilizer(2 samples); as a result, the mean was in the range of 8.5 % - 46.0% (massfraction), and the repeatability standard deviation and relative repeatability
standard deviation were 0.1 % - 0.5% (mass fraction) and 0.2 % - 2.0%,
respectively.
Additionally, the minimum limit of quantification of the test method isabout 0.05 % (mass fraction).
230

Table 1 Results and statistical analysis results from a collaborative study
for the organic carbon method
Sample name

Number of

Mean2)

sr4)

RSDr5)sR6)

RSDR7)

laboratories1)

(%)3)

(%)3)

(%)

(%)3)

(%)

0.38

4.0

0.42

2.8

0.94

4.6

0.62

1.8

0.73

1.9

Calcined sludge
9
9.45
0.17
1.8
fertilizer
Industrial sludge
8
15.13
0.20
1.3
fertilizer
Compost
9
20.50
0.76
3.7
Human waste
8
34.96
0.07
0.2
sludge fertilizer
Composted
9
38.20
0.27
0.7
sludge fertilizer
1) Number of laboratories used in analysis
2) Total mean (number of laboratories × 2 samples replicate analysis)
3) Mass fraction
4) Repeatability standard deviation
5) Repeatability relative standard deviation
6) Reproducibility standard deviation
7) Reproducibility relative standard deviation

Reference
1) Aiko YANO, Satono AKIMOTO, and Yuji SHIRAI: Determination of Organic Carbon
in Sludge Fertilizer and Compost by Hydrochloric Acid-Treated Combustion Method,
Research Report of Fertilizer, Vol.9, 2013. (9 - 19)(5) Flow sheet for organic carbon: The flow sheet for organic carbon in compost andsludge fertilizers is shown below:
0.05 ganalytical sample

Weigh to the order of 0.1 mg into a combustion vessel.

← Cover the analytical sample with 0.2 g of sea sandand drop a few drops of water.
← Drop 0.5mL - 0.7mL of hydrochloric acid (1+3) little by little← Drop 0.3mL of water
HeatingHeat to make it dry for 90 minutes, at 100 ºC

DryingHeat to make it dry for 30 minutes, at 105 ºC

Measure the content oforganic carbon
with a total nitrogentotal carbon analyzer
Figure Flow sheet for organic carbon by the combustion method
231

Reference: Chromatograms of a standard calibration curve and an analytical sample areshown below

1) Total carbon in a standard calibration curve (DL-Aspartic acid)2) Total carbon in an analytical sample (sludge fertilizer)
Reference figures

Chromatograms of organic carbon.

Measurement conditions for total nitrogen-total carbon analyzer by the combustion method
Combustion gas: Highly pure oxygen, purity no less than 99.99995 % (volume fraction),flow rate 200 mL/min
Carrier gas: Highly pure helium, purity no less than 99.9999 % (volume fraction), flow rate
80 mL/min
Separation column: A silica gel stainless column (Length: 1 m)
Detector: Thermal Conductivity Detector (TCD)
Measurement cycle: Purge time = 60 seconds, circulation combustion time = 300 seconds,
measurement time = 270 seconds
Current value of Detector: 160 mA
Temperature conditions: Reaction furnace temperature: 870 ºC
Reduction furnace temperature: 600 ºC
Column oven temperature: 70 ºC
Detector temperature: 100 ºC
232

4.11.2

Carbon-nitrogen ratio(1) Summary
Calculate carbon-nitrogen ratio (CN ratio) by dividing the organic carbon obtained in 4.11.1
by the total nitrogen obtained in 4.1.1.
(2) Calculation of carbon-nitrogen ratioa) Calculate the carbon-nitrogen ratio in an analytical sample by the following formula:
Carbon-nitrogen ratio in an analytical sample= O-C/T-N
O-C: Organic carbon (% (mass fraction)) (1) in the analytical sample obtained in
4.11.1
T-N: Total nitrogen (% (mass fraction)) (1) in the analytical sample obtained in
4.1.1
Note (1) O-C and T-N use raw data without rounding numerical value

233

4.12 Sulfur
4.12.1 Total sulfur content
4.12.1.a Titration analysis <Reference method>(1) Summary
This test method is applicable to fertilizers mainly containing ferrous sulfate among sulfur andits compound.
Dissolve an analytical sample in water and diluted sulfuric acid, add phosphate and thentitrate with a standard potassium permanganate solution.
(2) Reagents, etc.: Reagents and water are as shown below.
a) Water: Water of A3 specified in JIS K 0557.
b) Sulfuric acid: A JIS Guaranteed Reagent specified in JIS K 8951 or a reagent ofequivalent quality.
c) Phosphate: A JIS Guaranteed Reagent specified in JIS K 90051 or a reagent ofequivalent quality.
d) 0.02 mol/L potassium permanganate solution: Dissolve 3.16 g of potassium
permanganate specified in JIS K 8247 in about 800 mL of water to boil and add water tomake 1,000 mL and leave at rest 1 - 2 day (s). Further filter with a funnel type glass
filter (G4) and store in a colored bottle. Or, a reagent of equivalent quality (volumetricanalysis grade) that is commercially available.
Standardization: Dry sodium oxalate of reference materials for volumetric analysis
specified in JIS K 8005 at 200 ºC for 1 hour and let it stand to cool in a sulfate
desiccator, and then put about 0.3 g into a weighing dish to measure the mass to theorder of 0.1 mg. Add about 250 mL of sulfuric acid (1+20) cooled down to 25 ºC - 30
ºC after boiling in advance and dissolve. Add about 40 mL of 0.02 mol/L potassium
permanganate solution while gently shaking for about 1 minute. Heat up to 55 ºC - 60
ºC after the red color of potassium permanganate solution disappears. Titrate with 0.02
mol/L potassium permanganate solution while keeping the temperature and continue
titrating until the color of solution becomes light red (1). Calculate the factor of 0.02
potassium permanganate solution by the following formula.
Factor (f) of 0.02 mol/L potassium permanganate solution= (W × (A/100) × ((2/5)/133.999) × ((1,000/V1)/C)= W × (A/V1) × 1.4925
W: Mass (g) of sodium oxalate sampled
A: Purity (% (mass fraction)) of sodium oxalate sampled
V1: Volume (mL) of 0.02 mol/L potassium permanganate needed for titration
C: 0.02 mol/L potassium permanganate solution (0.02 mol/L)
Note (1) The endpoint is reached when the color of solution keeps as it is for 30 seconds
after coloring(3) Instruments: Instruments are as follows:a) Magnetic stirrer:(4) Test procedures(4.1) Measurement: Conduct measurement as shown below.
a) Weigh 0.5 g - 1 g of an analytical sample to the order of 0.1 mg, and transfer to a
200-mL tall beaker.
234b) Add about 50 mL of water and about 15 mL of sulfuric acid (1+5) and shake with amagnetic stirrer to dissolve.
c) Immediately add about 1 mL of phosphate, and then titrate with 0.02 mol/L potassium
permanganate solution until the color of solution becomes light red.
d) As a blank test, conduct the procedures in b) - c) using another sample digestion flask totitrate (2).
e) Calculate the total sulfur content in an analytical sample by the following formula.
Total sulfur content (% (mass fraction)) = (5 × 0.02 × f × (V2-V3)/1,000 × 80.064)/W
× 100= (f × (V2-V3))/W × 0.80064

W: Mass (g) of an analytical sample sampled
V2: Volume (mL) of 0.02 mol/L potassium permanganate solution needed fortitration
V3: Volume (mL) of 0.02 mol/L potassium permanganate solution needed fortitration of a blank test
f: Factor of 0.02 mol/L potassium permanganate solution
Note (2) Titrate using a brown burette.
Comment 1 Replicate testing was conducted using ferrous sulfate materials (5 samples);as a result, the mean was in the range of 5.57 % - 44.74 % (mass fraction),and the standard deviation and relative standard deviation were 0.01 % 0.04 % (mass fraction) and 0.02 % - 0.7 %, respectively.
References
1) Yasushi SUGIMURA and Shinjiro IZUKA: Method validation of Redox Titration for
Determination of Sulfur content (as sulfur trioxide) in Fertilizers of Ferrous sulfate andits mixture materials, Research Report of Fertilizer, Vol.3, 2010. (25 - 29)
2) JIS K 8978: Iron (II) sulfate heptahydrate (Reagent) (2008)(5)

Flow sheet for total sulfur content: The flow sheet for total sulfur content in sludge
fertilizers mainly containing ferrous sulfate is shown below.
0.5 g - 1g
Weigh to the order of 0.1 mg into a 200-mL tall beaker
analytical sample← About 50 mL of water← About 15 mL of sulfuric acid (1+5)
Dissolution
Shake to mix← About 1 mL of phosphate
0.02 mol/L potassium permanganate solution
Titration(until the solution becomes light red)
Figure Flow sheet for total sulfur content (raw material: ferrous sulfate)235

4.12.1.b Barium chloride gravimetric analysis(1) Summary
This test method is applicable to fertilizers mainly containing sulfur and sulfuric acid among
sulfur and its compound.
Dissolve an analytical sample in a potassium hydroxide-ethanol solution and add hydrogen
peroxide to oxidize, and then add thermal barium chloride to form the precipitate of barium
sulfate and obtain total sulfur content from the mass.
(2) Reagents: Reagents are as shown below.
a) Potassium hydroxide/ethanol solution: Dissolve 10 g of potassium hydroxide
specified in JIS K 8574 in 50 mL of ethanol (95) specified in JIS K 8102, further add 50
mL of water.
b) Hydrogen peroxide: A JIS Guaranteed Reagent (30 % (mass fraction)) specified in JIS
K 8230 or a reagent of equivalent quality.
c) Hydrochloric acid: A JIS Guaranteed Reagent specified in JIS K 8180 or a reagent ofequivalent quality.
d) Nitric acid: A JIS Guaranteed Reagent (HNO3 60 % (mass fraction)) specified in JIS K
8541 or a reagent of equivalent quality.
e) Barium chloride solution (100 g/L): Dissolve 100 g of barium chloride dihydrate
specified in JIS K 8155 in water to make 1,000 mL.
f) Silver nitrate solution (2 g/100mL): Dissolve 2 g of silver nitrate specified in JIS K
8550 in water to make 100 mL.
g) Phenolphthalein solution (1 g/100 mL): Dissolve 1 g of phenolphthalein specified in
JIS K 8799 in 100 mL of ethanol (95) specified in JIS K 8102.
(3)
a)
b)
c)

Apparatus and instruments: Apparatus and instruments are as follows:
Hot plate: A hot plate whose surface temperature can be adjusted up to 250 ºC.
Water bath: A water bath that can adjust test temperature to ± 2 ºC.
Crucible: After heating porcelain crucible or platinum crucible in an electric furnace at
800 ºC in advance, let it stand to cool in a desiccator and measure the mass to the orderof 1 mg.
d) Electric furnace: An electric furnace that can keep the test temperature to ± 5 ºC.

(4) Test procedures(4.1) Extraction: Conduct extraction as follows (1):a) Weigh 1 g - 5 g of an analytical sample to the order of 1 mg, and put it in a 200-mL tallbeaker.
b) Add about 50 mL of potassium hydroxide/ethanol solution, cover with a watch glassand heat on a hot plate to boil (2).
c) After standing to cool, transfer to a 250-mL volumetric flask and add water up to themarked line.
d) Filter with Type 3 filter paper (3) to make the sample solution.
Note (1) Omit extraction if fluid fertilizers are made from only sulfuric acid and allmaterials are dissolved(2) Until sulfur content is dissolved. About 5 minutes when raw materials, etc. are
not dissolved.
(3) Omit the procedures in d) when all materials are dissolved.
(4.2) Measurement: Conduct measurement as shown below.
236a) Transfer a predetermined amount (the equivalents of about 30 mg - 170 mg as SO3) of
the sample solution to a 300-mL tall beaker. (4)b) Add about 50 mL of water and about 5 mL of hydrogen peroxide and heat in a water
bath at 80 ºC - 90 ºC for about 1 hour while sometimes shaking (5).
c) After standing cool, add 1 - 2 drop(s) of phenolphthalein solution (1 g/100 mL) (6), andadd hydrochloric acid (2+1) until the color of solution disappears (7).
d) Further add hydrochloric acid (2+1), add water to make about 100 mL and boil on a
hotplate for about 5 minutes.
e) Immediately add about 6 mL of thermal barium chloride solution (100 g/L) (8) whilesometimes shaking in a water bath at 80 ºC - 90 ºC (9).
f) After standing to cool for a few minutes (10), add a few drops of thermal barium chloride
solution (100 g/L) and check that new precipitate of barium sulfate is no longer formed.
g) Further add about 2 mL of thermal barium chloride solution (100 g/L) while shaking (11).
h) After heating in a water bath at 80 ºC - 90 ºC for about 2 hours, stop the heat source ofwater bath and let it stand to cool for no less than 4 hours(5).
i) Filter with filter paper (Type 5 - C), wash a container with water and move the whole
precipitate to a filter paper.
j) Wash the precipitate and the filter paper (Type 5 - C) with water several times (12).
k) Put the precipitate together with the filter paper into a crucible.
l) Put the crucible into a drying apparatus and dry at about 120 ºC for 1 hour.
m) After standing to cool, put the crucible into an electric furnace and heat gently to becarbonized.
n) Ignite at about 800 ºC for 2 hours.
o) After ignition (13), move the crucible to a desiccator and let it stand to cool (14).
p) Measure the mass of crucible to the order of 0.1 mg.
q) Calculate the total sulfur content in an analytical sample by the following formula.
Total sulfur content (% (mass fraction)) = (A × 0.343)/(W×V2/V1) × 100= 34.3 × A × V1/(W ×V2)
A: Mass (g) of precipitate in p)
W: Mass (g) of an analytical sample
V1: Constant volume (mL) of sample solution
V2: Volume (mL) of sample solution transferred
Note (4) Weigh 1g - 5g of an analytical sample to the order of 0.1 mg if the fluid
fertilizers of the analytical sample are made from only sulfuric acid and allmaterials are dissolved.
(5) It can be suspended after the procedures end.
(6) A pH meter can be used for neutralization.
(7) Omit the procedures in c) if fluid fertilizers are made from only sulfuric acid andall materials are dissolved.
(8) Heated up to 70 ºC - 80 ºC in a water bath in advance.
(9) Add drop-by-drop.
(10) Until precipitate settles.
(11) Add barium chloride solution slightly excessively to reduce the solubility ofbarium sulfate.
(12) Wash the precipitate until a white turbidity is no longer formed when about 5 mLof nitric acid (1+2) and about 1 mL of silver nitrate solution (2 g/100 mL) areadded to about 20 mL of washing.
237

(13) To prevent a crucible from chipping, it is recommended to let it stand to cool
gently in an electric furnace until the temperature of an electric furnace falls to
no more than 200 ºC.
(14) Time to let it stand to cool in a desiccator should be constant. In the case of aporcelain crucible, it is about 45 - 60 minutes.
Comment 1 Replicate testing was conducted using sulfur and sulfur materials (6 samples);as a result, the mean was in the range of 3.28 %- 246.96 % (mass fraction),and the standard deviation and relative standard deviation were 0 % - 0.49 %(mass fraction) and 0 % - 0.30 %, respectively.
Additionally, table 1 shows results and analysis results from a collaborative
study for test method validation.
Table 1 Results and analysis results from a collaborative study
for total sulfur content with the barium chroride gravimetric analysis (analized as surfur (S))
Samplename

Number of

Mean

2)

Mean

sr

4)

RSDr

laboratories1)

(%)8)

(%)8)

(%)8)

(%)

3)

5)

sR

6)

RSDR

(%)8)

(%)

Surfur
8
8.32
3.33
0.02
0.7
0.05
material a
Surfur
10
12.71
5.09
0.03
0.6
0.14
material b
Surfur
9
247.6
99.17
0.24
0.2
1.39
material c
Surfur
8
245.6
98.37
0.18
0.2
0.30
material dSurfur
8
1.41
0.564
0.002
0.4
0.003
material e
Surfur
9
2.89
1.157
0.001
0.1
0.010
material f1) Number of laboratories used in analysis
2) Total mean as surfur trioxid (SO 3) (number of laboratories × number of repetition (2))3) Total mean as surfur (S) obtained by dividing the total mean in 2) by 2.4969
4) Repeatability standard deviation
5) Repeatability relative standard deviation
6) Reproducibility standard deviation
7) Reproducibility relative standard deviation
8) Mass fraction

7)

1.4
2.8
1.4
0.3
0.6
0.9

References
1) JIS K 8088: Sulfur (Regent) (2010)
2) JIS M 8217: Iron ores - Methods for determination of sulfur content (1994)
3) Edited by KANTO CHEMICAL CO., INC.: Technique on Chemical Analysis of Regent- Practical Basic Technique and Knowledge, p.112 - 120 (2009)4) Yasushi SUGIMURA: Validation of Gravimetric Analysis for Determination of Sulfur
Content (as Sulfur Trioxide) in Sulfur and its Compounds as Fertilizers, Research
Report of Fertilizer, Vol.4, 2011. (9 - 15)

238

(5)

Flow sheet for total sulfur content: The flow sheet for total sulfur content in fertilizers
mainly containing sulfur and sulfuric acid is shown below.

1 g -5 g
Weigh to the order of 0.1 mg into a 200-mL tall beaker
analytical sample← About 50 mL of potassium hydroxide/ethanol solution
Heating
Cover with a watch glass and boil
Standing to coolTransfer
250-mL volumetric flask, water← Water (up to the marked line)
Filtration
Type 3 filter paper
Aliquot
300-mL tall beaker(predetermined← About 50 mL of water← About 5 mL of hydrogen peroxide
Heating
80 ºC - 90 ºC, 1 hourStanding to cool
← 1 - 2 drop(s) of phenolphthalein solution (1 g/100 mL)
Neutralization
Hydrochloric acid (2+1) (until the solution becomes transparent)← About 1mL of hydrochloric acid (2+1)← Water (the liquid volume to reach about 100 mL)
HeatingBoil, for 5 minutes← About 6 mL of thermal barium chloride solution (100 g/L), while shaking
Standing to cool
For several minutes← A few drops of thermal barium chloride solution (100 g/L)
(Check that new precipitate is no longer formed)← About 2 mL of thermal barium chloride solution (100 g/L), while shaking
Heating
80 ºC - 90 ºC, 2 hourStanding to cool
Filtration

For no less than 4 hours, in a water bath whose heat source is stopped
Type 5-C filter paper

Transfer
Type 5-C filter paper, water← Wash with water (until reaction of chloric matters disappears in filtrate)
Put
Crucible
DryingDrying apparatus, about 120 ºC, 1 hourStanding to coolCharring
Incineration

Heat gently in an electric furnace
800 ºC, 2 hours

Standing to cool

Desiccator

Measurement

Measure the mass to the order of 0.1 mg

Figure Flow sheet for total sulfur content in fertilizers

239

4.12.1.c Transmitted light analysis <Reference method>(1) Summary
This test method is applicable to fertilizers mainly containing sulfur and sulfuric acid among
sulfur and its compound.
Dissolve an analytical sample in a potassium hydroxide-ethanol solution and add hydrogen
peroxide to oxidize, and then measure the absorbance from the intensity of transmitted light ofsuspension of barium sulfate formed by the reaction with barium chloride to obtain total
sulfur content.
(2) Reagents: Reagents are as shown below.
a) Potassium hydroxide/ethanol solution: Dissolve 10 g of potassium hydroxide
specified in JIS K 8574 in 50 mL of ethanol (95) specified in JIS K 8102, further add 50
mL of water.
b) Hydrogen peroxide: A JIS Guaranteed Reagent (H2O2 30 % (mass fraction)) specified
in JIS K 8230 or a reagent of equivalent quality.
c) Hydrochloric acid: A JIS Guaranteed Reagent specified in JIS K 8180 or a reagent ofequivalent quality.
d) Glycerin – ethanol solution (1+1): Add 250 mL of ethanol (95) specified in JIS K
8102 to 250 mL of glycerin specified in JIS K 8295.
e) Sodium chloride solution: Dilute 240 g of sodium chloride specified on JIS K 8150 inwater containing 20 mL of hydrochloric acid specified in JIS K 8180, further add water
to make 1,000 mL.
f) Barium chloride: Sieve barium chloride dihydrate specified in JIS K 8155 to makebarium chloride whose particle size is between 710 μm and 500 μm.
g) Sulfate standard solution (SO3 2 mg/mL): Heat potassium sulfate specified in JIS K
8962 until it becomes constant weight value at 800 ºC in advance, stand to cool it in a
desiccator, and then transfer 4.3531 g to a weighing dosh. Disolve with a small amountof water, move it into a 1,000-mL of volumetric flask and add water up to the markedline.
h) Sulfate standard solution (SO3 0.02 mg/mL – 0.1 mg/mL): Transfer 2mL – 10 mL ofsulfate standard solution (SO3 2 mg/mL) to 200-mL volumetric flasks step by step and
add water up to the marked line.
i) Phenolphthalein solution (1 g/100 mL): Dissolve 1 g of phenolphthalein specified in
JIS K 8799 in 100 mL of ethanol (95) specified in JIS K 8102.
(3)
a)
b)
c)
d)

Apparatus and instruments: Apparatus and instruments are as follows:
Hot plate: A hot plate whose surface temperature can be adjusted up to 250 ºC.
Water bath: A water bath that can adjust test temperature to ± 2 ºC.
Magnetic stirrer:
Spectrophotometer: A spectrophotometer specified in JIS K 0115

(4) Test procedures(4.1) Extraction: Conduct extraction as follows (1):a) Weigh 1 g - 2 g of an analytical sample to the order of 1 mg, and put it in a 200-mL tallbeaker.
b) Add about 50 mL of potassium hydroxide/ethanol solution, cover with a watch glassand heat on a hot plate to boil (2).
c) After standing to cool, transfer to a 250-mL volumetric flask and add water up to themarked line.
d) Filter with Type 3 filter paper (3) to make the extract.
240

Note (1) Omit extraction if fluid fertilizers are made from only sulfuric acid and allmaterials are dissolved(2) Until sulfur content is dissolved. About 5 minutes when raw materials, etc. are
not dissolved.
(3) Omit the procedures in d) when all materials are dissolved.
(4.2) Oxidation: Conduct oxidation as shown below.
a) Transfer a predetermined amount (the equivalents of about 5 mg - 200 mg as SO3) ofthe extract to a 300-mL tall beaker. (4)b) Add about 50 mL of water and about 5 mL of hydrogen peroxide and heat in a water
bath at 80 ºC - 90 ºC for about 1 hour while sometimes shaking (5).
c) After standing to cool, add 1 - 2 drop(s) of phenolphthalein solution (1 g/100 mL) (6),and add hydrochloric acid (2+1) until the color of solution disappears (7).
d) After standing to cool, transfer to a 200-mL volumetric flask and add water up to themarked line.
e) Filter with 0.3μm glass filter paper.
Note (4) Weigh 1g - 5g of an analytical sample to the order of 0.1 mg if the fluid
fertilizers of the analytical sample are made from only sulfuric acid and allmaterials are dissolved.
(5) It can be suspended after the procedures end.
(6) A pH meter can be used for neutralization.
(7) Omit the procedures in c) if fluid fertilizers are made from only sulfuric acid andall materials are dissolved.
(4.3) Precipitate formation: Form precipitate as shown below.
a) Transfer 50 mL of filtrate to a 100-mL Erlenmeyer flask with screw cap.
b) Add about 10 mL of glycerin – ethanol solution (1+1) and about 5 mL of sodium
chloride solution in the Erlenmeyer flask with screw cap.
c) Warm up on a water bath at 30ºC± 2 ºC.
d) After warming, add 0.30 g of barium chloride and stir with a magnetic stirrer for about
2 minutes.
e) Warm up on a water bath at 30ºC± 2 ºC for about 4 minutes.
f) After warming, stir with a magnetic stirrer for about 3 minutes to make the sample
solution.
g) As a blank test, conduct the procedures in a) - b) and e) – f) using another 100-mL
Erlenmeyer flask with screw cap to prepare the blank test solution.
(4.4) Measurement: Conduct measurement according to JIS K 0115 and as shown below.
Specific measurement procedures are according to the operation method of thespectrophotometer used in measurement.
a) Measurement conditions for the spectrophotometer: Set up the measurement
conditions for the spectrophotometer considering the following:
Detection wavelength: 450 nmb) Preparation of calibration curve
1) Transfer 50 mL of sulfate standard solution (SO3 0.02 mg/mL – 0.1 mg/mL) to a
100-mL Erlenmeyer flasks with screw cap and conduct the procedure (4.3 b) - f) to
make the SO3 1 mg/65 mL - 5 mg/65 mL sulfate standard solution for thecalibration curve preparation.

2412) Transfer 50 mL of water to another 100-mL Erlenmeyer flask with screw cap andconduct the same procedures as 1) to make the blank test solution for the calibration
curve preparation.
3) Measure absorbance at wavelength 450 nm of the sulfate standard solution for thecalibration curve preparation using the blank test solution for the calibration curve
preparation as the control(8)(9).
4) Prepare a curve for the relationship between the sulfate concentration and theabsorbance of the sulfate standard solutions for the calibration curve preparation.
c) Sample measurement
1) For the sample solution, conduct procedures similarly as in b) 4) to measureabsorbance.
2) For the blank test solution, conduct procedures similarly as in d) 1) to measureabsorbance, and correct the absorbance obtained for the sample solution.
3) Obtain the sulfate (SO3) content from the calibration curve, and calculate the total
sulfur content (SO3) in the analytical sample.
Note (8) Measure right after stirring because barium sulfate easily precipitates.
(9) A spectrophotometer with automatic sample introducing device is preferable.
Comment 1 The range of calibration curve with linearity is SO3 1 mg/65 mL - 5 mg/65
mL and the curve does not pass through the origin.
Comment 2 Replicate testing was conducted using sulfur and sulfuric acid materials (6samples); as a result, the mean was in the range of 3.21 % - 248% (massfraction), and the standard deviation and relative standard deviation were0.01 % - 6% (mass fraction) and 0.2 % - 2.4%, respectively. As a result of thereplicate testing using simple substance of sulfur fertilizer containing nomaterials, the constant value is 98.4 % - 99.4 % of the theoretical value of
sulfur.
.
References
1) JIS M 8001: General rule for test methods of reagents (2009)
2) JIS K 8088: Sulfur (Regent) (2010)
3) JAPAN Sewage Works Association: Sewage Sludge Analysis -2007-, p132 – 134 Tokyo(1964)
4) Edited by KANTO CHEMICAL CO., INC.: Technique on Chemical Analysis of Regent- Practical Basic Technique and Knowledge, p.131 - 135 (2009)5) Yasushi SUGIMURA: Method Validation of Turbidimetry for Determination of Sulfur
Content (as Sulfur Trioxide) in Fertilizers of Sulfur, Sulfuric acid and Its Mixture ,Research Report of Fertilizer Vol.6, 2013, (20 - 26)

242

(5)

Flow sheet for total sulfur content: The flow sheet for total sulfur content in fertilizers
mainly containing sulfur and sulfuric acid is shown below:
1g-2g
Weigh to the order of 0.1 mg into a 200-mL tall beaker
analytical sample← About 50 mL of potassium hydroxide/ethanol solution
Heating
Cover with a watch glass and boil
Standing to coolTransfer
250-mL volumetric flask, water← Water (up to the marked line)
Filtration
Type 3 filter paper
Aliquot
300-mL tall beaker(predetermined volume)← About 50 mL of water← About 5 mL of hydrogen peroxide
Heating
80 ºC - 90 ºC, 1 hourStanding to cool
← 1 - 2 drop(s) of phenolphthalein solution (1 g/100 mL)
Neutralization
Hydrochloric acid (2+1) (until the color of solution becomes transparent)
Transfer

200-mL volumetric flask, water

Filtration

0.3μm glass filter paper

Aliquot(50 mL)

Erlenmeyer flaskwith screw cap

Aliquot(50 mL)

Erlenmeyer flaskwith screw cap← About 10 mL of glycerin – ethanol solution (1+1) →← About 5 mL of sodium chloride solution →Warming
30 ºC← Barium chloride 0.30 g
Stirring
2 minutes
Warming

30 ºC, 4 minutes

Stirring

3 minutes

Sample solution

Warming

30 ºC

Stirring

3 minutes

Blank test solution

Measurement
Spectrophotometer(450 nm)

Measurement
Spectrophotometer(450 nm)

Figure Flow sheet for total sulfur content in fertilizers

2434.13 Iron
4.13.1 Water-soluble iron4.13.1.a Flame atomic absorption spectrometry(1) Summary
This test method is applicable to fertilizers that indicate iron content as a response modifier.
Extract by adding water to an analytical sample, spray in an acetylene-air flame and measure
the atomic absorption with iron at a wavelength of 248.3 nm to quantify water-soluble iron
(W-Fe).
(2) Reagents, etc.: Reagents and water are as shown below:a) Water: Water of A3 specified in JIS K 0557.
b) Hydrochloric acid: A reagent of harmful metal analysis grade, microanalysis grade orequivalents.
c) Iron standard solution (0.1 mg/mL): Iron standard solution (Fe 0.1 mg/mL) for
atomic absorption traceable to a specified reference material (national measurement
standard) according to Article 134 of the Measurement Act.
d) Iron standard solutions (0.5 µg/ mL - 5 µg/ mL) for the calibration curve
preparation (1): Transfer 2.5 mL - 25 mL of iron standard solution (0.1 mg/mL) to
500-mL volumetric flasks step-by-step, and add hydrochloric acid (1+23) up to themarked line.
e) Blank test solution for the calibration curve preparation (1): Hydrochloric acid
(1+23) used in the procedure in d).
Note (1) This is an example of preparation; prepare an amount as appropriate.
(3) Instruments: Instruments are as follows:a) Rotary shaker: A rotary shaker that can rotate upside down a 500-mL volumetric flask
at 30 - 40 revolutions/min.
b) Flame atomic absorption spectrometer: An atomic absorption spectrometer specified
in JIS K 0121 with the background correction (2) function.
1) Light source: An iron hollow cathode lamp (when the continuous source method as
the background correction method is used, the light source is a deuterium lamp.)
2) Gas: Gas for heating by flame(i) Fuel gas: acetylene(ii) Auxiliary gas: Air sufficiently free of dust and moisture.
Note (2) There are the continuous source method, the Zeeman method, the non-resonancespectrum method, and the self-reversal method, etc.
(4) Test procedures(4.1) Extraction: Conduct extraction as shown below.
a) Weigh 5.00 g of an analytical sample and transfer to a 500-mL volumetric flask.
b) Add about 400 mL of water, and shake to mix at 30 - 40 revolutions/min for about 30
minutes.
c) Add water up to the marked line.
d) Filter with Type 3 filter paper to make the sample solution.
Comment 1 In the procedure in a), it is also allowed to weigh 2.50 g of the analyticalsample and put it into a 250-mL volumetric flask.
Comment 2 The procedure in (4.1) is the same as the procedure in (4.1) in 4.9.2.a.
244

(4.2) Measurement: Conduct measurement according to JIS K 0121 and as shown below.
Specific measurement procedures are according to the operation method of the atomic
absorption spectrometer used in measurement.
a) Measurement conditions for the atomic absorption spectrometer: Set up themeasurement conditions for the atomic absorption spectrometer considering thefollowing:
Analytical line wavelength: 248.3 nmb) Calibration curve preparation
1) Spray the iron standard solutions for the calibration curve preparation and the blanktest solution for the calibration curve preparation into a flame, and read theindicated value at a wavelength of 248.3 nm.
2) Prepare a curve for the relationship between the iron concentration and theindicated value of the iron standard solutions for the calibration curve preparationand the blank test solution for the calibration curve preparation.
c) Sample measurement
1) Transfer a predetermined amount of the sample solution (the equivalents of 0.05 mg- 0.5 mg as Fe) to a 100-mL volumetric flask.
2) Add about 25 mL of hydrochloric acid (1 + 5), and add water up to the marked line.
3) Conduct similarly as in b) 1) to read the indicated value.
4) Obtain the iron content from the calibration curve, and calculate the water-solubleiron (W-Fe) in the analytical sample.
Comment 3 Table 1 shows results from a simultaneous analysis with the same samples(proficiency testing and external quality control testing) hosted by the Japan
Fertilizer Quality Assurance Committee analyzed by the robust method.
Table 1 Results and analysis results from simultaneous analysis with the same samples
for water-soluble iron hosted by the Japan Fertilizer Quality Assurance Committee 1)
2)

Year
2012

1)
2)
3)
4)
5)

SampleFluid mixed
fertilizer

Number of Median Μ
3)laboratories
(%)
71

0.243

NIQR

4)

RSDrob

3)

(%)

0.013

5.5

(%)

5)

Proficiency testing and external quality control testingMedian (M) agrees with the mean in normal distribution.
Mass fraction
Robust standard deviation (NIQR) agrees with standard deviation in normal distribution.
RSDrob stands for relative standard deviation obtained from the robust methodby the formula shown below:
RSDrob = (NIQR/M) × 100

Reference
1) Masayoshi KOSHINO: Second Revision of The Methods of Analysis of Fertilizers(Details), p.252, Yokendo, Tokyo (1988)

245

(5)

Flow sheet for water-soluble iron: The flow sheet for water-soluble iron in fertilizers
is shown below:
5.00 g analytical sample
500-mL volumetric flask.
← About 400 mL of water,
Shaking to mix
Rotary shaker (30 - 40 revolutions/min), for 30 minutes← Water (up to the marked line)
Filtration
Type 3 filter paper
Aliquot
100-mL volumetric flasks(predetermined amount)← 25 mL of hydrochloric acid (1+5)← Water (up to the marked line)
Measurement
Atomic absorption spectrometer (248.3nm)
Figure Flow sheet for water-soluble iron in fertilizers.

246

4.14 Molybdenum
4.14.1 Water-soluble molybdenum
4.14.1.a Sodium thiocyanate absorptiometric analysis(1) Summary
This test method is applicable to fertilizers that indicate molybdenum content as a response
modifier.
Extract by adding water to an analytical sample, add sulfuric acid (1+1) and perchloric acid,further add a sodium thiocyanate solution and a tin (II) chloride solution, and measure theabsorbance with thiocyanate complex formed by the reaction of reduced molybdenum (V)with thiocyanate ion to obtain water-soluble molybdenum (W-Mo).
(2) Reagents: Reagents are as shown below:a) Sulfuric acid: A JIS Guaranteed Reagent specified in JIS K 8951 or a reagent ofequivalent quality.
b) Perchloric acid: A JIS Guaranteed Reagent specified in JIS K 8223 or a reagent ofequivalent quality.
c) Iron (III) sulfate solution (1): Dissolve 5 g of iron (III) sulfate specified in JIS K 8981 inabout 10 mL of sulfuric acid (1+1) and a proper amount of water, and further add water
to make 100 mL.
d) Sodium thiocyanate solution (1): Dissolve 50 g of sodium thiocyanate specified in JIS
K 9002 in water to make 500 mL.
e) Tin (II) chloride solution (1): Dissolve 50 g of tin (II) chloride dihydrate specified in JIS
K 8136 in 200 mL of hydrochloric acid (1+1) while heating, then add water to make
500 mL, add a small amount of granulated tin specified in JIS K 8580 and store in acolored bottle.
f) Molybdenum standard solution (1 mg/mL) (1): After leaving at rest molybdenum (VI)oxide (2) in a desiccator for about 24 hours to dry, put it to a 1,500 g weighing dish.
Dissolve in a small amount of water, transfer to a 1,000 mL volumetric flask and add
about 5 g of sodium hydroxide specified in JIS K 8576 to dissolve and add water up to
the marked line.
g) Molybdenum standard solution (0.01 mg/mL): Dilute a predetermined amount ofmolybdenum standard solution (1 mg/mL) precisely 100 times with water.
Note (1) This is an example of preparation; prepare an amount as appropriate.
(2) A reagent of no less than 99.5 % (mass fraction) in purity is commercially soldas molybdenum (VI) oxide.
Comment 1 Instead of a molybdenum standard solution (1 mg/mL) in (2) f, a chemical
analysis grade molybdenum standard solution (1 mg/mL) traceable to a
specified reference material (national measurement standard) according to
Article 134 of the Measurement Act can be used.
(3) Instruments: Instruments are as follows:a) Rotary shaker: A rotary shaker that can rotate upside down a 500-mL volumetric flask
at 30 - 40 revolutions/min.
b) Spectrophotometer: A spectrophotometer specified in JIS K 0115.
(4) Test procedures(4.1) Extraction: Conduct extraction as shown below.
a) Weigh 5.00 g of an analytical sample and transfer to a 500-mL volumetric flask.
247b) Add about 400 mL of water, and shake to mix at 30 - 40 revolutions/min for about 30
minutes.
c) Add water up to the marked line.
d) Filter with Type 3 filter paper to make the sample solution.
Comment 2 In the procedure in a), it is also allowed to weigh 2.50 g of the analyticalsample and put it into a 250-mL volumetric flask.
Comment 3 The procedure in (4.1) is the same as the procedure in (4.1) in 4.9.2.a.
Comment 4 When the sample solution in d) contains organic matters that affect thedetermination, put a predetermined amount of the sample solution to a
100-mL tall beaker, add a small amount of sulfuric acid and nitric acid to heatand digest the organic matters until white smoke of sulfuric acid evolves.
After standing to cool, transfer the solution to a 100-mL volumetric flask andadd water up to the marked line to filter. The filtrate is used as the sample
solution in (4.2) a).
(4.2) Coloring: Conduct coloring as shown below.
a) Put a predetermined amount of the sample solution (the equivalents of 0.01 mg - 0.3 mg
as Mo) to a 100-mL volumetric flask.
b) Add about 5 mL of sulfuric acid (1+1), about 5 mL of perchloric acid and about 2 mL ofiron (III) sulfate solution.
c) Add about 16 mL of sodium thiocyanate solution and about 10 mL of tin (II) chloride
solution successively while shaking to mix and further add water up to the marked line
(3).
Note (3) When the solution becomes muddy, centrifuge after the procedure in c).
However if it is presumed that copper (I) thiocyanate caused the muddying,centrifuge after leaving at rest for 1 hour.
(4.3) Measurement: Conduct measurement according to JIS K 0121 and as shown below.
Specific measurement procedures are according to the operation method of thespectrophotometer used in measurement.
a) Measurement conditions for the spectrophotometer: Set up the measurement conditions
for the spectrophotometer considering the following:
Detection wavelength: 460 nmb) Preparation of calibration curve
1) Transfer 1 mL - 30 mL of molybdenum standard solution (0.01 mg/mL) to 100-mL
volumetric flasks step-by-step.
2) Conduct the same procedure as (4.2 b) - c) to make the 0.01 mg/100 mL - 0.3
mg/100 mL molybdenum standard solution for the calibration curve preparation.
3) Conduct the same procedures as 2) for another 100-mL volumetric flask to makethe blank test solution for the calibration curve preparation.
4) Measure absorbance at wavelength 460 nm of the molybdenum standard solution
for the calibration curve preparation using the blank test solution for the calibration
curve preparation as the control.
5) Prepare a curve for the relationship between the molybdenum concentration and theabsorbance of the molybdenum standard solutions for the calibration curve
preparation.
c) Measurement of a sample
1) Measure the absorbance of the solution in (4.2) c) by conducting the sameprocedure as b) 4).
248

2) Obtain the molybdenum (Mo) content from the calibration curve and calculate thewater-soluble molybdenum (W-Mo) in the analytical sample.
Comment 5 Table 1 shows results from a simultaneous analysis with the same samples(proficiency testing and external quality control testing) hosted by the Japan
Fertilizer Quality Assurance Committee analyzed by the robust method.
Table 1 Results and analysis results from simultaneous analysis with the same samples
for water-soluble molybdenum hosted by the Japan Fertilizer Quality Assurance Committee 1)
2)

1)
2)
3)
4)
5)

Year

Sample2012

Fluid mixed
fertilizer

Number of Median Μlaboratories
(%)3)
32

0.212

NIQR

4)

RSDrob

(%)3)

(%)

0.009

4.2

5)

Proficiency testing and external quality control testingMedian (M) agrees with the mean in normal distribution.
Mass fraction
Robust standard deviation (NIQR) agrees with standard deviation in normal distribution.
RSDrob stands for relative standard deviation obtained from the robust methodby the formula shown below:
RSDrob = (NIQR/M) × 100

Reference
1) Masayoshi KOSHINO: Second Revision of The Methods of Analysis of Fertilizers(Details), p.281 - 283, Yokendo, Tokyo (1988)(5)

Flow sheet for water-soluble molybdenum: The flow sheet for water-soluble
molybdenum in fertilizers is shown below:
5.00 g analytical sample

500-mL volumetric flask.

← About 400 mL of water
Shaking to mix
Rotary shaker (30 - 40 revolutions/min), for 30 minutes← Water (up to the marked line)
Filtration
Type 3 filter paper
Aliquot
100-mL volumetric flask(predetermined← About 5 mL of sulfuric acid (1+1)← About 5 mL of perchloric acid
← About 2 mL of iron (III) sulfate solution←16 mL of sodium thiocyanate solution (while shaking to mix)←About 10 mL of tin (II) chloride solution (while shaking to mix)
← Water (up to the marked line)
Leaving at rest
Until the red color of iron disappears
Measurement

Spectrophotometer (460 nm)

Figure Flow sheet for water-soluble molybdenum in fertilizers.
249

5.

Harmful components

5.1

Mercury

5.1.a Cold vapor atomic absorption spectrometry(1) Summary
The test method is applicable to fertilizers.
Pretreat an analytical sample with nitric acid- perchloric acid, and then reduce mercury (II) in
the solution with tin (II) chloride. Aerate this solution, and measure the atomic absorption forgenerated mercury vapor at a wavelength of 253.7 nm to quantify mercury.
(2) Reagents, etc.: Reagents and water are as shown below:a) Water: Water of A3 specified in JIS K 0557.
b) Nitric acid: A reagent of harmful metal analysis grade, microanalysis grade orequivalents.
c) Perchloric acid: A reagent of harmful metal analysis grade, microanalysis grade orequivalents.
d) Sulfuric acid: A reagent of harmful metal analysis grade, microanalysis grade orequivalents.
e) Tin (II) chloride solution: To 10 g of tin (II) chloride dihydrate (1) specified in JIS K
8136, add 60 mL of sulfuric acid (1+20), and stir while heating to dissolve. After
standing to cool, add water to make 100 mL.
f) L-cystein solution: To 10 mg of L-cystein (HSCH2CH(NH2)COOH) of no less than
98.0 % in purity, add 100 mL of water and 2 mL of nitric acid to dissolve, and further
add water to make 1,000 mL. Store in a refrigerator, and do not use after 6 months after
preparation.
g) Tri-n-butyl phosphate (2): A reagent of no less than 98 % in purity.
h) Mercury standard solution (1 mg/mL): Mercury standard solution (1 mg/mL) for
atomic absorption spectrometry traceable to a specified reference material (national
measurement standard) according to Article 134 of the Measurement Act.
i) Mercury standard solution (10 µg/mL): Dilute a predetermined amount of mercury
standard solution (1 mg/mL) with L-cystein solution to prepare mercury standard
solution (10 µg/mL). Store in a refrigerator, and do not use after 6 months after
preparation.
j) Mercury standard solution (0.1 µg/mL): Dilute a predetermined amount of mercury
standard solution (10 µg/mL) with L-cystein solution to prepare mercury standard
solution (0.1 µg/mL). Store in a refrigerator, and do not use after 1 month after
preparation.
Note (1) Use a reagent with low mercury content, such as for mercury analysis or for
harmful metal analysis.
(2) This is used as an antifoaming agent.
(3)
a)
b)
c)

Apparatus and instruments Apparatus and instruments are as follows:
Atomic absorption spectrometer or mercury atomic absorption spectrometer
Light source: A mercury hollow cathode lamp or a mercury lamp.
Hot plate or sand bath: A hot plate whose surface temperature can be adjusted up to
250 ºC. Adjust the amounts of gas and silica sand of a sand bath so that the sand bathtemperature can be set to 180 ºC - 200 ºC.
d) Sample digestion flask (3): A 100-mL borosilicate glass volumetric flask (180 mm totalheight, 13 mm mouth diameter)
250

Note (3) Distinguish the volumetric flask used in digestion as a sample digestion flask
and do not use it for any other purposes.
(4) Test procedures(4.1) Extraction: Conduct extraction as follows:a) Weigh 1.00 g of an analytical sample, and transfer to a sample digestion flask.
b) Add about 10 mL of nitric acid, and heat on a hot plate or sand bath for a short time. (4)c) After standing to cool, add about 10 mL of perchloric acid, and digest by heating on a
hot plate or sand bath at 180 ºC - 200 ºC for about 30 minutes - 1 hour(5).
d) After standing to cool, fill up with water to 100 mL to make the sample solution.
e) As a blank test, conduct the procedures in b) - d) using another sample digestion flask
to prepare the blank test solution.
Note (4) If it foams vigorously, leave at rest overnight.
(5) The sample solution and the blank test solution should be stored when they arecooled after the procedure in (4.1) c). After filling up the sample solution and theblank test solution with water, immediately conduct the procedure in (4.2).
(4.2) Measurement: Conduct measurement by cold vapor atomic absorption spectrometry
specified in JIS K 0121. Specific measurement procedures are according to theoperation method of the atomic absorption spectrometer used. An example ofmeasurement using a mercury atomic absorption spectrometer is shown below:a) Measurement conditions for the atomic absorption spectrometer: Set up themeasurement conditions for the atomic absorption spectrometer considering thefollowing:
Analytical line wavelength: 253.7 nmb) Calibration curve preparation
1) Transfer 1 mL - 20 mL of mercury standard solution (0.1 µg/mL) to 100-mL
volumetric flasks step-by-step, and add water up to the marked line. Transfer 5 mLof these solutions to respective reduction vessels, add 1 drop of tri-n-butyl
phosphate (6), to make mercury standard solutions for the calibration curve
preparation.
2) Add 5 mL of water to another reduction vessel, and add 1 drop of tri-n-butyl
phosphate (6), to make the blank test solution for the calibration curve preparation.
3) Connect the reduction vessel to the mercury atomic absorption spectrometer, andintroduce sulfuric acid (1+1) and tin (II) chloride solution, and circulate air.
4) Read the indicated value at a wavelength of 253.7 nm.
5) Prepare a curve for the relationship between the mercury content (µg) and theindicated value of the mercury standard solutions for the calibration curve
preparation and the blank test solution for the calibration curve preparation.
c) Sample measurement
1) Transfer 5 mL of a sample solution to respective reduction vessels, add 1 drop of
tri-n-butyl phosphate (6), and conduct similarly as in b) 3) - 4) to read the indicatedvalue.
2) Transfer 5 mL of the blank test solution to a reduction vessel, add 1 drop of
tri-n-butyl phosphate (6), and conduct similarly as in b) 2) - 4) to read the indicatedvalue, and correct the indicated value obtained for the sample solution.
3) Obtain the mercury content (µg) from the calibration curve, and calculate themercury concentration in the analytical sample.
Note (6) It is not required to add tri-n-butyl phosphate if not needed.
251

Comment 1 The mercury concentration in the analytical sample can also be corrected bysubjecting the blank test solution to the same procedures as in 1) and 3) andobtaining the mercury content in the blank test solution.
Comment 2 A recovery testing was conducted using industrial sludge fertilizer (1 sample),composted sludge fertilizer (3 samples) and human waste sludge fertilizer (1sample); as a result, the mean recovery at the concentration level of 2 mg/kg
and 0.2 mg/kg was 98.7 % - 101.6 % and 100.7 % - 105.4 %, respectively,
and the repeatability was 0.3 % - 1.4 % and 1.4 % - 2.5 % relative standard
deviation, respectively. Also, a recovery testing was conducted using soybean
meal and powdered soybean meal, rape seed meal and powdered rape seed
meal, and compound fertilizer (2 samples) and blended fertilizer; as a result,the mean recovery at the concentration level of 40 mg/kg and 0.5 mg/kg was
98.5 % - 101.5 % and 100.4 % - 103.3 %, respectively, and the repeatability
was 0.2 % - 2.1 % and 0.8 % - 2.8 % relative standard deviation, respectively.
In addition, the results and analysis results of a collaborative study for test
method validation are shown in Table 1.
Additionally, the minimum limit of quantification of the test method is about
0.01 mg/kg.
Table 1 Results and analysis results from a collaborative study
for mercury test method validation
3)
2)
Number of
RSDr
Mean
Sample name1)
(mg/kg)(%)
laboratories

RSDR
(%)

Human waste sludge fertilizer A

11

0.651

5.3

11.6

Human waste sludge fertilizer B

11

1.10

6.3

10.2

Composted sludge fertilizer A

11

0.489

6.8

10.2

Composted sludge fertilizer B

11

0.822

8.1

13.1

Composted sludge fertilizer C

9

0.182

10.6

10.6

1)
2)
3)
4)

4)

Number of laboratories used in analysis
Mean (n= number of laboratories x number of samples (2))
Repeatability relative standard deviation
Reproducibility relative standard deviation

References
1) Fumihiro ABE, Takeshi HASHIMOTO and Yasushi SUGIMURA: Determination of
mercury in sludge fertilizer - Improved decomposition method -, Research Report of
Fertilizer Vol.1 2008. (60 - 66)
2) Fumihiro ABE, Takeshi HASHIMOTO and Norio HIKICHI: Determination of
mercury in sludge fertilizer - Collaborative Test Results -, Research Report of
Fertilizer Vol.1 2008. (67 - 73)
3) Akira SHIMIZU, Kaori OKADA, Takeshi HASHIMOTO, Yasuto IDE and Toshiaki
HIROI: Determination of Mercury in Fertilizer- Expanding the scope of application of
improved method -, Research Report of Fertilizer Vol.2 2009. (12 - 17)

252

(5)

Flow sheet for mercury: The flow sheet for mercury in fertilizers is shown below:
1.00 gSample digestion flask
analytical sample← About 10 mL nitric acid
Heating
For a short timeStanding to coolRoom temperature← About 10 mL of perchloric acid
Heating on a hot plate or sand bath at 180 ºC - 200 ºC
Decomposition
for 30 minutes -1 hourStanding to cool
← Water (fill up to 100 mL)
Sample solution
Measurement

Mercury atomic absorption spectrometer

Figure Flow sheet for mercury in fertilizers

253

5.2

Arsenic

5.2.a Hydride generation atomic absorption spectrometry(1) Summary
The test method is applicable to fertilizers.
Pretreat an analytical sample with nitric acid-sulfuric acid-perchloric acid, and then generatearsenic hydride by the addition of sodium tetrahydroborate in the acidic condition withhydrochloric acid, introduce it with argon gas to a heated absorption cell, and measure theatomic absorption with arsenic at a wavelength of 193.7 nm to quantify arsenic.
(2) Reagents, etc.: Reagents and water are as shown below:a) Water: Water of A3 specified in JIS K 0557.
b) Nitric acid: A reagent of harmful metal analysis grade, microanalysis grade orequivalents.
c) Sulfuric acid: A reagent of harmful metal analysis grade, microanalysis grade orequivalents.
d) Perchloric acid: A reagent of harmful metal analysis grade, microanalysis grade orequivalents.
e) Hydrochloric acid: Hydrochloric acid for arsenic analysis specified in JIS K 8180, orof harmful metal analysis grade, microanalysis grade or equivalents.
f) Potassium iodide solution (1): Dissolve 20 g of potassium iodide specified in JIS K
8913 in water to make 100 mL.
g) Sodium hydroxide: A JIS Guaranteed Reagent specified in JIS K 8576 or a reagent ofequivalent quality.
h) Sodium tetrahydroborate solution (1): Dissolve 10 g of sodium tetrahydroborate(NaBH4) for atomic absorption spectrometry in sodium hydroxide solution (4 g/L) to
make 1 L.
i) Arsenic standard solution (0.1 mg/mL): Arsenic standard solution (0.1 mg/mL) for
atomic absorption spectrometry traceable to a specified reference material (national
measurement standard) according to Article 134 of the Measurement Act.
j) Arsenic standard solution (1 µg/mL): Dilute a predetermined amount of arsenic
standard stock solution (0.1 mg/mL) accurately with hydrochloric acid (1+100) to
prepare arsenic standard solution (1 µg/mL). Store in a refrigerator, and do not use after
6 months after preparation.
k) Arsenic standard solution (0.1 µg/mL): Dilute a predetermined amount of arsenic
standard solution (1 µg/mL) with hydrochloric acid (1+100) to prepare arsenic standard
solution (0.1 µg/mL)). Store in a refrigerator, and do not use after 1 month after
preparation.
Note (1)The concentrations of potassium iodide solution and sodium tetrahydroborate
solution vary depending on the instrument used.
(3) Instruments: Instruments are as shown below:a) Atomic absorption spectrometer: To an atomic absorption spectrometer specified in
JIS K 0121, connect a hydride generator and parts shown below. Also, an atomic
absorption spectrometer with a built-in hydride generator can be used.
1) Light source: An arsenic hollow cathode lamp or an arsenic high-intensity discharge
lamp.
2) Atomizer: Heated absorption cell (2)3) Gas: Gases for heating the heated absorption cell.
(i) Fuel gas: acetylene(ii) Auxiliary gas: Air sufficiently free of dust and moisture.
254b) Hydride generator: A batch-type or continuous-type hydride generator specified in JIS
K 0121. For continuous hydride generators, there is a method to introduce potassium
iodide solution on-line in addition to a sample solution, hydrochloric acid, and sodium
tetrahydroborate solution.
1) Argon: Argon of grade 2 specified in JIS K 1105 or equivalents.
c) Hot plate or sand bath: A hot plate whose surface temperature can be adjusted up to
350 ºC. Adjust the amounts of gas and silica sand of a sand bath so that the sand bathtemperature can be set to no less than 300 ºC.
Note (2)For cell heating, there are a method of electric heating and a method of flame
heating.

(4) Test procedures(4.1) Extraction: Conduct extraction as follows:a) Weigh 1.00 g - 2.00 g of an analytical sample, and transfer to a 200-mL - 300-mL tallbeaker.
b) Add about 10 mL of nitric acid and about 5 mL of sulfuric acid, cover the tall beakerwith a watch glass, and leave at rest overnight.
c) Heat mildly on a hot plate or sand bath at 170 ºC - 220 ºC for no less than 30 minutes.
After bubbles cease to form, set the temperature of the hot plate or sand bath to no lessthan 300 ºC, and heat until nitroxide (yellow-brown smoke) is no longer
generated(3)(4).
d) After standing to cool, add about 5 mL of perchloric acid.
e) Cover the tall beaker with a watch glass, and heat on a hot plate or sand bath at no lessthan 300 ºC for 2 - 3 hours to digest(5).
f) Slightly move the watch glass(6), and keep on heating on the hot plate or sand bath toconcentrate until the liquid volume becomes no more than 2 mL(7).
g) After standing to cool, add about 5 mL of hydrochloric acid (1+10) and about 20 mL ofwater, cover the tall beaker with a watch glass and heat mildly to dissolve.
h) After standing to cool, transfer to a 100-mL volumetric flask with water, add water up
to the marked line, and filter with Type 3 filter paper to make the sample solution.
i) As a blank test, conduct the procedures in b) - h) using another tall beaker to prepare theblank test solution.
Note (3)

(4)

(5)

(6)
(7)

Carbonization (degradation) of organic matters by sulfuric acid begins byheating when nitric acid no longer remains. In this state, As5+ may be reduced
to As3+ and evaporate; therefore stop heating immediately after the end of thegeneration of nitroxide (yellow-brown smoke).
Oxidation of organic matters by perchloric acid progresses extremely rapidly
and explosively. For that reason, add perchloric acid after fully degradingorganic matters with nitric acid to avoid danger.
When the white smoke of perchloric acid is generated, if the solution is coloredsuch as black-brown or brown, stop heating immediately, and after standing tocool, add nitric acid, and heat again to degrade remaining organic matters.
The watch glass can be removed.
The generation of arsenic hydride is inhibited by the presence of nitric acid;
therefore remove nitric acid by sufficiently generating the white smoke ofsulfuric acid.

Comment 1 The procedures in (4.1) are the same as in (4.1) in 5.2.b and 5.5.c. However,the sampling amount of the analytical sample in (4.1) a) in 5.5.c is 1.00 g.
255

Comment 2 When the analytical sample solidifies in the procedure in (4.1) b), moisten theanalytical sample with a small amount of water as necessary in advance.
(4.2)

Measurement: Conduct measurement according to JIS K 0121 and as shown below.
Specific measurement procedures are according to the operation method of the atomic
absorption spectrometer used. Two examples of measurement procedures with acontinuous hydride generator are shown below.
(4.2.1) Measurement (A): Leaving at rest after adding potassium iodide solution.
a) Measurement conditions for the atomic absorption spectrometer: Set up themeasurement conditions for the atomic absorption spectrometer considering thefollowing:
Analytical line wavelength: 193.7 nmb) Calibration curve preparation
1) Transfer 2.5 mL - 10 mL of arsenic standard solution (0.1 µg/mL) to 50-mL
volumetric flasks step-by-step.
2) Add 5 mL of hydrochloric acid and 5 mL of potassium iodide solution, leave at rest for
about 15 minutes, and then add water up to the marked line, to make 5 ng/mL -20
ng/mL arsenic standard solutions for the calibration curve preparation.
3) Conduct the procedure in 2) for another 50-mL volumetric flask to make the blank test
solution for the calibration curve preparation.
4) While letting argon flow, introduce hydrochloric acid (1+1) and sodium
tetrahydroborate solution, such as arsenic standard solutions for the calibration curve
preparation, to the hydride generator to generate arsenic hydride.
5) Separate arsenic hydride and liquid waste, and then introduce the gas containing
arsenic hydride to a heated absorption cell, and read the indicated value at awavelength of 193.7 nm.
6) Prepare a curve for the relationship between the arsenic concentration and theindicated value of the arsenic standard solutions for the calibration curve preparationand the blank test solution for the calibration curve preparation.
c) Sample measurement
1) Transfer a predetermined amount of the sample solution to a 50-mL volumetric flask,
and conduct procedures similarly as in b) 2) and b) 4) - 5) to read the indicated value.
2) Transfer a predetermined amount of the blank test solution to a 50-mL volumetricflask, and conduct procedures similarly as in b) 2) and b) 4) - 5) to read the indicatedvalue, and correct the indicated value obtained for the sample solution.
3) Obtain the arsenic content from the calibration curve, and calculate the arsenic
concentration in the analytical sample.
(4.2.2) Measurement (B): On-line introduction of potassium iodide solution.
a) Measurement conditions for the atomic absorption spectrometer: Set up themeasurement conditions for the atomic absorption spectrometer considering thefollowing:
Analytical line wavelength: 193.7 nmb) Calibration curve preparation
1) Transfer 5 mL - 25 mL of arsenic standard solution (0.1 µg/mL) to 50-mL volumetricflasks step-by-step, add water up to the marked line, to make the 10 ng/mL - 50 ng/mL
arsenic standard solution for the calibration curve preparation. Use water as the blanktest solution for the calibration curve preparation.
2) While letting argon flow, introduce arsenic standard solutions for the calibration curve
preparation and the blank test solution for the calibration curve preparation,
respectively, and further introduce potassium iodide solution, hydrochloric acid (1+1)
256and sodium tetrahydroborate solution to the hydride generator to generate arsenic
hydride.
3) Separate arsenic hydride generated and liquid waste, and then introduce the gas
containing arsenic hydride to a heated absorption cell, and read the indicated value at awavelength of 193.7 nm.
4) Prepare a curve for the relationship between the arsenic concentration and theindicated value of the arsenic standard solutions for the calibration curve preparationand the blank test solution for the calibration curve preparation.
c) Sample measurement
1) Transfer a predetermined amount of the sample solution to a 50-mL volumetric flask,
add water up to the marked line, and conduct similarly as in b) 2)-3) to read theindicated value.
2) Transfer a predetermined amount of the blank test solution to a 50-mL volumetricflask, add water up to the marked line, and conduct similarly as in b) 2)-3) to read theindicated value, and correct the indicated value obtained for the sample solution.
3) Obtain the arsenic content from the calibration curve, and calculate the arsenic
concentration in the analytical sample.
Comment 3 The coexistence of iron, nickel, and cobalt at over 5, 10, 80 fold amount ofarsenic, respectively, inhibits the generation of arsenic hydride. However, theinhibition of arsenic hydride generation even in the coexistence of iron at 1,000
fold amount can be removed by adding or introducing potassium iodide
solution.
Comment 4 The arsenic concentration in the analytical sample can also be corrected bysubjecting the blank test solution to the same procedures as in 1) and 3) andobtaining the arsenic content in the blank test solution.
Comment 5 A recovery testing was conducted using industrial sludge fertilizer, composted
sludge fertilizer (3 samples) and human waste sludge fertilizer; as a result, therecovery at the concentration level of 50 mg/kg and 5 mg/kg was 94.6 % 100.6 % and 99.9 % - 103.3 %, respectively, and the repeatability was 0.3 % 5.1 % and 0.4 % - 4.0 % relative standard deviation, respectively. Also, arecovery testing was conducted using processed slug phosphate fertilizer,
soybean
meal,
rape
seed
meal,
compoundfertilizer
andpotassium-magnesium-sulfate; as a result, the recovery at the concentrationlevel of 50 mg/kg and 5 mg/kg was 98.5 % - 109.8 % and 103.5 % - 107.9 %,respectively, and the repeatability was 0.4 % - 6.5 % and 0.5 % - 4.2 % relative
standard deviation, respectively.
In addition, the results and analysis results of a collaborative study for test
method validation are shown in Table 1.
Additionally, the minimum limit of quantification of the test method is about
0.1 mg/kg.

257

Table 1 Results and analysis results from a collaborative study
for arsenic test method validation
Mean2)

RSDr3)RSDR4)

laboratories(mg/kg)(%)(%)

Sewage sludge fertilizer

11

6.42

3.5

10.7

Human waste sludge fertilizer

10

4.62

4.9

7.0

Industrial sludge fertilizer

12

0.632

5.7

19.7

Calcined sludge fertilizer

12

5.08

4.1

9.5

Composted sludge fertilizer

10

1.23

6.1

11.4

Sample name

1)
2)
3)
4)

Number of1)

Number of laboratories used in analysis
Mean (n= number of laboratories x number of samples (2))
Repeatability relative standard deviation
Reproducibility relative standard deviation

References
1) Naoki ASAO, Yukie ISHIDA, Shinjiro IZUKA and Masakazu SAIKI: Determination of
Arsenic in Sludge Fertilizer - Improved Decomposition Method - Research Report of
Fertilizer Vol.1 2008, (74 - 81)2) Naoki ASAO, Shinjiro IZUKA and Norio HIKICHI: Determination of Arsenic in
Sludge Fertilizer - Collaborative Test Results - Research Report of Fertilizer Vol.1 2008,(82 - 89)
3) Yasushi SUGIMURA, Naoki ASAO and Shinjiro IZUKA: Determination of Arsenic in
Fertilizer- Expanding the scope of application of improved method -, Research Reportof Fertilizer Vol.2 2009. (18 - 24)

258

(5)

Flow sheet for arsenic: The flow sheet for arsenic in fertilizers is shown below:

1.00 g - 2.00 g
200-mL - 300-mL tall beaker
analytical sample← A small amount of water to moisten analytical sample(if needed).
← About 10 mL nitric acid
← About 5 mL sulfuric acid
Leaving at rest
Cover with a watch glass
overnight
HeatingHeat mildly on a hot plate or sand bathat 170 ºC - 220 ºC for no less than 30 minutes.

HeatingHeat on a hot plate or sand bath at no less than 300 ºC
until yellow-brown smoke is no longer generated

Standing to coolRoom temperature← About 5 mL of perchloric acid
Cover with a watch glass, and heat on a hot plate or
Heatingsand bath at no less than 300 ºC for 2 - 3 hours
Heating

Slightly move the watch glass, and concentrate
until it becomes no more than 2 mL

Standing to coolRoom temperature← About 5 mL of hydrochloric acid (1+10)← About 20 mL of water
Heating
Cover with a watch glass, and dissolve
Standing to coolRoom temperature

Transfer
100-mL volumetric flask← Water (up to the marked line)
Filtration
Type 3 filter paper
Sample solution
Measurement

Atomic absorption spectrometerwith a hydride generator

Figure Flow sheet for arsenic in fertilizers

259

5.2.bSilver diethyl dithiocarbamate absorptiometric analysis(1) Summary
This test method is applicable to fertilizers other than sulfur and its compound.
Pretreat an analytical sample with nitric acid - sulfuric acid - perchloric acid, and then put thepredetermined volume into an arsenic hydride generation bottle, and generate arsenic hydrideby adding potassium iodide solution, tin chloride solution and zinc successively in the acidic
condition with hydrochloric acid to react with silver diethyl dithiocarbamate in pyridine.
Measure the absorbance with silver diethyl dithiocarbamate solution, the coloring liquid, at awavelength of 510 nm to quantify arsenic.
(2) Reagents, etc.: Reagents and water are as shown below:a) Water: Water of A3 specified in JIS K 0557.
b) Nitric acid: A reagent of harmful metal analysis grade, microanalysis grade orequivalents.
c) Sulfuric acid: A reagent of harmful metal analysis grade, microanalysis grade orequivalents.
d) Perchloric acid: A reagent of harmful metal analysis grade, microanalysis grade orequivalents.
e) Hydrochloric acid: Hydrochloric acid for arsenic analysis specified in JIS K 8180, orof harmful metal analysis grade, microanalysis grade or equivalents.
f) Potassium iodide solution: Dissolve 20 g of potassium iodide specified in JIS K 8913
in water to make 100 mL.
g) Tin (II) chloride solution: Dissolve 15 g of tin (II) chloride dihydrate specified in JIS
K 8136 in 100 mL of hydrochloric acid (1+1), add a small amount of granular tin
specified in JIS K 8580 then store in a colored bottle.
h) Ascorbic acid: A JIS Guaranteed Reagent specified in JIS K 9502 or a reagent ofequivalent quality.
i) Zinc: A reagent of arsenic analysis grade specified in JIS K 8012 or equivalents. (1 mm- 1.5 mm particle diameter)j) Lead acetate glass wool: Glass wool air-dried after moisturizing with 100 mL of thesolution, where 10 g of lead acetate (II) trihydrate specified in JIS K 8374 is dissolvedin water.
k) Silver diethyldithiocarbamate solution: Dissolve 0.5 g of silver N, N-diethyl
dithiocarbamate specified in JIS K 9512 in 100 ml of pyridine specified in JIS K 8777,
then store in cool and dark placel) Arsenic standard solution (0.1 mg/mL): Arsenic standard solution (0.1 mg/mL) for
atomic absorption spectrometry traceable to a specified reference material (national
measurement standard) according to Article 134 of the Measurement Act.
m) Arsenic standard solution (1 µg/mL): Dilute a predetermined amount of arsenic
standard solution (0.1 mg/mL) accurately with water to prepare arsenic standard
solution (1 µg/mL).
(3) Instruments: Instruments are as shown below:a) Arsenic hydride generator: An arsenic hydride generator specified in 61.1 in JIS K
0102 or equivalents.
b) Spectrophotometer: A spectrophotometer specified in JIS K 0115.
c) Hot plate or sand bath: A hot plate whose surface temperature can be adjusted up to
350 ºC. Adjust the amounts of gas and silica sand of a sand bath so that the sand bathtemperature can be set to no less than 300 ºC.

260

(4) Test procedures(4.1) Extraction: Conduct extraction as follows:a) Weigh 1.00 g - 2.00 g of an analytical sample, and transfer to a 200-mL - 300-mL tallbeaker.
b) Add about 10 mL of nitric acid and about 5 mL of sulfuric acid, cover the tall beakerwith a watch glass, and leave at rest overnight.
c) Heat mildly on a hot plate or sand bath at 170 ºC - 220 ºC for no less than 30 minutes.
After bubbles cease to form, set the temperature of the hot plate or sand bath to no lessthan 300 ºC, and heat until nitroxide (yellow-brown smoke) is no longer generated(1) (2).
d) After standing to cool, add about 5 mL of perchloric acid.
e) Cover the tall beaker with a watch glass, and heat on a hot plate or sand bath at no lessthan 300 ºC for 2 - 3 hours to digest(3).
f) Slightly move the watch glass(4), and keep on heating on the hot plate or sand bath toconcentrate until the liquid volume becomes no more than 2 mL(5).
g) After standing to cool, add about 5 mL of hydrochloric acid (1+10) and about 20 mL ofwater, cover the tall beaker with a watch glass and heat mildly to dissolve.
h) After standing to cool, transfer to a 100-mL volumetric flask with water, add water up to
the marked line, and filter with Type 3 filter paper to make the sample solution.
i) As a blank test, conduct the procedures in b) - h) using another tall beaker to prepare theblank test solution.
Note (1)

(2)

(3)

(4)
(5)

Carbonization (degradation) of organic matters by sulfuric acid begins byheating when nitric acid no longer remains. In this state, As5+ may be reduced
to As3+ and evaporate; therefore stop heating immediately after the end of thegeneration of nitroxide (yellow-brown smoke).
Oxidation of organic matters by perchloric acid progresses extremely rapidly
and explosively. For that reason, add perchloric acid after fully degradingorganic matters with nitric acid to avoid danger.
When the white smoke of perchloric acid evolves, if the solution is coloredsuch as black-brown or brown, stop heating immediately, and after standing tocool, add nitric acid, and heat again to degrade remaining organic matters.
The watch glass can be removed.
The generation of arsenic hydride is inhibited by the presence of nitric acid;
therefore remove nitric acid by sufficiently generating the white smoke ofsulfuric acid.

Comment 1 The procedures in (4.1) are the same as in (4.1) in 5.2.a.
Comment 2 When the analytical sample solidifies in the procedure in (4.1) b), moisten theanalytical sample with a small amount of water as necessary in advance.
(4.2) Reaction: Conduct reaction as shown below.
a) Put a predetermined amount (the equivalents of 1 μg - 20 μg of As and liquid volume is
no more than 40 mL) of the sample solution into an arsenic hydride generation bottle.
b) Add water to make about 40 mL of liquid volume.
c) Add hydrochloric acid to make it equivalent to 10 mL of hydrochloric acid.
d) Add about 2 mL of potassium iodide solution, shake and leave at rest for a few minutes.
e) Add about 1 mL of tin (II) chloride solution, shake and leave at rest for about 10
minutes (6).
f) Connect arsenic hydride generation bottle, glass tube lightly stuffed with lead acetate
glass wool in advance and 5 mL of silver diethyl dithiocarbamate solution (7), andquickly put 2.5g of zinc into an arsenic hydride generation bottle.
261g) Leave at rest at room temperature (15ºC - 25ºC) for about 45 minutes, and allow
generated arsenic hydride to absorb into silver diethyl dithiocarbamate solution andcolor.
h) Transfer a predetermined amount of blank test solution into the arsenic hydride
generation bottle, conduct procedures similarly as in b) - g) to allow generated arsenic
hydride to absorb into silver diethyl dithiocarbamate solution and color.
Note (6)(7)

When a large amount of iron is contained, add 1 g of ascorbic acid and 2mL oftin (II) chloride solution and shake to mix to leave at rest for about 10 minutes
instead of the procedure in e).
Apply a small amount of silicone grease, etc. to the connecting parts of anarsenic hydride generation bottle, glass tube and arsenic hydride absorption
tube to maintain their airtightness.

(4.3) Measurement: Conduct measurement according to JIS K 0115 and as shown below.
Specific measurement procedures are according to the operation method of thespectrophotometer used.
a) Measurement conditions for the spectrophotometer: Set up the measurement
conditions for the spectrophotometer considering the following:
Detection wavelength: 510 nmb) Calibration curve preparation
1) Transfer 2.5 mL - 20 mL of arsenic standard solution (0.1 µg/mL) to arsenic
hydride generation bottles step-by-step.
2) Conduct procedures similarly as in (4.2) b) - g) and allow them to react.
3) For another arsenic hydride generation bottle, the silver diethyl dithiocarbamate
solution prepared similarly as in the procedure in 2) is used as the blank test
solution for the calibration curve preparation.
4) Measure the absorbance at wavelength 510 nm of the silver diethyl dithiocarbamate
solution of arsenic standard solution for the calibration curve preparation using theblank test solution for the calibration curve preparation as the control.
5) Prepare a curve for the relationship between the arsenic concentration and theindicated value of the arsenic standard solutions for the calibration curve
preparation and the blank test solution for the calibration curve preparation.
c) Sample measurement
1) For silver diethyl dithiocarbamate solution of (4.2) g), conduct procedures similarly
as in b) 4) to measure absorbance.
2) For silver diethyl dithiocarbamate solution of (4.2) h), conduct procedures similarly
as in b) 4) to measure absorbance, and correct the absorbance obtained for the
sample solution.
3) Obtain the arsenic content from the calibration curve, and calculate the arsenic
concentration in the analytical sample.
Comment 3 The arsenic concentration in the analytical sample can also be corrected bysubjecting the blank test solution to the same procedures as in 1) and 3) toobtain the arsenic content in the blank test solution.
Reference
1) Masayoshi KOSHINO: Second Revision of The Methods of Analysis of Fertilizers(Details), p.270 - 273, Yokendo, Tokyo (1988)

262

(5)

Flow sheet for arsenic: The flow sheet for arsenic in fertilizers is shown below:
1.00 g -2.00 g
200-mL - 300-mL tall beaker
analytical sample← A small amount of water to moisten analytical sample (if needed).
← About 10 mL nitric acid, about 5 mL sulfuric acid
Leaving at rest
Cover with a watch glass
overnight
HeatingHeat mildly on a hot plate or sand bathat 170 ºC - 220 ºC for no less than 30 minutes.

HeatingHeat on a hot plate or sand bath at no less than 300 ºC
until yellow-brown smoke is no longer generated

Standing to coolRoom temperature← About 5 mL of perchloric acid
Cover with a watch glass, and heat on a hot plate or
Heatingsand bath at no less than 300 ºC for 2 - 3 hours
Heating

Slightly move the watch glass, and concentrate
until it becomes no more than 2 mL

Standing to coolRoom temperature← About 5 mL of hydrochloric acid (1+10), about 20 mL of water
Heating
Cover with a watch glass, and dissolve
Standing to coolRoom temperature

Transfer
100-mL volumetric flask← Water (up to the marked line)
Filtration
Type 3 filter paper
Sample solution
Aliquot
Transfer into an arsenic hydride generation bottle(predetermined volume)← Water (until liquid volume becomes about 40 mL)← Hydrochloric acid (until hydrochloric acid becomes equivalent to 10 mL)← About 2 mL of potassium iodide solution
Shaking to mix
Leaving at rest
For a few minutes←About 1 mL of tin (II) chloride solution
Shaking to mix
Leaving at rest
For about 10 minutes←2.5 g of zinc
Generation ofarsenic hydrideººRoom temperature (15 C - 25 C),
about 45 minutes

Absorption

5 mL of silver diethyldithiocarbamate solution

Measurement

Wavelength 510 nm

Figure Flow sheet for arsenic in fertilizers
263

5.3

Cadmium

5.3.a Flame atomic absorption spectrometry(1) Summary
The test method is applicable to fertilizers.
Pretreat an analytical sample with incineration and nitric acid-hydrochloric acid (1+3), spray
into an acetylene-air flame, and measure the atomic absorption with cadmium at a wavelengthof 228.8 nm to quantify cadmium.
(2) Reagents, etc.: Reagents and water are as shown below:a) Water: Water of A3 specified in JIS K 0557.
b) Nitric acid: A reagent of harmful metal analysis grade, microanalysis grade orequivalents.
c) Hydrochloric acid: A reagent of harmful metal analysis grade, microanalysis grade orequivalents.
d) Cadmium standard solution (0.1 mg/mL): Cadmium standard solution (0.1 mg/mL)
for atomic absorption spectrometry traceable to a specified reference material (national
measurement standard) according to Article 134 of the Measurement Act.
e) Cadmium standard solution (10 µg/mL): Transfer 10 mL of cadmium standard
solution (0.1 mg/mL) to a 100-mL volumetric flask, and add hydrochloric acid (1+23)
up to the marked line.
f) Cadmium standard solutions (0.05 µg/mL - 0.5 µg/mL) for the calibration curve
preparation (1) (2): Transfer 2.5 mL - 25 mL of cadmium standard solution (10 µg/mL)
to 500-mL volumetric flasks step-by-step, and add hydrochloric acid (1+23) up to themarked line.
g) Blank test solution for the calibration curve preparation (1) (2): Hydrochloric acid
(1+23) used in the procedures in e) and f).
Note (1)(2)

This is an example of preparation; prepare an amount as appropriate.
Store at room temperature, and do not use after 6 months after preparation.

(3) Instruments: Instruments are as shown below:a) Flame atomic absorption spectrometer: An atomic absorption spectrometer specified
in JIS K 0121 with the background correction (3) function.
1) Light source: A cadmium hollow cathode lamp (when the continuous source method
as the background correction method is used, the light source is a deuterium lamp.)
2) Gas: Gas for heating by flame(i) Fuel gas: acetylene(ii) Auxiliary gas: Air sufficiently free of dust and moisture.
b) Electric furnace: An electric furnace that can be adjusted to 450 ºC ± 5 ºC.
c) Hot plate or sand bath: A hot plate whose surface temperature can be adjusted up to
250 ºC. Adjust the amounts of gas and silica sand of a sand bath so that the sand bathtemperature can be set to 250 ºC.
Note (3)There are the continuous source method, the Zeeman method, thenon-resonance spectrum method, and the self-reversal method, etc.

(4) Test procedures(4.1) Extraction: Conduct extraction as follows:a) Weigh 5.00 g of an analytical sample, and put it in a 200-mL - 300-mL tall beaker.
b) Put the tall beaker in an electric furnace, and heat gently to char(4).
c) Ignite at 450 ºC ± 5 ºC to incinerate (5).
264d) After standing to cool, moisten the residue with a small amount of water, and add about
10 mL of nitric acid and about 30 mL of hydrochloric acid.
e) Cover the tall beaker with a watch glass, and heat on a hot plate or a sand bath to digest.
f) Slightly move the watch glass (6), and continue heating on the hot plate or sand bath toconcentrate until nearly dried up.
g) After standing to cool, add 25 mL - 50 mL of hydrochloric acid (1+5) (7) to the digest,cover the tall beaker with the watch glass, and heat quietly to dissolve.
h) After standing to cool, transfer the solution to a 100-mL - 200-mL volumetric flask withwater, add water up to the marked line, and filter with Type 3 filter paper to make thesample solution.
i) As a blank test, conduct the procedures in b) - h) using another tall beaker to prepare theblank test solution.
Note (4)
(5)
(6)
(7)

Example of the charring procedure: Heat at about 250 ºC until smoke is nolonger formed.
Example of ignition period: 8 - 16 hours
The watch glass can be removed.
Add hydrochloric acid (1+5) so that the hydrochloric acid concentration of thesample solution will be hydrochloric acid (1+23). For example, when a 100-mL
volumetric flask is used in the procedure in h), about 25 mL of hydrochloric
acid (1+5) should be added.

Comment 1 Do not conduct the procedures in (4.1) b)-c) in the case of fertilizers notcontaining organic matters.
Comment 2 The procedures in (4.1) are the same as in (4.1) in 4.9.1.a.
(4.2) Measurement: Conduct measurement according to JIS K 0121 and as shown below.
Specific measurement procedures are according to the operation method of the atomic
absorption spectrometer used in measurement.
a) Measurement conditions for the atomic absorption spectrometer: Set up themeasurement conditions for the atomic absorption spectrometer considering thefollowing:
Analytical line wavelength: 228.8 nmb) Calibration curve preparation
1) Spray the cadmium standard solutions for the calibration curve preparation and theblank test solution for the calibration curve preparation into a flame, and read theindicated value at a wavelength of 228.8 nm.
2) Prepare a curve for the relationship between the cadmium concentration and theindicated value of the cadmium standard solutions for the calibration curve
preparation and the blank test solution for the calibration curve preparation.
c) Sample measurement
1) Subject the sample solution (8) to the same procedure as in b) 1) to read the indicatedvalue.
2) Subject the blank test solution to the same procedure as in b) 1) to read theindicated value, and correct the indicated value obtained for the sample solution.
3) Obtain the cadmium content from the calibration curve, and calculate the cadmium
concentration in the analytical sample.
Note (8)If there is a possibility that the cadmium concentration in the sample solution
will exceed the maximum limit of the calibration curve, dilute a predeterminedamount with hydrochloric acid (1+23).
265

Comment 3 The cadmium concentration in the analytical sample can also be corrected bysubjecting the blank test solution to the same procedures as in 1) and 3) toobtain the cadmium content in the blank test solution.
Comment 4 A recovery testing was conducted using industrial sludge fertilizer andcomposted sludge fertilizer (5 samples); as a result, the recovery at theconcentration level of 5 mg/kg and 0.5 mg/kg was 97.5 % - 99.2 % and 96.7 %- 99.7 %, respectively, and the repeatability was 0.3 % - 1.9 % and 1.0 - 2.4 %relative standard deviation, respectively.
In addition, the results and analysis results of a collaborative study for test
method validation are shown in Table 1.
Additionally, the minimum limit of quantification of the test method is about
0.1 mg/kg.
Table 1 Results and analysis results from a collaborative study
for cadmium test method validation
Number of Mean2)
RSDr3)
Sample name1)laboratories(mg/kg)(%)

RSDR4)(%)

Sewage sludge fertilizer a

10

1.50

5.5

6.4

Sewage sludge fertilizer b

10

3.35

1.2

4.2

Composted sludge fertilizer a

10

1.96

1.0

4.4

Composted sludge fertilizer b

11

3.81

1.9

3.2

Composted sludge fertilizer c
10
1.80
3.5
1) Number of laboratories used in analysis
2) Mean (n= number of laboratories x number of samples (2))3) Repeatability relative standard deviation
4) Reproducibility relative standard deviation

4.9

References
1) Yoshinari SAKAKIBARA, Manabu MATSUZAKI and Tadao AMANO: Determinationof Cadmium, Lead, Nickel and Chromium in Sludge Fertilizer - Improved
Decomposition Method - Research Report of Fertilizer Vol.1 2008. (41 - 49)
2) Yoshinari SAKAKIBARA and Manabu MATSUZAKI: Determination of Cadmium,
Lead, Nickel and Chromium in Sludge Fertilizer - Collaborative Test Results - Research
Report of Fertilizer Vol.1 2008. (50 - 59)
3) Hisanori ARAYA and Yoshimi TAKEBA: Validation of atomic absorption
spectrometry for determination of cadmium, lead, nickel and chromium in calcinedsludge fertilizer - Research Report of Fertilizer Vo3. 2010. (30 - 42)

266

(5)

Flow sheet for cadmium: Flow sheet for cadmium in fertilizers is shown below:
5.00 ganalytical sample
Charring
Incineration

200-mL - 300-mL tall beaker.
Heat gently
Ignite at 450 ºC ± 5 ºC

Standing to coolRoom temperature← A small amount of water← About 10 mL of nitric acid
← About 30 mL of hydrochloric acid
Heating
Cover with a watch glass, and digest
Heating

Slightly move a watch glass to remove acid

Standing to coolRoom temperature← About 25 mL - 50 mL of hydrochloric acid (1+5)
Heating
Cover with a watch glass, and dissolve
Standing to coolRoom temperature

Transfer
100-mL - 200-mL volumetric flask, water← Water (up to the marked line)
Filtration
Type 3 filter paper
Measurement

Atomic absorption spectrometer (228.8 nm)

Figure Flow sheet for cadmium in fertilizers

267

5.3.b ICP Atomic Emission Spectrometry <Reference method>(1) Summary
The test method is applicable to sludge fertilizers.
Pretreat an analytical sample with incineration and nitric acid-hydrochloric acid (1+3), andthen introduce it to an ICP Atomic Emission Spectrometer (“ICP-AES”) and measure theemission with cadmium at a wavelength of 228.802 nm to quantify cadmium.
(2) Reagents, etc.: Reagents and water are as shown below:a) Water: Water of A3 specified in JIS K 0557.
b) Nitric acid: A reagent of harmful metal analysis grade, microanalysis grade orequivalents.
c) Hydrochloric acid: A reagent of harmful metal analysis grade, microanalysis grade orequivalents.
d) Cadmium standard solution (0.1 mg/mL): Cadmium standard solution (0.1 mg/mL)
for atomic absorption spectrometry traceable to a specified reference material (national
measurement standard) according to Article 134 of the Measurement Act.
e) Cadmium standard solutions (0.25µg/mL) (1) (2): Dilute a predetermined amount of
cadmium standard solution (0.1 mg/mL) with hydrochloric acid (1+23) to prepare
cadmium standard solution (0.25 µg/mL)
Note (1)(2)

This is an example of preparation; prepare an amount as appropriate.
Store at room temperature, and do not use after 6 months after preparation.

(3) Instruments: Instruments are as shown below:a) ICP Atomic Emission Spectrometer: An Atomic Emission Spectrometer specified in
JIS K 0116.
1) Gas: Argon gas specified in JIS K 1105 of no less than 99.5 % (volume fraction) inpurityb) Electric furnace: An electric furnace that can keep the test temperature at  5 ºCc) Hot plate or sand bath: A hot plate whose surface temperature can be adjusted up to
250 ºC. Adjust the amounts of gas and silica sand of a sand bath so that the sand bathtemperature can be set to 250 ºC.
(4) Test procedures(4.1) Extraction: Conduct extraction as follows:a) Weigh 5.00 g of an analytical sample, and put it in a 200-mL - 300-mL tall beaker.
b) Put the tall beaker in an electric furnace, and heat gently to char(3).
c) Ignite at 450 ºC ± 5 ºC to incinerate (4).
d) After standing to cool, moisten the residue with a small amount of water, and add about
10 mL of nitric acid and about 30 mL of hydrochloric acid.
e) Cover the tall beaker with a watch glass, and heat on a hot plate or a sand bath to digest.
f) Slightly move the watch glass (5), and continue heating on the hot plate or sand bath toconcentrate until nearly dried up.
g) After standing to cool, add 25 mL - 50 mL of hydrochloric acid (1+5) (5) to the digest,cover the tall beaker with the watch glass, and heat quietly to dissolve.
h) After standing to cool, transfer the solution to a 100-mL - 200-mL volumetric flask withwater, add water up to the marked line, and filter with Type 3 filter paper to make thesample solution.
i) As a blank test, conduct the procedures in b) - h) using another tall beaker to prepare theblank test solution.
268

Note (3)
(4)
(5)
(6)

Example of the charring procedure: Heat at about 250 ºC until smoke is nolonger formed.
Example of ignition period: 8 - 16 hours
The watch glass can be removed.
Add hydrochloric acid (1+5) so that the hydrochloric acid concentration of thesample solution will be hydrochloric acid (1+23). For example, when a 100-mL
volumetric flask is used in the procedure in h), about 25 mL of hydrochloric
acid (1+5) should be added.

Comment 1 Do not conduct the procedures in (4.1) b) - c) in the case of fertilizers notcontaining organic matters.
Comment 2 The procedures in (4.1) are the same as in (4.1) in 4.9.1.a.
(4.2) Measurement: Conduct measurement (Standard Addition Method) according to JIS K
0116 and as shown below. Specific measurement procedures are according to theoperation method of the ICP Atomic Emission Spectrometry used in measurement.
a) Measurement conditions for the ICP Atomic Emission Spectrometer: Set up themeasurement conditions for the ICP Atomic Emission Spectrometer considering thefollowing:
Analytical line wavelength: 228.802 nmb) Calibration curve preparation and Sample measurement.
1) Put 5mL of sample solution into three 10-mL volumetric flasks respectively.
2) Add 2mL and 4 mL of cadmium standard solution (0.25 μg/mL) to volumetricflasks of 1) above, and further add hydrochloric acid (1+23) to the marked line to
make the sample solutions of Standard Addition Method.
3) Add hydrochloric acid (1+23) up to the marked line of the remaining volumetricflask of 1) above to make the sample solution without the standard solution.
4) Spray the sample solution of Standard Addition Method and the sample solution
without the standard solution into the induction plasma, and read the indicatedvalue at a wavelength of 228.802 nm.
5) Put 5 mL of blank test solution into a 10-mL volumetric flask, conduct the sameprocedures as in 3) - 4) to read the indicated value, and correct the indicated value
obtained from the respective sample solutions.
6) Prepare a curve for the relationship between the added cadmium concentration andthe corrected indicated value of the sample solution for Standard Addition Method
and sample solution without standard solution.
7) Obtain the cadmium content from the intercept of the calibration curve to calculate
the concentration of cadmium in the analytical sample
Comment 3 The cadmium concentration in the analytical sample can also be corrected bysubjecting the blank test solution to the same procedures as in 1) - 4) and 6) 7) to obtain the cadmium content in the blank test solution.
Comment 4 Simultaneous measurement of multiple elements by ICP-AES is possible. In
this case, mix a predetermined amount of copper standard solution (0.1mg/mL), zinc standard solution (0.1 mg/mL), cadmium standard solution (0.1mg/mL), nickel standard solution (0.1 mg/mL), chromium standard solution(0.1 mg/mL) and lead standard solution (0.1 mg/mL), dilute with hydrochloric
acid (1 +23) to prepare mixture standard solution (Cu 25μg/mL, Zn 25 μg/mL,
Cd 0.25μg/mL, Ni 2.5 μg/mL, Cr 2.5 μg/mL and Pb 2.5 μg/mL) (1) (2). Use themixture solution instead of cadmium standard solution (0.25 μg/mL) in (4.2) b)
2). After that, conduct the same procedures as in (4.2) b) to calculate the269

concentration of respective elements in the analytical sample. Note that the
wavelength for measurement of respective elements is Cu 324.754 nm, Zn
206.191 nm, Cd 228.802 nm, Ni 231.604 nm, Cr 205.552 nm and Pb 220.351
nm.
In addition, the respective element concentrations for respective standard
addition samples are shown in the below Table.
Table The additive amount of mixed standard solution
and the additive concentration of respective elements for respective sample solutions
Additive amount Cd
Pb
Ni
Cr
Cu
Zn
(mL) of mixed(mg/L) (mg/L) (mg/L) (mg/L) (mg/L) (mg/L)
standard solution
Sample solution without
0
0
0
0
0
0
0
standard solution
Sample solution 1 byStandard Addition Method

2

0.05

0.5

0.5

0.5

5.0

5.0

Sample solution 2 byStandard Addition Method

4

0.1

1.0

1.0

1.0

10

10

Comment 5 For each one sample of sewage sludge fertilizer, human waste sludge fertilizer,
industrial sludge fertilizer, mixed sludge fertilizer, calcined sludge fertilizer andcomposted sludge fertilizer, the repeatability obtained from triplicatesmeasurement is 0.8 % - 4.1 % relative standard deviation.
Additionally, the minimum limit of quantification of the test method is about
0.2 mg/kg.
Reference
1) Masahiro ECHI, Tomoe INOUE, Megumi TABUCHI, Tetsuya NOMURA :Simultaneous Determination of Cadmium, Lead, Nickel, Chromium, Copper and Zincin Sludge Fertilizer using Inductively Coupled Plasma-Atomic Emission Spectrometry(ICP-AES), Research Report of Fertilizer Vol.4 2011. (36 - 48)

270

(5)

Flow sheet for cadmium test method: The flow sheet for cadmium test method in
fertilizers is shown below:
5.00 ganalytical sample
Charring
Incineration

200-mL - 300-mL tall beaker.
Heat gently
Ignite at 450 ºC ± 5 ºC

Standing to coolRoom temperature← A small amount of water← About 10 mL of nitric acid
← About 30 mL of hydrochloric acid
Heating
Cover with a watch glass, and digest
Heating

Slightly move a watch glass to remove acid

Standing to coolRoom temperature← 25 mL - 50 mL of hydrochloric acid (1+5)
Heating
Cover with a watch glass, and dissolve
Standing to coolRoom temperature

Transfer
100-mL - 200-mL volumetric flask, water← Water (up to the marked line)
Filtration
Type 3 filter paper
Aliquot 5 mL
10-mL volumetric flask, 3 flasks← 0, 2 and 4 mL of cadmium standard solution (0.25 μg / mL)← Hydrochloric acid (1+23) ( up to the marked line)
Measurement
ICP Atomic Emission Spectrometer (228.802 nm)
Figure Flow sheet for cadmium in fertilizers

271

5.4

Nickel

5.4.a Flame atomic absorption spectrometry(1) Summary
The test method is applicable to fertilizers.
Pretreat an analytical sample with incineration and nitric acid-hydrochloric acid (1+3), spray
into an acetylene-air flame, and measure the atomic absorption with nickel at a wavelength of
232.0 nm to quantify nickel.
(2) Reagents, etc.: Reagents and water are as shown below:a) Water: Water of A3 specified in JIS K 0557.
b) Nitric acid: A reagent of harmful metal analysis grade, microanalysis grade orequivalents.
c) Hydrochloric acid: A reagent of harmful metal analysis grade, microanalysis grade orequivalents.
d) Nickel standard solution (0.1 mg/mL): Nickel standard solution (0.1 mg/mL) for
atomic absorption spectrometry traceable to a specified reference material (national
measurement standard) according to Article 134 of the Measurement Act.
e) Nickel standard solutions (0.5 µg/mL - 5 µg/mL) for the calibration curve
preparation (1) (2): Transfer 2.5 mL - 25 mL of nickel standard solution (0.1 mg/mL) to
500-mL volumetric flasks step-by-step, and add hydrochloric acid (1+23) to the markedline.
f) Blank test solution for the calibration curve preparation (1) (2): Hydrochloric acid
(1+23) used in the procedure in e).
Note (1) This is an example of preparation; prepare an amount as appropriate.
(2) Store at room temperature, and do not use after 6 months after preparation.
(3) Instruments: Instruments are as shown below:a) Flame atomic absorption spectrometer: An atomic absorption spectrometer specified
in JIS K 0121 with the background correction (3) function.
1) Light source: A nickel hollow cathode lamp (when the continuous source method
as the background correction method is used, the light source is a deuterium lamp.)
2) Gas: Gas for heating by flame(i) Fuel gas: acetylene(ii) Auxiliary gas: Air sufficiently free of dust and moisture.
b) Electric furnace: An electric furnace that can be adjusted to 450 ºC ± 5 ºC.
c) Hot plate or sand bath: A hot plate whose surface temperature can be adjusted up to
250 ºC. Adjust the amounts of gas and silica sand of a sand bath so that the sand bathtemperature can be set to 250 ºC.
Note (3) There are the continuous source method, the Zeeman method, the non-resonancespectrum method, and the self-reversal method, etc.
(4) Test procedures(4.1) Extraction: Conduct extraction as follows:a) Weigh 5.00 g of an analytical sample, and put it in a 200-mL - 300-mL tall beaker.
b) Put the tall beaker in an electric furnace, and heat gently to char(4).
c) Ignite at 450 ºC ± 5 ºC to incinerate (5).
d) After standing to cool, moisten the residue with a small amount of water, and add about
10 mL of nitric acid and about 30 mL of hydrochloric acid.
e) Cover the tall beaker with a watch glass, and heat on a hot plate or a sand bath to digest.
272

Slightly move the watch glass (6), and continue heating on the hot plate or sand bath toconcentrate until nearly dried up.
g) After standing to cool, add 25 mL - 50 mL of hydrochloric acid (1+5) (7) to the digest,cover the tall beaker with the watch glass, and heat quietly to dissolve.
h) After standing to cool, transfer the solution to a 100-mL - 200-mL volumetric flask withwater, add water up to the marked line, and filter with Type 3 filter paper to make thesample solution.
i) As a blank test, conduct the procedures in b) - h) using another tall beaker to prepare theblank test solution.
f)Note (4) Example of the charring procedure: Heat at about 250 ºC until smoke is nolonger formed.
(5) Example of ignition period: 8 - 16 hours(6) The watch glass can be removed.
(7) Add hydrochloric acid (1+5) so that the hydrochloric acid concentration of thesample solution will be hydrochloric acid (1+23). For example, when a 100-mL
volumetric flask is used in the procedure in h), about 25 mL of hydrochloric acid(1+5) should be added.
Comment 1 Do not conduct the procedures in (4.1) b) - c) in the case of fertilizers notcontaining organic matters.
Comment 2 The procedures in (4.1) are the same as in (4.1) in 4.9.1.a.
(4.2) Measurement: Conduct measurement according to JIS K 0121 and as shown below.
Specific measurement procedures are according to the operation method of the atomic
absorption spectrometer used in measurement.
a) Measurement conditions for the atomic absorption spectrometer: Set up themeasurement conditions for the atomic absorption spectrometer considering thefollowing:
Analytical line wavelength: 232.0 nmb) Calibration curve preparation
1) Spray the nickel standard solutions for the calibration curve preparation and theblank test solution for the calibration curve preparation into a flame, and read theindicated value at a wavelength of 232.0 nm.
2) Prepare a curve for the relationship between the nickel concentration and theindicated value of the nickel standard solutions for the calibration curve preparationand the blank test solution for the calibration curve preparation.
c) Sample measurement
1) Subject the sample solution (8) to the same procedure as in b) 1) to read the indicatedvalue.
2) Subject the blank test solution to the same procedure as in b) 1) to read theindicated value, and correct the indicated value obtained for the sample solution.
3) Obtain the nickel content from the calibration curve, and calculate the nickel
concentration in the analytical sample.
Note (8) If there is a possibility that the nickel concentration in the sample solution will
exceed the maximum limit of the calibration curve, dilute a predeterminedamount with hydrochloric acid (1+23).

273

Comment 3 The nickel concentration in the analytical sample can also be corrected bysubjecting the blank test solution to the same procedures as in 1) and 3) toobtain the nickel content in the blank test solution.
Comment 4 A recovery testing was conducted using industrial sludge fertilizer andcomposted sludge fertilizer (5 samples); as a result, the recovery at theconcentration level of 300 mg/kg and 30 mg/kg was 98.5 % - 100.3 % and97.1 % - 99.9 %, respectively, and the repeatability was 1.3 % - 3.3 % and
2.1 % - 9.4 % relative standard deviation, respectively.
In addition, the results and analysis results of a collaborative study for test
method validation are shown in Table 1.
Additionally, the minimum limit of quantification of the test method is about
1 mg/kg.
Table 1 Results and analysis results from a collaborative study
for nickel test method validation
Sample name

Number of

Mean2)

RSDr3)RSDR4)

(%)
1.1

(%)
4.6

1)

laboratories

Sewage sludge fertilizer a

11(mg/kg)
56.9

Sewage sludge fertilizer b

11

21.8

2.2

3.9

Composted sludge fertilizer a

11

28.9

1.3

6.4

Composted sludge fertilizer b

11

28.5

1.8

4.4

Composted sludge fertilizer c
12
58.3
1.6
1) Number of laboratories used in analysis
2) Mean (n= number of laboratories x number of samples (2))3) Repeatability relative standard deviation
4) Reproducibility relative standard deviation

4.4

References
1) Yoshinari SAKAKIBARA, Manabu MATSUZAKI and Tadao AMANO: Determinationof Cadmium, Lead, Nickel and Chromium in Sludge Fertilizer - Improved
Decomposition Method - Research Report of Fertilizer Vol.1 2008. (41 - 49)
2) Yoshinari SAKAKIBARA and Manabu MATSUZAKI: Determination of Cadmium,
Lead, Nickel and Chromium in Sludge Fertilizer - Collaborative Test Results - Research
Report of Fertilizer Vol.1 2008. (50 - 59)
3) Hisanori ARAYA and Yoshimi TAKEBA: Validation of Atomic Absorption
Spectrometry for Determination of Cadmium, Lead, Nickel and Chromium in Calcined
Sludge Fertilizer – Using Decomposition Method for Inorganic Fertilizer. Research
Report of Fertilizer, Vo3, 2010 (30 - 42)

274

(5)

Flow sheet for nickel: The flow sheet for nickel in fertilizers is shown below:
5.00 ganalytical sample

Charring
Incineration

200-mL - 300-mL tall beaker.

Heat gently
Ignite at 450 ºC ± 5 ºC

Standing to coolRoom temperature← A small amount of water← About 10 mL of nitric acid
← About 30 mL of hydrochloric acid
Heating
Cover with a watch glass, and digest
Heating

Slightly move a watch glass to remove acid

Standing to coolRoom temperature← About 25 mL - 50 mL of hydrochloric acid (1+5)
Heating
Cover with a watch glass, and dissolve
Standing to coolRoom temperature

Transfer
100-mL - 200-mL volumetric flask, water← Water (up to the marked line)
Filtration
Type 3 filter paper
Measurement

Atomic absorption spectrometer (232.0 nm)

Figure Flow sheet for nickel in fertilizers

275

5.4.b ICP Atomic Emission Spectrometry <Reference method>(1) Summary
The test method is applicable to sludge fertilizers.
Pretreat an analytical sample with incineration and nitric acid-hydrochloric acid (1+3), andthen introduce it to an ICP Atomic Emission Spectrometer (“ICP-AES”) and measure theemission with nickel at a wavelength of 231.604 nm to quantify nickel.
(2) Reagents, etc.: Reagents and water are as shown below:a) Water: Water of A3 specified in JIS K 0557.
b) Nitric acid: A reagent of harmful metal analysis grade, microanalysis grade orequivalents.
c) Hydrochloric acid: A reagent of harmful metal analysis grade, microanalysis grade orequivalents.
d) Nickel standard solution (0.1 mg/mL): Nickel standard solution (0.1 mg/mL) for
atomic absorption spectrometry traceable to a specified reference material (national
measurement standard) according to Article 134 of the Measurement Act.
e) Nickel standard solutions (2.5 µg/mL) (1) (2):Dilute a predetermined amount of nickel standard solution (0.1 mg/mL) withhydrochloric acid (1+23) to prepare nickel standard solution (2.5 µg/mL)
Note (1) This is an example of preparation; prepare an amount as appropriate.
(2) Store at room temperature, and do not use after 6 months after preparation.
(3) Instruments: Instruments are as shown below:a) ICP Atomic Emission Spectrometer: An Atomic Emission Spectrometer specified in
JIS K 0116.
1) Gas: Argon gas specified in JIS K 1105 of no less than 99.5 % (volume fraction) inpurityb) Electric furnace: An electric furnace that can keep the test temperature at  5 ºCc) Hot plate or sand bath: A hot plate whose surface temperature can be adjusted up to
250 ºC. Adjust the amounts of gas and silica sand of a sand bath so that the sand bathtemperature can be set to 250 ºC.
(4) Test procedures(4.1) Extraction: Conduct extraction as follows:a) Weigh 5.00 g of an analytical sample, and put it in a 200-mL - 300-mL tall beaker.
b) Put the tall beaker in an electric furnace, and heat gently to char(3).
c) Ignite at 450 ºC ± 5 ºC to incinerate (4).
d) After standing to cool, moisten the residue with a small amount of water, and add about
10 mL of nitric acid and about 30 mL of hydrochloric acid.
e) Cover the tall beaker with a watch glass, and heat on a hot plate or a sand bath to digest.
f) Slightly move the watch glass (5), and continue heating on the hot plate or sand bath toconcentrate until nearly dried up.
g) After standing to cool, add 25 mL - 50 mL of hydrochloric acid (1+5) (6) to the digest,cover the tall beaker with the watch glass, and heat quietly to dissolve.
h) After standing to cool, transfer the solution to a 100-mL - 200-mL volumetric flask withwater, add water up to the marked line, and filter with Type 3 filter paper to make thesample solution.
i) As a blank test, conduct the procedures in b) - h) using another tall beaker to prepare theblank test solution.
276

Note (3) Example of the charring procedure: Heat at about 250 ºC until smoke is nolonger formed.
(4) Example of ignition period: 8 - 16 hours(5) The watch glass can be removed.
(6) Add hydrochloric acid (1+5) so that the hydrochloric acid concentration of thesample solution will be hydrochloric acid (1+23). For example, when a 100-mL
volumetric flask is used in the procedure in h), about 25 mL of hydrochloric acid(1+5) should be added.
Comment 1 Do not conduct the procedures in (4.1) b) - c) in the case of fertilizers notcontaining organic matters.
Comment 2 The procedures in (4.1) are the same as in (4.1) in 4.9.1.a.
(4.2) Measurement: Conduct measurement (Standard Addition Method) according to JIS K
0116 and as shown below. Specific measurement procedures are according to theoperation method of the ICP Atomic Emission Spectrometer used in measurement.
a) Measurement conditions for the ICP Atomic Emission Spectrometer: Set up themeasurement conditions for the ICP Atomic Emission Spectrometer considering thefollowing:
Analytical line wavelength: 231.604 nmb) Calibration curve preparation and Sample measurement.
1) Put 5 mL of sample solution to three 10-mL volumetric flasks respectively.
2) Add 2 mL and 4 mL (0.25 μg/mL) of nickel standard solution to volumetric flasks of1) above, then add hydrochloric acid (1+23) to the marked line to make the sample
solution of Standard Addition Method.
3) Add hydrochloric acid (1+23) to the marked line of the remaining volumetric flask of1) above to make the sample solution without the standard solution.
4) Spray the sample solution of Standard Addition Method and sample solution without
standard solution into the induction plasma, and read the indicated value at awavelength of 231.604 nm.
5) Add 5 mL of blank test solution to a 10-mL volumetric flask, conduct the sameprocedures as in 3) - 4) to read the indicated value, and correct the indicated value
obtained from the respective sample solutions.
6) Prepare a curve for the relationship between the added nickel concentration and thecorrected indicated value of the sample solution for Standard Addition Method andthe sample solution without standard solution.
7) Obtain the nickel content from the intercept of the calibration curve to calculate theconcentration of nickel in the analytical sample.
Comment 3 The nickel concentration in the analytical sample can also be corrected bysubjecting the blank test solution to the same procedures as in 1) - 4) and 6) 7) to obtain the nickel content in the blank test solution.
Comment 4 Simultaneous measurement of multiple elements by ICP-AES is possible. In
this case, mix a predetermined amount of copper standard solution (0.1mg/mL), zinc standard solution (0.1 mg/mL), cadmium standard solution (0.1mg/mL), nickel standard solution (0.1 mg/mL), chromium standard solution(0.1 mg/mL) and lead standard solution (0.1 mg/mL), dilute withhydrochloric acid (1 +23) to prepare mixture standard solution (Cu 25 μg/mL,
Zn 25 μg/mL, Cd 0.25 μg/mL, Ni 2.5 μg/mL, Cr 2.5 μg/mL and Pb 2.5
μg/mL) (1) (2). Use the mixture solution instead of nickel standard solution (2.5277μg/mL) in (4.2) b) 2). After that, conduct the same procedures as in (4.2) b) to
calculate the concentration of respective elements in the analytical sample.
Note that the wavelength for measurement of respective elements is Cu
324.754 nm, Zn 206.191 nm, Cd 228.802 nm, Ni 231.604 nm, Cr 205.552 nm
and Pb 220.351 nm. In addition, the respective element concentrations forrespective standard addition samples are shown in the below Table.
Table The additive amount of mixed standard solution
and the additive concentration of respective elements for respective sample solutions
Additive amount
Cd
Pb
Ni
Cr
Cu
Zn
(mL) of mixed
standard solution (mg/L) (mg/L) (mg/L) (mg/L) (mg/L) (mg/L)
Sample solution without
standard solution
Sample solution 1 byStandard Addition Method
Sample solution 2 byStandard Addition Method

Comment 5

0

0

0

0

0

0

0

2

0.05

0.5

0.5

0.5

5.0

5.0

4

0.1

1.0

1.0

1.0

10

10

For each one sample of sewage sludge fertilizer, human waste sludge fertilizer,
industrial sludge fertilizer, mixed sludge fertilizer, calcined sludge fertilizer
and composted sludge fertilizer, the repeatability obtained from triplicatesmeasurement is 1.0 % - 2.6 % relative standard deviation.
Additionally, the minimum limit of quantification of the test method is about
8 mg/kg.

Reference
1) Masahiro ECHI, Tomoe INOUE, Megumi TABUCHI, Tetsuya NOMURA:
Simultaneous Determination of Cadmium, Lead, Nickel, Chromium, Copper and Zincin Sludge Fertilizer using Inductively Coupled Plasma-Atomic Emission Spectrometry(ICP-AES), Research Report of Fertilizer Vol.4 2011. (36 - 48)278

(5) Flow sheet for nickel: The flow sheet for nickel in fertilizers is shown below:
5.00 ganalytical sample
Charring
Incineration

200-mL - 300-mL tall beaker.
Heat gently
Ignite at 450 ºC ± 5 ºC

Standing to coolRoom temperature← A small amount of water← About 10 mL of nitric acid
← About 30 mL of hydrochloric acid
Heating
Cover with a watch glass, and digest
Heating

Slightly move a watch glass to remove acid

Standing to coolRoom temperature← 25 mL - 50 mL of hydrochloric acid (1+5)
Heating
Cover with a watch glass, and dissolve
Standing to coolRoom temperature

Transfer
100-mL - 200-mL volumetric flask, water← Water (up to the marked line)
Filtration
Type 3 filter paper
Aliquot 5 mL
10-mL volumetric flask, 3 flasks← 0, 2 and 4 mL of nickel standard solution (0.25 μg / mL)← Hydrochloric acid (1+23) ( up to the marked line)
Measurement
ICP Atomic Emission Spectrometer (231.604 nm)
Figure Flow sheet for nickel in fertilizers

279

5.5

Chromium

5.5.a Flame atomic absorption spectrometry (Fertilizers containing organic matters)(1) Summary
The test method is applicable to fertilizers containing organic matters.
Pretreat an analytical sample with incineration and nitric acid-hydrochloric acid (1+3), spray
into an acetylene-air flame, and measure the atomic absorption with chromium at awavelength of 357.9 nm or 359.3 nm to quantify chromium.
(2) Reagents, etc.: Reagents and water are as shown below:a) Water: Water of A3 specified in JIS K 0557.
b) Nitric acid: A reagent of harmful metal analysis grade, microanalysis grade orequivalents.
c) Hydrochloric acid: A reagent of harmful metal analysis grade, microanalysis grade orequivalents.
d) Interference suppressor solution (1): Dissolve 100 g of potassium disulfate specified in
JIS K 8783 in water to make 1,000 mL.
e) Chromium standard solution (0.1 mg/mL): Chromium standard solution (0.1 mg/mL)
for atomic absorption spectrometry traceable to a specified reference material (national
measurement standard) according to Article 134 of the Measurement Act.
f) Chromium standard solutions (0.5 µg/mL - 5 µg/mL) for the calibration curve
preparation(1) (2): Transfer 2.5 mL - 25 mL of chromium standard solution (0.1 mg/mL)
to 500-mL volumetric flasks step-by-step, add about 50 mL of interference suppressor
solution(3), and further add hydrochloric acid (1+23) to the marked line.
g) Blank test solution for the calibration curve preparation (1) (2): Transfer about 50 mLof interference suppressor solution (3) to a 500-mL volumetric flask, and addhydrochloric acid (1+23) used in the procedure in f) to the marked line.
Note (1) This is an example of preparation; prepare an amount as appropriate.
(2) Store at room temperature, and do not use after 6 months after preparation.
(3) Add interference suppressor solution that is 1/10 volume of the volume to beprepared.
(3) Instruments: Instruments are as shown below:a) Flame atomic absorption spectrometer: An atomic absorption spectrometer specified
in JIS K 0121 with the background correction (4) function.
1) Light source: A chromium hollow cathode lamp (when the continuous source
method as the background correction method is used, the light source is a deuterium
lamp.)
2) Gas: Gas for heating by flame(i) Fuel gas: acetylene(ii) Auxiliary gas: Air sufficiently free of dust and moisture.
b) Electric furnace: An electric furnace that can be adjusted to 450 ºC ± 5 ºC.
c) Hot plate or sand bath: A hot plate whose surface temperature can be adjusted up to
250 ºC. Adjust the amounts of gas and silica sand of a sand bath so that the sand bathtemperature can be set to 250 ºC.
Note (4) There are the continuous source method, the Zeeman method, the non-resonancespectrum method, and the self-reversal method, etc.
(4) Test procedures(4.1) Extraction: Conduct extraction as follows:
280

a)
b)
c)
d)

Weigh 5.00 g of an analytical sample, and put it in a 200-mL - 300-mL tall beaker.
Put the tall beaker in an electric furnace, and heat gently to char(5).
Ignite at 450 ºC ± 5 ºC to incinerate (6).
After standing to cool, moisten the residue with a small amount of water, and add about
10 mL of nitric acid and about 30 mL of hydrochloric acid.
e) Cover the tall beaker with a watch glass, and heat on a hot plate or a sand bath to digest.
f) Slightly move the watch glass (7), and continue heating on the hot plate or sand bath toconcentrate until nearly dried up.
g) After standing to cool, add 25 mL - 50 mL of hydrochloric acid (1+5)(8) to the digest,cover the tall beaker with the watch glass, and heat quietly to dissolve.
h) After standing to cool, transfer the solution to a 100-mL - 200-mL volumetric flask withwater, and add water up to the marked line, and filter with Type 3 filter paper to makethe sample solution.
i) As a blank test, conduct the procedures in b) - h) using another tall beaker to prepare theblank test solution.
Note (5) Example of the charring procedure: Heat at about 250 ºC until smoke is nolonger formed.
(6) Example of ignition period: 8 - 16 hours(7) The watch glass can be removed.
(8) Add hydrochloric acid (1+5) so that the hydrochloric acid concentration of thesample solution will be hydrochloric acid (1+23). For example, when a 100-mL
volumetric flask is used in the procedure in h), about 25 mL of hydrochloric acid(1+5) should be added.
Comment 1 The procedures in (4.1) are the same as in (4.1) in 4.9.1.a.
(4.2) Measurement: Conduct measurement according to JIS K 0121 and as shown below.
Specific measurement procedures are according to the operation method of the atomic
absorption spectrometer used in measurement.
a) Measurement conditions for the atomic absorption spectrometer: Set up themeasurement conditions for the atomic absorption spectrometer considering thefollowing:
Analytical line wavelength: 357.9 nm or 359.3 nm (9)b) Calibration curve preparation
1) Spray the chromium standard solutions for the calibration curve preparation and theblank test solution for the calibration curve preparation into a flame(10), and read theindicated value at a wavelength of 357.9 nm or 359.3 nm(9).
2) Prepare a curve for the relationship between the chromium concentration and theindicated value of the chromium standard solutions for the calibration curve
preparation and the blank test solution for the calibration curve preparation.
c) Sample measurement
1) Transfer 25 mL (11) of the sample solution to a 100-mL volumetric flask.
2) Add about 10 mL of interference suppressor solution (3), and add hydrochloric acid
(1+23) to the marked line.
3) Subject to the same procedure as in b) 1) to read the indicated value.
4) Subject the blank test solution to the same procedure as in 1) - 2) and b) 1) to readthe indicated value, and correct the indicated value obtained for the sample
solution.
5) Obtain the chromium content from the calibration curve, and calculate thechromium concentration in the analytical sample.
281

Note (9)When background correction is conducted by the Zeeman method, 359.3 nm is
recommended as the analytical line wavelength.
(10) Use low-fuel acetylene-air flame. Acetylene-nitrous oxide flame can also be
used.
(11) If there is a possibility that the chromium concentration in the sample solution
will exceed the maximum limit of the calibration curve, decrease the amount tobe transferred.

Comment 2 In an acetylene-air flame, sensitivity is enhanced in high-fuel flame, butinterference by coexisting substances such as iron and nickel will also beenhanced.
In an acetylene-nitrous oxide flame, such interference hardly affects theresult.
Comment 3 The chromium concentration in the analytical sample can also be corrected bysubjecting the blank test solution to the same procedures as in 1) - 3) and 5) toobtain the chromium content in the blank test solution.
Comment 4 A recovery testing was conducted using industrial sludge fertilizer andcomposted sludge fertilizer (5 samples); as a result, the recovery at theconcentration level of 500 mg/kg and 50 mg/kg was 97.5 % - 100.0 % and95.9 % - 101.9 %, respectively, and the repeatability was 0.6 % - 2.7 % and1.0 % - 6.7 % relative standard deviation, respectively.
In addition, the results and analysis results of a collaborative study for test
method validation are shown in Table 1.
Additionally, the minimum limit of quantification of the test method is about
1 mg/kg.
Table 1 Results and analysis results from a collaborative study
for chromium test method validation
3)
2)
Number of
RSDr
Mean
Sample name1)laboratories(mg/kg)
(%)

RSDR
(%)

Sewage sludge fertilizer a

12

33.6

5.3

15.6

Sewage sludge fertilizer b

12

26.3

4.9

18.7

Composted sludge fertilizer a

11

41.3

2.1

11.0

Composted sludge fertilizer b

12

30.2

5.5

13.8

Composted sludge fertilizer c
12
85.0
6.4
1) Number of laboratories used in analysis
2) Mean (n= number of laboratories x number of samples (3))3) Repeatability relative standard deviation
4) Reproducibility relative standard deviation

12.5

4)

References
1) Yoshinari SAKAKIBARA, Manabu MATSUZAKI and Tadao AMANO: Determinationof Cadmium, Lead, Nickel and Chromium in Sludge Fertilizer - Improved
Decomposition Method - Research Report of Fertilizer Vol.1 2008. (41 - 49)
2) Yoshinari SAKAKIBARA and Manabu MATSUZAKI: Determination of Cadmium,
Lead, Nickel and Chromium in Sludge Fertilizer - Collaborative Test Results - Research
Report of Fertilizer Vol.1 2008. (50 - 59)2823) Yoshinari SAKAKIBARA, Chie INOUE: Validation of determination methods forchromium in sludge fertilizer - Evaluation of measurement procedure, Research Reportof Fertilizer Vol.2 2009. (130 - 136)
(5)

Flow sheet for chromium: The flow sheet for chromium in fertilizers containing
organic matters is shown below:
5.00 ganalytical sample
Charring
Incineration

200-mL - 300-mL tall beaker.
Heat gently
Ignite at 450 ºC ± 5 ºC

Standing to coolRoom temperature← A small amount of water, moisten the residue← About 10 mL of nitric acid
← About 30 mL of hydrochloric acid
Heating
Cover with a watch glass, and digest
Heating

Slightly move a watch glass to remove acid

Standing to coolRoom temperature← 25 mL - 50 mL of hydrochloric acid (1+5)
Heating
Cover with a watch glass, and dissolve
Standing to coolRoom temperature

Transfer
100-mL - 200-mL volumetric flask, water← Water (up to the marked line)
Filtration
Type 3 filter paper
Aliquot 25 mL
100-mL volumetric flasks← About 10 mL of interference suppressor solution← Hydrochloric acid (1+23) ( up to the marked line)
Measurement
Atomic absorption spectrometer (357.9 nm or 359.3 nm)
Figure Flow sheet for chromium in fertilizers containing organic matters283

5.5.b Flame atomic absorption spectrometry (Fertilizers not containing organic matters)(1) Summary
The test method is applicable to fertilizers not containing organic matters.
Pretreat an analytical sample with phosphate - nitric acid - sulfuric acid, and then spray into
an acetylene-air flame, and measure the atomic absorption with chromium at a wavelength of
357.9 nm or 359.3 nm to quantify chromium.
(2) Reagents, etc.: Reagents and water are as shown below:a) Water: Water of A3 specified in JIS K 0557.
b) Phosphate: A JIS Guaranteed Reagent specified in JIS K 9005 or a reagent ofequivalent quality.
c) Nitric acid: A reagent of harmful metal analysis grade, microanalysis grade orequivalents.
d) Sulfuric acid: A reagent of harmful metal analysis grade, microanalysis grade orequivalents.
e) Hydrochloric acid: A reagent of harmful metal analysis grade, microanalysis grade orequivalents.
f) Interference suppressor solution (1): Dissolve 100 g of potassium disulfate specified in
JIS K 8783 in water to make 1,000 mL.
g) Chromium standard solution (0.1 mg/mL): Chromium standard solution (0.1 mg/mL)
for atomic absorption spectrometry traceable to a specified reference material (national
measurement standard) according to Article 134 of the Measurement Act.
h) Chromium standard solutions (0.5 µg/mL - 5 µg/mL) for the calibration curve
preparation(1) (2): Transfer 2.5 mL - 25 mL of chromium standard solution (0.1 mg/mL)
to 500-mL volumetric flasks step-by-step, add about 50 mL of interference suppressor
solution(3), and further add hydrochloric acid (1+23) to the marked line.
i) Blank test solution for the calibration curve preparation (1) (2): Transfer about 50 mLof interference suppressor solution (3) to a 500-mL volumetric flask, and addhydrochloric acid (1+23) used in the procedure in h) to the marked line.
Note (1) This is an example of preparation; prepare an amount as appropriate.
(2) Store at room temperature, and do not use after 6 months after preparation.
(3) Add interference suppressor solution that is 1/10 volume of the volume to beprepared.
(3) Instruments: Instruments are as shown below:a) Flame atomic absorption spectrometer: An atomic absorption spectrometer specified
in JIS K 0121 with the background correction (4) function.
1) Light source: A chromium hollow cathode lamp (when the continuous source
method as the background correction method is used, the light source is a deuterium
lamp.)
2) Gas: Gas for heating by flame(i) Fuel gas: acetylene(ii) Auxiliary gas: Air sufficiently free of dust and moisture.
b) Hot plate or sand bath: A hot plate whose surface temperature can be adjusted up to
350 ºC. Adjust the amounts of gas and silica sand of a sand bath so that the sand bathtemperature can be set to about 300 ºC.
Note (4) There are the continuous source method, the Zeeman method, the non-resonancespectrum method, and the self-reversal method, etc.
284

(4) Test procedures(4.1) Extraction: Conduct extraction as follows:a) Weigh 1.00 g of an analytical sample, and put it in a 200-mL tall beaker.
b) Moisten the analytical sample with a small amount of water, and add about 5mL - 10
mL of phosphate and about 10 mL of nitric acid.
c) Cover the tall beaker with a watch glass, and heat on a hot plate or a sand bath for 10
minutes to digest.
d) Slightly move the watch glass (5), and continue heating on the hot plate or sand bath tovaporize nitric acid.
e) After standing to cool, add 5 mL - 10 mL of nitric acid, cover the tall beaker with thewatch glass, and heat on the hot plate or sand bath to digest. (6)f) After standing to cool, add 5 mL of sulfuric acid, cover the tall beaker with the watch
glass and heat on the hot plate or sand bath until white smoke of sulfuric acid evolves (7).
g) After standing to cool, add gradually 15 mL – 20 mL of water, cover the tall beaker withthe watch glass, and heat gently (8).
h) After standing to cool, transfer the solution to a 100-mL volumetric flask with water,
add water up to the marked line, and filter with Type 3 filter paper to make the sample
solution.
i) As a blank test, conduct the procedures in b) - h) using another tall beaker to prepare theblank test solution.
Note (5) The watch glass can be removed.
(6) Heat sufficiently to digest.
(7) For about 30 minutes at 300 ºC.
(8) The solution may be scattered by the sudden addition of water and heating.
(4.2) Measurement: Conduct measurement according to JIS K 0121 and as shown below.
Specific measurement procedures are according to the operation method of the atomic
absorption spectrometer used in measurement.
a) Measurement conditions for the atomic absorption spectrometer: Set up themeasurement conditions for the atomic absorption spectrometer considering thefollowing:
Analytical line wavelength: 357.9 nm or 359.3 nm (9)b) Calibration curve preparation
1) Spray the chromium standard solutions for the calibration curve preparation and theblank test solution for the calibration curve preparation into a flame(10), and read theindicated value at a wavelength of 357.9 nm or 359.3 nm(9).
2) Prepare a curve for the relationship between the chromium concentration and theindicated value of the chromium standard solutions for the calibration curve
preparation and the blank test solution for the calibration curve preparation.
c) Sample measurement
1) Transfer 25 mL (11) of the sample solution to a 100-mL volumetric flask.
2) Add about 10 mL of interference suppressor solution (3), and add hydrochloric acid
(1+23) up to the marked line.
3) Subject to the same procedure as in b) 1) to read the indicated value.
4) Subject the blank test solution to the same procedure as in 1) - 2) and b) 1) to readthe indicated value, and correct the indicated value obtained for the sample
solution.
5) Obtain the chromium content from the calibration curve, and calculate thechromium concentration in the analytical sample.
285

Note (9)When background correction is conducted by the Zeeman method, 359.3 nm is
recommended as the analytical line wavelength.
(10) Use low-fuel acetylene-air flame. Acetylene-nitrous oxide flame can also be
used.
(11) If there is a possibility that the chromium concentration in the sample solution
will exceed the maximum limit of the calibration curve, decrease the amount tobe transferred.

Comment 1 In an acetylene-air flame, sensitivity is enhanced in high-fuel flame, butinterference by coexisting substances such as iron and nickel will also beenhanced.
In an acetylene-nitrous oxide flame, such interference hardly affects theresult.
Comment 2 A replicate testing was conducted using calcined sludge fertilizer (5samples); as a result, in the range of 71.0 mg/kg - 113.1 mg/kg measurement
value, the repeatability was 2.1 % - 3.8 % relative standard deviation.
Additionally, the minimum limit of quantification of the test method is about
1 mg/kg.
Reference
1) Masayoshi KOSHINO: Second Revision of The Methods of Analysis of Fertilizers(Details), p.213 - 216, Yokendo, Tokyo (1988)

286

(5)

Flow sheet for chromium test method: The flow sheet for test method of chromium in
inorganic fertilizers and composted sludge fertilizers, etc.
1.00 g200-mL tall beaker.
analytical sample← A small amount of water, moisten the analytical sample← About 5 mL - 10 mL of phosphate← About 10 mL of nitric acid
Heating
Cover with a watch glass, and digest for 10 minutes
Heating

Slightly move the watch glass, vaporize nitric acid

Standing to coolRoom temperature← 5 mL - 10 mL of nitric acid
Heating
Cover with a watch glass, and digest
Standing to coolRoom temperature← About 5 mL of nitric acid
Heating
Cover with watch glass, until white smoke evolves
Standing to coolRoom temperature← About 15 mL – 20 mL of water
Heating
Cover with a watch glass, gently
Standing to coolRoom temperature

Transfer
100-mL volumetric flask, water← Water (up to the marked line)
Filtration
Type 3 filter paper
Aliquot 25 mL
100-mL volumetric flask← About 10 mL of interference suppressor solution← Hydrochloric acid (1+23) ( up to the marked line)
Measurement
Atomic absorption spectrometer (357.9 nm or 359.3 nm)
Figure Flow sheet for chromium in inorganic fertilizers

287

5.5.c Flame atomic absorption spectrometry (Calcined sludge fertilizer, etc.)(1) Summary
The test method is applicable to calcined sludge fertilizers, etc.
Pretreat an analytical sample with nitric acid - sulfuric acid - perchloric acid, and then spray
into an acetylene-air flame, and measure the atomic absorption with chromium at awavelength of 357.9 nm or 359.3 nm to quantify chromium.
(2) Reagents, etc.: Reagents and water are as shown below:a) Water: Water of A3 specified in JIS K 0557.
b) Nitric acid: A reagent of harmful metal analysis grade, microanalysis grade orequivalents.
c) Sulfuric acid: A reagent of harmful metal analysis grade, microanalysis grade orequivalents.
d) Perchloric acid: A reagent of harmful metal analysis grade, microanalysis grade orequivalents.
e) Hydrochloric acid: A reagent of harmful metal analysis grade, microanalysis grade orequivalents.
f) Interference suppressor solution (1): Dissolve 100 g of potassium disulfate specified in
JIS K 8783 in water to make 1,000 mL.
g) Chromium standard solution (0.1 mg/mL): Chromium standard solution (0.1 mg/mL)
for atomic absorption spectrometry traceable to a specified reference material (national
measurement standard) according to Article 134 of the Measurement Act.
h) Chromium standard solution (0.01 mg/mL) (1): Put 10 mL of chromium standard
solution (0.1 mg/mL) to 100-mL volumetric flasks and add hydrochloric acid (1+23) up
to the marked line.
i) Chromium standard solutions (0.05 µg/mL - 5 µg/mL) for the calibration curve
preparation (1) (2): Transfer 2.5 mL - 25 mL of chromium standard solution (0.01mg/mL) to 500-mL volumetric flasks step-by-step, add about 50 mL of interference
suppressor solution (3), and further add hydrochloric acid (1+23) up to the marked line.
j) Blank test solution for the calibration curve preparation (1) (2): Transfer about 50 mLof interference suppressor solution (3) to a 500-mL volumetric flask, and addhydrochloric acid (1+23) used in the procedure in h) up to the marked line.
Note (1) This is an example of preparation; prepare an amount as appropriate.
(2) Store at room temperature, and do not use after 6 months after preparation.
(3) Add interference suppressor solution that is 1/10 volume of the volume to beprepared.
(3) Instruments: Instruments are as shown below:a) Flame atomic absorption spectrometer: An atomic absorption spectrometer specified
in JIS K 0121 with the background correction (4) function.
1) Light source: A chromium hollow cathode lamp (when the continuous source
method as the background correction method is used, the light source is a deuterium
lamp.)
2) Gas: Gas for heating by flame(i) Fuel gas: acetylene(ii) Auxiliary gas: Air sufficiently free of dust and moisture.
b) Hot plate or sand bath: A hot plate whose surface temperature can be adjusted up to
350 ºC. Adjust the amounts of gas and silica sand of a sand bath so that the sand bathtemperature can be set to no less than 300 ºC.
288

Note (4) There are the continuous source method, the Zeeman method, the non-resonancespectrum method, and the self-reversal method, etc.
(4) Test procedures(4.1) Extraction: Conduct extraction as follows:a) Weigh 1.00 g of an analytical sample, and put it in a 200-mL - 300-mL tall beaker.
b) Add about 10 mL of nitric acid and about 5 mL of sulfuric acid, cover the tall beakerwith a watch glass, and leave at rest overnight.
c) Heat mildly on a hot plate or sand bath at 170 ºC - 220 ºC for no less than 30 minutes.
After bubbles cease to form, set the temperature of the hot plate or sand bath to no lessthan 300 ºC (5), and heat until nitrogen oxides (yellow-brown smoke) no longer evolve(6).
d) After standing to cool, add about 5 mL of perchloric acid.
e) Cover the tall beaker with a watch glass, and heat on the hot plate or sand bath at no lessthan 300 ºC for 2 - 3 hours to digest(7).
f) Slightly move the watch glass(8), and keep on heating on the hot plate or sand bath toconcentrate until the liquid volume becomes no more than 2 mL(7).
g) After standing to cool, add about 5 mL of hydrochloric acid (1+10) and about 20 mL ofwater, cover the tall beaker with a watch glass and heat mildly to dissolve.
h) After standing to cool, transfer to a 100-mL volumetric flask with water, add water up to
the marked line, and filter with Type 3 filter paper to make the sample solution.
i) As a blank test, conduct the procedures in b) - h) using another tall beaker to prepare theblank test solution.
Note (5) If there is vigorous bumping, increase the temperature gradually.
(6) Oxidation of organic matters with perchloric acid progresses extremely rapidly
and explosively. For that reason, add perchloric acid after fully degradingorganic matters with nitric acid to avoid danger.
(7) When the white smoke of perchloric acid is generated, if the solution is coloredsuch as black-brown or brown, stop heating immediately, and after standing tocool, add nitric acid, and heat again to degrade remaining organic matters.
(8) If there is no possibility of bumping, the watch glass can be removed.
Comment 1 The procedures in (4.1) are the same as in (4.1) in 5.2.a.
Comment 2 When the analytical sample solidifies in the procedure in (4.1) b), moisten theanalytical sample with a small amount of water as necessary in advance.
Comment 3 In some cases, about 10 minutes heating in procedure in (4.1) g) is required.
(4.2) Measurement: Conduct measurement according to JIS K 0121 and as shown below.
Specific measurement procedures are according to the operation method of the atomic
absorption spectrometer used in measurement.
a) Measurement conditions for the atomic absorption spectrometer: Set up themeasurement conditions for the atomic absorption spectrometer considering thefollowing:
Analytical line wavelength: 357.9 nm or 359.3 nm (9)b) Calibration curve preparation
1) Spray the chromium standard solutions for the calibration curve preparation and theblank test solution for the calibration curve preparation into a flame(10), and read theindicated value at a wavelength of 357.9 nm or 359.3 nm(9).
2) Prepare a curve for the relationship between the chromium concentration and theindicated value of the chromium standard solutions for the calibration curve
preparation and the blank test solution for the calibration curve preparation.
289c) Sample measurement
1) Transfer 25 mL of the sample solution to a 100-mL volumetric flask.
2) Add about 10 mL of interference suppressor solution (3), and add hydrochloric acid
(1+17) up to the marked line.
3) Subject to the same procedure as in b) 1) to read the indicated value.
4) Subject the blank test solution to the same procedure as in 1) - 2) and b) 1) to readthe indicated value, and correct the indicated value obtained for the sample
solution.
5) Obtain the chromium content from the calibration curve, and calculate thechromium concentration in the analytical sample.
Note (9)When background correction is conducted by the Zeeman method, 359.3 nm is
recommended as the analytical line wavelength.
(10) Acetylene-nitrous oxide flame can also be used.

Comment 3 In an acetylene-air flame, sensitivity is enhanced in high-fuel flame, butinterference by coexisting substances such as iron and nickel will also beenhanced.
In an acetylene-nitrous oxide flame, such interference hardly affects theresult.
Comment 4 The chromium concentration in the analytical sample can also be corrected bysubjecting the blank test solution to the same procedures as in 1) - 3) and 5) toobtain the chromium content in the blank test solution.
Comment 5 A replicate testing was conducted using calcined sludge fertilizer (5 samples);as a result, in the range of 82.4 mg/kg - 123.6 mg/kg mean, standard deviation
and relative standard deviation are 0 mg/kg - 3.4 mg/kg and 0 % - 2.8 %,respectively. In addition, the results and analysis results of a collaborative
study for test method validation are shown in Table 1.
Additionally, the minimum limit of quantification of the test method is about
6 mg/kg.
Table 1 Results and analysis results from a collaborative study
for chromium test method validation
3)
2)
Number of
RSDr
Mean
Sample name(%)
laboratories1) (mg/kg)

4)

RSDR
(%)

Calcined sludge fertilizer 1

10

107

5.0

9.7

Calcined sludge fertilizer 2

9

136

3.4

3.6

Calcined sludge fertilizer 3

9

182

1.1

2.6

Calcined sludge fertilizer 4

9

213

1.1

3.9

Calcined sludge fertilizer 5
9
117
1.8
1) Number of laboratories used in analysis
2) Mean (n= number of laboratories x number of samples (2))3) Repeatability relative standard deviation
4) Reproducibility relative standard deviation

4.0

Reference
1) Hisanori ARAYA, Yoshimi TAKEBA and Toshiaki HIROI: Evaluation of Digest
Method for Determination of Chromium in Calcined Sludge Fertilizer by Atomic
Absorption Spectrometry - Research Report of Fertilizer Vol.4, 2011. (23 - 29).
2902) Hisanori ARAYA, Yasuharu KIMURA and Yoshimi TAKEBA: Evaluation of Digest
Method for Determination of Chromium in Calcined Sludge Fertilizer by Atomic
Absorption Spectrometry: A Collaborative Study Research Report of Fertilizer Vol.5,
2012. (41 - 47).
(5)

Flow sheet for chromium test method: The flow sheet for test method of chromium incalcined sludge fertilizer
1.00 g200-mL - 300-mL tall beaker.
analytical sample← A small amount of water, moisten the analytical sample (as necessary)← About10 mL of nitric acid
← About 5 mL of sulfuric acid
Leaving at rest
Cover with a watch glass
overnight
HeatingHeatingHeat gently on a hot plate or a sand bath at 170 ºC - 220 ºC
for no less than 30 minutes
Heat on a hot plate or sand bath at no less than 300 ºC,
until yellow-brown smoke no longer evolves.

Standing to coolRoom temperature← About 5 mL of perchloric acid
Cover with a watch glass,
Heatingdigest on a hot plate or a sand bath at 300 ºC for 2-3 hours
Heating

Slightly move the watch glass,concentrate until it becomes no more than 2mL

Standing to coolRoom temperature← About 5 mL of nitric acid
← About 20 mL of water
Heating
Cover with watch glass, dissolve
Standing to coolRoom temperature

Transfer
100-mL volumetric flask, water← Water (up to the marked line)
Filtration
Type 3 filter paper
Sample solution
Aliquot 25 mL
100-mL volumetric flask← About 10 mL of interference suppressor solution← Hydrochloric acid (1+17) ( up to the marked line)
Measurement
Atomic absorption spectrometer (357.9 nm or 359.3 nm)
Figure Flow sheet for chromium in calcined sludge fertilizers, etc.

291

5.5.d ICP Atomic Emission Spectrometry <Reference method>(1) Summary
The test method is applicable to sludge fertilizers, etc. (except for calcined sludge fertilizer)
Pretreat an analytical sample with incineration, nitric acid - hydrochloric acid (1+3), and thenintroduce it to an ICP Atomic Emission Spectrometer (“ICP-AES”) and measure the emission
with chromium at a wavelength of 205.552 nm to quantify chromium.
(2) Reagents, etc.: Reagents and water are as shown below:a) Water: Water of A3 specified in JIS K 0557.
b) Nitric acid: A reagent of harmful metal analysis grade, microanalysis grade orequivalents.
c) Hydrochloric acid: A reagent of harmful metal analysis grade, microanalysis grade orequivalents.
d) Chromium standard solution (0.1 mg/mL): Chromium standard solution (0.1 mg/mL)
for atomic absorption spectrometry traceable to a specified reference material (national
measurement standard) according to Article 134 of the Measurement Act.
e) Chromium standard solutions (2.5 µg/mL) (1) (2):Dilute a predetermined amount of chromium standard solution (0.1 mg/mL) withhydrochloric acid (1+23) to prepare chromium standard solution (2.5 µg/mL)
Note (1) This is an example of preparation; prepare an amount as appropriate.
(2) Store at room temperature, and do not use after 6 months after preparation.
(3) Instruments: Instruments are as shown below:a) ICP Atomic Emission Spectrometer: An Atomic emission spectrometer specified in
JIS K 0116.
1) Gas: Argon gas specified in JIS K 1105 of no less than 99.5 % (volume fraction) inpurityb) Electric furnace: An electric furnace that can keep the test temperature at  5 ºCc) Hot plate or sand bath: A hot plate whose surface temperature can be adjusted up to
250 ºC. Adjust the amounts of gas and silica sand of a sand bath so that the sand bathtemperature can be set to 250 ºC.
(4) Test procedures(4.1) Extraction: Conduct extraction as follows:a) Weigh 5.00 g of an analytical sample, and put it in a 200- mL - 300-mL tall beaker.
b) Put the tall beaker in an electric furnace, and heat gently to char.(3)c) Ignite at 450 ºC ± 5 ºC to incinerate (4).
d) After standing to cool, moisten the residue with a small amount of water, and add about
10 mL of nitric acid and about 30 mL of hydrochloric acid.
e) Cover the tall beaker with a watch glass, and heat on a hot plate or a sand bath to digest.
f) Slightly move the watch glass (5), and continue heating on the hot plate or sand bath toconcentrate until nearly dried up.
g) After standing to cool, add 25 mL - 50 mL of hydrochloric acid (1+5)(6) to the digest,cover the tall beaker with the watch glass, and heat quietly to dissolve.
h) After standing to cool, transfer the solution to a 100-mL - 200-mL volumetric flask withwater, add water up to the marked line, and filter with Type 3 filter paper to make thesample solution.
i) As a blank test, conduct the procedures in b) - h) using another tall beaker to prepare theblank test solution.
292

Note (3) Example of the charring procedure: Heat at about 250 ºC until smoke is nolonger formed.
(4) Example of ignition period: 8 - 16 hours(5) The watch glass can be removed.
(6) Add hydrochloric acid (1+5) so that the hydrochloric acid concentration of thesample solution will be hydrochloric acid (1+23). For example, when a 100-mL
volumetric flask is used in the procedure in h), about 25 mL of hydrochloric acid(1+5) should be added.
Comment 1 The procedures in (4.1) are the same as in (4.1) in 4.9.1.a.
(4.2) Measurement: Conduct measurement (Standard Addition Method) according to JIS K
0116 and as shown below. Specific measurement procedures are according to theoperation method of the ICP Atomic Emission Spectrometer used in measurement.
a) Measurement conditions for the ICP Atomic Emission Spectrometer: Set up themeasurement conditions for the ICP Atomic Emission Spectrometer considering thefollowing:
Analytical line wavelength: 205.552 nmb) Calibration curve preparation and Sample measurement.
1) Put 5 mL of sample solution into three 10-mL volumetric flasks respectively.
2) Add 2 mL and 4 mL of chromium standard solution (0.25 μg/mL) to volumetricflasks of 1) above, then add hydrochloric acid (1+23) to the marked line to makethe sample solutions of Standard Addition Method.
3) Add hydrochloric acid (1+23) to the marked line of the remaining volumetric flaskof 1) above to make the sample solution without standard solution.
4) Spray the sample solution of Standard Addition Method and sample solution
without standard solution into the induction plasma, and read the indicated value ata wavelength of 205.552 nm.
5) Add 5 mL of blank test solution to a 10-mL volumetric flask, conduct the sameprocedures as in 3) - 4) to read the indicated value, and correct the indicated value
obtained from the respective sample solutions.
6) Prepare a curve for the relationship between the added chromium concentration andthe corrected indicated value of the sample solution for Standard Addition Method
and the sample solution without standard solution.
7) Obtain the chromium content from the intercept of the calibration curve to calculate
the concentration of chromium in the analytical sample.
Comment 2 The chromium concentration in the analytical sample can also be corrected bysubjecting the blank test solution to the same procedures as in 1) - 4) and 6) 7) to obtain the chromium content in the blank test solution.
Comment 3 Simultaneous measurement of multiple elements by ICP-AES is possible. In
this case, mix a predetermined amount of copper standard solution (0.1mg/mL), zinc standard solution (0.1 mg/mL), cadmium standard solution (0.1mg/mL), nickel standard solution (0.1 mg/mL), chromium standard solution(0.1 mg/mL) and lead standard solution (0.1 mg/mL), dilute withhydrochloric acid (1 +23) to prepare mixture standard solution (Cu 25 μg/mL,
Zn 25 μg/mL, Cd 0.25 μg/mL, Ni 2.5 μg/mL, Cr 2.5 μg/mL and Pb 2.5
μg/mL) (1) (2). Use the mixture solution instead of chromium standard solution(2.5 μg/mL) in (4.2) b) 2). After that, conduct the same procedures as in (4.2)b) to calculate the concentration of respective elements in the analyticalsample. Note that the wavelength for measurement of respective elements is293

Cu 324.754 nm, Zn 206.191 nm, Cd 228.802 nm, Ni 231.604 nm, Cr 205.552
nm and Pb 220.351 nm.
In addition, the respective element concentrations for respective standard
addition samples are shown in the below Table.
Table The additive amount of mixed standard solution
and the additive concentration of respective elements for respective sample solutions
Additive amount Cd
Pb
Ni
Cr
Cu
Zn
(mL) of mixed
standard solution (mg/L) (mg/L) (mg/L) (mg/L) (mg/L) (mg/L)
Sample solution without
0
0
0
0
0
0
0
standard solution
Sample solution 1 by
2
0.05
0.5
0.5
0.5
5.0
5.0
Standard Addition Method
Sample solution 2 by
4
0.1
1.0
1.0
1.0
10
10
Standard Addition Method

Comment 4 For each one sample of sewage sludge fertilizer, human waste sludge fertilizer,
industrial sludge fertilizer, mixed sludge fertilizer, calcined sludge fertilizer
and composted sludge fertilizer, the repeatability obtained from triplicatesmeasurement is 0.9 % - 2.5 % relative standard deviation.
Additionally, the minimum limit of quantification of the test method is about
4 mg/kg.
Reference
1) Masahiro ECHI, Tomoe INOUE, Megumi TABUCHI, Tetsuya NOMURA :Simultaneous Determination of Cadmium, Lead, Nickel, Chromium, Copper and Zincin Sludge Fertilizer using Inductively Coupled Plasma-Atomic Emission Spectrometry(ICP-AES), Research Report of Fertilizer Vol.4 2011. (36 - 48)

294

(5)

Flow sheet for chromium: The flow sheet for chromium in fertilizers is shown below:
5.00 ganalytical sample
Charring
Incineration

200-mL - 300-mL tall beaker.
Heat gently
Ignite at 450 ºC ± 5 ºC

Standing to coolRoom temperature← A small amount of water, moisten the residue← About 10 mL of nitric acid
← About 30 mL of hydrochloric acid
Heating
Cover with a watch glass, and digest for 30 minutes
Heating

Slightly move a watch glass to remove acid

Standing to coolRoom temperature← 25 mL - 50 mL of hydrochloric acid (1+5)
Heating
Cover with a watch glass, and dissolve
Standing to coolRoom temperature

Transfer
100-mL - 200-mL volumetric flask, water← Water (up to the marked line)
Filtration
Type 3 filter paper
Aliquot 5 mL
10-mL volumetric flask, 3 flasks← 0, 2 and 4 mL of chromium standard solution (2.5 μg / mL) respectively← Hydrochloric acid (1+23) ( up to the marked line)
Measurement
ICP Atomic Emission Spectrometer (205.552 nm)
Figure Flow sheet for chromium in fertilizers

295

5.6

Lead

5.6.a Flame atomic absorption spectrometry(1) Summary
The test method is applicable to fertilizers.
Pretreat an analytical sample with incineration and nitric acid-hydrochloric acid (1+3), spray
into an acetylene-air flame, and measure the atomic absorption with lead at a wavelength of
217.0 nm or 283.3 nm to quantify lead.
(2) Reagents, etc.: Reagents and water are as shown below:a) Water: Water of A3 specified in JIS K 0557.
b) Nitric acid: A reagent of harmful metal analysis grade, microanalysis grade orequivalents.
c) Hydrochloric acid: A reagent of harmful metal analysis grade, microanalysis grade orequivalents.
d) Lead standard solution (0.1 mg/mL): Lead standard solution (0.1 mg/mL) for atomic
absorption spectrometry traceable to a specified reference material (national
measurement standard) according to Article 134 of the Measurement Act.
e) Lead standard solutions (0.5 µg/mL - 5 µg/mL) for the calibration curve
preparation (1) (2): Transfer 2.5 mL - 25 mL of lead standard solution (0.1 mg/mL) to
500-mL volumetric flasks step-by-step, and add hydrochloric acid (1+23) to the markedline.
f) Blank test solution for the calibration curve preparation (1) (2): Hydrochloric acid
(1+23) used in the procedure in e).
Note (1) This is an example of preparation; prepare an amount as appropriate.
(2) Store at room temperature, and do not use after 6 months after preparation.
(3) Instruments: Instruments are as shown below:a) Flame atomic absorption spectrometer: An atomic absorption spectrometer specified
in JIS K 0121 with the background correction (3) function.
1) Light source: A lead hollow cathode lamp (when the continuous source method as
the background correction method is used, the light source is a deuterium lamp.)
2) Gas: Gas for heating by flame(i) Fuel gas: acetylene(ii) Auxiliary gas: Air sufficiently free of dust and moisture.
b) Electric furnace: An electric furnace that can be adjusted to 450 ºC ± 5 ºC.
c) Hot plate or sand bath: A hot plate whose surface temperature can be adjusted up to
250 ºC. Adjust the amounts of gas and silica sand of a sand bath so that the sand bathtemperature can be set to 250 ºC.
Note (3) There are the continuous source method, the Zeeman method, the non-resonancespectrum method, and the self-reversal method, etc.
(4) Test procedures(4.1) Extraction: Conduct extraction as follows:a) Weigh 5.00 g of an analytical sample, and put it in a 200-mL- 300-mL tall beaker.
b) Put the tall beaker in an electric furnace, and heat gently to char (4).
c) Ignite at 450 ºC±5 ºC to incinerate (5).
d) After standing to cool, moisten the residue with a small amount of water, and add about
10 mL of nitric acid and about 30 mL of hydrochloric acid.
e) Cover the tall beaker with a watch glass, and heat on a hot plate or a sand bath to digest.
296

Slightly move the watch glass (6), and continue heating on the hot plate or sand bath toconcentrate until nearly dried up.
g) After standing to cool, add 25 mL -50 mL of hydrochloric acid (1+5) (7) to the digest,cover the tall beaker with the watch glass, and heat quietly to dissolve.
h) After standing to cool, transfer the solution to a 100-mL - 200-mL volumetric flask withwater, add water up to the marked line, and filter with Type 3 filter paper to make thesample solution.
i) As a blank test, conduct the procedures in b) - h) using another tall beaker to prepare theblank test solution.
f)Note (4) Example of the charring procedure: Heat at about 250 ºC until smoke is nolonger formed.
(5) Example of ignition period: 8 - 16 hours(6) The watch glass can be removed.
(7) Add hydrochloric acid (1+5) so that the hydrochloric acid concentration of thesample solution will be hydrochloric acid (1+23). For example, when a 100-mL
volumetric flask is used in the procedure in h), about 25 mL of hydrochloric acid(1+5) should be added.
Comment 1 Do not conduct the procedures in (4.1) b) - c) in the case of fertilizers notcontaining organic matters.
Comment 2 The procedures in (4.1) are the same as in (4.1) in 4.9.1.a.
(4.2) Measurement: Conduct measurement according to JIS K 0121 and as shown below.
Specific measurement procedures are according to the operation method of the atomic
absorption spectrometer used in measurement.
a) Measurement conditions for the atomic absorption spectrometer: Set up themeasurement conditions for the atomic absorption spectrometer considering thefollowing:
Analytical line wavelength: 217.0 nm or 283.3 nmb) Calibration curve preparation
1) Spray the lead standard solutions for the calibration curve preparation and the blanktest solution for the calibration curve preparation into a flame, and read theindicated value at a wavelength of 217.0 nm or 283.3 nm.
2) Prepare a curve for the relationship between the lead concentration and theindicated value of the lead standard solutions for the calibration curve preparationand the blank test solution for the calibration curve preparation.
c) Sample measurement
1) Subject the sample solution (8) to the same procedure as in b) 1) to read the indicatedvalue.
2) Subject the blank test solution to the same procedure as in b) 1) to read theindicated value, and correct the indicated value obtained for the sample solution.
3) Obtain the lead content from the calibration curve, and calculate the leadconcentration in the analytical sample.
Note (8) If there is a possibility that the lead concentration in the sample solution will
exceed the maximum limit of the calibration curve, dilute a predeterminedamount with hydrochloric acid (1+23).

297

Comment 3 The lead concentration in the analytical sample can also be corrected bysubjecting the blank test solution to the same procedures as in 1) and 3) toobtain the lead content in the blank test solution.
Comment 4 A recovery testing was conducted using industrial sludge fertilizer andcomposted sludge fertilizer (5 samples); as a result, the recovery at theconcentration level of 100 mg/kg and 10 mg/kg was 99.1 % - 100.6 % and97.5 % - 99.6 %, respectively, and the repeatability was 0.3 % - 1.4 % and
0.4 % - 3.0 % relative standard deviation, respectively.
In addition, the results and analysis results of a collaborative study for test
method validation are shown in Table 1.
Additionally, the minimum limit of quantification of the test method is about
1 mg/kg.
Table 1 Results and analysis results from a collaborative study
for lead test method validation
Number of Mean2) RSDr3) RSDR4)
Sample name1)laboratories (mg/kg)(%)(%)
Sewage sludge fertilizer a

10

25.2

4.6

3.9

Sewage sludge fertilizer b

11

29.4

3.7

4.3

Composted sludge fertilizer a

10

18.6

3.2

5.0

Composted sludge fertilizer b

10

22.2

1.8

7.0

Composted sludge fertilizer c
11
86.8
1.3
1) Number of laboratories used in analysis
2) Mean (n= number of laboratories x number of samples (2))3) Repeatability relative standard deviation
4) Reproducibility relative standard deviation

4.0

References
1) Yoshinari SAKAKIBARA, Manabu MATSUZAKI and Tadao AMANO: Determinationof Cadmium, Lead, Nickel and Chromium in Sludge Fertilizer - Improved
Decomposition Method - Research Report of Fertilizer Vol.1 2008. (41 - 49)
2) Yoshinari SAKAKIBARA and Manabu MATSUZAKI: Determination of Cadmium,
Lead, Nickel and Chromium in Sludge Fertilizer - Collaborative Test Results - Research
Report of Fertilizer Vol.1 2008. (50 - 59)
3) Hisanori ARAYA and Yoshimi TAKEBA: Determination of Cadmium, Lead, Nickel
and Chromium in Calcined Sludge Fertilizer - Using Decomposition Method forInorganic Fertilizer- Research Report of Fertilizer Vol.3 2010. (30 - 42)

298

(5)

Flow sheet for lead: The flow sheet for lead in fertilizers is shown below:
5.00 ganalytical sample
Charring
Incineration

200-mL - 300-mL tall beaker.
Heat gently
Ignite at 450 ºC ± 5 ºC

Standing to coolRoom temperature← A small amount of water, moisten the residue← About 10 mL of nitric acid
← About 30 mL of hydrochloric acid
Heating
Cover with a watch glass, and digest
Heating

Slightly move a watch glass to remove acid

Standing to coolRoom temperature← About 25 mL - 50 mL of hydrochloric acid (1+5)
Heating
Cover with a watch glass, and dissolve
Standing to coolRoom temperature

Transfer
100-mL - 200-mL volumetric flask, water← Water (up to the marked line)
Filtration
Type 3 filter paper
Measurement

Atomic absorption spectrometer (217.0 nm or 283.3 nm)

Figure Flow sheet for lead in fertilizers

299

5.6.b ICP Atomic Emission Spectrometry <Reference method>(1) Summary
The test method is applicable to sludge fertilizers.
Pretreat an analytical sample with incineration and nitric acid-hydrochloric acid (1+3),
introduce it to an ICP Atomic Emission Spectrometer (“ICP-AES”) and measure the emission
with lead at a wavelength of 220.351 nm to quantify lead.
(2) Reagents, etc.: Reagents and water are as shown below:a) Water: Water of A3 specified in JIS K 0557.
b) Nitric acid: A reagent of harmful metal analysis grade, microanalysis grade orequivalents.
c) Hydrochloric acid: A reagent of harmful metal analysis grade, microanalysis grade orequivalents.
d) Lead standard solution (0.1 mg/mL): Lead standard solution (0.1 mg/mL) for atomic
absorption spectrometry traceable to a specified reference material (national
measurement standard) according to Article 134 of the Measurement Act.
e) Lead standard solutions (2.5 µg/mL) (1) (2):Dilute a predetermined amount of lead standard solution (0.1 mg/mL) with hydrochloric
acid (1+23) to prepare lead standard solution (2.5 µg/mL).
Note (1) This is an example of preparation; prepare an amount as appropriate.
(2) Store at room temperature, and do not use after 6 months after preparation.
(3) Instruments: Instruments are as shown below:a) ICP Atomic Emission Spectrometer: An atomic emission spectrometer specified in
JIS K 0116.
1) Gas: Argon gas specified in JIS K 1105 of no less than 99.5 % (volume fraction) inpurityb) Electric furnace: An electric furnace that can keep the test temperature at  5 ºCc) Hot plate or sand bath: A hot plate whose surface temperature can be adjusted up to
250 ºC. Adjust the amounts of gas and silica sand of a sand bath so that the sand bathtemperature can be set to 250 ºC.
(4) Test procedures(4.1) Extraction: Conduct extraction as follows:a) Weigh 5.00 g of an analytical sample, and put it in a 200-mL - 300-mL tall beaker.
b) Put the tall beaker in an electric furnace, and heat gently to char.(3)c) Ignite at 450 ºC ± 5 ºC to incinerate (4).
d) After standing to cool, moisten the residue with a small amount of water, and add about
10 mL of nitric acid and about 30 mL of hydrochloric acid.
e) Cover the tall beaker with a watch glass, and heat on a hot plate or a sand bath to digest.
f) Slightly move the watch glass (5), and continue heating on the hot plate or sand bath toconcentrate until nearly dried up.
g) After standing to cool, add 25 mL - 50 mL of hydrochloric acid (1+5) (6) to the digest,cover the tall beaker with the watch glass, and heat quietly to dissolve.
h) After standing to cool, transfer the solution to a 100-mL - 200-mL volumetric flask withwater, add water up to the marked line, and filter with Type 3 filter paper to make thesample solution.
i) As a blank test, conduct the procedures in b) - h) using another tall beaker to prepare theblank test solution.
300

Note (3) Example of the charring procedure: Heat at about 250 ºC until smoke is nolonger formed.
(4) Example of ignition period: 8 - 16 hours(5) The watch glass can be removed.
(6) Add hydrochloric acid (1+5) so that the hydrochloric acid concentration of thesample solution will be hydrochloric acid (1+23). For example, when a 100-mL
volumetric flask is used in the procedure in h), about 25 mL of hydrochloric acid(1+5) should be added.
Comment 1 Do not conduct procedure (4.1) b) - c) in the case of fertilizers not containorganic matter
Comment 2 The procedures in (4.1) are the same as in (4.1) in 4.9.1.a.
(4.2) Measurement: Conduct measurement (Standard Addition Method) according to JIS K
0116 and as shown below. Specific measurement procedures are according to theoperation method of the ICP Atomic Emission Spectrometer used in measurement.
a) Measurement conditions for the ICP Atomic Emission Spectrometer: Set up themeasurement conditions for the ICP Atomic Emission Spectrometer considering thefollowing:
Analytical line wavelength: 220.351 nmb) Calibration curve preparation and Sample measurement.
1) Put 5 mL of sample solution into three 10-mL volumetric flasks respectively.
2) Add 2 mL and 4 mL of lead standard (0.25 μg/mL) solution to volumetric flasks of1) above, then add hydrochloric acid (1+23) to the marked line to make the sample
solution of Standard Addition Method.
3) Add hydrochloric acid (1+23) to the marked line of the remaining volumetric flaskof 1) above, and use this solution as the sample solution without standard solution.
4) Spray the sample solution of Standard Addition Method and sample solution
without standard solution into the induction plasma, and read the indicated value ata wavelength of 220.351 nm.
5) Add 5 mL of blank test solution to a 10-mL volumetric flask, conduct the sameprocedures as in 3) - 4) to read the indicated value, and correct the indicated value
obtained from the respective sample solutions.
6) Prepare a curve for the relationship between the added lead concentration and thecorrected indicated value of the sample solutions of Standard Addition Method andthe sample solution without standard solution.
7) Obtain the lead content from the intercept of the calibration curve, and calculate thelead concentration in the analytical sample.
Comment 3 The lead concentration in the analytical sample can also be corrected bysubjecting the blank test solution to the same procedures as in 1) - 4) and 6)-7) to obtain the lead content in the blank test solution.
Comment 4 Simultaneous measurement of multiple elements by ICP-AES is possible. In
this case, mix a predetermined amount of copper standard solution (0.1mg/mL), zinc standard solution (0.1 mg/mL), cadmium standard solution (0.1mg/mL), nickel standard solution (0.1 mg/mL), chromium standard solution(0.1 mg/mL) and lead standard solution (0.1 mg/mL), dilute withhydrochloric acid (1 +23) to prepare mixture standard solution (Cu 25 μg/mL,
Zn 25 μg/mL, Cd 0.25 μg/mL, Ni 2.5 μg/mL, Cr 2.5 μg/mL and Pb 2.5
μg/mL) (1) (2). Use the mixture solution instead of lead standard solution (2.5301μg/mL) in (4.2) b) 2). After that, conduct the same procedures as in (4.2) b) to
calculate the concentration of respective elements in the analytical sample.
Note that the wavelength for measurement of respective elements is Cu
324.754 nm, Zn 206.191 nm, Cd 228.802 nm, Ni 231.604 nm, Cr 205.552 nm
and Pb 220.351 nm In addition, the respective element concentrations forrespective standard addition samples are shown in the below Table.
Table The additive amount of mixed standard solution
and the additive concentration of respective elements for respective sample solutions
Additive amount Cd
Pb
Ni
Cr
Cu
Zn
(mL) of mixed(mg/L) (mg/L) (mg/L) (mg/L) (mg/L) (mg/L)
standard solution
Sample solution without
0
0
0
0
0
0
0
standard solution
Sample solution 1 by
2
0.05
0.5
0.5
0.5
5.0
5.0
Standard Addition Method
Sample solution 2 by
4
0.1
1.0
1.0
1.0
10
10
Standard Addition Method

Comment 5

For each one sample of sewage sludge fertilizer, human waste sludge fertilizer,
industrial sludge fertilizer, mixed sludge fertilizer, calcined sludge fertilizer
and composted sludge fertilizer, the repeatability obtained from triplicatesmeasurement is 0.9 % - 3.3 % relative standard deviation.
Additionally, the minimum limit of quantification of the test method is about
5 mg/kg.

Reference
1) Masahiro ECHI, Tomoe INOUE, Megumi TABUCHI, Tetsuya NOMURA :Simultaneous Determination of Cadmium, Lead, Nickel, Chromium, Copper and Zincin Sludge Fertilizer using Inductively Coupled Plasma-Atomic Emission Spectrometry(ICP-AES), Research Report of Fertilizer Vol.4 2011. (36 - 48)

302

(5)

Flow sheet for lead: The flow sheet for lead in fertilizers is shown below:
Standing to coolRoom temperature← A small amount of water← About 10 mL of nitric acid
← About 30 mL of hydrochloric acid
Heating
Cover with a watch glass, and digest
Heating

Slightly move a watch glass to remove acid

Standing to coolRoom temperature← 25 mL - 50 mL of hydrochloric acid (1+5)
Heating
Cover with a watch glass, and dissolve
Standing to coolRoom temperature

Transfer
100-mL - 200-mL volumetric flask, water← Water (up to the marked line)
Filtration
Type 3 filter paper
Aliquot 5 mL
10-mL volumetric flask, 3 flasks← 0, 2 and 4 mL of lead standard solution (2.5 μg / mL) respectively← Hydrochloric acid (1+23) ( up to the marked line)
Measurement
ICP Atomic Emission Spectrometer (220.351 nm)
Figure Flow sheet for lead in fertilizers

303

5.7 Sulfamic acid (amidosulfuric acid)5.7.a Ion Chromatography <Reference method>(1) Summary
The test method is applicable to ammonium sulfate.
Add water to an analytical sample to extract sulfamic acid, introduce it to an Ion
Chromatograph (IC) or a High Performance Liquid Chromatograph (HPLC) to isolate it with
an ion exchange column, then measure the sulfamic acid with an electric conductivity detector
to quantify sulfamic acid.
Sulfamic acid and ammonium thiocyanate can be simultaneously quantified by using thismethod. (Refer to Comment 4).
(2)
a)
b)
c)
d)
e)
f)

Reagents, etc.: Reagents and water are as shown below:
Water: Water of A3 specified in JIS K 0557 or equivalent quality
Phthalic acid: A reagent of no less than 98 % (mass fraction) in purity.
p-hydroxybenzoic acid: A reagent of no less than 95 % (mass fraction) in purity.
1-sodium octane sulfonate: A reagent of no less than 98 % (mass fraction) in purity.
1-sodium hexane sulfonate: A reagent of no less than 98 % (mass fraction) in purity.
Boric acid: A JIS Guaranteed Reagent specified in JIS K 8863 or a reagent ofequivalent quality.
g) Elute (1) (2): Weigh 0.083 g of phthalic acid, 0.552 g of p-hydroxybenzoic acid, 0.195 gof 1-sodium octane sulfonate, 0.376 g of 1-sodium hexane sulfonate and 6.183 g ofboric acid to a 1-L volumetric flask, add about 500 mL of water to dissolve and add
water up to the marked line. Filter with membrane type filter (pore size: no more than
0.5 μm) made of hydrophilic PTFEh) Sulfamic acid standard solution (1,000 mg/L) (1): Put 0.1 g of sulfamic acid, referencematerial for volumetric analysis (HOSO2NH2: dried for 48 hours in a silica gel
desiccator), in a weighing dish and measure the mass to the order of 0.1 mg. Add asmall amount of water to dissolve, then transfer to a 100-mL volumetric flask and add
water up to the marked line.
i) Sulfamic acid standard solution (10 mg/L) (1): At the time of usage, put 10 mL ofsulfamic acid standard solution (1,000 mg/L) to a 100-mL volumetric flask and add
water up to the marked line.
j) Sulfamic acid standard solution for the calibration curve preparation (0.3 mg/L - 3
mg/L): At the time of usage, put 3 mL - 30 mL of sulfamic acid standard solution (10mg/L) to 100-mL volumetric flasks step-by-step and add water up to the marked line.
Note (1) This is an example of preparation; prepare an amount as appropriate.
(2) The concentration of prepared solutions is phthalic acid 0.5 mmol/L,
p-hydroxybenzoic acid 4.0 mmol/L, 1-sodium octane sulfonate 0.9 mmol/L,
1-sodium hexane sulfonate 2.0 mmol/L and boric acid 100 mmol/L.

(3) Apparatus and instruments: Apparatus and instruments are as shown below:a) Ion Chromatograph (IC) or High Performance Liquid Chromatograph (HPLC):IC specified in JIS K 0127 or HPLC specified in JIS K 0124 that satisfies following
requirements.
1) Column: A 4-mm inner diameter 100-mm long stainless steel column tube filledwith hydrophilic methacrylate-gel, to which 5-μm particle diameter class 4
ammonium group chemically bonds (3).
2) Column bath: A column bath whose temperature can be adjusted to 55 ºC - 60 ºC.
3) Detection unit: An electric conductivity detector
304b) Membrane filters: Pore size is no more than 0.5 μm, made of hydrophilic PTFE
Note (3) A column is commercially sold under the name of Shodex IC NI-424, etc.
(4) Test procedures(4.1) Extraction: Conduct extraction as follows:a) Weigh 1.00 g of an analytical sample, and put it in a 100-mL volumetric flask.
b) Add about 50 mL of water, stopple the volumetric flask, and shake to dissolve, and then
add water up to the marked line.
c) Transfer a predetermined amount of the solution, and dilute 12.5 times exactly withwater.
d) Filter with a membrane filter (pore size: no more than 0.5 μm) to make the sample
solution.
(4.2) Measurement: Conduct measurement according to JIS K 0127 or JIS K 0124 and as
shown below. Specific measurement procedures are according to the operation methodof the Ion Chromatograph (IC) or the High Performance Liquid Chromatograph (HPLC)
used in measurement.
a) Measurement conditions for the Ion Chromatograph (IC) or High Performance
Liquid Chromatograph (HPLC): Set up the measurement conditions considering thefollowing example of measurement conditions.
1) Column: A hydrophilic methacrylate-gel column (4-mm inner diameter, 100-mm
long, 5-μm particle diameter) to which class 4 ammonium group chemically bonds.
2) Column bath temperature: 58 ºC3) Elute: Prepared by the procedures in (2) g)
4) Flow: 1 mL/min
5) Injection volume: 20 μL
6) Detection unit: An electric conductivity detectorb) Calibration curve preparation
1) Inject 20 μL of respective standard solutions for the calibration curve preparation toan IC or HPLC, and record the chromatogram of electric conductivity to obtainpeak area.
2) Prepare a curve for the relationship between the concentration and the peak area ofelectric conductivity of respective standard solutions for the calibration curve
preparation.
Prepare a calibration curve when the sample is measured.
Comment 1 In the measurement of sample solution, there is a possibility that the recovery
rate becomes lower than actual due to the influence of matrix if theconcentration is calculated with peak height. Therefore prepare a relationshipline with peak area when calibration curve is prepared.
c) Sample measurement
1) Subject 20 μL of sample solution to the same procedure as in b) 1)
2) Obtain the sulfamic acid content from calibration curve by peak area to calculate
the concentration of sulfamic acid in the analytical sample.
Comment 2 Calculate the concentration by the peak area similarly as the calibration curve
preparation to prevent the influence of matrix.
305

Comment 3 Note that it takes time to stabilize the baseline due to the usage of theion-pairing reagent in the elute. It is recommended to take about 120 minutes
for stabilization time before starting measurement.
Comment 4 It is possible for the simultaneous measurement of sulfamic acid andammonium thiocyanate in this testing method. In that case, mix apredetermined amount of sulfamic acid standard solution (1,000 mg/L) andammonium thiocyanate standard solution (1,000 mg/L), dilute with water toprepare mixture standard solution (10 mg/L) (1) and use it instead of respective
standard solutions (10 mg/L) in (2) i). After that, conduct the same procedurein (4.2) b) to calculate the respective concentrations of materials subjected tomeasurement in the analytical sample.
Comment 5 A recovery testing of ammonium sulfate (3 brands) was conducted. As aresult, the average recovery rate at additive level of 0.25 % (mass fraction)and 0.075 % (mass fraction) was 94.4 % - 103.5 %, and the repeatability was
0.7 % - 2.3 % relative standard deviation.
Additionally, the minimum limit of quantification of the test method is about
0.04 % (mass fraction).
(5)

Flow sheet for testing method: The flow sheet for sulfamic acid in ammonium sulfate
is shown below:
1.00 gWeigh into a 100-mL volumetric flask← About 50 mL of water
Shaking to mix

Stopple the volumetric flask and dissolve← Water (up to the marked line)
Dilution

Dilute 12.5 times, water

Filtration

Membrane filter (no more than 0.5-µm)

Measurement

Ion Chromatograph

Figure Flow sheet for sulfamic acid in ammonium sulfate

Reference
1) Toshiaki HIROI and Yuji SHIRAI: Simultaneous Determination of Sulfamic Acid andAmmonium Thiocyanate in Ammonium Sulfate by Nonsuppressed Ion Chromatography,
Research Report of Fertilizer,Vol.5, 2012, (1 - 23)306

Reference:

The IC chromatogram of sulfamic acid and thiocyanic acid of the standard
solution for the calibration curve preparation and sample solution (ammonium
sulfate) are shown below.

12(A) Mixture standard solution (the equivalents of 60 ng as sulfamic acid and ammonium
thiocyanate (3 mg/L, 20 μL), respectively)
Enlarged diagram of peak for sufamic acid112(B) Sample solution (the equivalents of 0.25 % (mass fraction) as sulfamic acid andammonium thiocyanate added in ammonium sulfate (2,500 μg/g), respectively)
Reference diagram: IC chromatogram of sulfamic acid and ammonium
thiocyanate.
(Peak: 1. Sulfamic acid, 2. Ammonium thiocyanate)
IC measurement conditions
Column: Shodex IC NI-424 (4.6-mm inner diameter, 100-mm long,
5μm-particle diameter)
Other conditions are according to the example of measurement conditions in(4.2) a)
307

5.8
5.8.a

Sulfurized cyanide (ammonium thiocyanate)
Ion Chromatography <Reference method>(1) Summary
The test method is applicable to ammonium sulfate.
Add water to an analytical sample to extract ammonium thiocyanate, introduce it to an Ion
Chromatograph (IC) or a High Performance Liquid Chromatograph (HPLC) to isolate it with
an ion exchange column, and then measure the thiocyanic acid with an electric conductivity
detector to quantify ammonium thiocyanate.
Sulfamic acid and ammonium thiocyanate can be simultaneously quantified by using thismethod. (Refer to Comment 4).
(2)
a)
b)
c)
d)
e)
f)

Reagents, etc.: Reagents and water are as shown below:
Water: Water of A3 specified in JIS K 0557 or equivalent quality
Phthalic acid: A reagent of no less than 98 % (mass fraction) in purity.
p-hydroxybenzoic acid: A reagent of no less than 95 % (mass fraction) in purity.
1-sodium octane sulfonate: A reagent of no less than 98 % (mass fraction) in purity.
1-sodium hexane sulfonate: A reagent of no less than 98 % (mass fraction) in purity.
Boric acid: A JIS Guaranteed Reagent specified in JIS K 8863 or a reagent ofequivalent quality.
g) Elute (1) (2): Weigh 0.083 g of phthalic acid, 0.552 g of p-hydroxybenzoic acid, 0.195 gof 1-sodium octane sulfonate, 0.376 g of 1-sodium hexane sulfonate, and 6.183 g ofboric acid to a 1-L volumetric flask, add about 500 mL of water to dissolve and add
water up to the marked line. Filter with membrane type filter (pore size: no more than
0.5 μm) made of hydrophilic PTFEh) Ammonium thiocyanate standard solution (1,000 mg/L) (1): Put 0.1 g of ammonium
thiocyanate (3), specified in JIS K 9000 in weighing dish, and measure the mass to theorder of 0.1 mg. Add a small amount of water to dissolve, then transfer to a 100-mL
volumetric flask and add water up to the marked line.
i) Ammonium thiocyanate standard solution (10 mg/L) (1): At the time of usage, put 10
mL of ammonium thiocyanate standard solution (1,000 mg/L) to a 100-mL volumetricflask and add water up to the marked line.
j) Ammonium thiocyanate standard solution for the calibration curve preparation(0.3 mg/L - 3 mg/L): At the time of usage, put 3 mL - 30 mL of sulfamic acid standard
solutions (10 mg/L) to 100-mLvolumetric flasks step-by-step and add water up to themarked line.
Note (1) This is an example of preparation; prepare an amount as appropriate.
(2) The concentrations of prepared solutions are phthalic acid 0.5 mmol/L,
p-hydroxybenzoic acid 4.0 mmol/L, 1-sodium octane sulfonate 0.9 mmol/L,
1-sodium hexane sulfonate 2.0 mmol/L, and boric acid 100 mmol/L,
respectively.
(3) It is recommended to store in a desiccator because of deliquescence.

(3) Apparatus and instruments: Apparatus and instruments are as shown below:a) Ion Chromatograph (IC) or High Performance Liquid Chromatograph (HPLC):IC specified in JIS K 0127 or HPLC specified in JIS K 0124 that satisfies following
requirements.
1) Column: A 4-mm inner diameter 100-mm long stainless steel column tube filledwith hydrophilic methacrylate-gel, to which 5-μm particle diameter class 4
ammonium group chemically bonds (4).
308

2) Column bath: A column bath whose temperature can be adjusted to 55 ºC - 60 ºC.
3) Detection unit: An electric conductivity detectorb) Membrane filters: Pore size is no more than 0.5 μm, made of hydrophilic PTFE
Note (4) The column is commercially sold under the name of Shodex IC NI-424 etc.
(4) Test procedures(4.1) Extraction: Conduct extraction as follows:a) Weigh 1.00 g of an analytical sample, and put it in a 100-mL volumetric flask.
b) Add about 50 mL of water, stopple the volumetric flask, and shake to dissolve, and then
add water up to the marked line.
c) Transfer a predetermined amount of the solution, and dilute 12.5 times exactly withwater.
d) Filter with a membrane filter (pore size no more than 0.5 μm) to make the sample
solution.
(4.2) Measurement: Conduct measurement according to JIS K 0127 or JIS K 0124 and as
shown below. Specific measurement procedures are according to the operation methodof the Ion Chromatograph (IC) or the High Performance Liquid Chromatograph (HPLC)
method used in measurement.
a) Measurement conditions for the Ion Chromatograph (IC) or High Performance
Liquid Chromatograph (HPLC): Set up the measurement conditions considering thefollowing example of measurement conditions.
1) Column: A hydrophilic methacrylate-gel column (4-mm inner diameter, 100-mm
long, 5-μm particle diameter) to which class 4 ammonium group chemically bonds.
2) Column bath temperature: 58 ºC3) Elute: Prepared by the procedures in (2) g)
4) Flow: 1 mL/min
5) Injection volume: 20 μL
6) Detection unit: An electric conductivity detectorb) Calibration curve preparation
1) Inject 20 μL of respective standard solutions for the calibration curve preparation
into an IC or HPLC, and record the chromatogram of electric conductivity to obtainpeak area.
2) Prepare a curve for the relationship between the concentration and the peak area ofelectric conductivity of respective standard solutions for the calibration curve
preparation.
Prepare a calibration curve when the sample is measured.
Comment 1 In the measurement of sample solution, there is a possibility that the recovery
rate becomes lower than actual due to the influence of matrix if theconcentration is calculated with peak height. Therefore prepare a relationshipline with peak area when calibration curve is prepared.
c) Sample measurement
1) Subject 20 μL of sample solution to the same procedure as in b) 1)
2) Obtain the ammonium thiocyanate content from calibration curve by peak area tocalculate the concentration of ammonium thiocyanate in the analytical sample.

309

Comment 2 Calculate the concentration by the peak area to prevent the influence of
matrix similarly as the calibration curve preparation,
Comment 3 Note that it takes time to stabilize the baseline due to the usage of theion-pairing reagent in the elute. It is recommended to take about 120 minutes
for stabilization time before starting measurement.
Comment 4 It is possible for the simultaneous measurement of ammonium thiocyanateand sulfamic acid in this testing method. In that case, mix a predeterminedamount of sulfamic acid standard solution (1,000 mg/L) and ammonium
thiocyanate standard solution (1,000 mg/L), dilute with water to prepare
mixture standard solution (10 mg/L) (1) and use it instead of respective
standard solutions (10 mg/L) in (2) i). After that, conduct similarly as theprocedure in (4.2) b), and calculate the respective concentrations of materials
subjected to measurement in the analytical sample.
Comment 5 A recovery testing of ammonium sulfate (3 brands) was conducted, as a result,the average recovery rate at additive level of 0.25 % (mass fraction) and0.075 % (mass fraction) was 93.9 % - 103.7 %, and the repeatability was
0.6 % - 5.9 % relative standard deviation.
Additionally, the minimum limit of quantification of the test method is about
0.04 % (mass fraction).
Reference
1) Toshiaki HIROI and Yuji SHIRAI: Simultaneous Determination of Sulfamic Acid andAmmonium Thiocyanate in Ammonium Sulfate by Nonsuppressed Ion Chromatography,
Research Report of Fertilizer,Vol.5, 2012, (1 - 23)(5)

Flow sheet for testing method: The flow sheet for ammonium thiocyanate inammonium sulfate is shown below:
1.00 gWeigh into a 100-mL volumetric flask← About 50 mL of water
Shaking to mix

Stopple the volumetric flask and dissolve← Water (up to the marked line)
Dilution

Dilute 12.5 times, water

Filtration

Membrane filter (no more than 0.5-µm)

Measurement

Ion Chromatograph

Figure flow sheet for ammonium thiocyanate in ammonium sulfate

310Reference

The IC chromatogram of sulfamic acid and thiocyanic acid of the standard
solution for the calibration curve preparation and the sample solution(ammonium sulfate) are shown below.

12(A) Mixture standard solution (the equivalents of 60 ng as sulfamic acid and ammonium
thiocyanate (3 mg/L, 20 μL), respectively)
Enlarged diagram of peak for sufamic acid

1

1
2
1

(B)

Sample solution (the equivalents of 0.25% (mass fraction) as sulfamic acid andammonium thiocyanate added in ammonium sulfate (2,500 μg/g), respectively)
Reference diagram: IC chromatogram of sulfamic acid and thiocyanic acid(Peak: 1. Sulfamic acid, 2. Thiocyanic acid)IC measurement conditions
Column: Shodex IC NI-424 (4.6-mm inner diameter, 100-mm long, 5-μm particle
diameter)Other conditions are according to the example of measurement conditions in (4.2)a)
311

5.8.bHigh Performance Liquid Chromatography <Reference method>(1) Summary
This method is applicable to fertilizers.
Add water to an analytical sample, extract sulfurized cyanide (hereinafter referred to as“ammonium thiocyanate”) and adjust pH as necessary. Introduce it into a High Performance
Liquid Chromatograph (HPLC), isolate with a silica gel column to which amino groupchemically bonds or a vinyl alcohol polymer column to which amino group chemically bonds,
and measure at wavelength 210 nm to quantify ammonium thiocyanate.
With this method, nitrous acid and ammonium thiocyanate can be determined
simultaneously. (Refer to Comment 4)(2) Reagents: Reagents and water are as shown below:a) Water: Water of A3 specified in JIS K 0557or equivalent quality.
b) Sodium hydroxide: A JIS Guaranteed Reagent specified in JIS K 8576 or a reagent ofequivalent quality.
c) Disodium hydrogenphosphate dodecahydrate: A JIS Guaranteed Reagent specified
in JIS K 9019 or a reagent of equivalent quality.
d) Sodium dihydrogenphosphate dihydrate: A JIS Guaranteed Reagent specified in JIS
K 9009 or a reagent of equivalent quality.
e) Sodium perchlorate monohydrate: A JIS Guaranteed Reagent specified in JIS K 8227
or a reagent of equivalent quality.
f) Ammonium thiocyanate standard solution (1 mg/mL) (1): Put 0.1 g of ammonium
thiocyanate specified in JIS K 9000 in a weighing dish and measure the mass to theorder of 0.1 mg. Add a small amount of water to dissolve, then transfer to a
100-mLvolumetric flask and add water up to the marked line.
g) Ammonium thiocyanate standard solution (100 μg/mL) (1): At the time of usage, put
10 mL of ammonium thiocyanate standard solution (1 mg/mL) to a 100-mL volumetricflask and add water up to the marked line.
h) Ammonium thiocyanate standard solution for the calibration curve preparation (1μg/mL - 20 μg/mL): At the time of usage, put 1 mL - 20 mL of ammonium thiocyanate
standard solution (100 μg/mL) to 100-mL volumetric flasks step-by-step and add water
up to the marked line.
Note (1) This is an example of preparation; prepare an amount as appropriate.
(3) Apparatus and instruments: Apparatus and instruments are as shown below:a) High Performance Liquid Chromatograph (HPLC): HPLC specified in JIS K 0124
that satisfies following requirements.
1) Column: A 4-mm to 6-mm inner diameter 150-mm to 250-mm long stainless steel
column tube filled with silica gel (2) or poly vinyl alcohol, to which 5-μm particle
diameter amino group chemically bonds .
2) Column bath: A column bath whose temperature can be adjusted to 30 ºC - 45 ºC.
3) Detector: An absorptiometric detector that can measure at wavelength around 210
nm.
b) Magnetic stirrerc) Centrifugal separator: A centrifugal separator that can centrifuge at 2,000 × g.
d) High speed centrifugal separator: A centrifugal separator that can centrifuge at
8,000×g.
e) pH test paper: A pH test paper infiltrated with indicator and dried, which can measure
the value from pH 1 to pH 11 and a color change chart with the pH interval value 1 is
attached.
312Note (2) Remaining silanol group of silica gel affects the measurement of ion in somecases. Therefore use a column which does not affect the measurement of
thiocyanic acid by treating the silanol group. As an example of the treatment,
silica gel is to be entirely covered with the uniform membrane of silicone
polymer.
Comment 1

Comment 2

A column is sold under the production names of CAPCELL PAK NH2
UG80 and Asahipak NH2P-50 4E from Shiseido and Showa Denko
respectively.
pH test paper is sold under the production name of UNIV Shikenshi fromADVANTEC(4) Test procedures(4.1) Extraction: Conduct extraction as shown below.
(4.1.1) Powdery analytical samplea) Weigh 1.00 g of an analytical sample, and put it into a 200-mL ground-in stopper
Erlenmeyer flask.
b) Add 100 mL of water and stir it with a magnetic stirrer for about 10 minutes.
c) After allowing to stand still, transfer supernatant solution to a 50-mL ground-in stopper
centrifugal precipitate tube.
d) Centrifuge at 2,000 × g centrifugal force for about five minutes (3) to make supernatantas the extract.
Note (3) 18.7-cm of rotor radius and 3,000 rpm of revolutions makes about 2,000 × g
centrifugal force.
(4.1.2) Fluid analytical samplea) Weigh 1.00 g of an analytical sample, and put it in a 100-mL volumetric flask.
b) Add about 50 mL of water, stopple the volumetric flask and shake to mix and dissolve.
c) Add water up to the marked line to make the extract.
(4.2) pH adjustment: Conduct pH adjustment as shown below.
a) Transfer a small amount of the extract to confirm pH value using a pH-test paper.
b) If the pH value in a) is pH 5 or more, transfer the extract to a 1.5-mL ground-in stopper
centrifugal precipitate tube(4) and conduct the procedure in f) to prepare sample solution.
c) If the pH value in a) is pH 4 or less, transfer 40 mL of the extract to a 100-mL beaker.
d) Add sodium hydroxide solution (5 mg/mL), adjust it to pH 5 to pH 7 with a pH meterand transfer to a 50-mL volumetric flask with water.
e) Add water to the marked line and transfer to a 1.5-mL ground-in stopper centrifugal
precipitate tube (4).
f) Centrifuge at 8,000 × g centrifugal force for about five minutes (5) to make supernatantas the sample solution.
Note (4) The ground-in stopper centrifugal precipitate tube should be made ofpolypropylene, etc. to not affect the measurement.
(5) 7-cm of rotor radius and 10,000 rpm of revolutions makes about 8,000 × g
centrifugal force.

313

Comment 3

Instead of procedures in (4.2) b) and e) - f), it is allowed to filter with amembrane filter (aperture diameter: no more than 0.5-µm) made ofhydrophilic PTFE and the filtrate can be the sample solution.

(4.3) Measurement: Conduct measurement according to JIS K 0124 and as shown below.
Specific measurement procedures are according to the operation method of High
Performance Liquid Chromatograph (HPLC) used in measurement.
a) Measurement conditions for High Performance Liquid Chromatograph (HPLC):
Set up the measurement conditions for High Performance Liquid Chromatograph(HPLC) considering the following example:
1) Column: A silica gel column (4-mm - 6-mm inner diameter, 150-mm - 250-mm
long, 5-µm particle diameter) to which amino group chemically bonds or a vinyl
alcohol polymer column (4-mm - 6-mm inner diameter, 150-mm - 250-mm long,
5-µm particle diameter) to which amino group chemically bonds.
2) Temperature of Column bath: 30 ºC - 40 ºC3) Eluent (1): Dissolve 1.79 g of disodium hydrogenphosphate dodecahydrate, 0.78 g of
sodium dihydrogenphosphate dihydrate and 14.04 g of sodium perchloratemonohydrate in water to make 1,000 mL. Filter with a membrane filter (aperture
diameter: no more than 0.5-µm) made of hydrophilic PTFE.
4) Flow rate: 0.9 mL/min
5) Injection rate: 10 μL
6) Detector: An absorptiometric detector, measurement wavelength: 210 nmb) Calibration curve preparation
1) Inject 10 µL of respective standard solutions for the calibration curve preparation to
HPLC, record chromatogram at wavelength 210 nm and obtain the peak area.
2) Prepare a curve for the relationship between the concentration and the peak area atwavelength 210 nm of the standard solutions for the calibration curve preparation.
c) Sample measurement
1) Subject 10 µL of the sample solution to the same procedure as in b) 1).
2) Obtain the ammonium thiocyanate content from the peak area using the calibration
curve to calculate the concentration of ammonium thiocyanate in the analyticalsample.
Comment 4

This test method enables the simultaneous measurement of nitrous acid andammonium thiocyanate. In this case, mix a predetermined amount of nitrousacid standard solution (1 mg/mL) and ammonium thiocyanate standard
solution (1 mg/mL), dilute with water to prepare mixture standard solution(100 μg/mL) (1) and use it instead of (2) h) ammonium thiocyanate standard
solution (100 μg/mL). After that, conduct the same procedure in (4.3) b) to
calculate the respective concentrations of materials subjected tomeasurement in the analytical sample.

314

(5)

Flow sheet for testing method: The flow sheet for ammonium thiocyanate in fertilizers
is shown below:

1.00 g200-mL ground-in stopper Erlenmeyer flask
analytical sample (powdery)← 100 mL of water
Extraction
Stir to mix, 10 minites
Centrifugal separation

Ground-in stopper centrifugal precipitate tube,
2,000 ×g , 5 minutes

1.00 ganalytical sample (fluid)

100-mL volumetric flask← 50 mL - 70 mL of water
Extraction

Shake to mix← Water (up to the marked line)

pH value confirmation

pH test paper

pH 5 or more
pH 4 or less
Aliquot (40 mL)
pH adjustment (pH 5 - pH 7)

100-mL beaker
Sodium hydroxide solution (5 mg/mL)Transfer
100-mL volumetric flask, water← Water (up to the marked line)
Centrifugal separation

Measurement
FigureGround-in stopper centrifugal precipitate tube,
8,000 ×g , 5 minutes
High Performance Liquid Chromatograph

Flow sheet for ammonium thiocyanate in fertilizers

3155.9 Nitrous acid
5.9.a High Performance Liquid Chromatography <Reference method>(1) Summary
This method is applicable to fertilizers.
Add water to an analytical sample, extract nitrous acid and adjust pH as necessary. Introduce
it into a High Performance Liquid Chromatograph (HPLC), isolate with a silica gel column towhich amino group chemically bonds or a vinyl alcohol polymer column to which aminogroup chemically bonds, and measure at wavelength 210 nm to quantify nitrous acid.
With this method, nitrous acid and ammonium thiocyanate can be measured simultaneously.
(Refer to Comment 4)(2) Reagents: Reagents and water are as shown below:a) Water: Water of A3 specified in JIS K 0557or equivalent quality.
b) Sodium hydroxide: A JIS Guaranteed Reagent specified in JIS K 8576 or a reagent ofequivalent quality.
c) Disodium hydrogenphosphate dodecahydrate: A JIS Guaranteed Reagent specified
in JIS K 9019 or a reagent of equivalent quality.
d) Sodium dihydrogenphosphate dihydrate: A JIS Guaranteed Reagent specified in JIS
K 9009 or a reagent of equivalent quality.
e) Sodium perchlorate monohydrate: A JIS Guaranteed Reagent specified in JIS K 8227
or a reagent of equivalent quality.
f) Nitrous Acid standard solution (1 mg/mL) (1): Put 0.147 g of sodium nitrite specified
in JIS K 8019 in a weighing dish and measure the mass to the order of 0.1 mg. Add asmall amount of water to dissolve, then transfer to a 100-mL volumetric flask and add
water up to the marked line.
g) Nitrous acid standard solution (100 μg/mL) (1): At the time of usage, put 10 mL ofnitrous acid standard solution (1 mg/mL) to a 100-mL volumetric flask and add water
up to the marked line.
h) Nitrous acid standard solution for the calibration curve preparation (1 μg/mL - 20
μg/mL): At the time of usage, put 1 mL - 20 mL of nitrous acid standard solution (100μg/mL) to 100-mL volumetric flasks step-by-step and add water up to the marked line.
Note (1) This is an example of preparation; prepare an amount as appropriate.
(3) Apparatus and instruments: Apparatus and instruments are as shown below:a) High Performance Liquid Chromatograph (HPLC): HPLC specified in JIS K 0124
that satisfies following requirements.
1) Column: A 4-mm to 6-mm inner diameter 150-mm to 250-mm long stainless steel
column tube filled with silica gel (2) or poly vinyl alcohol, to which 5-μm particle
diameter amino group chemically bonds .
2) Column bath: A column bath whose temperature can be adjusted to 30 ºC - 45 ºC.
3) Detector: An absorptiometric detector that can measure at wavelength around 210
nm.
b) Magnetic stirrerc) Centrifugal separator: A centrifugal separator that can centrifuge at 2,000 × g.
d) High speed centrifugal separator: A centrifugal separator that can centrifuge at
8,000×g.
e) pH test paper: A pH test paper infiltrated with indicator and dried, which can measure
the value from pH 1 to pH 11 and a color change chart with the pH interval value 1 is
attached.
316

Note (2) Remaining silanol group of silica gel affects the measurement of ion in somecases. Therefore use a column which does not affect the measurement of nitrousacid by treating the silanol group. As an example of the treatment, silica gel isentirely covered with the uniform membrane of silicone polymer.

Comment 1

Comment 2

A column is sold under the production names of CAPCELL PAK NH2
UG80 and Asahipak NH2P-50 4E from Shiseido and Showa Denko
respectively.
pH test paper is sold under the production name of UNIV Shikenshi fromADVANTEC(4) Test procedures(4.1) Extraction: Conduct extraction as shown below.
(4.1.1) Powdery analytical samplea) Weigh 1.00 g of an analytical sample, and put it into a 200-mL ground-in stopper
Erlenmeyer flask.
b) Add 100 mL of water and stir it with a magnetic stirrer for about 10 minutes.
c) After allowing to stand still, transfer supernatant solution to a 50-mL ground-in stopper
centrifugal precipitate tube.
d) Centrifuge at 2,000 × g centrifugal force for about five minutes (3) to make supernatantas the extract.
Note (3) 18.7-cm of rotor radius and 3,000 rpm of revolutions makes about 2,000 × g
centrifugal force.
(4.1.2) Fluid analytical samplea) Weigh 1.00 g of an analytical sample, and put it in a 100-mL volumetric flask.
b) Add about 50 mL of water, stopple the volumetric flask and shake to mix and dissolvec) Add water up to the marked line to make the extract.
(4.2) pH adjustment: Conduct pH adjustment as shown below.
a) Transfer a small amount of extract to confirm pH value using a pH-test paper.
b) If the pH value in a) is pH 5 or more, transfer the extract to a 1.5-mL ground-in stopper
centrifugal precipitate tube (4) and conduct the procedure in f) to prepare sample
solution.
c) If the pH value in a) is pH 4 or less, transfer 40 mL of the extract to a 100-mL beaker.
d) Add sodium hydroxide solution (5 mg/mL), adjust it to pH 5 to pH 7 with a pH meterand transfer to a 50-mL volumetric flask with water.
e) Add water to the marked line and transfer to a 1.5-mL ground-in stopper centrifugal
precipitate tube (4).
f) Centrifuge at 8,000 × g centrifugal force for about five minutes (5) to make supernatantas the sample solution.
Note (4) The ground-in stopper centrifugal precipitate tube should be made ofpolypropylene, etc. to not affect the measurement.
(5) 7-cm of rotor radius and 10,000 rpm of revolutions makes about 8,000 × g
centrifugal force.

317

Comment 3

Instead of procedures in (4.2) b) and e) - f), it is allowed to filter with amembrane filter (aperture diameter: no more than 0.5-µm) made ofhydrophilic PTFE and the filtrate can be the sample solution.

(4.3) Measurement: Conduct measurement according to JIS K 0124 and as shown below.
Specific measurement procedures are according to the operation method of High
Performance Liquid Chromatograph (HPLC) used in measurement.
a) Measurement conditions for High Performance Liquid Chromatograph (HPLC):
Set up the measurement conditions for High Performance Liquid Chromatograph(HPLC) considering the following example:
1) Column: A silica gel column (4-mm - 6-mm inner diameter, 150-mm - 250-mm
long, 5-µm particle diameter) to which amino group chemically bonds or a vinyl
alcohol polymer column (4-mm - 6-mm inner diameter, 150-mm - 250-mm long,
5-µm particle diameter) to which amino group chemically bonds.
2) Temperature of Column bath: 30 ºC - 40 ºC3) Eluent (1): Dissolve 1.79 g of disodium hydrogenphosphate dodecahydrate, 0.78 g of
sodium dihydrogenphosphate dihydrate and 14.04 g of sodium perchloratemonohydrate in water to make 1,000 mL. Filter with a membrane filter (aperture
diameter: no more than 0.5-µm) made of hydrophilic PTFE.
4) Flow rate: 0.9 mL/min
5) Injection rate: 10 μL
6) Detector: An absorptiometric detector, measurement wavelength: 210 nmb) Calibration curve preparation
1) Inject 10 µL of respective standard solutions for the calibration curve preparation to
HPLC, record chromatogram at wavelength 210 nm and obtain the peak area.
2) Prepare a curve for the relationship between the concentration and the peak area atwavelength 210 nm of the standard solutions for the calibration curve preparation.
c) Sample measurement
1) Subject 10 µL of the sample solution to the same procedure as in b) 1).
2) Obtain the nitrous acid content by the peak area using the calibration curve tocalculate the concentration of nitrous acid in the analytical sample.
Comment 4

This test method enables the simultaneous measurement of nitrous acid andammonium thiocyanate. In this case, mix a predetermined amount of nitrousacid standard solution (1 mg/mL) and ammonium thiocyanate standard
solution (1 mg/mL), dilute with water to prepare mixture standard solution(100 μg/mL) (1) and use it instead of (2) h) nitrous acid standard solution(100 μg/mL). After that, conduct the same procedure in (4.3) b) to calculate
the respective concentrations of materials subjected to measurement in theanalytical sample.

318

(5)

Flow sheet for testing method: The flow sheet for nitrous acid in fertilizers is shown
below:
1.00 g200-mL ground-in stopper Erlenmeyer flask
analytical sample (powdery)← 100 mL of water
Extraction
Stir to mix, 10 minites
Centrifugal separation

Ground-in stopper centrifugal precipitate tube,
2,000 ×g , 5 minutes

1.00 ganalytical sample (fluid)

100-mL volumetric flask← 50 mL - 70 mL of water
Extraction

Shake to mix← Water (up to the marked line)

pH value confirmation

pH test paper

pH 5 or more
pH 4 or less
Aliquot (40 mL)
pH adjustment (pH 5 - pH 7)

100-mL beaker
Sodium hydroxide solution (5 mg/mL)Transfer
100-mL volumetric flask, water← Water (up to the marked line)
Centrifugal separation

Measurement
FigureGround-in stopper centrifugal precipitate tube,
8,000 ×g , 5 minutes
High Performance Liquid Chromatograph

Flow sheet for nitrous acid in fertilizers

319

6.

Testing relating to the other limitations

6.1

Dicyandiamide nitrogen

6.1.a High Performance Liquid Chromatography(1) Summary
The test method is applicable to nitrolime and fertilizers containing nitrolime.
Add methanol to an analytical sample to extract dicyandiamide (Dd), introduce it to a High
Performance Liquid Chromatograph (HPLC), isolate with an amino propyl silica gel column
and measure at wavelength 215 nm to calculate dicyandiamide nitrogen (Dd-N).
(2) Reagents: Reagents are as shown below:a) Methanol: A JIS Guaranteed Reagent specified in JIS K 8891 or a reagent of equivalentquality.
b) Methanol: Methanol used in eluent of HPLC is HPLC analysis grade or a reagent ofequivalent quality.
c) Acetonitrile: A reagent of HPLC grade or equivalents.
d) Dicyandiamide standard solution (1 mg/mL) (1): Put 0.1 g of dicyandiamide [C2H4N4](2)
to a weighing dish and measure the mass to the order of 0.1 mg. Add a small amountof methanol to dissolve, transfer to a 100-mL volumetric flask and add the solvent up to
the marked line. Store in a refrigerator, and do not use after 6 months after preparation.
e) Dicyandiamide standard solution (100 µg/ mL): Put 10 mL of dicyandiamide
standard solution (1 mg/ mL) to a 100-mL volumetric flask and add methanol up to themarked line.
f) Dicyandiamide standard solution (10 µg/ mL - 50 µg/ mL) for the calibration curve
preparation: At the time of usage, put 5 mL - 25 mL of dicyandiamide standard
solution (100 µg/ mL) to 50-mL volumetric flasks step-by-step and add methanol up to
the marked line.
g) Dicyandiamide standard solution (1 µg/mL - 10 µg/mL) for the calibration curve
preparation: At the time of usage, put 2.5 mL - 25 mL of dicyandiamide standard
solution (20 µg/mL) for the calibration curve preparation to 50-mL volumetric flasks
step-by-step and add methanol up to the marked line.
Note (1) This is an example of preparation; prepare an amount as appropriate.
(2) A reagent of no less than 98 % (mass fraction) in purity as dicyanamide iscommercially sold.
Comment 1 Dicyandiamide is commercially sold as dicyanodiamide by Wako Pure
Chemical Industries, Ltd. and Kanto Chemical Co., Inc.
(3) Instruments: Instruments are as shown below:a) High Performance Liquid Chromatograph: HPLC specified in JIS K 0124 thatsatisfies the following requirements.
1) Column: A column of 4 mm - 6 mm inner diameter and 150 mm - 250 mm longstainless steel column tube filled with silica gel, to which amino or amino propyl
chemically bonds.
2) Column bath: A column bath whose temperature can be adjusted to at 30 ºC - 45ºC.
3) Detector: An absorptiometric detector that can measure at wavelength around 215
nm.
b) Shaking apparatusc) Centrifugal separator: A centrifugal separator that can centrifuge at 8,000×g.
320Comment 2 A column is sold under production names such as Hibar LiChrosorb NH2,
Inertsil NH2, Unison UK-Amino, Mightysil NH2, Shim-pack CLC-NH2,
Shodex NH-5A, Unisil Q NH2, etc.
(4) Test procedures(4.1) Extraction: Conduct extraction as shown below.
a) Weigh 1.00 g of an analytical sample, and put it in a 200-mL - 300-mL ground-in
stopper Erlenmeyer flask.
b) Immediately add 100 mL of methanol (3) and shake to mix by using a shaking apparatus
for about 10 minutes.
c) After allowing to stand still, transfer supernatant solution to a 1.5 mL of ground-in
stopper centrifugal precipitate tube (4).
d) Centrifuge by 8,000 × g for about five minutes (5)e) 1 mL of the supernatant is used as the sample solution.
Note (3)(4)(5)

Add methanol immediately as the determined value becomes higher than usual if
it is left in air.
The ground-in stopper centrifugal precipitate tube should be made ofpolypropylene, etc. to not affect the measurement.
7-cm of rotor radius and 10,000 rpm of revolutions makes about 8,000 × g
centrifugal force.

Comment 3 Instead of the procedures in (4.1) c) - e), it is allowed to filter with amembrane filter (aperture diameter: no more than 0.5-µm) made of PTFE andthe filtrate can be the sample solution.
(4.2) Measurement: Conduct measurement according to JIS K 0124 and as shown below.
Specific measurement procedures are according to the operation method of High
Performance Liquid Chromatograph (HPLC) used in measurement.
a) Measurement conditions for High Performance Liquid Chromatograph (HPLC):
An example of measurement conditions for High Performance Liquid Chromatograph(HPLC) is shown below. Set up the measurement conditions considering it:1) Column: A silica gel column (4-mm - 6-mm inner diameter, 150-mm - 250-mm
long, 5-µm particle diameter) to which amino or amino propyl chemically bonds.
2) Temperature of Column bath: 30 ºC - 40 ºC3) Eluent: Acetonitrile - methanol (6+1）
4) Flow rate: 1 mL/ min5) Detector: An absorptiometric detector, measurement wavelength: 215 nmb) Calibration curve preparation
1) Inject 10 µL of respective dicyandiamide standard solutions for the calibration
curve preparation to HPLC, record chromatogram at wavelength 215 nm, andobtain the peak area or the height.
2) Prepare a curve for the relationship between the concentration and the peak area or
the height at wavelength 215 nm of the dicyandiamide standard solutions for thecalibration curve preparation.
c) Sample measurement
1) Subject 10 µL of the sample solution to the same procedure as in b) 1).
2) Obtain dicyandiamide (Dd) content from the calibration curve to calculate theconcentration of dicyandiamide (Dd) in the analytical sample.
3213) Calculate the dicyandiamide nitrogen (Dd - N) by the following formula.
Dicyandiamide nitrogen (Dd - N) (% (mass fraction)) in an analytical sample= A × (MW1/MW2)= A × 0.6664
A: Dicyandiamide (Dd) (% (mass fraction)) in an analytical sample
MW1: 4 atomic weight of nitrogen (56.027)
MW2: Molecular weight of dicyandiamide (84.080)
Comment 4:

Recovery testing was conducted using nitrolime (3 samples) and blended
fertilizer containing nitrolime (2 samples), as a result, the recovery rate ofdicyandiamide at concentration level of 6 and 0.6 % (mass fraction) was
94.9 % - 105.1 % and 95.6 % - 103.5 %, and the repeatability relative
standard deviation were 0.7 % - 2.0 % and 0.4 % - 1.7 %, respectively.
In addition, the results and analysis results of a collaborative study for test
method validation are shown in Table 1
Additionally, the minimum limit of quantification of this test method isabout 0.01 % (mass fraction).

Table 1 Results and analysis results from a collaborative study
for the test method validation of dicyandiamide nitrogen
Number of

Mean2)

sr4)

RSDr5)sR6)

RSDR7)

laboratories

1)

(%)3)

(%)3)

(%)

(%)3)

(%)

Nitrolime 1

90.03210.00103.2

0.0012

3.8

Nitrolime 2

10

0.159

0.002

1.3

0.006

3.8

Nitrolime 3

11

0.245

0.002

0.7

0.008

3.3

Samplename

Blended
11
0.124
0.001
0.7
0.002
fertilizers 1
Blebded
11
0.410
0.007
1.6
0.008
fertilizers 2
1) Number of laboratories used in analysis
2) Mean (n= number of laboratories x number of samples (2))3) Mass fraction
4) Repeatability standard deviation
5) Repeatability relative standard deviation
6) Reproducibility standard deviation
7) Reproducibility relative standard deviation

2.0
1.9

References
1) Masakazu SAIKI and Miyuki ASAO: Validation of High Performance Liquid
Chromatography for Determination of Dicyandiamide in Nitrolime, Research Report of
Fertilizers, Vol.2, 2009. (25 - 31)2) Masakazu SAIKI and Masayuki YOSHIMOTO: Determination of Dicyandiamide in
Nitrolime by High Performance Liquid Chromatography: A Collaborative Study,
Research Report of Fertilizers, Vol.2, 2009. (32 - 37)

322

(5)

Flow sheet for dicyandiamide nitrogen: The flow sheet for dicyandiamide nitrogen innitrolime and fertilizers containing nitrolime is shown below:
1.00 g200-mL - 300-mL ground-in stopper Erlenmeyer flask
analytical sample← 100 mL of methanol
Extraction
Shaking, 30 minutes
Centrifugal separation
Measurement

Ground-in stopper centrifugal precipitate tube,
8,000 ×g , 5 minutes
High Performance Liquid Chromatograph

Figure Flow sheet for dicyandiamide nitrogenin nitrolime and fertilizers containing nitrolime

HPLC chromatographs of dicyandiamide standard solution and sample solution(nitrolime) for the calibration curve preparation are shown below.

0.10
0.09

Dd

0.09

0.08

0.07

0.07

0.06

0.06

AU

0.08

0.05

0.05

0.04

0.04

0.03

0.03

0.02

0.02

0.01

0.01

0.00
0

1

2

3

4

5 6
min

7

8.458

0.10

AU

0.11

8.541

0.11

8

Dd

Reference

0.00
0

9 10 111) Standard solution

1

2

3

4

5 6
min

7

8

9 10 112) Sample solution

Reference diagram: HPLC chromatogram of dicyandiamide

1) Dicyandiamide standard solution (the equivalents of 100 ng of dicyandiamide (10 µg/
mL, 10 µL))
2) Sample solution (nitrolime)
The measurement conditions of HPLC
Column: Hibar LiChrosorb NH2 (4.6-mm inner diameter, 25-cm long, 5-µm
particle diameter)
Other conditions are according to the example of the measurement condition of
HPLC in (4.2) a).

323

7.

Nitrification inhibitor

7.1

2-amino-4-chloro-6 -methylpyrimidine (AM)7.1.aHigh Performance Liquid Chromatography(1) Summary
This testing method is applicable to fertilizers containing
2-amino-4-chloro-6-methylpyrimidine (AM).
Add methanol - water (1+1) to an analytical sample to extract
2-amino-4-chloro-6-methylpyrimidine, introduce it into a High Performance Liquid
Chromatograph (HPLC), isolate with an octadecyl silylation silica gel column, and measure atwavelength 295 nm to calculate 2-amino-4-chloro-6-methylpyrimidine.
(2) Reagents, etc.: Reagents and water are as shown below.
a) Water: Water of A3 specified in JIS K 0557 or equivalents.
b) Methanol: A JIS Guaranteed Reagent specified in JIS K 8891 or a reagent of equivalentquality.
c) Methanol: Methanol used in eluent of HPLC is a regent of HPLC analysis grade orequivalents.
d) 2-amino-4-chloro-6-methylpyrimidine standard solution (1 mg/mL)(1): Put 0.1 g of
2-amino-4-chloro-6-methylpyrimidine [C5H6ClN3](2) in a weighing dish and measure themass to the order of 0.1 mg. Add methanol - water (1+1) to dissolve, transfer to a
100-mL volumetric flask and add the same solvent up to the marked line. Store in arefrigerator, and do not use after 6 months after preparation.
e) 2-amino-4-chloro-6-methylpyrimidine standard solution (100 µg/mL): In the case ofusage, transfer 10 mL of 2-amino-4-chloro-6-methylpyrimidine standard solution (1mg/mL) to a 100-mL volumetric flask and add methanol - water (1+1) up to the markedline.
f) 2-amino-4-chloro-6-methylpyrimidine standard solution (10 µg/mL - 50 µg/mL) forthe calibration curve preparation: In the case of usage, transfer 5 mL - 25 mL of
2-amino-4-chloro-6-methylpyrimidine standard solution (100 µg/mL) to 50-mL
volumetric flasks step-by-step and add methanol - water (1+1) up to the marked line.
g) 2-amino-4-chloro-6-methylpyrimidine standard solution （1 µg/mL - 10 µg/mL）forthe calibration curve preparation: In the case of usage, transfer 2.5 mL - 25 mL of
2-amino-4-chloro-6-methylpyrimidine standard solution (20 µg/mL) for the calibration
curve preparation to 50-mL volumetric flasks step-by-step and add methanol - water(1+1) up to the marked line.
Note (1) This is an example of preparation; prepare an amount as appropriate.
(2) A reagent of no less than 98 % (mass fraction) in purity is commercially sold as
2-amino-4-chloro-6-methylpyrimidine.
Comment 1:2-amino-4-chloro-6-methylpyrimidine is commercially sold by Wako Pure
Chemical Industries, Ltd. and Kanto Chemical Co., Inc.

(3) Instruments: Instruments are as shown below:a) High Performance Liquid Chromatograph: HPLC specified in JIS K 0124 thatsatisfies the following requirements.
1) Column: A 4-mm - 6-mm inner diameter 150-mm - 250-mm long stainless steel
column tube filled with silica gel, to which octadecyl chemically bonds.
2) Column bath: A column bath whose temperature can be adjusted to 30 ºC - 45 ºC.
324

3) Detector: An absorptiometric detector that can measure at wavelength around 295
nm.
b) Magnetic stirrerc) Centrifugal separator: A centrifugal separator that can centrifuge at 2,000 × g.
d) High speed centrifugal separator: A centrifugal separator that can centrifuge at
8,000×g.
e) Acidic alumina cartridge column: Link a 10-mL cylinder to a column (3) that is filledwith 500 mg - 1 g of acidic alumina, put 3 mL of methanol and let it flow down.
Note (3) A cartridge with a 3-mL - 6-mL column filled with 500-mg - 1-g of silica gel canbe used.
Comment 2

Comment 3

A column is sold under production names such as Inertsil ODS, Mightysil
RP-18, L-column ODS, Shim-pack VP-ODS, Silica C18M 4D, Puresil C18,COSMOSIL 5C18-MS-II, etc.
An acidic alumina cartridge is commercially sold under production names
such as Bond Elut AL-A, Sep-Pak Alumina-A, Supelclean LC-Alumina-A.

(4) Test procedures(4.1) Extraction: Conduct extraction as shown below.
a) Weigh 1.00 g of an analytical sample, and put it into a 200-mL ground-in stopper
Erlenmeyer flask.
b) Add 100 mL of methanol - water (1+1) and stir it by using a magnetic stirrer for about
30 minutes.
c) After allowing to stand still, transfer supernatant solution to a 50-mL ground-in stopper
centrifugal precipitate tube.
d) Centrifuge it at 2,000 × g centrifugal force for about five minutes (4) and use thesupernatant as the extract (5).
Note (4) 18.7-cm of rotor radius and 3,000 rpm of revolutions makes about 2,000 × g
centrifugal force.
(5) If there is a possibility that the 2-amino-4-chloro-6-methylpyrimidine in thesample solution exceeds the maximum limit of the calibration curve, dilute apredetermined amount of extract with methanol water (1+1).
(4.2) Cleanup: Conduct cleanup as shown below:a) Transfer the extract to an acidic alumina cartridge column.
b) Dispose of about the first 3 mL of effluent and then transfer about the next 2 mL to atest tube.
c) Transfer the effluent to a 1.5-mL ground-in stopper centrifugal precipitate tube (6).
d) Centrifuge at 8,000×g centrifugal force for about five minutes (7) and use thesupernatant as the sample solution.
Note (6) The ground-in stopper centrifugal precipitate tube should be made ofpolypropylene, etc. to not affect the measurement.
(7) 7-cm of rotor radius and 10,000 rpm of revolutions makes about 8,000 × g
centrifugal force.

325

Comment 4

Comment 5

Instead of the procedures in (4.2) c) - e), it is allowed to filter with amembrane filter (aperture diameter: no more than 0.5-µm) made of PTFE
and the filtrate can be the sample solution.
The test is possible by the following procedures in the case of fertilizers notcontaining organic matters.
The procedures in (4.1) c) - d) and (4.2) a) - b) are omitted and “Transfer
effluent” in (4.2) c) is replaced with the “After allowing to stand still,transfer supernatant” to operate.

(4.3) Measurement: Conduct measurement according to JIS K 0124 and as shown below.
Specific measurement procedures are according to the operation method of High
Performance Liquid Chromatograph (HPLC) used in measurement.
a) Measurement conditions for High Performance Liquid Chromatograph (HPLC):
Set up the measurement conditions for High Performance Liquid Chromatograph(HPLC) considering the following example:
1) Column: A silica gel column (4-mm - 6-mm inner diameter, 150-mm - 250-mm
long, 5-µm particle diameter) to which octadecyl chemically bonds.
2) Temperature of column bath: 30 ºC - 40 ºC3) Eluent: Methanol - water (4+6）4) Flow rate: 1 mL/min
5) Detector: An absorptiometric detector, measurement wavelength: 295 nmb) Calibration curve preparation
1) Inject 10 µL of respective 2-amino-4-chloro-6-methylpyrimidine standard solutions
for the calibration curve preparation to HPLC, record chromatogram at wavelength
295 nm and obtain the peak area or height.
2) Prepare a curve for the relationship between the concentration and the peak area orheight at wavelength 295 nm of the 2-amino-4-chloro-6-methylpyrimidine standard
solutions for the calibration curve preparation.
c) Sample measurement
1) Subject 10 µL of the sample solution to the same procedure as in b) 1).
2) Obtain 2-amino-4-chloro-6-methylpyrimidine content from the calibration curve tocalculate the concentration of 2-amino-4-chloro-6-methylpyrimidine in the analyticalsample.
Comment 6

Recovery testing was conducted using compound fertilizer (1 sample) andblended fertilizer (2 samples), as a result, the mean recovery rate of
2-amino-4-chloro-6-methylpyrimidine at concentration level of 1.0 %,0.4 % and 0.1 % (mass fraction) was 99.1 % - 100.5 %, 99.3 % - 101.6 %and 100.2 % - 100.7 %, and repeatability relative standard deviation were
0.4 % - 1.8 %, 1.2 % - 2.5 % and 0.8 % - 3.0 %, respectively. Additionally,
the minimum limit of quantification of this test method is about 0.005 %(mass fraction).

Reference
1) Yuji SHIRAI: The volumetric analysis of 2-amino-4-chloro-6-methylpyrimidine infertilizers with High Performance Liquid Chromatograph, Validation Report of
Fertilizers (in Japanese), 44 (3), 26 - 41(1991)

326

(5)

Flow sheet for 2-amino-4-chloro-6-methylpyrimidine: The flow sheet for
2-amino-4-chloro-6-methylpyrimidine in fertilizers is shown below:
1.00 ganalytical sample

200-mL of ground-in stopper Erlenmeyer flask← 100 mL of methanol-water (1+1)
Extraction
Shaking, 30 minutes
Centrifugal separation
CleanupCentrifugal separation
Measurement
FigureGround-in stopper centrifugal precipitate tube,
2,000 × g , 5 minutes
Acidic alumina cartridge column
Ground-in stopper centrifugal precipitate tube,
8,000 × g , 5 minutes
High Performance Liquid Chromatograph

Flow sheet for 2-amino-4-chloro-6-methylprimidine in fertilizers.

Reference: HPLC chromatogram of 2-amino-4-chloro-6-methylpyrimidine standard solution
for the calibration curve preparation is shown below.

Reference diagram:

HPLC chromatogram of
2-amino-4-chloro-6-methylpyrimidine standard solution

Measurement conditions for HPLC
Column: Mightysil RP-18 GP(4.6-mm inner diameter, 150-mm long, 5-µm particle diameter)
2-amino-4-chloro-6-methylpyrimidine standard solution (the equivalents of 100 ng)
Other conditions are according to the examples of the measurement conditions of(4.3) a) HPLC.

327

7.2

1-amidino-2-thiourea (ASU)

7.2.aHigh Performance Liquid Chromatography(1)
SummaryThis testing method is applicable to fertilizers containing 1-amidino-2-thiourea (ASU). Add
water to an analytical sample to extract 1-amidino-2-thiourea, introduce it into a High
Performance Liquid Chromatograph (HPLC), isolate with an octadecyl silylation silica gelcolumn, and measure at wavelength 262 nm to calculate 1-amidino-2-thiourea.
(2) Reagents, etc.: Reagents and water are as shown below.
a) Water: Water of A3 specified in JIS K 0557 or equivalents.
b) Methanol: Methanol used in eluent of HPLC is a regent of HPLC analysis grade orequivalents.
c) 1-sodium hexasulfonate: A reagent of ion pair chromatography analysis grade orequivalents.
d) Acetic acid: A reagent of HPLC analysis grade or equivalents.
e) 1-amidino-2-thiourea standard solution (1 mg/mL)(1): Put 0.1 g of
1-amidino-2-thiourea [C2H6N4S](2) in a weighing dish and measure the mass to the orderof 0.1 mg. Add water to dissolve, transfer to a 100-mL volumetric flask and add water
up to the marked line. Store in a refrigerator, and do not use after 6 months after
preparation.
f) 1-amidino-2-thiourea standard solution (100 µg/mL): In the case of usage, transfer
10 mL of 1-amidino-2-thiourea standard solution (1 mg/mL) to a 100-mL volumetricflask and add water up to the marked line.
g) 1-amidino-2-thiourea standard solution (10 µg/mL- 50 µg/mL) for the calibration
curve preparation: In the case of usage, transfer 5 mL - 25 mL of
1-amidino-2-thiourea standard solution (100 µg/mL) to 50-mL volumetric flasks
step-by-step and add water up to the marked line.
h) 1-amidino-2-thiourea standard solution (1 µg/mL- 10 µg/mL) for the calibration
curve preparation: In the case of usage, transfer 2.5 mL - 25 mL of
1-amidino-2-thiourea standard solution (20 µg/mL) for the calibration curve preparation
to 50-mL volumetric flasks step-by-step and add water up to the marked line.
Note (1) This is an example of preparation; prepare an amount as appropriate.
(2) A reagent of no less than 98 % (mass fraction) in purity is commercially sold as
1-amidino-2-thiourea.
Comment 1

1-amidino-2-thiourea is sold under the name of guanylthiourea by Tokyo
Chemical Industry Co., Ltd, and under the name of amidino thiourea byKanto Chemical Co., Inc.

(3) Instruments: Instruments are as shown below.
a) High Performance Liquid Chromatograph (HPLC): HPLC specified in JIS K 0124
that satisfies the following requirements.
1) Column: A column of 4-mm - 6-mm inner diameter 150-mm - 250-mm longstainless steel column tube filled with silica gel, to which octadecyl chemicallybonds.
2) Column bath: A column bath whose temperature can be adjusted to 30 ºC - 45 ºC.
3) Detector: An absorptiometric detector that can measure at wavelength around 262
nm.
b) Magnetic stirrer
328c) High speed centrifugal separator: A centrifugal separator that can centrifuge at 8,000
× g.
Comment 2:

A column is sold under the production names such as Inertsil ODS,
Mightysil RP-18, L-column ODS, Shim-pack VP-ODS, Silica C18M 4D,
Puresil C18, COSMOSIL 5C18-MS-II.

(4) Test procedures(4.1) Extraction: Conduct extraction as shown below.
a) Weigh 1.00 g of an analytical sample, and put it into a 200-mL ground-in stopper
Erlenmeyer flask.
b) Add 100 mL of water and stir it with a magnetic stirrer for about 10 minutes.
c) After allowing to stand still, transfer supernatant (3) solution to a 1.5-mL ground-in
stopper centrifugal precipitate tube (4).
d) Centrifuge at 8,000 × g centrifugal force for about five minutes (5) to make supernatantas the sample solution.
Note (3) If there is a possibility that the 1-amidino-2-thiourea concentration in the sample
solution exceeds the maximum limit of the calibration curve, dilute apredetermined amount of supernatant with water.
(4) The ground-in stopper centrifugal precipitate tube should be made ofpolypropylene, etc. to not affect the measurement.
(5) 7-cm of rotor radius and 10,000 rpm of revolutions makes about 8,000 × g
centrifugal force.
Comment 3

Instead of procedures in (4.1) c) - d), it is allowed to filter with a membranefilter (aperture diameter: no more than 0.5-µm) made of hydrophilic PTFE
and the filtrate can be the sample solution.

(4.2) Measurement: Conduct measurement according to JIS K 0124 and as shown below.
Specific measurement procedures are according to the operation method of High
Performance Liquid Chromatograph (HPLC) used in measurement.
a) Measurement conditions for High Performance Liquid Chromatograph (HPLC):
Set up the measurement conditions for High Performance Liquid Chromatograph(HPLC) considering the following example:
1) Column: A silica gel column (4-mm - 6-mm inner diameter, 150-mm - 250-mm
long, 5-µm particle diameter) to which octadecyl chemically bonds.
2) Temperature of Column bath: 30 ºC - 45 ºC3) Eluent: Dissolve 0.94 g of sodium 1-hexasulfonic acid in 1,000 mL of methanol
water (2+8), adjust to pH 3.15 with acetic acid and filter with a membrane filter(aperture diameter: no more than 0.5-µm) made of hydrophilic PTFE (1).
4) Flow rate: 1 mL/min
5) Detector: An absorptiometric detector, measurement wavelength: 262 nmb) Calibration curve preparation
1) Inject 10 µL of respective 1-amidino-2-thiourea standard solutions for the calibration
curve preparation to HPLC, record chromatogram at wavelength 262 nm and obtain
the peak area or height.
2) Prepare a curve for the relationship between the concentration and the peak area orheight at wavelength 262 nm of the 1-amidino-2-thiourea standard solutions for thecalibration curve preparation.
329c) Sample measurement
1) Subject 10 µL of the sample solution to the same procedure as in b) 1).
2) Obtain the 1-amidino-2-thiourea content from the calibration curve to calculate theconcentration of 1-amidino-2-thiourea in the analytical sample.
Comment 4

Recovery testing with triplicates measurement was conducted using
compound fertilizer (2 samples), as a result, the mean recovery rate of
1-amidino-2-thiourea at concentration level of 1.0 %, 0.5 % and 0.25 %(mass fraction) were 99.0 % - 104.3 %, 97.7 % - 100.7 % and 99.7 % 101.3 %, and repeatability relative standard deviation were 1.0 % - 3.9 %,1.5 % - 2.6 % and 8.2 % - 8.4 %, respectively.
In addition, the results and analysis results of a collaborative study for test
method validation are shown in Table 1.
Additionally, the minimum limit of quantification of this test method isabout 0.005 % (mass fraction)

Table 1 Results and analysis results from a collaborative study
for the test method validation of 1-amidino-2-thiourea (ASU)
2)

Sample name

Number oflaboratories1)

Mean
3)
(%)

sr

4)

5)

6)

(%)

(%)

RSDR
(%)

3)

sR

7)

RSDr
(%)

3)

Compound fertilizer 1

10

0.093

0.009

9.1

0.010

11.2

Compound fertilizer 2

10

0.246

0.021

8.6

0.021

8.6

Compound fertilizer 3

10

0.511

0.018

3.6

0.025

4.9

Compound fertilizer 4

10

0.759

0.039

5.1

0.040

5.3

Compound fertilizer 5

10

1.020

0.039

3.8

0.044

4.3

1)
2)
3)
4)
5)
6)
7)

Number of laboratories used in analysis
Mean (n= number of laboratories × number of samples (2))
Mass fraction
Repeatability standard deviation
Repeatability relative standard deviation
Reproducibility standard deviation
Reproducibility relative standard deviation

Reference
1) Kazunori CHIBA: Analysis method of 1-amidino-2-thiourea (ASU) of Nitrification
inhibitor in fertilizers with High Performance Liquid Chromatograph, Validation Reportof Fertilizers (in Japanese), 43 (4), 15 - 22 (1990)
2) Shigehiro KAI and Erina WATABE: Determination of 1-Amidino-2-thiourea as a
Nitrification Inhibitor in Compound Fertilizer by High Performance Liquid
Chromatography: A Collaborative Study, Research Report of Fertilizers, Vol.6, 2013.
(36 - 42)

330

(5)

Flow sheet for 1-amidino-2-thiourea: The flow sheet for 1-amidino-2-thiourea in
fertilizers is shown below:
1.00 g analytical sample
200-mL of ground-in stopper Erlenmeyer flask← 100 mL of water
Extraction
Shaking, 10 minutes
Centrifugal separation
Measurement
FigureReference

Ground-in stopper centrifugal precipitate tube,
8,000 × g , 5 minutes
High Performance Liquid Chromatograph

Flow sheet for 1-amidino-2-thiourea in fertilizers.

HPLC chromatogram of 1-amidino-2-thiourea standard solution for thecalibration curve preparation is shown below.

Reference diagram: HPLC chromatogram of 1-amidino-2-thiourea standard solution
Measurement conditions for HPLC
Column: Mightysil RP-18 GP (4.6-mm inner diameter, 150-mm long, 5-µm particle
diameter)
1-amidino-2-thiourea standard solution (the equivalents of 200 ng)
Other conditions are shown according to the examples of the measurement conditions of(4.2) a) HPLC.

331

7.3
7.3.a

4-amino-1,2,4-triazole hydrochloride (ATC)High Performance Liquid Chromatography(1) Summary
This testing method is applicable to fertilizers containing 4-amino-1,2,4-triazolehydrochloride (ATC) but not containing organic matters.
Add methanol to an analytical sample to extract 4-amino-1,2,4-triazole hydrochloride,
introduce it into a High Performance Liquid Chromatograph (HPLC), isolate with anaminopropyl silica gel column, and measure at wavelength 220 nm to calculate
4-amino-1,2,4-triazole hydrochloride.
(2) Reagents: Reagents are as shown below.
a) Methanol: A JIS Guaranteed Reagent specified in JIS K 8891 or a reagent of equivalentquality.
b) Methanol: Methanol used in eluent of HPLC is a regent of HPLC analysis grade orequivalents.
c) Acetonitrile: Acetonitrile used in eluent of HPLC is a regent of HPLC analysis grade orequivalents.
d) 4-amino-1,2,4-triazole standard solution (1 mg/mL)(1)(2): Transfer 0.1 g of
4-amino-1,2,4-triazole [C2H4N4](3) to a weighing dish and measure the mass to the orderof 0.1 mg. Add methanol to dissolve, transfer to a 100-mL amber volumetric flask andadd methanol to the marked line. Store in a refrigerator, and do not use after 6 months
after preparation.
e) 4-amino-1,2,4-triazole standard solution (100 µg/mL):In the case of usage, transfer
10 mL of 4-amino-1,2,4-triazole standard solution (1 mg/mL) to a 100-mL volumetricflask and add methanol up to the marked line.
f) 4-amino-1,2,4-triazole standard solution (10 µg/mL - 50 µg/mL) for the calibration
curve preparation: In the case of usage, transfer 5 mL - 25 mL of
4-amino-1,2,4-triazole standard solution (100 µg/mL) to 50-mL volumetric flasks
step-by-step and add methanol up to the marked line.
g) 4-amino-1,2,4-triazole standard solution (1 µg/mL - 10 µg/mL) for the calibration
curve preparation: In the case of usage, transfer 2.5 mL - 25 mL of
4-amino-1,2,4-triazole standard solution for the calibration curve preparation (20µg/mL) to 50-mL volumetric flasks step-by-step and add methanol up to the marked
line.
Note (1) The solution contains 1.434 mg/mL as 4-amino-1,2,4-triazole hydrochloride.
(2) This is an example of preparation; prepare an amount as appropriate.
(3) A reagent of no less than 98 % (mass fraction) in purity is commercially sold as
4-amino-1,2,4-triazole.
Comment 1

4-amino-1,2,4-triazole is sold under production names such as
4-amino-1,2,4-triazole by Wako Pure Chemical Industries., Ltd. and Tokyo
Chemical Industry Co., Ltd, and 4-amino-4H-1,2,4-triazole by Kanto
Chemical Co., Inc.

(3) Instruments: Instruments are as shown below:a) High Performance Liquid Chromatograph (HPLC): HPLC specified in JIS K 0124
that satisfies the next requirements.
1) Column: A 4-mm - 6-mm inner diameter 150-mm - 250-mm long stainless steel
column tube filled with silica gel, to which amino or aminopropyl chemically bonds.
332

2) Column bath: A column bath whose temperature can be adjusted to 30 ºC - 45 ºC.
3) Detector: An absorptiometric detector that can measure at wavelength around 220
nm.
b) Magnetic stirrerc) High speed centrifugal separator: A centrifugal separator that can centrifuge at 8,000
× g.
Comment 2

A column is commercially sold under the names such as Hibar LiChrosorb
NH2, Inertsil NH2, Unison UK-Amino, Mightysil NH2, Shim-pack
CLC-NH2, Shodex NH-5A, Unisil Q NH2.

(4) Test procedures(4.1) Extraction: Conduct extraction as shown below.
a) Weigh 1.00 g of an analytical sample, and put it to a 200-mL ground-in stopper
Erlenmeyer flask.
b) Add 100 mL of methanol and stir it with using a magnetic stirrer for about 10 minutes.
c) After allowing to stand still, transfer the supernatant solution to a 1.5-mL ground-in
stopper centrifugal precipitate tube (4)d) Centrifuge by 8,000 × g centrifugal force for about five minutes (5) to make thesupernatant as the sample solution.
Notes (4) The ground-in stopper centrifugal precipitate tube should be made ofpolypropylene, etc. to not affect the measurement.
(5) 7 cm of rotor radius and 10,000 rpm of revolutions makes about 8,000 × g
centrifugal force.
Comment 3

Instead of procedures in (4.1) c) - d), it is allowed to filter with a membranefilter (aperture diameter: no more than 0.5-µm) made of PTFE and thefiltrate can be the sample solution.

(4.2) Measurement: Conduct measurement according to JIS K 0124 and as shown below.
Specific measurement procedures are according to the operation method of High
Performance Liquid Chromatograph (HPLC) used in measurement.
a) Measurement conditions for High Performance Liquid Chromatograph (HPLC):
Set up the measurement conditions for High Performance Liquid Chromatograph(HPLC) considering the following example:
1) Column: A silica gel column (4-mm - 6-mm inner diameter, 150-mm - 250-mm
long, 5-µm particle diameter) to which amino or aminopropyl chemically bonds.
2) Temperature of Column bath: 30 ºC - 40 ºC3) Eluent: Acetonitrile-methanol (9+1)4) Flow rate: 1 mL/min
5) Detector: An absorptiometric detector, measurement wavelength: 220 nmb) Calibration curve preparation
1) Inject 10 µL of respective 4-amino-1,2,4-triazole standard solutions for thecalibration curve preparation to HPLC, record chromatogram at wavelength 220 nm
and obtain the peak area or height.
2) Prepare a curve for the relationship between the concentration and the peak area orheight at wavelength 220 nm of the 4-amino-1,2,4-triazole standard solutions for thecalibration curve preparation.
333c) Sample measurement
1) Subject 10 µL of the sample solution to the same procedure as in b) 1).
2) Obtain the amount of 4-amino-1,2,4-triazole from the calibration curve to calculate
the concentration of 4-amino-1,2,4-triazole in the analytical sample.
3) Calculate the 4-amino-1,2,4-triazole hydrochloride by the following formula.
4-amino-1,2,4-triazole hydrochloride in the analytical sample (% (mass fraction))＝A × 1.434
A: 4-amino-1,2,4-triazole in the analytical sample (% (mass fraction))
Comment 4

Recovery testing was conducted using compound fertilizer (2 samples), as aresult, the mean recovery rate of 4-amino-1,2,4-triazole hydrochloride atconcentration level of 0.5 %, 0.3 % and 0.2 % (mass fraction) were 100.2 %- 104.9 %, 100.8 % - 103.0 % and 100.7 % - 104.2 %, and repeatability
relative standard deviation were 0.8 % - 5.1 %, 3.3 % - 7.4 % and 0.9 % 8.4 %, respectively. Additionally, the minimum limit of quantification ofthis test method is about 0.005 % (mass fraction)

Reference
1) Koichi SAKAGAMI: Analysis methods of 4-amino-1,2,4-triazole hydrochloride withHigh Performance Liquid Chromatography, Validation Report of Fertilizers (inJapanese), 40 (4), 9 - 16 (1987)(5)

Flow sheet for 4-amino-1,2,4-triazole hydrochloride: The flow sheet for
4-amino-1,2,4-triazole hydrochloride in fertilizers is shown below:
1.00 g analytical sample
200-mL ground-in stopper Erlenmeyer flask← 100 mL of Methanol
Extraction
Shaking, 10 minutes
Centrifugal separation
Measurement
FigureGround-in stopper centrifugal precipitate tube,
8,000 × g , 5 minutes
High Performance Liquid Chromatograph

Flow sheet for 4-amino-1,2,4-triazole hydrochloride in fertilizers.

334

7.4
7.4.a

N-2,5-dichlorophenyl succinamic acid (DCS)High Performance Liquid Chromatography(1) Summary
This testing method is applicable to fertilizers containing N-2,5-dichlorophenyl succinamic
acid (DCS) but not containing organic matters.
Add methanol - phosphate (996+4) and water to an analytical sample to extract
N-2,5-dichlorophenyl succinamic acid (DCS), introduce it into a High Performance Liquid
Chromatograph (HPLC), isolate with octadecyl silylation silica gel column, and measure atwavelength 246 nm to calculate N-2,5-dichlorophenyl succinamic acid (DCS).
(2) Reagents, etc.: Reagents and water are as shown below.
a) Water: Water of A3 specified in JIS K 0557 or water with equivalent quality.
b) Methanol: A JIS Guaranteed Reagent specified in JIS K 8891 or a reagent of equivalentquality.
c) Methanol: Methanol used in eluent of HPLC is a regent of HPLC analysis grade orequivalents.
d) N-2,5-dichlorophenyl succinamic acid standard solution(1 mg/mL) (1) : Transfer 0.1
g of N-2,5-dichlorophenyl succinamic acid [C10H9Cl2NO3] to a weighing dish andmeasure the mass to the order of 0.1 mg. Add methanol to dissolve, transfer to a
100-mL volumetric flask and add methanol to the marked line. Store in a refrigerator,and do not use after 6 months after preparation.
e) N-2,5-dichlorophenyl succinamic acid standard solution(100 µg/mL): In the case ofusage, transfer 10 mL of N-2,5-dichlorophenyl succinamic acid standard solution (1mg/mL) to a 100-mL volumetric flask and add methanol up to the marked line.
f) N-2,5-dichlorophenyl succinamic acid standard solution (10 µg/mL - 50 µg/mL) forthe calibration curve preparation: In the case of usage, transfer 5 mL - 25 mL of
N-2,5-dichlorophenyl succinamic acid standard solution (100 µg/mL) to 50-mL
volumetric flasks step-by-step and add methanol up to the marked line.
g) N-2,5-dichlorophenyl succinamic acid standard solution (1 µg/mL - 10 µg/mL) forthe calibration curve preparation: In the case of usage, transfer 2.5 mL - 25 mL of
N-2,5-dichlorophenyl succinamic acid standard solution for the calibration curve
preparation (20 µg/mL) to 50-mL volumetric flasks step-by-step and add methanol up to
the marked line.
Note (1) This is an example of preparation; prepare an amount as appropriate.
(3) Instruments: Instruments are as shown below:a) High Performance Liquid Chromatograph (HPLC): HPLC specified in JIS K 0124
that satisfies the following requirements.
1) Column: A 4-mm - 6-mm inner diameter 150-mm - 250-mm long stainless steel
column tube filled with silica gel, to which octadecyl chemically bonds.
2) Column bath: A column bath whose temperature can be adjusted to 30 ºC - 45 ºC.
3) Detector: An absorptiometric detector that can measure at wavelength around 246
nm.
b) Magnetic stirrerc) High speed centrifugal separator: A centrifugal separator that can centrifuge at 8,000
× g.
Comment 1

A column is sold under the production names such as Inertsil ODS,
Mightysil RP-18, L-column ODS, Shim-pack VP-ODS, Silica C18M 4D,
Puresil C18, COSMOSIL 5C18-MS-II.
335

(4) Test procedures(4.1) Extraction: Conduct extraction as shown below.
a) Weigh 1.00 g of an analytical sample, and put it into a 200-mL ground-in stopper
Erlenmeyer flask.
b) Add 100 mL of methanol - phosphate (996+4) and stir it with a magnetic stirrer for
about 30 minutes.
c) After allowing to stand still, transfer supernatant solution (2) to 1.5-mL ground-in stopper
centrifugal precipitate tube (3).
d) Centrifuge by 8,000 × g centrifugal force for about five minutes (4) to make supernatantas the sample solution.
Note (2) If there is a possibility that the N-2,5-dichlorophenyl succinamic acidconcentration in the sample solution exceeds the maximum limit of thecalibration curve, dilute a predetermined amount of the outflow solution withmethanol.
(3) The ground-in stopper centrifugal precipitate tube should be made ofpolypropylene, etc. to not affect the measurement.
(4) 7-cm of rotor radius and 10,000 rpm of revolutions makes about 8,000 × g
centrifugal force.
Comment 2

Instead of procedures in (4.1) c) - d), it is allowed to filter with a membranefilter (aperture diameter: no more than 0.5-µm) made of hydrophilic PTFE
and the filtrate can be the sample solution.

(4.2) Measurement: Conduct measurement according to JIS K 0124 and as shown below.
Specific measurement procedures are according to the operation method of High
Performance Liquid Chromatograph (HPLC) used in measurement.
a) Measurement conditions for High Performance Liquid Chromatograph (HPLC):
Set up the measurement conditions for High Performance Liquid Chromatograph(HPLC) considering the following example:
1) Column: A silica gel column (4-mm - 6-mm inner diameter, 150-mm - 250-mm
long, 5-µm particle diameter) to which octadecyl chemically bonds.
2) Temperature of Column bath: 30 ºC - 40 ºC3) Eluent: Methanol - water (5) (55 + 45)
4) Flow rate: 0.8 mL/min
5) Detector: Absorptiometric detector, measurement wavelength: 246 nm
Note (5) Adjust the water used to pH 3 with phosphate in advance.
b) Calibration curve preparation
1) Inject 10 µL of respective N-2,5-dichlorophenyl succinamic acid standard solutions
for the calibration curve preparation to HPLC, record chromatogram at wavelength
246 nm and obtain the peak area or height.
2) Prepare a curve for the relationship between the concentration and the peak area orheight at wavelength 246 nm of the N-2,5-dichlorophenyl succinamic acid standard
solution for the calibration curve preparation.
c) Sample measurement
1) Subject 10 µL of the sample solution to the same procedure as in b) 1).
2) Obtain the N-2,5-dichlorophenyl succinamic acid content from the calibration curve
to calculate the concentration of N-2,5-dichlorophenyl succinamic acid in theanalytical sample.
336

Comment 3

Recovery testing was conducted using compound fertilizer (2 samples) andblended fertilizer (1 sample), as a result, the mean recovery rate of
N-2,5-dichlorophenyl succinamic acid at concentration level of 0.4 %, 0.2 %and 0.1 % (mass fraction) were 100.9 % - 101.4 %, 100.8 % - 101.4 % and101.2 % - 103.4 %, and repeatability relative standard deviation were 0.5 %- 1.0 %, 1.1 % - 2.0 % and 0.8 % - 1.1 %, respectively. Additionally, theminimum limit of quantification of this test method is about 0.005 % (massfraction)

Reference
1) Akira KUBO: Analysis methods of N-2,5-dichlorophenyl succinamic acid (DCS) of
Nitrification suppression materials in fertilizers with High Performance Liquid
Chromatograph, Validation Report of Fertilizers (in Japanese), 44 (4), 25 - 36 (1991).
(5)

Flow sheet for N-2,5-dichlorophenyl succinamic acid: The flow sheet for
N-2,5-dichlorophenyl succinamic acid in fertilizers is shown below:
1.00 g analytical sample
200-mL ground-in stopper Erlenmeyer flask← 100 mL of methanol phosphate (996+4)
Extraction
Shaking, 30 minutes
Centrifugal separation
Measurement
FigureGround-in stopper centrifugal precipitate tube,
8,000 × g , 5 minutes
High Performance Liquid Chromatograph

Flow sheet for N-2,5-dichlorophenyl succinamic acid in fertilizers.

337

Reference: HPLC chromatogram of N-2,5-dichlorophenyl succinamic acid standard solution
for the calibration curve preparation is shown below.

Reference diagram: HPLC chromatogram of N-2,5-dichlorophenyl succinamic acid
Measurement conditions for HPLC
Column: Mightysil RP-18 GP (4.6-mm inner diameter, 150-mm long, 5-µm particle
diameter)
N-2,5-dichlorophenyl succinamic acid standard solution (the equivalents of 100 ng)
Other conditions are according to the examples of the measurement conditions of (4.2) a)
HPLC.

338

7.5
7.5.a

Dicyandiamide (Dd)High Performance Liquid Chromatography(1) Summary
This testing method is applicable to fertilizers containing dicyandiamide (Dd).
Add water to an analytical sample, leave at rest for a little while and add methanol to extract
dicyandiamide. After removing interfering substances with a silica gel cartridge column,
introduce it into a High Performance Liquid Chromatograph (HPLC), isolate with anaminopropyl silica gel column, and measure at wavelength 215 nm to calculate
dicyandiamide.
(2) Reagents, etc.: Reagents and water are as shown below.
a) Water: Water of A3 specified in JIS K 0557 or water with equivalent quality.
b) Methanol: A JIS Guaranteed Reagent specified in JIS K 8891 or a reagent of equivalentquality.
c) Methanol: Methanol used in eluent of HPLC is a regent of HPLC analysis grade orequivalents.
d) Acetonitrile: A regent of HPLC analysis grade or equivalents.
e) Dicyandiamide standard solution (1 mg/mL) (1): Transfer 0.1 g of dicyandiamide[C2H4N4](2) to a weighing dish and measure the mass to the order of 0.1 mg. Add a small
amount methanol to dissolve, transfer to a 100-mL volumetric flask and add the same
solvent up to the marked line. Store in a refrigerator, and do not use after 6 months after
preparation.
f) Dicyandiamide standard solution (100 µg/mL): In the case of usage, transfer 10 mLof dicyandiamide standard solution (1 mg/mL) to a 100-mL volumetric flask and add
methanol up to the marked line.
g) Dicyandiamide standard solution (10 µg/mL - 50 µg/mL) for the calibration curve
preparation: In the case of usage, transfer 5 mL - 25 mL of dicyandiamide standard
solution (100 µg/mL) to 50-mL volumetric flasks step-by-step and add methanol up to
the marked line.
h) Dicyandiamide standard solution (1 µg/mL - 10 µg/mL) for the calibration curve
preparation: In the case of usage, transfer 2.5 mL - 25 mL of dicyandiamide standard
solution for the calibration curve preparation (20 µg/mL) to 50-mL volumetric flasks
step-by-step and add methanol up to the marked line.
i) Sodium sulfate: A JIS Guaranteed Reagent specified in JIS K 8987 or a reagent ofequivalent quality.
Note (1) This is an example of preparation; prepare an amount as appropriate.
(2) A reagent of no less than 98 % in purity is commercially sold as dicyandiamide.
Comment 1

Dicyandiamide is commercially sold as Dicyanodiamide by Wako Pure
Chemical Industries., Ltd and Kanto Chemical Co., Inc.

(3) Instruments: Instruments are as shown below.
a) High Performance Liquid Chromatograph (HPLC): HPLC specified in JIS K 0124
that satisfies the following requirements.
1) Column: A 4-mm - 6-mm inner diameter 150-mm - 250-mm long stainless steel
column tube filled with silica gel, to which octadecyl chemically bonds.
2) Column bath: A column bath whose temperature can be adjusted to 30 ºC - 45 ºC.
3) Detector: An absorptiometric detector that can measure at wavelength around 215
nm.
339b) Shaking apparatusc) Centrifugal separator: A centrifugal separator that can centrifuge at 2,000 × g.
d) High speed centrifugal separator: A centrifugal separator that can centrifuge at 8,000× g.
e) Silica gel cartridge column: Link a 10-mL cylinder to the column (3) filled with 500 mg- 1 g of silica gel, add 3 mL of methanol to let it flow down.
Note (3) A cartridge with a 3-mL - 6-mL column that is filled with 500 mg - 1 g of silica
gel can be used.
Comment 2

Comment 3

A column is commercially sold under production names such as Hibar
LiChrosorb NH2 、 Inertsil NH2, Unison UK-Amino, Mightysil NH2,
Shim-pack CLC-NH2, Shodex NH-5A, Unisil Q NH2.
A silica gel cartridge column is commercially sold under production names
such as Sep-Pak Plus Silica, InertSep Si.

(4) Test procedures(4.1) Extraction: Conduct extraction as shown below.
a) Weigh 1.00 g of an analytical sample, and put it into a 200-mL ground-in stopper
Erlenmeyer flask.
b) Add 1 mL of water (4) and leave at rest for 5 minutes.
c) Add 100 mL of methanol and shake to mix with a shaking apparatus for about 10
minutes.
d) Add an appropriate amount of sodium sulfate (5).
e) After allowing to stand still, transfer supernatant solution to a 50-mL ground-in stopper
centrifugal precipitate tube.
f) Centrifuge at 2,000 × g centrifugal force for about five minutes (6) to make thesupernatant as the extract (7).
Note (4) Mix well until the whole sample comes in contact with water.
(5) About 5 g - 10 g.
(6) 18.7-cm of rotor radius and 3,000 rpm of revolutions makes about 2,000 × g
centrifugal force.
(7) If there is a possibility that the concentration of dicyandiamide in the sample
solution exceeds the maximum limit of the calibration curve, dilute apredetermined amount of extract with methanol.
(4.2) Cleanup: Conduct cleanup as shown belowa) Transfer extract to a silica gel cartridge column.
b) Dispose of about the first 3 mL of effluent and then transfer about the next 2 mL to atest tube.
c) Transfer effluent to a 1.5- mL of ground-in stopper centrifugal precipitate tube (8).
d) Centrifuge at 8,000 × g centrifugal force for about five minutes (9) to make supernatantas the sample solution.
Note (8) The ground-in stopper centrifugal precipitate tube should be made ofpolypropylene, etc. to not affect the measurement.
(9) 7-cm of rotor radius and 10,000 rpm of revolutions makes about 8,000 × g
centrifugal force.

340

Comment 4

Instead of the procedures in (4.2) c) - d), it is allowed to filter with amembrane filter (aperture diameter: no more than 0.5-µm) made of PTFE
and the filtrate can be the sample solution.

(4.3) Measurement: Conduct measurement according to JIS K 0124 and as shown below.
Specific measurement procedures are according to the operation method of High
Performance Liquid Chromatograph (HPLC) used in measurement.
a) Measurement conditions for High Performance Liquid Chromatograph (HPLC):
Set up the measurement conditions for High Performance Liquid Chromatograph(HPLC) considering the following example:
1) Column: A silica gel column (4-mm - 6-mm inner diameter, 150-mm - 250-mm
long, 5-µm particle diameter) to which amino or aminopropyl chemically bonds.
2) Temperature of column bath: 30 ºC - 40 ºC3) Eluent: Acetonitrile – methanol (6+1)
4) Flow rate: 0.5 mL/min - 1 mL/min
5) Detector: An absorptiometric detector, measurement wavelength: 215 nmb) Calibration curve preparation
1) Inject 10 µL of respective dicyandiamide standard solutions for the calibration curve
preparation to HPLC, record chromatogram at wavelength 215 nm and obtain thepeak area or height.
2) Prepare a curve for the relationship between the concentration and the peak area orheight at wavelength 215 nm of the dicyandiamide standard solution for thecalibration curve preparation.
c) Sample measurement
1) Subject 10 µL of the sample solution to the same procedure as in b) 1).
2) Obtain the dicyandiamide content from the calibration curve to calculate theconcentration of dicyandiamide in the analytical sample.
Comment 5:

Recovery testing was conducted using inorganic compound fertilizer (2samples) and organic compound fertilizer (3 samples), as a result, the mean
recovery at the concentration level of 2 % and 0.2% (mass fraction) were101.2 % - 102.6 % and 98.4 % - 100.6 %, and repeatability relative standard
deviation were 0.5 % - 1.8 % and 0.2 % - 1.3 %, respectively.
In addition, the results and analysis results of a collaborative study for test
method validation are shown in Table 1.
Additionally, the minimum limit of quantification of this test method isabout 0.01 % (mass fraction).

341

Table 1 Results and analysis results from a collaborative study
for the test method validation of dicyandiamide
4)
5)
Number ofsr
RSDr
Mean2)
Sample name1)laboratories
(%)
(%)3)
(%)3)

6)

sR

7)

RSDR
(%)

Compound fertilizer 1

11

0.263

0.009

3.2

(%)3)
0.019

Compound fertilizer 2

11

2.04

0.04

1.7

0.07

3.2

Compound fertilizer 3

13

0.548

0.011

2.0

0.033

6.0

Compound fertilizer 4

12

0.423

0.013

3.2

0.022

5.2

Compound fertilizer 5

12

1.02

0.01

1.4

0.04

4.3

1)
2)
3)
4)
5)
6)
7)

7.4

Number of laboratories used in analysis
Mean (n= number of laboratories × number of samples (2))
Mass fraction
Repeatability standard deviation
Repeatability relative standard deviation
Reproducibility standard deviation
Reproducibility relative standard deviation

References
1) Masakazu SAIKI: Development of High Performance Liquid Chromatography for
Determination of Dicyandiamide as a Nitrification Inhibitor in Fertilizer. Research
Report of Fertilizer, Vol.3, 2010 (43 - 50).
2) Masakazu SAIKI: Determination of Dicyandiamide as a Nitrification Inhibitor in
Fertilizer by High Performance Liquid Chromatography: Collaborative Study, Research
Report of Fertilizer, Vol.4, 2011 (16 - 22).
(5)

Flow sheet for dicyandiamide: The flow sheet for dicyandiamide in fertilizers isshown
1.00 g200-mL ground-in stopper Erlenmeyer flask
analytical sample← 100 mL of water
Leave at rest
5 minutes
Shaking
10 minutes← An adequate amount of sodium sulfate
Ground-in stopper centrifugal precipitate tube,
Centrifugal separation
2,000 × g , 5 minutes
CleanupCentrifugal separation
Measurement
FigureSilica gel cartridge column
1.5-mL ground-in stopper centrifugal precipitate tube,
8,000 × g , 5 minutes
High Performance Liquid Chromatograph

Flow sheet for dicyandiamide in fertilizers

342

0.18

0.18

0.16

0.16

0.12

AU

0.12

AU

0.14

8.605

0.14

8.607

0.20

Dd

0.20

Dd

Reference: HPLC chromatogram of dicyandiamide standard solution for the calibration curve
preparation and sample solution (compound fertilizer) are shown below.

0.10

0.10

0.08

0.08

0.06

0.06

0.04

0.04

0.02

0.02

0.00

0.00
0

2

4

6

8
min

10

12

14

0

1) Standard solution
Reference diagram

2

4

6

8
min

10

12

14

2) Sample solution
HPLC chromatogram of dicyandiamide

1) Dicyandiamide standard solution (the equivalents of 100 ng of dicyandiamide(10 µg/mL, 10 µL))
2) Sample solution (compound fertilizer)
Measurement conditions for HPLC
Column: Inertsil NH2 (4.6-mm inner diameter, 250-mm long, 5-µm particle
diameter)Temperature of Column bath: 30 ºC
Flow rate: 0.5 mL/min
Other conditions are according to the examples of the measurement
conditions of (4.3) a) HPLC.

343

7.6
7.6.a

2-sulfanilamide thiazole (ST)High Performance Liquid Chromatography(1) Summary
This testing method is applicable to fertilizers containing 2-sulfanilamide thiazole (ST).
Add methanol - water (1+1) to an analytical sample to extract 2-sulfanilamide thiazole,
introduce it into a High Performance Liquid Chromatograph (HPLC), isolate with an
octadecyl silylation silica gel column, and measure at wavelength 285 nm to calculate
2-sulfanilamide thiazole.
(2) Reagents, etc.: Reagents and water are as shown below.
a) Water: Water of A3 specified in JIS K 0557 or water with equivalent quality..
b) Methanol: A JIS Guaranteed Reagent specified in JIS K 8891 or a reagent of equivalentquality.
c) Methanol: Methanol used in eluent of HPLC is a reagent of HPLC analysis grade orequivalents.
d) 2-sulfanilamide thiazole standard solution (1 mg/mL) (1) :Transfer 0.1 g of2-sulfanilamide thiazole [C9H9N3O2S2](2) to a weighing dish and measure the mass tothe order of 0.1 mg. Add water to dissolve, transfer to a 1,000-mL volumetric flask andadd methanol - water (1+1) to the marked line. Store in a refrigerator, and do not use
after 6 months after preparation.
e) 2-sulfanilamide thiazole standard solution (100 µg/mL): In the case of usage, transfer
10 mL of 2-sulfanilamide thiazole standard solution (100 µg/mL) to a 100-mL
volumetric flask and add methanol - water (1+1) up to the marked line.
f) 2-sulfanilamide thiazole standard solution (10 µg/mL - 50 µg/mL) for thecalibration curve preparation: In the case of usage, transfer 5 mL - 25 mL of
2-sulfanilamide thiazole standard solution (100 µg/mL) to 50-mL volumetric flasks
step-by-step and add methanol - water (1+1) up to the marked line.
g) 2-Sulfanilamide thiazole standard solution（1 µg/mL - 10 µg/mL）for the calibration
curve preparation: In the case of usage, transfer 2.5 mL - 25 mL of 2-sulfanilamide
thiazole standard solution for the calibration curve preparation (20 µg/mL) to 50-mL
volumetric flasks step-by-step and add methanol - water (1+1) up to the marked line.
Note (1) This is an example of preparation; prepare an amount as appropriate.
(2) A reagent of no less than 98 % (mass fraction) in purity is commercially sold as
2-sulfanilamide thiazole.
Comment 1:

2-sulfanilamide thiazole is sold under the production name of sulfathiazoleby Tokyo Chemical Industry Co., Ltd, Wako Pure Chemical Industries., Ltd
and Kanto Chemical Co., Inc.

(3) Instruments: Instruments are as shown below:a) High Performance Liquid Chromatograph: HPLC specified in JIS K 0124 thatsatisfies the following requirements.
1) Column: A 4-mm - 6-mm inner diameter 150-mm - 250-mm long stainless steel
column tube filled with silica gel, to which octadecyl chemically bonds.
2) Column bath: A column bath whose temperature can be adjusted to 30 ºC - 45 ºC.
3) Detector: An absorptiometric detector that can measure at wavelength around 285
nm.
b) Magnetic stirrerc) Centrifugal separator: A centrifugal separator that can centrifuge at 2,000 × g.
344d) High speed centrifugal separator: A centrifugal separator that can centrifuge at 8,000× g.
e) Acidic alumina cartridge column: Link a 10-mL cylinder to column (3) that is filledwith 500 mg - 1 g of acidic alumina and add 3 mL of methanol to let it flow down.
Note (3) A cartridge with a 3-mL - 6-mL column filled with 500 mg - 1 g of silica gel canbe used.
Comment 2

Comment 3

A column is sold under production names such as Inertsil ODS, Mightysil
RP-18, L-column ODS, Shim-pack VP-ODS, Silica C18M 4D, Puresil C18,COSMOSIL 5C18-MS-II, etc.
An acidic alumina cartridge is commercially sold under production names
such as Bond Elut AL-A, Sep-Pak Alumina-A, Supelclean LC-Alumina-A.

(4) Test procedures(4.1) Extraction: Conduct extraction as shown below.
a) Weigh 1.00 g of an analytical sample, and put it into a 200-mL ground-in stopper
Erlenmeyer flask.
b) Add 100 mL of methanol - water (1+1) and stir it with a magnetic stirrer for about 15
minutes.
c) After allowing to stand still, transfer supernatant solution to a 50-mL ground-in stopper
centrifugal precipitate tube.
d) Centrifuge at 2,000 × g centrifugal force for about five minutes (4) to make thesupernatant as the extract (5).
Note (4) 18.7-cm of rotor radius and 3,000 rpm of revolutions makes about 2,000 × g
centrifugal force.
(5) If there is a possibility that the 2-2-sulfanilamide thiazole sulfanilamide thiazole
concentration in the sample solution exceeds the maximum limit of thecalibration curve, dilute a predetermined amount of the extract with methanol.
(4.2) Cleanup: Conduct cleanup as shown below:a) Transfer extract to an acidic alumina cartridge column.
b) Dispose of about first 3 mL of effluent and then transfer about the next 2 mL to a test
tube.
c) Transfer effluent to a 1.5-mL ground-in stopper centrifugal precipitate tube (6).
d) Centrifuge at 8,000 × g centrifugal force for about five minutes (7) to make thesupernatant as the sample solution.
Note (6) The ground-in stopper centrifugal precipitate tube should be made ofpolypropylene, etc. to not affect the measurement.
(7) 7-cm of rotor radius and 10,000 rpm of revolutions makes about 8,000 × g
centrifugal force.
Comment 4

Comment 5

Instead of the procedures in (4.2) c) - d), it is allowed to filter with amembrane filter (aperture diameter: no more than 0.5-µm) made of PTFE
and the filtrate can be the sample solution.
The test is possible by the following procedures in the case of fertilizers notcontaining organic matters.

345

The procedures in (4.1) c) - d) and (4.2) a) - b) are omitted and “Transfer
effluent” in (4.2) c) is replaced with the “After allowing to stand still,transfer supernatant” to operate.
(4.3) Measurement: Conduct measurement according to JIS K 0124 and as shown below.
Specific measurement procedures are according to the operation method of High
Performance Liquid Chromatograph (HPLC) used in measurement.
a) Measurement conditions for High Performance Liquid Chromatograph (HPLC):
Set up the measurement conditions for High Performance Liquid Chromatograph(HPLC) considering the following example:
1) Column: A silica gel column (4-mm - 6-mm inner diameter, 150-mm - 250-mm
long, 5-µm particle diameter) to which octadecyl chemically bonds.
2) Temperature of Column bath: 30 ºC - 40 ºC3) Eluent: Methanol - water (2+8)4) Flow rate: 1 mL/min
5) Detector: An absorptiometric detector, measurement wavelength: 285 nmb) Calibration curve preparation
1) Inject 10 µL of respective 2-sulfanilamide thiazole standard solutions for thecalibration curve preparation to HPLC, record chromatogram at wavelength 285 nm
and obtain the peak area or height.
2) Prepare a curve for the relationship between the concentration and the peak area orheight at wavelength 285 nm of the 2-sulfanilamide thiazole standard solutions forthe calibration curve preparation.
c) Sample measurement
1) Subject 10 µL of the sample solution to the same procedure as in b) 1).
2) Obtain the 2-sulfanilamide thiazole content from the calibration curve to calculate
the concentration of 2-sulfanilamide thiazole in the analytical sample.
Comment 6

Recovery testing was conducted using compound fertilizer (1 sample) andblended fertilizer (2 sample), as a result, the mean recovery rate of
2-sulfanilamide thiazole at the concentration level of 1.0 %, 0.4 % and
0.1 % (mass fraction) were 101.2 % - 102.1 %, 99.6 % - 101.7 % and99.4 % - 101.0 %, and repeatability relative standard deviation were 0.5 % 1.2 %, 2.1 % - 3.2 % and 1.0 % - 3.4 %, respectively.
Additionally, the minimum limit of quantification of this test method isabout 0.005 % (mass fraction).

Reference
1) Yuji SHIRAI: The volumetric analysis of 2-sulfanilamide thiazole in fertilizers withHigh Performance Liquid Chromatograph, Validation Report of Fertilizers (in Japanese),
44 (1), 10 - 20 (1991)

346

(5)

Flow sheet for 2-sulfanilamide thiazole: The flow sheet for 2-sulfanilamide thiazole infertilizers is shown below:
1.00 g analytical sample
200-mL of ground-in stopper Erlenmeyer flask← 100 mL of methanol - water (1+1)
Extraction
Shaking to mix, 15 minutes
Centrifugal separation
CleanupCentrifugal separation
Measurement
FigureGround-in stopper centrifugal precipitate tube,
2,000 × g , 5 minutes
Acidic alumina cartridge column
Ground-in stopper centrifugal precipitate tube,
8,000 × g , 5 minutes
High Performance Liquid Chromatograph

Flow sheet for 2-sulfanilamide thiazole in fertilizers.

347

Reference: HPLC chromatogram of 2-sulfanilamide thiazole standard solution for thecalibration curve preparation is shown below.

Reference diagram: HPLC chromatogram of 2-sulfanilamide thiazole
Measurement conditions for HPLC
Column: Mightysil RP-18 GP (4.6-mm inner diameter, 150-mm long, 5-µm
particle diameter)
2-sulfanilamide thiazole standard solution (the equivalents of 200 ng)
Other conditions are according to the examples of the measurement conditions of(4.3) a) HPLC.

348

8.

Others

8.1

Melamine and its degradation products

8.1.a

Gas Chromatography/Mass Spectrometry <Reference method>(1) Summary
Extract melamine and its derivative substances (hereinafter referred to as “melamine
derivations”) in organic matters and fertilizers containing organic matters with
diethylamine-water-acetonitrile (1+4+5) and derivatize with BSTFA-TMCS (99+1) and thenmeasure with a gas chromatography/mass spectrometer.
Comment 1

The structural formula of melamine and its degradation products is shown in
the figure 1. During the production process of melamine, a by-product that
replaces “-NH2” of R1-R3 with “–OH” is formed in some cases.

R1
N
R2

N
N

R3

R1
M e l a m i n e NH2
A m m e l i n e OH
A m m e l i d e OH
C y a n u r i c a cOH
id

R2
NH2
NH2
OH
OH

R3
NH2
NH2
NH2
OH

MW
126.12
127.10128.09129.07

Figure 1 Structural formula of melamine and its degradation products(2) Reagents, etc.: Reagents and water are as shown below:a) Water: Water of A3 specified in JIS K 0557or equivalent quality.
b) Acetonitrile: A reagent of agricultural chemicals residue/PCB testing grade(concentration: no less than 300) specified in JIS K 8039 or a reagent of equivalentquality.
c) Diethylamine: A guaranteed reagent or a reagent of equivalent quality.
d) Pyridine (dehydration) (1): A reagent of organic synthesis grade of no less than 99.5 %(mass fraction) in purity and no more than 0.05 mg/mL in moisture or a reagent ofequivalent quality.
e) Derivatization reagent (2): Bis (trimethylsilyl) trifluoroacetamide trimethylchlorosilane (99+1).
f) Melamine derivations standard solution (0.5 mg/mL): Put about 0.05 g of melamine[C3H6N6] (3), ammeline [C3H5N5O] (3), ammelide [C3H4N4O2] (3) and cyanuric acid
[C3H3N3O3] (3) into a weighing dish and measure the mass to the order of 0.1 mg.
Dissolve in a small amount of diethylamine - water (1+4), transfer to a 100 mL
volumetric flask respectively, and add the solvent up to the marked line.
g) Mixture standard solution (50 µg/mL) (3): Put 5 mL of respective melamine
derivations standard solutions (0.5 mg/mL) to 50-mL volumetric flasks and add
diethylamine - water - acetonitrile (1+4+5) up to the marked line.
Note (1) After it is opened once, add a proper amount of sodium sulfate (anhydrous) andseal tightly to store.
(2) A mixed derivatization reagent is commercially sold under the name of
BSTFA-TMCS (99+1).
(3) The respective standard reagents of melamine, ammeline, ammelide andcyanuric acid are commercially sold.

349

Comment 2
Comment 3

BSTFA-TMCS (99+1) is sold as 1-mL ampoule by SUPELCO. After it isopened once, use it on the same day.
The standard reagent of melamine, ammeline, ammelide and cyanuric acidare sold by Wako Pure Chemical Industries., Ltd and Kanto Chemical Co.,
Inc. and Hayashi Pure Chemical Industries., Ltd.

(3) Instruments: Instruments are as shown below.
a) Gas Chromatograph/Mass Spectrometer (GC/MS): GC/MS specified in JIS K 0123
that satisfies the following requirements.
1) Gas Chromatograph(i) Sample injector: An injector that enables split less system.
(ii) Capillary column: A capillary column (0.25-mm - 0.32-mm inner diameter and30-m long) made of fused silica. 5 % phenyl 95 % methyl polysiloxane
chemically bonds to the inner surface of a capillary column with 0.25 µm
thickness. The column is according to the specification of mass spectrometer.
(iii) Carrier gas: High purity helium of no less than 99.999 % (volume fraction) inpurity.
2) Mass Spectrometer(i) Ionization method: Electron-Impact ionization (EI) method(ii) Ion detection method: Selected Ion Monitoring (SIM) methodb) Ultrasonic generator: An ultrasonic washer can be used.
c) High speed centrifugal separator: A centrifugal separator that can centrifuge at 8,000
× g.
d) Concentrator: A Centrifugal evaporator that can be adjusted to 70 ºC ± 2 ºC.
e) Water bath: A water bath that can be adjusted to 70 ºC ± 2 ºC.
Comment 4

A capillary column is commercially sold under the names such as DB-5ms,
Rtx-5ms, HP-5ms, SLB-5ms, BPX-5, CP-Sil 8CB low Bleed/MS and
TC-5HT for GC/MS.

(4) Test procedures(4.1) Extraction: Conduct extraction as shown below.
a) Weigh 0.50 g of an analytical sample, and put it into a 200-mL - 300-mL ground-in
stopper Erlenmeyer flask.
b) Add 160 mL - 200 mL of diethylamine - water - acetonitrile (1+4+5), and subject toultra-sonication for about 30 minutes using an ultrasonic generator.
c) Transfer about 1.5 mL to a 1.5-mL ground-in stopper centrifugal precipitate tube (4), andcentrifuge at 8,000 × g for about 5 minutes(5).
d) Transfer 1 mL of the supernatant solution to 5-mL - 50-mL volumetric flasks, add
diethylamine - water - acetonitrile (1+4+5) up to the marked line to make the extract.
Note (4) Confirm that the tube is made of polypropylene, etc. to not affect testing results.
(5) 7-cm of rotor radius and 10,000 rpm of revolutions makes about 8,000 × g
centrifugal force.
Comment 5 Grind until it completely passes through a sieve of 500 µm aperture toprepare the test sample.
Comment 6 Weigh 0.5 g of an analytical sample, extract with 200 mL of diethylamine water - acetonitrile (1+4+5). If it is diluted 50 times in the procedure in d),
the quantitative range of melamine derivations in the analytical sample is
0.2 % - 10 % (mass fraction). In the case of measuring melamine derivations
350

which do not reach the quantitative range, make the dilution factor in theprocedure in d) decrease. In addition, if the contents of melamine derivations
exceeds 10 % (mass fraction) respectively, the sampling volume of ananalytical sample should be reduced.
(4.2) Derivatization: Conduct derivatization as shown below.
a) Transfer 0.2 mL of the extract to a 5-mL - 10-mL test tube with a screw stopper.
b) Place a test tube in a concentrator, concentrate under reduced pressure at 70 ºC ± 2 ºC
and vaporize the solvent completely (6).
c) Add 0.3 mL of pyridine (dehydration) (1) and 0.2 mL of derivatization reagent (2) to theresidue to mix, and then stopple to seal tightly.
d) After heating in a water bath at 70 ºC ± 2 ºC for about 45 minutes (7), let it stand to cool
to make the sample solution (8).
Note (6) A spraying type concentrator can be used.
(7) If moisture remains after the procedure in b) or the reagent used in the procedurein c) contains moisture, the reaction of the derivatization in d) does not advanceenough in some cases.
(8) If necessary, transfer the sample solution into a 1.5-mL ground-in stopper
centrifugal precipitate tube (4) to centrifuge at 8,000 × g for about 5 minutes (5).
(4.3) Measurement: Conduct measurement according to JIS K 0123 and as shown below.
Specific measurement procedures are according to the operation method of the gas
chromatograph/ mass spectrometer used in measurement.
a) Measurement conditions for the gas chromatograph/ mass spectrometer: Set up themeasurement conditions considering the following example of measurement conditions
for gas chromatograph/mass spectrometer.
1) Gas chromatograph:(i) Sample injection method: split less injection method (1min)(ii) Temperature of sample injector: 280 ºC(iii) Capillary column: A capillary column (0.25-mm - 0.32-mm inner diameter,
30-m long, 0.25 µm layer thickness) made of fused silica. 5 % phenyl 95 %methyl polysiloxane chemically bonds to the inner surface of the capillarycolumn.
(iv) Temperature of column bath: 100 ºC (1 min) → (15 ºC /min) → 320 ºC (3 min)(v) Temperature of GC/MS: 250 ºC(vi) Carrier gas: helium, flow rate: 1.5 mL/min
2) Mass spectrometer:(i) Ionization method: Electron-Impact ionization (EI) method(ii) Ionization voltage: 70 V(iii) Temperature of ion source: 230 ºC(iv) Ion detection method: Selected Ion Monitoring (SIM) method(v) Measurement of ion: As shown in table 1b) Calibration curve preparation
1) Transfer 5 mL of mixture standard solution (50 µg/mL) to a 50-mL volumetric flask
and add diethylamine - water - acetonitrile (1+4+5) up to the marked line to make themixture standard solution (5 µg/mL).
2) Transfer 1 mL - 20 mL of the mixture standard solution (5 µg/ mL) to 50 mL
volumetric flasks step-by-step and add diethylamine - water - acetonitrile (1+4+5) up
to the marked line to make the mixture standard solution (0.1 µg/mL - 2 µg/mL).
351

3) Conduct the procedures of (4.2) b) - d) for the mixture standard solution (0.1 µg/mL- 2 µg/mL) to make the mixture standard solution, the equivalents of 0.04 µg/mL 0.8 µg/mL, for calibration curve.
4) Inject 1 µL of respective mixture standard solutions for calibration curve to GC/MS
and record the chromatogram of ion (m/z) for determination and ion (m/z) for
validation of materials subjected to measurement to obtain the peak area and height.
5) Calculate the peak area ratio or height ratio of ion (m/z) for determination and ion(m/z) for validation of respective materials subjected to measurement. .
6) Prepare a curve for the relationship between the concentration of material subjected
to measurement and the peak area or height of ion (m/z) for determination ofrespective mixture solutions for the calibration curve preparation.
c) Sample measurement
1) Subject 1 µL of the sample solution to the same procedure as in b) 4) - 5) (9).
2) Obtain the content of respective materials subjected to measurement from thecalibration curve to calculate the concentration of respective materials subjected tomeasurement in the analytical sample.
Note (9) Confirm that the ratio against the peak area ratio or height ratio of the standard
solution is within the range of about ± 30 %. In addition, the peak area ratio orheight ratio depends on the concentration.
Comment 7

If the variation of sensitivity of melamine derivations is observed, conduct
measurement by the following method of a) or b).
a) In the procedure in (4.3) c) 1, inject the sample solution into GC/MS
predetermined times, and then correct the calibration curve according to(4.3) b) 4) - 6).
b) Add 2,6-diamino-4-chloropyrimidine (the equivalents of 0.5 µg) as aninternal reference material to the standard solution and the sample
solution, conduct the same procedures as (4.2) c) - d), (4.3) b) 4) - 6) andc) 1). However, prepare the calibration curve from the peak area ratio orheight ratio of ion (m/z) for determination of respective materials
subjected to measurement and internal reference material, and calculate
the concentration of respective materials subjected to measurement in theanalytical sample.
Table 1 Fragment ion of materials to be measured
Materials
Measured fragment ion (m /z)
to be measured Determination Validation Validation Validation
342
Melamine
344
327
285
328
Ammeline
345
343
285
344
Ammelide
346
329
214
345
Cyanuric acid
347
330
215
288
DACP(I.S.)
289
290
273

Comment 8

Validation
213
214
198
188
275

Recovery testing of melamine derivations was conducted using soybean
meal, fish meal, fish waste processed fertilizer, mixed organic fertilizer,
blended fertilizer and compound fertilizer, as a result, the mean recovery
rate at additive level of 10 and 0.2 % (mass fraction) were 92.1 % - 102.9 %and 90.3 % - 102.2 %, and the repeatability were 0.9 % - 10.5 % and 0.6 % 9.1 % relative standard deviation.
352

Additionally, the minimum limit of quantification of melamine derivationsof the test method is about 0.01 % (mass fraction).
Reference
1) Yuji SHIRAI, Jun OKI: Validation of Gas Chromatography/Mass Spectrometry for
Determination of Melamine and Its Degradation Products in Fertilizers, Research
Report of Fertilizer, Vol.1, 2008, (114 - 121)
(5) Flow sheet for melamine derivations: The flow sheet for melamine derivations in
fertilizers is shown below.
0.50 g analytical sample

200-mL - 300-mL ground-in stopper Erlenmeyer flask← 160 mL - 200 mL of diethylamine - water - acetonitrile（1+4+5）
Extraction
Centrifugal separation
Dilution
0.2 mL Aliquot

Ultrasonic extraction for 30 minutes
8,000 × g, 5 minutes
Transfer 1 mL to 5-mL - 50-mL volumetric flasks,
add diethylamine - water - acetonitrile（1+4+5）up to the marked line
5-mL - 10-mL test tube with a screw stopper

Vacuumconcentration

Centrifugal evaporator (70 ºC ± 2 ºC)/Drying up← 0.3 mL of pyridine (dehydration)← 0.2 mL of derivatization reagent (BSTFA-TMCS (99+1))
Stopple and seal.
Derivatization

70 ºC ± 2 ºC, 45 minutes

Standing to cool
Measurement
Figure 2

Gas Chromatograph/Mass Spectrometer

Flow sheet for melamine derivations in fertilizers

353

Reference↓

Total Ion Chromatogram (TIC) of GC/MS of mixture standard solution for
calibration curve preparation of melamine derivations is shown below.
a

↓b

↓c

↓d

Figure 3 Total Ion Chromatogram (TIC) of GC/MS for melamine derivations
Measurement conditions of GC/MS
Capillary column: Rtx-5ms (0.25-mm inner diameter, 30-m long, 0.25 µm
layer thickness)
Other conditions are according to the example of measurement conditions forgas chromatography/mass spectrometer in (4.3) a.
Peak name of respective total ion chromatogramsa) Cyanuric acidb) Ammelidec) Ammelined) Melamine
Sample and amount injected into GC/MS
Injected sample: Mixture standard solutions (the equivalents of 2 µg/mL) forthe calibration curve preparation of respective melamine derivations.
Injected amount: 1 µL (the equivalents of 2 ng of respective melamine
derivations)354

8.1.bHigh Performance Liquid Chromatography (Nitrolime) <Reference method>(1) Summary
This method is applicable to nitrolime.
Add acetonitrile - water - diethylamine (5+4+1) to an analytic sample, extract melamine,
introduce it into a High Performance Liquid Chromatograph (HPLC), isolate with a silica gelcolumn to which carbamoyl chemically bonds, and measure at wavelength 214 nm to
calculate melamine. However it is difficult to extract cyanuric acid that is melamine
derivations from nitrolime by this method and the recovery testing for ammeline andammelide does not achieve good results, so that they are excluded from components subjected
to measurement.
(2) Reagents, etc.: Reagents and water are as shown below.
a) Water: Water of A3 specified in JIS K 0557 or equivalent quality.
b) Acetonitrile: A JIS Guaranteed Reagent specified in JIS K 8032 or a reagent ofequivalent quality.
c) Acetonitrile: Acetonitrile used in eluent of HPLC is a regent of HPLC analysis grade orequivalents.
d) Diethylamine: A guaranteed reagent or a reagent of equivalent quality.
e) Phosphate buffer solution (1): Dissolve 0.237 g of disodium hydrogen-phosphate
specified in JIS K 9020 and 0.520 g of sodium dihydrogenphosphate dihydrate specified
in JIS K 9009 in water to make 1,000 mL (2). If it is used for eluent of HPLC, filter witha membrane filter (pore size: no more than 0.5-µm) made of hydrophilic PTFE.
f) Melamine derivations standard solution (0.5 mg/mL): Put about 0.05g of melamine[C3H6N6](3), ammeline [C3H5N5O](3)and ammelide [C3H4N4O2](3) to weighing dishes
respectively and measure the mass to the order of 0.1 mg. Dissolve in a small amount ofdiethylamine - water (1+4), transfer to respective 100 mL volumetric flasks, and add thesolvent up to the marked line.
g) Mixture standard solution (50 µg/mL) (1): Transfer 5 mL of respective melamine
derivations standard solutions (0.5 mg/mL) to a 50-mL volumetric flask and add
acetonitrile - phosphate buffer solution (4+1) up to the marked line.
h) Mixture standard solution for calibration curve preparation (2 µg/mL - 20
µg/mL): In the case of usage, transfer 2 mL - 20 mL of mixture standard solution (50µg /mL) to 50-mL volumetric flasks step-by-step and add acetonitrile - phosphate buffer
solution (4+1) up to the marked line.
i) Mixture standard solution for calibration curve preparation (0.1 µg/mL - 2µg/mL): In the case of usage, transfer 1 mL - 20 mL of mixture standard solution (50µg/ mL) to 50-mL volumetric flasks step-by-step, add acetonitrile - phosphate buffer
solution (4+1) up to the marked line.
Note (1) This is an example of preparation; prepare an amount as appropriate.
(2) pH of phosphate buffer solution becomes 6.7 ± 0.2.
(3) Standard reagents as melamine, ammeline and ammelide are commercially soldrespectively.
Comment 1

The standard reagents of melamine, ammeline and ammelide are sold by
Wako Pure Chemical Industries., Ltd, Kanto Chemical Co., Inc., Hayashi
Pure Chemical Industries, Ltd and Tokyo Chemical Industry Co., Ltd.

(3) Instruments: Instruments are as shown below.
a) High Performance Liquid Chromatograph (HPLC): HPLC specified in JIS K 0124
that satisfies the following requirements.
355

1) Column: A 4-mm - 6-mm inner diameter 150-mm - 250-mm long stainless steel
column tube filled with silica gel, to which carbamoyl chemically bonds.
2) Column bath: A column bath whose temperature can be adjusted to 30 ºC - 45 ºC.
3) Detector: An absorptiometric detector that can measure at wavelength around 214nm.
b) Ultrasonic generator: An ultrasonic washer can be used.
c) Centrifugal separator: A centrifugal separator that can centrifuge at 2,000 × g.
d) High speed centrifugal separator: A centrifugal separator that can centrifuge at 8,000
× g.
Comment 2

A column is sold under production name of TSKgel Amide-80, etc.

(4) Test procedures(4.1) Extraction: Conduct extraction as shown below.
a) Weigh 0.50 g of an analytical sample, and put it into a 200-mL ground-in stopper
Erlenmeyer flask.
b) Add 100 mL of acetonitrile - water - diethylamine (5+4+1) and conduct ultra-sonication
for about 30 minutes using an ultrasonic generator.
c) After allowing to stand still, transfer 50 mL of the supernatant solution to a ground-in
stopper centrifugal precipitate tube.
d) Centrifuge at 2,000 × g for about 5minutes (4) to make supernatant as the extract.
e) Transfer 5 mL of the extract (5) into a 50-mL volumetric flask, and add acetonitrile phosphate buffer solution (4+1) up to the marked line to dilute.
f) Transfer 1.5 mL of dilution liquid to a 1.5-mL ground-in stopper centrifugal precipitate
tube (6).
g) Centrifuge at 8,000 × g for about 5 minutes (7) to make supernatant as the sample
solution
Note (4) 18.7-cm of rotor radius and 3,000 rpm of revolutions makes about 2,000 × g
centrifugal force.
(5) If there is a possibility that the concentration of melamine derivations in thesample solution exceeds the maximum limit of the calibration curb, the amountof supernatant solution to be transferred should be1 mL - 2.5 mL.
(6) The ground-in stopper centrifugal precipitate tube should be made ofpolypropylene, etc. to not affect the measurement.
(7) 7cm of rotor radius and 10,000 rpm of revolutions makes about 8,000 × g
centrifugal force.
Comment 3

Instead of the procedures in (4.2) f) - g), it is allowed to filter with amembrane filter (pore size: no more than 0.5-µm) made of hydrophilic
PTFE and the filtrate can be the sample solution.

(4.2) Measurement: Conduct measurement according to JIS K 0124 and as shown below.
Specific measurement procedures are according to the operation method of the High
Performance Liquid Chromatograph (HPLC) used in measurement.
a) Measurement conditions of High Performance Liquid Chromatograph (HPLC):
Set up the measurement conditions considering the following example of measurement
conditions for High Performance Liquid Chromatograph (HPLC).
1) Column: A silica gel column (4-mm - 6-mm inner diameter, 150-mm - 250-mm
long, 5-µm particle diameter column) to which carbamoyl chemically bonds.
2) Temperature of column bath: 30 ºC - 40 ºC
3563) Eluent: Acetonitrile - phosphate buffer solution (4+1)
4) Flow rate: 1 mL/min
5) Detector: An absorptiometric detector, measurement wavelength: 214 nmb) Calibration curve preparation
1) Inject 10 µL of respective mixture standard solutions for calibration curve
preparation into HPLC, record chromatogram at wavelength 214 nm and obtain thepeak area and height.
2) Prepare a curve for the relationship between the concentration and the peak area orheight at wavelength 214 nm of respective mixture solutions for the calibration curve
preparation.
c) Sample measurement
1) Subject 10 µL of the sample solution to the same procedure as in b) 1).
2) Obtain the melamine derivations content from the calibration curve to calculate theconcentration of melamine derivations in the analytical sample.
Comment 4

Recovery testing was conducted using 3 brands of nitrolime, as a result, therecovery rate of melamine at concentration level of 4 % and 0.4 % were95.7 % - 103.2 % and 93.6 % - 102.5 %, and the repeatability were 0.8 % 2.3 % and 0.5 % - 1.0 % relative standard deviation.
Additionally, the minimum limit of quantification of the test method isabout 0.01 % (mass fraction).

Reference
1) Etsuko BANDO, Toshiaki HIROI, Masahiro ECHI and Yuji SHIRAI: Validation ofHigh Performance Liquid Chromatography (HPLC) for Determination of Melamine and
Its Related Substances in Calcium Cyanamid, Research Report of Fertilizer,Vol.5, 2012,(24 - 30)
(5) Flow sheet for melamine derivations: The flow sheet for melamine derivations in
nitrolime is shown below.
0.50 g analytical sample
200-mL ground-in stopper Erlenmeyer flask←100 mL of acetonitrile - water - diethylamine (5+4+1）
Extraction
Ultrasonic extraction for 30 minutes
Centrifugal separation
DilutionCentrifugal separation
Measurement

2,000 × g for 5 minutes
Transfer 5 mL to a 50-mL volumetric flask,
add acetonitrile - phosphate buffer solution (4+1) up to the marked line
8,000 × g for 5 minutes
High Performance Liquid Chromatograph

Figure Flow sheet for melamine derivations in fertilizers

357

Reference HPLC chromatogram of the mixture standard solution for calibration curve
preparation of melamine derivations is shown below.

Reference diagram

HPLC chromatogram of melamine derivations

The names of materials for respective peaks(1) cyanuric acid (reference) (2) ammelide (3) melamine (4) ammeline
Measurement conditions of HPLC
Column: TSKgel Amide-80 (4.6-mm inner diameter, 250-mm long, 5-µm particle
diameter)Mixture standard solutions (the equivalents of 100 ng (10 µg/mL, 10 µL)) for
calibration curve preparation of respective melamine derivations
Other conditions are according to the examples of the measurement conditions in(4.2) a) HPLC.

358

8.1.cHigh Performance Liquid Chromatographymatters)(Fertilizers not containing organic(1) Summary
This method is applicable to fertilizers not containing organic matters.
Add hydrochloric acid (1+15) to an analytic sample, extract melamine and its degradation
products (hereinafter referred to as “melamine derivations”), introduce it into a High
Performance Liquid Chromatograph (HPLC), isolate with a silica gel column to which
carbamoyl chemically bonds, and measure at wavelength 214 nm to calculate melamine
derivations.
(2) Reagents, etc.: Reagents and water are as shown below.
a) Water: Water of A3 specified in JIS K 0557.
b) Acetonitrile: A JIS Guaranteed Reagent specified in JIS K 8032 or a reagent ofequivalent quality. In addition, acetonitrile used in eluent of HPLC is a regent of HPLC
analysis grade.
c) Hydrochloric acid: A JIS Guaranteed Reagent or a reagent of equivalent quality.
d) Phosphate buffer solution (1): Dissolve 0.237 g of disodium hydrogen-phosphate
specified in JIS K 9020 and 0.520 g of sodium dihydrogenphosphate dihydrate specified
in JIS K 9009 in water to make 1,000 mL (2). If it is used for eluent of HPLC, filter witha membrane filter (pore size: no more than 0.5-µm) made of hydrophilic PTFE.
e) Melamine derivations standard solution (0.5 mg/mL): Put about 0.05g of melamine[C3H6N6](3), ammeline [C3H5N5O](3), ammelide [C3H4N4O2](3) and cyanuric acid
[C3H3N3O3] (3) to weighing dishes respectively and measure the mass to the order of 0.1
mg. Dissolve them with a small amount of hydrochloric acid (1+15), transfer to 100 mL
volumetric flasks respectively, and add the solutions up to the marked line.
f) Mixture standard solution (50 µg/mL) (1): Transfer 5 mL of respective melamine
derivations standard solutions (0.5 mg/mL) to 50-mL volumetric flasks and add
acetonitrile - phosphate buffer solutions (4+1) up to the marked line.
g) Mixture standard solution for calibration curve preparation (1 µg/mL - 5 µg/mL):
In the case of usage, transfer 1 mL - 5 mL of mixture standard solution (50 µg /mL) to
50-mL volumetric flasks step-by-step and add acetonitrile - phosphate buffer solutions(4+1) up to the marked line.
h) Mixture standard solution for calibration curve preparation (0.05 µg/mL – 0.5µg/mL): In the case of usage, transfer 2.5 mL - 25 mL of mixture standard solution (1µg/ mL) to 50-mL volumetric flasks step-by-step, add acetonitrile - phosphate buffer
solutions (4+1) up to the marked line.
Note (1) This is an example of preparation; prepare an amount as appropriate.
(2) pH of phosphate buffer solution becomes 6.7 ± 0.2.
(3) Standard reagents as melamine, ammeline, ammelide and cyanuric acid arecommercially sold respectively.
Comment 1

The standard reagents of melamine, ammeline, ammelide and cyanuric acidare sold by Wako Pure Chemical Industries., Ltd, Kanto Chemical Co., Inc.,
Hayashi Pure Chemical Industries, Ltd and Tokyo Chemical Industry Co.,Ltd.

(3) Instruments: Instruments are as shown below.
a) High Performance Liquid Chromatograph (HPLC): HPLC specified in JIS K 0124
that satisfies the following requirements.
359

1) Column: A 4-mm - 6-mm inner diameter 150-mm - 250-mm long stainless steel
column tube filled with silica gel, to which carbamoyl chemically bonds.
2) Column bath: A column bath whose temperature can be adjusted to 40 ºC ± 1 ºC.
3) Detector: An absorptiometric detector that can measure at wavelength around 214nm.
b) Ultrasonic generator: An ultrasonic washer can be used.
c) Centrifugal separator: A centrifugal separator that can centrifuge at 2,000 × g.
d) High speed centrifugal separator: A centrifugal separator that can centrifuge at 8,000
× g.
Comment 2

Column is sold under production name of TSKgel Amide-80, etc. A column
which has actually isolated melamine, ammeline, ammelide and cyanuric
acid should be used.

(4) Test procedures(4.1) Extraction: Conduct extraction as shown below.
a) Weigh 0.50 g of an analytical sample, and put it into a 200-mL ground-in stopper
Erlenmeyer flask.
b) Add 100 mL of hydrochloric acid (1+15) and conduct ultra-sonication for about 30
minutes using an ultrasonic generator.
c) After allowing to stand still, transfer supernatant solution to a 50-mL ground-in stopper
centrifugal precipitate tube.
d) Centrifuge at 2,000 × g for about 5minutes (4) to make supernatant as the extract.
e) Transfer 5 mL of the extract (5) into a 50-mL volumetric flask, and add acetonitrile phosphate buffer solution (4+1) up to the marked line to dilute.
f) Transfer dilution liquid to a 1.5-mL ground-in stopper centrifugal precipitate tube (6).
g) Centrifuge at 8,000 × g for about 5 minutes (7) to make supernatant as the sample
solution
Note (4) 18.7cm of rotor radius and 3,000 rpm of revolutions makes about 2,000 × g
centrifugal force.
(5) If there is a possibility that the concentration of melamine derivations in thesample solution exceeds the maximum limit of the calibration curb, the amountof supernatant solution to be transferred should be1 mL - 2.5 mL.
(6) The ground-in stopper centrifugal precipitate tube should be made ofpolypropylene, etc. to not affect the measurement.
(7) 7cm of rotor radius and 10,000 rpm of revolutions makes about 8,000 × g
centrifugal force.
Comment 3

Instead of the procedures in (4.1) f) - g), it is allowed to filter with amembrane filter (pore size: no more than 0.5-µm) made of hydrophilic
PTFE and the filtrate can be the sample solution.

(4.2) Measurement: Conduct measurement according to JIS K 0124 and as shown below.
Specific measurement procedures are according to the operation method of the High
Performance Liquid Chromatograph (HPLC) used in measurement.
a) Measurement conditions of High Performance Liquid Chromatograph (HPLC):
Set up the measurement conditions considering the following example of measurement
conditions for High Performance Liquid Chromatograph (HPLC).
1) Column: A silica gel column (4-mm - 6-mm inner diameter, 150-mm - 250-mm
long, 5-µm particle diameter column) to which carbamoyl chemically bonds.
3602) Temperature of column bath: 40 ºC ± 1 ºC
3) Eluent: Acetonitrile - phosphate buffer solution (4+1)
4) Flow rate: 1 mL/min
5) Detector: An absorptiometric detector, measurement wavelength: 214 nmb) Calibration curve preparation
1) Inject 10 µL of respective mixture standard solutions for calibration curve
preparation into HPLC, record chromatogram at wavelength 214 nm and obtain thepeak area or height.
2) Prepare a curve for the relationship between the concentration and the peak area orheight at wavelength 214 nm of respective mixture solutions for the calibration curve
preparation.
c) Sample measurement
1) Subject 10 µL of the sample solution to the same procedure as in b) 1).
2) Obtain the melamine derivations content from the calibration curve to calculate theconcentration of respective melamine derivations in the analytical sample.
Comment 4

Recovery testing was conducted using 3 brands of nitrolime, 1 brand ofcompound fertilizer containing nitrolime, 2 brands of compound fertilizers
not containing nitrolime, 1 brand of ammonium sulfate and 1 brand of urea,
as a result, the recovery rate of melamine derivations at concentration levelof 4 % (mass fraction) and 0.1 % (mass fraction) were 90.5 % - 106.3 % and92.2 % - 107.0 %, and the repeatability were 0.5 % - 4.7 % and 0.3 % 4.2 % as relative standard deviation.
In addition, the results and analysis results of a collaborative study for test
method validation are shown in Table 1.
Additionally, the minimum limit of quantification of the test method isabout 0.02 % (mass fraction) for melamine and cyanuric acid and about
0.01 % (mass fraction) for ammeline and ammelide. In the case of ammelideand cyanuric acid, the sufficient reproducibility was observed in the range of
0.188 % - 1.10 % and 0.105 % - 1.15 % respectively.

361

Table 1 Results and analysis results from a collaborative study
for the test method validation of melamine deivations
2)
Agrichemical
Number of
sr3)
RSDr4)sR5)
Mean
Sample namename(%)
laboratories1)
(%)7)
(%)7)
(%)7)
9
2.83
0.04
1.4
0.12
Melamine
Nitrolime 1
10
0.391
0.003
0.8
0.023
Nitrolime 2
Compound fertilizer
9
0.845
0.019
2.2
0.036
containing nitrolime
11
0.198
0.005
2.6
0.012
Compounf ferttilizer
10
0.03430.00154.5
0.0040
Ammonium sulfate
9
1.60
0.02
1.3
0.06
Ammeline
Nitrolime 1
10
0.105
0.001
1.3
0.002
Nitrolime 2
Compound fertilizer
9
0.629
0.027
4.3
0.023
containing nitrolime
11
0.195
0.004
2.1
0.009
Compounf ferttilizer
10
0.03460.00133.7
0.0024
Ammonium sulfate
9
1.10
0.02
2.1
0.08
Ammelide
Nitrolime 1
11
0.361
0.008
2.2
0.023
Nitrolime 2
Compound fertilizer
9
0.188
0.004
2.2
0.014
containing nitrolime
11
0.718
0.028
3.9
0.052
Compounf ferttilizer
11
0.03450.00318.9
0.0056Ammonium sulfate
9
1.15
0.06
4.8
0.09
Cyanuric acid Nitrolime 1
10
0.390
0.018
4.5
0.029
Nitrolime 2
Compound fertilizer
9
0.105
0.003
2.9
0.014
containing nitrolime
9
0.788
0.026
3.2
0.054
Compounf ferttilizer
10
0.03650.00154.2
0.0067Ammonium sulfate
1) Number of laboratories used in analysis
2) Total mean (number of laboratories × 2 samples replicate analysis)
3) Repeatability standard deviation
4) Repeatability relative standard deviation
5) Reproducibility standard deviation
6) Reproducibility relative standard deviation
7) Mass fraction

RSDR6)
(%)
4.3
5.8
4.2
6.2
11.6
3.8
2.3
3.7
4.5
6.9
7.6
6.5
7.5
7.2
16.1
7.7
7.4
13.2
6.8
18.3

Reference
1) Etsuko BANDO and Yuji SHIRAI: Validation of High Performance Liquid
Chromatography (HPLC) for Determination of Melamine and Its Related Substances in
Fertilizer, Research Report of Fertilizer,Vol.6, 2013, (27 - 35)362

(5) Flow sheet for melamine derivations: The flow sheet for melamine derivations in
fertilizers is shown below.
0.50 g analytical sample
200-mL ground-in stopper Erlenmeyer flask←100 mL of hydrochloric acid (1+15）
Extraction
Ultrasonic extraction for 30 minutes
Centrifugal separation
DilutionCentrifugal separation
Measurement

2,000 × g for 5 minutes
Transfer 5 mL to a 50-mL volumetric flask,
add acetonitrile - phosphate buffer solution (4+1) up to the marked line
8,000 × g for 5 minutes
High Performance Liquid Chromatograph

Figure 2 Flow sheet for melamine derivations in fertilizers

Reference HPLC chromatogram of the mixture standard solution for calibration curve
preparation of melamine derivations is shown below.

Reference diagram HPLC chromatogram of melamine derivations
The names of materials for respective peaks(1) cyanuric acid(2) ammelide (3) melamine (4) ammeline
Measurement conditions of HPLC
Column: TSKgel Amide-80 (4.6-mm inner diameter, 250-mm long, 5-µm particle
diameter)Mixture standard solutions (respective equivalents of 10 ng (1 µg/mL, 10 µL)) for
calibration curve preparation of respective melamine derivations
Other conditions are according to the examples of the measurement conditions in(4.2) a) HPLC.

363

8.2

Clopyralid and its degradation products

8.2.a High Performance Liquid Chromatography/Tandem Mass Spectrometry<Reference method>(1) Summary
This method is applicable to compost and composted sludge fertilizers.
Extract clopyralid, aminopyralid and picloram with methanol under alkaline condition, refinewith a cleanup cartridge by taking advantage of characteristics that the behavior of elution
varies between acidity and alkaline, and then measure with a High Performance Liquid
Chromatograph Tandem Mass Spectrometer.
Comment 1

Structural formulas of clopyralid, aminopyralid and picloram are as shownin Figure 1.

Figure 1 Structural formula of clopyralid, aminopyralid and picloram(2) Reagents, etc.: Reagents and water are as shown below:a) Water: Water of A3 specified in JIS K 0557or equivalent quality. Note that water of A4
or equivalent quality should be used as the eluent which is introduced to LC-MS/MS.
b) Acetonitrile: A regent of agricultural chemicals residue/PCB testing grade(concentration: no less than 300) specified in JIS K 8039 or a reagent of equivalentquality.
c) Methanol: A JIS Guaranteed Reagent specified in JIS K 8891 or a reagent of equivalentquality.
d) Methanol: Methanol used in eluent of LC/MS/MS is a regent of LC-MS analysis grade
or equivalents.
e) Sodium hydroxide: A JIS Guaranteed Reagent specified in JIS K 8576 or a reagent ofequivalent quality.
f) Hydrochloric acid: A JIS Guaranteed Reagent specified in JIS K 8180 or a reagent ofequivalent quality.
g) Ammonia solution: A JIS Guaranteed Reagent of 25 % (mass fraction) specified in JIS
K 8085 or a reagent of equivalent quality.
h) Formic acid: A JIS Guaranteed Reagent specified in JIS K 8264 or a reagent ofequivalent quality.
i) Ammonia solution (0.0025 % (mass fraction)) (1): Add 0.1 mL of ammonia solution to
1,000 mL of water.
j) Respective agrichemical standard solutions (0.1 mg/mL)(1): Put about 0.01 g ofclopyralid [C6H3C12NO2](2), aminopyralid [C6H4C12N2O2](2), picloram [C6H3C13N2O2] (2)to weighing dishes and measure the mass to the order of 0.1 mg. Dissolve with a small
amount of acetonitrile, transfer to 100-mL volumetric flasks and add the solvent up to
the marked line.

364k) Mixture standard solution (250 ng/mL): Dilute a predetermined amount of respective
agrichemical standard solutions (0.1 mg/mL) with formic acid (1+1,000) to prepare
mixture standard solution (250 ng/mL).
l) Mixture standard solution for calibration curve preparation (5 ng/mL - 50 ng/mL):
In the case of usage, transfer 2.5 mL - 25 mL of mixture standard solution (100 ng/mL)
to 50 mL volumetric flasks step-by-step, and add formic acid (1+1,000) up to themarked line.
m) Mixture standard solution for calibration curve preparation (0.5 ng/mL - 5
ng/mL): In the case of usage, transfer 2.5 mL - 25 mL of mixture standard solution (10ng/mL) to 50-mL volumetric flasks step-by-step, and add formic acid (1+1,000) up to
the marked line.
Notes (1) This is an example of preparation; prepare an amount as appropriate.
(2) A standard reagent is commercially sold.
Comment 2 Standard reagents of clopyralid, aminopyralid and picloram are sold by Wako
Pure Chemical Industries., Ltd, Kanto Chemical Co., Inc. and Hayashi Pure
Chemical Ind., Ltd.
(3) Apparatus and instruments: Apparatus and instruments are as shown below:a) High Performance Liquid Chromatograph/Mass Spectrometer (LC-MS/MS):
LC/MS/MS specified in JIS K 0136 that satisfies the following requirements.
1) High Performance Liquid Chromatograph:(i) Column bath: A column bath whose temperature can be adjusted at 30 ºC - 45ºC.
(ii) Column: A 2-mm - 3-mm inner diameter 50-mm - 150-mm long 1.6-µm 2.2-µm particle diameter stainless steel column tube filled with silica gel, towhich octadecyl chemically bonds. The specification is according to the mass
spectrometer specification.
2) Mass Spectrometer:(i)
Ionization method: Electro Spray Ionization (ESI)(ii)
Ion detection method: Selected Reaction Monitoringb) Shaking apparatusc) Manifoldd) Centrifugal separator: A centrifugal separator that can centrifuge at 1,300 × g.
e) High speed centrifugal separator: A centrifugal separator that can centrifuge at 8,000
× g.
f) Concentrator: An evaporator that can adjust to 40 ºC ± 2 ºC.
g) Copolymer cartridge column: A divinylbenzene-N-vinylpyrrolidone copolymer minicolumn (200 mg)
Comment 3
Comment 4

A column is sold under the production names such as ACQUITY UPLCHSS C18.
A copolymer cartridge is sold under the production names such as Oasis
HLB 6cc (200 mg).

(4) Test procedures(4.1) Extraction: Conduct extraction as shown below.
a) Weigh 5.00 g of an analytical sample and put it into a 200-mL - 300-mL ground-in
stopper Erlenmeyer flask.
365b) Add 1 mL of sodium hydroxide solution (40 g/L) and 99 mL of methanol, shake to mix
with a shaking apparatus for about 30 minutes.
c) After allowing to stand still, transfer the supernatant solution to a 50-mL ground-in
stopper centrifugal precipitate tube.
d) Centrifuge at 1,300 × g for about 5 minutes (3) to make supernatant as the sample
solution.
Note (3) 13-cm of rotor radius and 3,000 rpm of revolutions makes about 1,300 × g
centrifugal force.
Comment 5

Grind until it completely passes through a sieve of 500 µm aperture toprepare the test sample.

(4.2) Cleanup (1): Conduct cleanup (1) as shown below.
a) Wash a cartridge column quickly with about 5 mL of methanol and 5 mL of water inadvance.
b) Place a 50-mL round-bottom flask under the cartridge column, transfer 5 mL of theextract to the cartridge column and allow the extract to overflow quickly until thesurface of the liquid reaches the top of the packing materials.
c) Add about 5 mL of sodium hydroxide solution (0.4 g/L) - methanol (1+1) to thecartridge column 2 times and allow the liquid to overflow in the same manner in b).
Note (4) If there is a possibility to foam by the concentration procedure in (4.3) b), a
100-mL round-bottom flask can be used.
(4.3) Cleanup (2): Conduct cleanup (2) as shown below.
a) Wash a new cartridge column quickly with about 5 mL of acetonitrile and 5 mL ofhydrochloric acid (1+120) in advance.
b) After conducting vacuum concentration of the effluent in (4.2) c) until no more than 5
mL on a water bath at no more than 40 °C, add 3 mL of hydrochloric acid (1 +11).
c) Put the concentrated effluent into the cartridge column and allow the effluent quickly to
overflow until the surface of liquid reaches the top of packing materials.
d) Wash a round-bottom flask with about 5 mL of hydrochloric acid (1+120) 2 times andadd washing into the cartridge successively.
e) Then, add about 5 mL of hydrochloric acid (1+120) - acetonitrile (9+1) and about 5 mLof water into the cartridge successively and allow the liquid to overflow quickly.
f) Place a 5-mL volumetric flask under the cartridge column, add 4 mL of ammonia
solution (0.0025 % (mass fraction)) - acetonitrile (9+1) to the cartridge column andallow clopyralid, aminopyralid and picloram to elute quickly.
g) Add formic acid (1+1,000) up to the marked line (5) and transfer it to 1.5-mL ground-in
stopper centrifugal precipitate tube.
h) Centrifuge at 8,000 × g centrifugal force for about 5 minutes (7) to make supernatant as
the sample solution.
Note (5) If there is a possibility that the clopyralid, aminopyralid and picloram
concentration in the sample solution exceed the maximum limit of thecalibration curve, dilute a predetermined amount of effluent with formic acid (1+1,000).
(6) The ground-in stopper centrifugal precipitate tube should be made ofpolypropylene, etc. to not affect the measurement.
366

(7) 7-cm of rotor radius and 10,000 rpm of revolutions makes about 8,000 × g
centrifugal force.

Comment 6

The procedures in (4.2) and (4.3) should be quickly carried out by using asuction apparatus.

(4.4) Measurement: Conduct measurement according to JIS K 0136 and as shown below.
Specific measurement procedures are according to the operation method of the High
Performance Liquid Chromatograph/Mass Spectrometer used in measurement.
a) Measurement conditions of High Performance Liquid Chromatograph/Mass
Spectrometer: Set up the measurement conditions considering the following example
of measurement conditions for High Performance Liquid Chromatograph/Mass
Spectrometer.
1) High Performance Liquid Chromatograph:(i) Column: A silica gel column (2-mm - 3-mm inner diameter, 50-mm - 150-mm
long, 1.6-µm - 2.2-µm particle diameter column) to which octadecyl
chemically bonds.
(ii) Flow rate: 0.2 mL/min - 0.5 mL/min(iii) Eluent: A: Formic acid (1+1,000)
B: methanol(iv) Gradient: 0 min (5 % B) → 5 min (60 % B) → 6 min (95 % B) → 7 min (5 %B)(v) Temperature of column bath: 40 ºC(vi) Injection volume: 5 µL
2) Mass Spectrometer:(i) Ionization method: Electro Spray Ionization (ESI) method(ii) Mode: Positive(iii) Capillary voltage: 1.0 kV(iv) Ion source temperature: 120 ºC(v) Desolvation temperature: 400 ºC(vi) Cone voltage: Shown in Table 1(vii) Collision energy: Shown in Table 1(viii) Monitor ion: Shown in Table 1
Table 1 Monitor ion, etc. of respective agrichemicals
Substance Precussor ion Product ion
Product ion Cone Collision energy Collision energy
name(Determination) (Validation) voltage (Determination)(Validation)
(m/z)
(m/z)
(m/z)
(v)
(eV)
(eV)
Clopyralid
192
146
110
20
20
30
Aminipyralid
Oicrloram

207
241

161
195

189
223

22
28

22
22

16
16

b) Calibration curve preparation
1) Inject 5 μL of respective mixture standard solutions for calibration curve into the
LC-MS/MS, record the chromatogram of ion (m/z) for determination and ion (m/z)
for validation of clopyralid, aminopyralid and picloram and obtain respective peakareas or heights.
2) Calculate the peak area ratio or height ratio of ion (m/z) for determination and ion(m/z) for validation of clopyralid, aminopyralid and picloram.
367

3) Prepare a curve for the relationship between the concentration of respective
agrichemicals and the peak area or height of ion (m/z) for determination of respective
mixture standard solutions for the calibration curve preparation.
c) Sample measurement
1) Subject 5 µL of the sample solution to the same procedure as in b) 2) - 3) (8).
2) Obtain the content of material subjected to measurement from the calibration curve
to calculate the concentration of material subjected to measurement in the analyticalsample.
Note (8) Confirm that the ratio against the peak area ratio or height ratio of the standard
solution is within the range of about ± 30 %. In addition, the peak area ratio orheight ratio depends on the concentration.
Comment 7

Additive recovery testing of clopyralid, aminopyralid and picloram wasconducted using cow dung compost (2 kinds), composted sludge fertilizers
containing cow manure (2 kinds) and composted sludge fertilizers
containing pig manure (1 kind), as a result, the mean recovery rates atadditive level of 1000 µg/kg, 400 µg/kg and 40 µg/kg were 78.1 % - 90.0 %,81.0 % - 117.6 % and 71.2 % - 101.3 % and the repeatability were 1.5 % 8.7 %, 1.1 % - 5.1 % and 2.4 % - 10.7 % relative standard deviation.
Additionally, the minimum limits of quantification of clopyralid,aminopyralid and picloram of the test method are about 10 µg/ kg
respectively.

Reference
1) Toshiharu YAGI, Yuko SEKINE, Yuji SHIRAI: Determination of Clopyralid in
Fertilizer by Liquid Chromatography/Tandem Mass Spectrometry (LC/MS/MS),
Research Report of Fertilizer Vol. 3, 2010, (51 - 59)

368

(5)

Flow sheet for clopyralid and its derivative substances: The flow sheet for clopyralid
and its derivative substances in fertilizer is shown below

5.00 g analytical sample
200-mL - 300-mL ground-in stopper Erlenmeyer flask← 1 mL of sodium hydroxide solution (4 g/L)
← 99 mL of methanol
shaking
30 minutes
Centrifugal separation
50 mL centrifugal precipitate tube, 1,300 × g, 5 minutes← 5 mL of extract
copolymer cartridge column (1)(Wash with about 5 mL of methanol, then 5 mL of water in advance）
Cleanup (1)Cleanup procedures should be quickly carried out
by using a suction apparatus.
[Collection, 50-mL eggplant-shaped flask]← About 5 mL of sodium hydroxide solution (0.04 w/v %) - methanol [1+1] × 2 times[Collection, same as above vessel]
Vacuum40 ºCconcentration← 3 mL of hydrochloric acid (1+11)
copolymer cartridge column (1)(Wash with about 5 mL of acetonitrile,then 5 mL of hydrochloric acid in advance)
Cleanup (2)Clean up procedures should be quickly carried out
by using a suction apparatus.
← Wash the vessel 2 times with about 5mL of hydrochloric acid (1+120)← Wash with about 5mL of hydrochloric acid (1+120) -acetonitrile [9+1]← Wash with about 5mL of water← 4 mL of ammonia solution (0.0025 w/w %)- acetonitrile [9+1][Elute, 5-mL volumetric flask]
Filling up← Formic acid (1+1,000) (up to the marked line)
1.5-mL ground-in stoppered centrifugal precipitate tube,
Centrifugal separation
8,000 × g , 5 minutes
Measurement
Figure

LC-MS/MS

Flow sheet for clopyralid and its derivative substances in fertilizers

369

References

Selected Reaction Monitoring chromatograms of clopyralid standard
solution for calibration curve preparation and sample solution (cow dung
compost) are shown below

Peak No.

2

3

Intensity / arb.units

1

0

5

Retention Time / min

1) Standard solution
Peak2) Sample solution

No1: Picloram
No2: Aminopyralid
No3: Clopyralid

Reference diagram: SRM
chromatograms
agrichemicals

ofrespective

Mixture standard solution (the equivalents of 200 pg as respective
agrichemicals)
Measurement conditions of LC-MS/MS
Column: ACQUITY UPLC HSS C18 (2.1-mm inner diameter, 100-mm long,
1.8-µm particle diameter)
Other conditions are according to the examples of the measurement conditions in(4.4) a) LC-MS/MS.

370

8.3

Residue agrichemicals multi component analysis

8.3.a High Performance Liquid Chromatography/Tandem Mass Spectrometry(1) Compounds subjected to analysis: Abamectin: Abamectin B1a, ivermectin: 22,
23-dihydro avermectin B1a (Another name: ivermectin B1a), eprinomectin: eprinomectin B1a,rotenone: rotenone, piperonylbutoxide: piperonylbutoxide, pyrethrin: pyrethrin I and pyrethrinII(2) Summary
This method is applicable to fluid home garden-use mixed fertilizer and fluid mixed fertilizer.
Dissolve respective agricultural chemicals in fertilizers with acetonitrile and water, andextract. Refine by using 2 kinds of cleanup cartridge, and then measure with a High
Performance Liquid Chromatograph/Mass Spectrometer.
(3) Reagents, etc.: Reagents and water are as shown below:a) Water: Water of A3 specified in JIS K 0557or equivalent quality.
b) Acetonitrile: A regent of agricultural chemicals residue/PCB testing grade(concentration: no less than 300) specified in JIS K 8039 or a reagent of equivalentquality.
c) Methanol: A JIS Guaranteed Reagent specified in JIS K 8891 or a reagent of equivalentquality.
d) Methanol: Methanol used in eluent of HPLC is a regent of LC/MS analysis grade orequivalents.
e) Ethyl acetate: A JIS Guaranteed Reagent specified in JIS K 8361 or a reagent ofequivalent quality.
f) Toluene: A JIS Guaranteed Reagent specified in JIS K 8680 or a reagent of equivalentquality.
g) Ammonium formate: A JIS Guaranteed Reagent (no less than 95 % (mass fraction) inpurity) or a reagent of equivalent quality.
h) Ammonium formate solution (0.1 mol/L) (1): Add 6.306 g of ammonium formate into
1,000 mL of wateri) Ammonium formate solution (0.1 mmol/L) (1): Add 1mL formic acid ammonium
solution (0.1 mol/L) into 1,000 mL waterj) Formic acid: A JIS Guaranteed Reagent specified in JIS K 8264 or a reagent ofequivalent quality.
k) Formic acid solution (0.1 v/ v %) (1): Add 1 mL of formic acid to 1,000 mL of waterl) Acetonitrile formate solution (0.1 v/v %) (1): Add 1 mL of formic acid to 1,000 mL ofacetonitrilem) Respective agricultural chemicals standard solutions (0.1 mg/mL) (1): Put about 0.01
g of abamectin [C48H72O14] (2), ivermectin [C48H74O14] (2), eprinomectin [C50H75NO14] (2),rotenone [C23H22O6] (2), piperonylbutoxide [C19H30O5] (2) and pyrethrin [pyrethrin I:
C21H28O3 and pyrethrin II: C22H28O5] (2) to a weighing dish, and measure the mass to theorder of 0.1 mg. Dissolve with a small amount of methanol, transfer to a 100-mL
volumetric flask and add the solvent up to the marked line. (However 0.1 mg/mL ofpyrethrin contains total of pyrethrin I/II)n) Mixture standard solution (10 µg/mL): Transfer 10 mL of respective agriculturalchemicals standard solutions to a 100-mL volumetric flask and add methanol up to themarked line.
o) Mixture standard solution (1,000 ng/mL): Transfer 10 mL of mixture standard
solution (10 µg/mL) to a 100-mL volumetric flask and add methanol up to the marked
line.
371

p) Mixture standard solution for calibration curve preparation (50 ng/mL - 500
ng/mL): In the case of usage, transfer 2.5 mL - 25 mL of mixture standard solution(1,000 ng/ mL) to 50-mL volumetric flasks step-by-step, and add methanol up to themarked line.
q) Mixture standard solution for calibration curve preparation (5 ng/mL - 50 ng/mL):
In the case of usage, transfer 2.5 mL - 25 mL of mixture standard solution (100 ng/mL)
to 50-mL volumetric flasks step-by-step, and add methanol up to the marked line.
Note (1) This is an example of preparation; prepare an amount as appropriate.
(2) A standard reagent is commercially sold.
Comment 1

A standard reagent of respective agricultural chemicals is sold by Wako
Pure Chemical Industries., Ltd, Kanto Chemical Co., Inc. and Hayashi Pure
Chemical Industries., Ltd.

(4) Apparatus and instruments: Apparatus and instruments are as shown below.
a) High Performance Liquid Chromatograph/Mass Spectrometer (LC/MS/MS):
LC/MS/MS specified in JIS K 0136 that satisfies the following requirements.
1) High Performance Liquid Chromatograph:(i) Column bath: A column bath whose temperature can be adjusted at 30 ºC - 45ºC.
(ii) Column: A 2-mm - 3-mm inner diameter 50-mm - 150-mm long 1.6-µm 3.0-µm particle diameter stainless steel column tube filled with silica gel, towhich octadecyl chemically bonds. The specification is according to the mass
spectrometer specification (3).
2) Mass Spectrometer:(i) Ionization method: Electro Spray Ionization (ESI)(ii) Ion detection method: Selected Reaction Monitoringb) Ultrasonic generator: An ultrasonic washer can be used.
c) Concentrator: An evaporator that can keep test temperature ± 2 ºCd) Porous diatomaceous earth cartridge column: A column that is filled with the porous
diatomaceous earth (capacity: 5 mL) (4)e) Graphite carbon-NH2 laminate cartridge column: A 6-mL cylinder on which 500 mg
of graphite carbon and 500 mg of aminopropyl silylation silica gel is laminated (5).
Note (3) The column is sold under the names of ACQUITY UPLC HSS C18, etc.
(4) The column is sold under the names of Chem Elut (5 mL), etc.
(5) The column is sold under the names of Envi-carb/LC-NH2 (500 mg/500 mg, 6mL), etc.
(5) Test procedures(5.1) Extraction: Conduct extraction as shown below.
a) Put about 5.00 mL (6) of an analytical sample into a 10-mL volumetric flask.
b) Add 3 mL of acetonitrile to the same volumetric flask, and add water up to the marked
line to shake to mix well.
c) Conduct ultra-sonication for about 30 minutes using an ultrasonic generator (7) to makethe extract.
Note (6) After measuring the specific gravity of sample, calculate the concentration ofmaterials subjected to measurement in the analytical sample.
372

(7) Note that the volume of solution may expand as a result of ultra-sonication. It is
recommended to leave it at room temperature for a while when it expands.
Comment 2

The specific gravity (density) can be calculated by placing a 10-mL
volumetric flask on an electric balance, aligning the scale to zero,transferring 5.00 mL of the analytical sample to the volumetric flask andreading the weighing value.

(5.2) Cleanup (1): Conduct cleanup (1) as shown below.
a) Put 5 mL of extract into a porous diatomaceous earth cartridge column and keep it in thecolumn for about 5 minutes.
b) Place a 100-mL round-bottom flask under the same cartridge column, add about 5 mLof ethyl acetate into the same cartridge column 4 times successively and allow thesolution to elute until the surface of solution reaches the top of packing materials (8).
c) After conducting vacuum concentration of elute in a water bath of no more than 40 ºC
until most of the elute dries up, send a nitrogen gas to dry up the elute (9), and add 2 mLof acetonitrile-toluene (3+1) to dissolve the residue.
Note (8) Confirm the solution to elute before conducting the testing.
(9) There is a possibility for agricultural chemicals to vaporize if it is dried up
excessively.
(5.3) Cleanup (2): Conduct cleanup (2) as shown below.
a) Wash the graphite carbon-NH2 laminate cartridge column with about 10 mL ofacetonitrile - toluene (3+1) in advanceb) Place a 100-mL round-bottom flask under the same cartridge column, put the solution in(5.2) c) to the same cartridge column, and allow the solution to overflow until thesurface of solution reaches the top of packing materials.
c) Wash the vessel with about 5 mL of acetonitrile-toluene (3+1) 5 times and add washing
to the same cartridge successively to allow it to overflow.
d) After conducting vacuum concentration of elute in a water bath of no more than 40 ºC
until most of the elute dries up, send a nitrogen gas to dry up the elute (10), and add 2 mLof methanol to dissolve the residue. Transfer a predetermined amount of the solution
precisely and dilute with methanol exactly 5 times to make the solution as the sample
solution.
Note (10) There is a possibility for agricultural chemicals to vaporize if it is dried up
excessively.
(11) If there is a possibility that the concentration of agricultural chemicals in thesample solution exceeds the maximum limit of the calibration curve, dilute apredetermined amount of the sample solution with methanol.
(5.4) Measurement: Conduct measurement according to JIS K 0136 and as shown below.
Specific measurement procedures are according to the operation method of High
Performance Liquid Chromatograph/Mass Spectrometer used in measurement.
a) The measurement conditions of High Performance Liquid Chromatograph/Mass
spectrometer: Set up the measurement conditions for High Performance Liquid
Chromatograph/Mass Spectrometer considering the following:
1) High Performance Liquid Chromatograph:

373

(i)

Column: A silica gel column (2-mm - 3-mm inner diameter, 50-mm - 150-mm
long, 1.6-µm - 3.0-µm particle diameter column) to which octadecyl
chemically bonds.
(ii) Flow rate: 0.2 mL/min - 0.5 mL/min(iii) Eluent:
A: Ammonium formate solution (0.1 mmol/L) - formic acidsolution (0.1 v/v %) [1+1]
B: Acetonitrile formate solution (0.1 v/v %)(iv) Gradient: 0 min (50 % B) → 15 min (95 % B) → 20 min (98% B) → 30 min(50 % B)(v) Temperature of column bath: 40 ºC(vi) Injection volume: 5 µL
2) Mass Spectrometer:(i) Ionization method: Electro Spray Ionization (ESI) method(ii) Mode: Positive(iii) Capillary voltage: 3.0 kV(iv) Ion source temperature: 120 ºC(v) Desolvation temperature: 400 ºC(vi) Cone voltage: Shown in table 1(vii) Collision energy: Shown in table 1(viii) Monitor ion: Shown in table 1
Table 1 Monitoring ion conditions, etc. of respective agrichemicals
Precursor
Product ion
Product ion
Cone voltage
Agrichemicals
ion(determination) (validation)(V)(m/z )(m/z )(m/z )
Abamectin B1a
891
305
567
20
Ivermectin B1a
893
307
551
25
Eprinomectin B1a
915
186
298
20
Rotenone
395
213
192
35
Piperonylbutoxide
356
177
147
20
PyrethrinⅠ
329
161
133
20
PyrethrinⅡ
373
161
133
20

Collisionenergy
(eV)
25
25
20
25
15
10
10

b) Calibration curve preparation
1) Inject 5 μL of respective mixture standard solutions for calibration curve preparation
into the LC/MS/MS, record the chromatogram of ion (m/z) for determination and ion(m/z) for validation of materials subjected to measurement and obtain respective peakareas or heights.
2) Calculate the peak area ratio or height ratio of ion (m/z) for determination and ion(m/z) for validation of materials subjected to measurement.
3) Prepare a curve for the relationship between the concentration of materials subjected
to measurement and the peak area or height of ion (m/z) for determination ofrespective mixture standard solutions for the calibration curve preparation.
Prepare a calibration curve when the sample is measured.
c) Sample measurement
1) Subject 5 µL of the sample solution to the same procedure as in b) 2) - 3) (12).
2) Obtain the content of materials subjected to measurement from the calibration curveof the peak area or height to calculate the concentration of materials subjected tomeasurement in the analytical sample.
374

Note (12) Confirm that the ratio against the peak area ratio or height ratio of the standard
solution is within the range of about ± 30 %. In addition, the peak area ratio orheight ratio depends on the concentration.
(5.5) Calculation
Calculate the respective concentration of agricultural chemicals in the analytical sample
by the following formula.
Respective concentration of agricultural chemicals in the analytical sample (µg/kg)= (A × B × 10)/C
A: Concentration (ng/mL) of respective materials subjected to measurement in
the final sample solution obtained from the calibration curve
B: Dilution factor in the case that the final sample solution is further diluted
because it exceeds the upper limit of the calibration curve.
C: Specific gravity of the analytical sample (density) (g/mL)
Comment 3

Recovery testing was conducted using fluid home garden-use mixed
fertilizer (3 kinds) and fluid mixed fertilizer (2 kinds), as a result, the mean
recovery rate at additive level of 4,000 µg/kg and 400 µg/kg (pyrethrin,
however, is 4,000 µg/kg and 400 µg/kg as a total amount of pyrethrin I/II)
were 77.0 % - 107.9 % respectively, and the repeatability was 0.1 % - 9.1 %relative standard deviation.
In addition, the results and analysis results of a collaborative study for test
method validation are shown in Table 2.
Additionally, the minimum limit of quantification of the test method isabout 10 µg/ kg.

375

Table 2 Analysis results of a collaborative study for the testing method validation of multicomponent analysis of pesticide residue（µg/kg）（%）

RSDr3)
（%）

286.8

333.3

86.1

13.3

14.4

8

358.9

416.7

86.1

13.4

14.8

Home garden-use mixed fertilizer3
Fluid mixed fertilizer1

8

425.8

500.0

85.2

8.6

11.6

8

288.6

333.3

86.6

7.1

8.5

Fluid mixed fertilizer2

8

405.5

500.0

81.1

7.1

7.2

Home garden-use mixed fertilizer1

8

298.9

333.3

89.7

14.9

15.0

Home garden-use mixed fertilizer2

8

382.5

416.7

91.8

14.1

19.3

Home garden-use mixed fertilizer3
Fluid mixed fertilizer1

8

431.1

500.0

86.2

9.8

10.9

8

298.8

333.3

89.6

10.1

12.8

Fluid mixed fertilizer2

8

405.2

500.0

81.0

3.8

5.8

Eprinomectin B1a Home garden-use mixed fertilizer1

8

293.5

333.3

88.1

7.0

10.4

Home garden-use mixed fertilizer2

8

361.9

416.7

86.9

9.2

14.3

Home garden-use mixed fertilizer3
Fluid mixed fertilizer1

8

425.3

500.0

85.1

7.0

10.0

8

277.3

333.3

83.2

9.0

12.0

Fluid mixed fertilizer2

8

398.2

500.0

79.6

7.5

11.6

Home garden-use mixed fertilizer1

8

276.8

333.3

83.1

5.7

7.8

Home garden-use mixed fertilizer2

8

353.5

416.7

84.8

9.8

12.5

Home garden-use mixed fertilizer3
Fluid mixed fertilizer1

8

426.6

500.0

85.3

6.6

8.5

8

263.5

333.3

79.1

11.0

12.3

Fluid mixed fertilizer2

8

385.2

500.0

77.0

5.7

12.1

Piperonylbutoxide Home garden-use mixed fertilizer1

8

318.2

333.3

95.5

8.1

13.2

Home garden-use mixed fertilizer2

8

395.6

416.7

94.9

8.4

13.6

Home garden-use mixed fertilizer3
Fluid mixed fertilizer1

8

450.3

500.0

90.1

4.6

9.3

8

299.7

333.3

89.9

7.4

11.0

Number of

Agrichemicals

Sample name

Abamectin B1a

Home garden-use mixed fertilizer1

laboratories
8Home garden-use mixed fertilizer2

Ivermectin B1a

Rotenone

Pyrethrin I

Pyrethrin II1)

Mean2)（µg/kg）

Additive amount

Recovery

RSDR4)（%）

Fluid mixed fertilizer2

8

435.8

500.0

87.2

5.8

7.4

Home garden-use mixed fertilizer1

8

160.7

186.0

86.4

9.3

11.9

Home garden-use mixed fertilizer2

8

202.2

232.5

87.0

12.6

12.8

Home garden-use mixed fertilizer3
Fluid mixed fertilizer1

8

228.6

279.0

81.9

5.4

8.8

8

158.2

186.0

85.1

6.8

10.4

Fluid mixed fertilizer2

8

223.1

279.0

80.0

8.5

9.1

Home garden-use mixed fertilizer1

8

131.1

147.3

89.0

6.5

9.7

Home garden-use mixed fertilizer2

8

163.2

184.2

88.6

10.8

13.6

Home garden-use mixed fertilizer3
Fluid mixed fertilizer1

8

182.0

221.0

82.4

5.4

8.9

8

126.2

147.3

85.7

7.8

11.4

Fluid mixed fertilizer2

8

180.2

221.0

81.5

6.3

8.3

1) Number of laboratories used in analysis
2) Mean (n= number of laboratories x number of samples (2))3) Repeatability (relative standard deviation)4) Reproducibility (relative standard deviation)

Reference
1) Toshiharu YAGI, Masayuki YAMANISHI, Yuji SHIRAI: Simultaneous Determinationof Agricultural chemicals in Fluid Fertilizer by Liquid Chromatography/Tandem Mass
Spectrometry, Research Report of Fertilizer,Vol.4, 2011, (36 - 48)2) Toshiharu YAGI, Masayuki YAMANISHI, Yuji SHIRAI and Masato SHIBATA:
Simultaneous Determination of Six Kind of Agricultural Chemicals in Fluid Fertilizer
by Liquid Chromatograph-Tandem Mass Spectrometer (LC-MS/MS): A Collaborative
Study, Research Report of Fertilizer,Vol.5, 2012, (48 - 59)376

(6) Flow sheet for simultaneous analysis of 6 kinds of agrichemicals: The flow sheet for
simultaneous analysis of 6 kinds of agrichemicals in fertilizer is shown below.
5.00 mL analytical sample
10-mL volumetric flask← About 3 mL of acetonitrile← Water (up to the marked line)
Ultra sonication
5 minutes
Aliquot (5 mL)
Keep the extract for 5 minutes
in the cartridge column (1)← About 5 mL of ethyl acetate ×4 times[elute, 100-mL round-bottom flask ]Vacuum40 ºCconcentration← Dissolve with about 2 mL of acetonitrile-toluene(3+1)
Cartridge column (2)Cleanup (2)(wash with about 5 mL of acetonitrile-toluene (3+1)
× 2 times in advance)[100-mL round-bottom flask]← Wash the vessel 5 times with about 5 mL of acetonitrile-toluene (3+1)[overflow, the same vessel]
Vacuum40 ºCconcentration← 5 mL of methanol
Dilution
Dilute the solution 5 times
Cleanup (1)Measurement

LC/MS/MS

Figure Flow sheet for simultaneous analysis of 6 kinds of agrichemicals in fertilizers

377

Reference

Selected Reaction Monitoring chromatograms of mixture standard solution
for calibration curve preparation and sample solution (fluid homegarden-use mixed fertilizer) are shown below.

Peak No.
1

1

3
4
5
6
7

2
3
4
Intensity / arb.units

2

5
6

7

0

2 0

Retention Time / min

Peak No.1:
No.2:
No.3:
No.4:
No.5:
No.6:
No.7:

2

AbamectinB1a
IvermectinB1a
Eprinomectin B1a
Rotenone
Piperonylbutoxide
Pyrethrin I
Pyrethrin II

1) Mixture standard solution
Reference diagram

Retention Time / min
0 52) Sample solution

Selected Reaction Monitoring chromatograms ofrespective agricultural chemicals

1) Mixture standard solution (the equivalents of 2,500 pg of respective agriculturalchemicals)(For pyrethrin, the equivalents of 2,500 pg of the total of pyrethrin I/II)
2) Sample solution (fluid home garden-use mixed fertilizer, additive of the equivalents of
400 µg/ kg in the sample)(For pyrethrin, the equivalents of 400 µg/kg of the total of pyrethrin I/II)
Measurement conditions of LC/MS/MS
Column: ACQUITY UPLC HSS C18 (2.1-mm inner diameter, 100-mm long,
1.8-µm particle diameter)
Flow rate: 0.2 mL/min
Other conditions are according to the examples of the measurement conditions of(5.4) a) LC/MS/MS.
378

0Appendix.

The procedure to validate characteristics of testing methods(1) Purposes
This article explains the procedure to validate characteristics of testing methods which will belisted in the Testing Methods of Fertilizers. In addition, when testing institutes conduct a test
which is not included in the Testing Methods of Fertilizers, a procedure to evaluate thevalidity of the test method should conform to a method stipulated in this article.
Additionally, this article targets chemical testing methods. However, this article is notapplicable to the extraction method of the content of effective figures (acid- , alkaline- ,citrate- and water-soluble) in a powdery sample or a solidified fertilizer.
Comment 1 The contents of effective figures (acid- , alkaline- , citrate- and water-soluble)
are stipulated in a notification of the Ministry of Agriculture, Forestry and
Fisheries. In addition, the change of measurement conditions such as an
extraction temperature may affect an observed value in some cases. Therefore,
no changes will be implemented in the extraction method of the contents ofeffective figures in a powdery fertilizer and a solidified fertilizer for thepresent and the application of this article is limited to the change of ameasurement method (including refining of extract, etc.).
(2) Definition of terminology
The definition of terminology in this article is as shown below.
a) Selectivity: Capability to accurately measure components subjected to analysis underthe existence of materials which seem to exist in a sample.
b) Trueness: The degree of agreement between the mean obtained from multiple
measurement results and the true value (1).
c) Precision: The degree of agreement among the independent measurement results which
are repeatedly measured under the determined conditions.
d) Repeatability: The precision of the measurement results of analytical samples, which
are regarded to be all identical, obtained under condition (repeatability conditions) thatindependent measurement results are measured in a short time, using the same method,in the same laboratory, by the same operator and with the same instrument.
e) Intermediate precision: The precision of a measurement result of analytical samples,
which are regarded to be all identical, obtained under condition (intermediate
conditions) that independent measurement results are measured, using the same method,in the same laboratory and in different factors (such as different time and a different
operator).
f) Reproducibility: The precision of a measurement result of analytical samples, which
are regarded to be all identical, obtained under condition (reproducibility conditions)that independent measurement results are measured, using the same method, in different
laboratories, by different operators and with different instruments.
g) Minimum Limit of Quantification (LOQ): The quantifiable lowest volume orminimum concentration of a component subjected to analysis which is contained in ananalytical sample.
h) Minimum Limit of Detection (LOD): The detectable lowest volume or minimum
concentration of a component subjected to analysis which is contained in an analyticalsample.
i) Reference material: A material which is uniform and stable enough for one or moreprescribed properties, and is made suitable for the purpose of use in a measurement
process.
379

j)Certified reference material: A reference material, whose values of one or moreprescribed properties are characterized by a reasonable metrological procedure, having acertificate of attestation on which the characteristics of prescribed properties and theiruncertainty and metrological traceability are stated.
k) Blank sample: An analytical sample not containing components subjected to analysis(2).
i) Addition sample: An analytical sample the content of whose components subjected toanalysis is known, or an analytical sample to which reference materials are added (3) (4)or compounded (3).
m) Natural contamination sample: An analytical sample prepared from fertilizers which
naturally contain the components subjected to analysis such as harmful components.
n) Distribution sample: An analytical sample prepared from fertilizers(5) which are
manufactured in a fertilizer production factory, etc.
o) Surrogate: A material which is added to an analytical sample in order to conduct a
pre-process operation, correct yields in respective steps of analysis operation andconfirm recovery, whose chemical structure is identical or similar to a target
component.
p) SN ratio: Intensity ratio of a signal (response value) S originated from the analysis
target and a signal (usually noise) N based on the other factors.
Note (1) In reality, the certified value of a certified reference material, the chemicalcomposition of a compound, the added content of a reference material, etc. andothers.
(2) Regents, etc. containing a target matrix can be used in the case that there is no
distribution fertilizer used as a blank sample for a recovery test and theconfirmation of the minimum limit of quantification, etc.
(3) Mix a component subjected to analysis with a mortar, etc. to sufficientuniformity(4) In the case of adding standard solution, vaporize the solvent sufficiently
conducting measures such as letting it stand for one night.
(5) A fertilizer containing components subjected to analysis whose formation orform changed due to a chemical or physical process (a granulation process, etc.).
References
1) JIS K 0211: Technical terms for analytical chemistry (General part) (2013)
2) JIS K 0214: Technical terms for analytical chemistry (Chromatography part) (2013)
3) JIS Q 0035: Reference materials−General and statistical principles for certification(2008)
4) JIS Z 8101-2: Statistics−Vocabulary and symbols−Part 2: Statistical quality control
terms (1999)
5) JIS Z 8402-1: Accuracy (trueness and precision) of measurement methods and results Part 1: General principles and definitions (1999)
6） ALINORM 09/32/23 Joint FAO/WHO Food Standards Programme: Repot of theThirtieth Session of the Codex Committee on Methods of Analysis and Sampling,
Codex Almentarius Commission Thirty-second Session (2009)
7） ICH Harmonised Tripartite Guideline, Validation of Analytical Procedures: Text andMethodology Q2(R1), International Conference on Harmonisation of TechnicalRequirements for Registration of Pharmaceuticals for Human Use (ICH) (2005)(3)

Validation method
380

Test necessary items of (3.1) to (3.8) in a planned manner and estimate performanceparameters from the obtained results.
Confirm whether the estimated values of performance parameters are suitable to target values(performance norm) respectively, and evaluate that the test method is validated if they are all
suitable.
(3.1) Scope of application
As a result of a validation test in a single laboratory and a collaborative study, if the result
is suitable up to reproducibility, the test method is evaluated as a validated test method as far
as the kind of a fertilizer used in the test and the range of concentration are concerned.
Therefore a laboratory where the said test is conducted can use the performance(reproducibility, etc.) as a validated method through implementing internal quality control,etc.
As a result of a validation test in a single laboratory, if the result is suitable to trueness,
repeatability and intermediate precision, etc., the test method is evaluated as a validated test
method as far as the laboratory where the test was conducted and as far as the kind of a
fertilizer used in the test and the range of concentration are concerned. Therefore anotherlaboratory which wants to introduce the test method is required to carry out the validation
anew in an individual laboratory with the above test method.
(3.2) Selectivity(3.2.1) Case of Chromatography
Conduct a procedure for a blank sample and confirm that there is no peak (interference peak)which affects the measurement of components subjected to analysis (6). In addition, in the caseof the simultaneous measurement of multi components, confirm that adjacent peaks are
sufficiently separated (6).
Note (6) Resolution (R) should be 1.0 or more at minimum though 1.5 or more ispreferable.
Comment 2

Resolution (R) is used as a separation indicator of peaks. If Resolution (R) is
1.5 or more, the adjacent two peaks are sufficiently separated and they do
not affect a measurement, whether a peak height or a peak area is used. If
Resolution (R) is 1.0 or more, a measurement using a peak height is not a
problem though the adjacent two peaks may overlap to some degree.
Resolution (R) can be obtained using a peak width by the formula (1a). In
addition, if the peak is a normal distribution, it can be obtained using a peakwidth at half height by the formula (1b). With the data processing device ofa chromatograph, the formula (1b) is often used to obtain Resolution (R).

381

(3.2.2) Case of a method other than Chromatography (7)Conduct a procedure for a blank sample and confirm that there is no response which
originates from other components than a component subjected to analysis and can be a factorof positive error of a quantification value (8).
Note (7) A test method such as Molecular absorption spectrometry, Atomic absorption
spectrometry or Titration analysis which does not isolate with a measurement
instrument.
(8) Absorbance, titer, etc.
References
1) AOAC Official Methods of Analysis Appendix K: Guidelines for Dietary Supplements
and Botanicals, AOAC INTERNATIONAL (2012)
2) JIS K 0114: General rules for gas chromatography (2012)
3) JIS K 0124: General rules for high performance liquid chromatography (2011)
4) The notification by the Director of Evaluation and Licensing Division, Pharmaceuticaland Food Safety Bureau, the Ministry of Health, Labour and Welfare: “Guideline on
Bioanalytical Method Validation in Pharmaceutical Development”, July 11, 2013,
Yaku-shoku-Sinsa-Hatsu-0711 No.1 (2013)(3.3) Calibration curve
Measure respective standard solutions for the calibration curve preparation of theconcentration or the content (9) of level 6 to 8 a few times (10) to make a figure plotting theobtained signals (11) as a function of the concentration or the content of a component subjected
to analysis and evaluate its linearity visually using the figure.
If linearity is recognized, calculate the inclination (b) and the intercept (a) of a calibration
curve, its confidence interval and the coefficient of determination (r2) using a statisticalmethod such as the calculation of a regression equation by the least square method. Moreover
make the plot of residuals (12) in respective levels.
Note (9) Blank test solution for the calibration curve preparation can be included.
(10) In order to avoid nonlinear confusion due to the variation of sensitivity, etc.,
conduct measurements randomly for each replicate determination.
(11) Absorbance, fluorescence intensity, peak height, peak area, etc.
(12) The difference between a signal obtained by measurement and a signal
estimated using a regression equation.
Comment 3
Comment 4

Comment 5

It is recommended that the 95% confidence interval of an intercept (a)includes the origin (0).
Though it is usable if the coefficient of determination (r2) is 0.99 or more, it
is recommended that the coefficient of determination (r2) is 0.999 or more
for a precise analysis. If it is less than 0.99, use the equation of a higher
order or study the conversion of a numerical value.
The mean of residuals is 0 and the residuals indicate a random pattern.

References
382

1) AOAC Official Methods of Analysis Appendix K: Guidelines for Dietary Supplements
and Botanicals, AOAC INTERNATIONAL (2012)
2) Thompson, M., Ellison, S.L.R, Wood, R., Harmonized guidelines for single-laboratory
validation of methods of analysis, Pure & Appl. Chem. 74 (5), 835–855 (2002)
3) CLSI EP9 A2 Ed. 2, Method Comparison and Bias Estimation Using Patient Samples,
Clinical and Laboratory Standards Institute (2002)(3.4) TruenessAs the estimation method of trueness, the methods are recommended in the following order.
(1) Use of a certified reference material (3.4.1), (2) Comparison with an observed value by avalidated method (3.4.2) and (3) Recovery test (3.4.3).
In addition, if a surrogate is used, it is recommended that a recovery is about 40% or more.
(3.4.1) Use of a certified reference materialWith regard to a component which has matrixes similar to a fertilizer subjected to test and canuse a certified reference material containing components subjected to measurement of theconcentration in a measurement level, conduct repeatability testing using 3 or more analyticalsamples (n) according to the test method of the certified reference material. As a result, themean of the observed values should be within the warning level to the certified value(characteristic value) or the absolute value of the difference between the mean of the observed
values and the certified value (characteristic value) should not exceed 2 times of the standard
uncertainty composed of respective uncertainties of the mean of the observed values and thecertified value (13).
Comment 6

A warning limit is given using the formula (2) which is obtained from acollaborative study for the characterization of a certified reference material.

A warning limit for a certified value (μ)µ: Certified value: Reproducibility standard deviation in a collaborative study: Repeatability standard deviation in a collaborative study (14)n: The number of analytical samples to repeatability test
: Pure between-laboratory standard deviation in a collaborative study
Note (13) The evaluation procedure of the difference between a measurement result and acertified value (characteristic value) is shown in Reference 1.
(14) It may be expressed as within-laboratory standard deviation ( ) in some cases.
(3.4.2) Use of another validated test method
For a component for which a certified reference material is not usable but another validated
test method (hereinafter referred to as “a standard test method”) is applicable, confirm that thecondition a) or b) is satisfied.
a) In case 12 or more samples are available: Conduct respective tests of 12 or more test
samples composed of addition samples, natural contamination samples or distribution
samples according to a new test method and a standard test method, create the correlation
383

chart of observed values with two methods for each sample and calculate the inclination(b) and the intercept (a) of a regression line, and a correlation coefficient (r). Further
confirm a prediction interval.
However, in case that the width between the minimum and the maximum observed value
is small, conduct the paired samples t-test to confirm that a significant difference is notobserved.
Comment 7 It is recommended that the 95% confidence interval of an inclination (b)includes 1, the 95% confidence interval of an intercept (a) includes the origin(0) and the correlation coefficient (r) is no less than 0.99.
b)

In case fewer samples are available: With regard to 3 or more test samples ofdifferent concentration, conduct respective repeatability addition tests using 4 analyticalsamples according to the new test method and a standard test method, confirm thehomoscedasticity of the results of 2 groups and conduct a t-test for each concentration toconfirm that significant difference is not observed under the two-sided significant level of
5%.

(3.4.3) In case neither certified reference material nor other validated test methods areusableFor 3 or more test samples of different concentration, conduct respective repeatability tests
using 3 analytical samples and evaluate by obtaining the recovery using the mean of theobserved values. The criteria of the trueness are shown in a Separate sheet.
References
1) AOAC Official Methods of Analysis Appendix K: Guidelines for Dietary Supplements
and Botanicals, AOAC INTERNATIONAL (2012)
2) Thompson, M., Ellison, S.L.R, Wood, R.,: Harmonized guidelines for single-laboratory
validation of methods of analysis, Pure & Appl. Chem. 74 (5), 835–855 (2002)
3) Linsinger, T.,: Comparison of a measurement result with the certified value, European
Reference Materials' application note 1, European Commission - Joint Research Centre
Institute for Reference Materials and Measurements (IRMM) (2010)
4) Joint FAO/WHO Food Standards Programme: Procedural manual Twenty-second
edition, Codex Almentarius Commission (2014)
5) ICH Harmonised Tripartite Guideline: Validation of Analytical Procedures: Text andMethodology Q2(R1), International Conference on Harmonisation of TechnicalRequirements for Registration of Pharmaceuticals for Human Use (ICH) (2005)(3.5) Precision
Evaluate reproducibility and repeatability by a collaborative study (3.5.1). Or evaluate anintermediate precision and repeatability by a repeatability test (3.5.2).
(3.5.1) Reproducibility and repeatability by a collaborative study
The number of laboratories to obtain effective data should be 8 or more (15). Conduct
undisclosed duplicate collaborative studies for 5 or more kinds of samples with different
concentration. Obtain reproducibility and repeatability from the observed values (16) toevaluate.
Criteria to evaluate these precisions are shown in a Separate sheet.
Note (15) In case the number of laboratories which have required facility/instruments islimited, this should be 5 or more.
384

(16) The calculation method is shown in Reference 2.
(3.5.2) Intermediate precision and repeatability by a repeatability test in a single
laboratory on different days
Conduct a duplicate test (17) per test day for 5 to 7 days using two analytical samples ofdifferent concentration which is included in a normal range (18). Obtain intermediate precisionand repeatability from the observed values (19) to evaluate.
Criteria to evaluate these precisions are shown in Separate sheet.
Note (17) The data of internal quality control can be used.
(18) It is not necessary for the same tester to conduct a test through 5 to 7 days.
(19) The calculation method is shown in Reference 2.
References
1) AOAC Official Methods of Analysis Appendix K: Guidelines for Dietary Supplements
and Botanicals, AOAC INTERNATIONAL (2012)
2) Thompson, M., Ellison, S.L.R, Wood, R.,: Harmonized guidelines for single-laboratory
validation of methods of analysis, Pure & Appl. Chem. 74 (5), 835-855 (2002)
3) AOAC Official Methods of Analysis Appendix D: Guidelines for Collaborative Study
Procedures To Validate Characteristics of a Method of Analysis, AOAC
INTERNATIONAL (2005)4) Horwitz，W.: Protocol for the Design，Conduct and Interpretation of
Method-Performance Studies，Pure & Appl. Chem.，67 (2), 331-343 (1995)(3.6) Minimum Limit of Quantification (LOQ)
Estimate Minimum Limit of Quantification according to (3.6.1) to (3.6.3). Prepare test
samples which include the concentration estimated to be near the Minimum Limit of
Quantification step by step as necessary. And conduct respective repeatability tests using 3
analytical samples and define the concentration of a prepared test sample as the Minimum
Limit of Quantification, where the mean of the obtained values using the prepared test sample
is suitable to the target value of trueness.
Comment 8 In case permissible content and equivalent level is 1.0 mg/kg or more, theMinimum Limit of Quantification (LOQ) of harmful components andrestricted components, etc. should be no more than 1/5 of the permissible
content and equivalent level. In case permissible content and equivalent levelis no more than 1.0 mg/kg, the Minimum Limit of Quantification (LOQ)
should be no more than 2/5 of the permissible content. Moreover, it isrecommended that the Minimum Limit of Quantification of main
components/major components and material components should be no morethan 1/5 of minimum volume to be contained and the minimum content of a
distribution fertilizer. In addition, in case the Minimum Limit of
Quantification exceeds 1/5 of these minimum volumes, conduct the above
mentioned repeatability test, confirm the Minimum Limit of Quantificationand state clearly the fact in the applicable range of a test method.
Comment 9 There are some methods to estimate Minimum Limit of Quantification. The
methods differ depending on whether they are based on an instrument
analysis or not and depending on instruments used. A method different fromthe methods shown in (3.6.1) to (3.6.3) is allowed. However, the definition ofa method and Minimum Limit of Quantification by the method should beclearly stated.
385

(3.6.1) Estimation method by a repeatability test
With regard to a test sample with concentration near Minimum Limit of Quantification,
conduct a repeatability test using 7 to 10 analytical samples, obtain repeatability standard
deviation and estimate Minimum Limit of Quantification in an analytical sample by theformula (3).

(3.6.2) Estimation method using a calibration curve
In case a calibration curve is linear, estimate Minimum Limit of Quantification (LOQ) in ananalytical sample by the formula (4) using the standard deviation of the residuals of acalibration curve or estimated signals in concentration 0 and the inclination of a calibration
curve.

(3.6.3) Estimation method using an SN ratio
In a test method such as Chromatography, etc. which has a baseline noise, calculate from a
concentration in an analytical solution whose SN ratio is 10 to 1 at the peak and estimate
Minimum Limit of Quantification (LOQ) in an analytical sample.
References
1) ICH Harmonised Tripartite Guideline, Validation of Analytical Procedures: Text andMethodology Q2(R1), International Conference on Harmonisation of TechnicalRequirements for Registration of Pharmaceuticals for Human Use (ICH) (2005)
2）The notification by the Director of Evaluation and Licensing Division, Pharmaceuticaland Food Safety Bureau, the Ministry of Health, Labour and Welfare: The text
(operation method) on Bioanalytical Method Validation”, October 28, 1997, Iyaku-Shin
No.338 (1997)(3.7) Minimum Limit of Detection (LOD)
Estimate Minimum Limit of Detection according to (3.7.1) to (3.7.3).
Comment 10

There are some methods to estimate Minimum Limit of Detection. The
methods differ depending on whether they are based on an instrument
analysis or not and depending on instruments used. A method different from386

the methods shown in (3.7.1) to (3.7.3) is allowed. However, the definitionof the method and Minimum Limit of Detection by the method should beclearly stated.
(3.7.1) Estimation method by a repeatability test
With regard to a test sample or a blank sample with concentration near Minimum Limit of
Quantification, conduct repeatability tests using 7 to 10 analytical samples, obtainrepeatability standard deviation and estimate Minimum Limit of Detection in an analyticalsample by the formula (5).

(20)

Note (20) In case of a repeatability test using 7 analytical samples, the value is 1.94. Incase of using 10 analytical samples, the value is 1.83.
(3.7.2) Estimation method using a calibration curve
In case a calibration curve is linear, estimate Minimum Limit of Detection (LOD) in ananalytical sample by the formula (6) using the standard deviation of the residuals of acalibration curve or estimated signals in concentration 0 and the inclination of a calibration
curve.

(3.7.3) Estimation method using an SN ratio
In a test method such as Chromatography, etc. which has a baseline noise, calculate from a
concentration in an analytical solution whose SN ratio is 3 to 1 at the peak and estimate
Minimum Limit of Detection (LOD) in an analytical sample.
References
1) ICH Harmonised Tripartite Guideline, Validation of Analytical Procedures: Text andMethodology Q2(R1), International Conference on Harmonisation of TechnicalRequirements for Registration of Pharmaceuticals for Human Use (ICH) (2005)
2）The notification by the Director of Evaluation and Licensing Division, Pharmaceuticaland Food Safety Bureau, the Ministry of Health, Labour and Welfare: The text
(operation method) on Bioanalytical Method Validation”, October 28, 1997, Iyaku-Shin
No.338 (1997)387

(3.8) Robustness
Robustness should be studied when an analysis method is developed, and the estimation
method depends on the type of analysis method to be developed. Robustness expresses thereliability of an analysis method when its analysis conditions are intentionally changed. If an
observed value tends to be easily affected by the variation of an analysis condition, it isnecessary to consider a method to control an analysis condition appropriately or to state thefact as a precaution in a testing method. The evaluation of robustness enables theestablishment of a series of parameters such as Resolution related to system conformance.
Similarly, the confirmation of these parameters ensures that the validation of an analysis
method is maintained in a daily analysis.
Typical variation factors are as follows.
(3.8.1) Common variation factors: Typical variation factors common to various kinds oftest methods are as follows.
a) Extraction time, extraction temperatureb) Stability of a test solution in respective stepsc) Regent’s grade(3.8.2)
Variation factors in Chromatography, etc.: Typical variation factors ofmeasurements by Chromatography or refining by solid phase extraction are as follows.
a) Change of a column or a cartridge (A different lot or a different brand)b) Influence by the variation of pH and composition of an eluent or a washc) Temperatured) Flow ratee) Influence of a matrix and effect of dilution
References
1) ICH Harmonised Tripartite Guideline, Validation of Analytical Procedures: Text andMethodology Q2(R1), International Conference on Harmonisation of TechnicalRequirements for Registration of Pharmaceuticals for Human Use (ICH) (2005)
2）The notification by the Director of Evaluation and Licensing Division, Pharmaceuticaland Food Safety Bureau, the Ministry of Health, Labour and Welfare: The text
(operation method) on Bioanalytical Method Validation ”, October 28, 1997, Iyaku-Shin
No.338 (1997)
3) Thompson, M., Ellison, S.L.R, Wood, R.: Harmonized guidelines for single-laboratory
validation of methods of analysis, Pure & Appl. Chem. 74 (5), 835–855 (2002)

388

Reference 1:

A procedure to compare an observed value and a certified value

Obtain the total mean (m) of the replication test results and the certified value (μ), and theabsolute value ( ) of the difference of the two values by the formula (R1.1). Next, obtainthe standard uncertainty () of the certified value of a certified reference material by theformula (R1.2), and obtain the standard uncertainty ( ) of the total mean by the formula(R1.3). Calculate the combined standard uncertainty () ofby the formula (R1.4)
using the obtainedand. Further, calculate an expanded uncertainty () by theformula (R1.5) using the coverage factor (k = 2).
Compareandto confirm that the criterion (the formula (R1.6)) is satisfied, that is,is no more than.

The combined standard uncertaintyofCriterion： The total mean of observed valuesµ： A certified value： The expanded uncertainty of a certified value： The coverage factor of an expanded uncertainty of a standard reference material： Repeatability standard deviation： The number of repeatability test samples： The coverage factor of an expanded uncertainty of389

Reference 2:
repeatability

Calculation of a reproducibility or an intermediate precision and a(1) Structure of an observed value
An observed value (xij) in Table 1, as is shown in the formula (R2.1), consists of a true value(μ), a variation (β) by a factor and a variation (e) by an accidental error under repeatability
conditions (hereinafter referred to as “an accidental error”). When p laboratories conduct acollaborative study in which respective laboratories conduct repeatability tests using nsamples, the formula (R2.2) is introduced on the assumption that the distribution of β isequivalent to N (0,) which depends on a pure between-laboratory variation and the
distribution of e is equivalent to N (0,) which depends on an accidental error. In addition,
when the same laboratory conducts replicate tests for p days using n samples on respective
test days, the formula (R2.3) is introduced on the assumption that the distribution of β isequivalent to N (0,) which depends on test days variation (factor T) and the distributionof e is equivalent to N (0,) which depends on an accidental error.
＋
＋
＋

＋
＋

：

Table 1 Test results of collaborative studies or repeatability tests on different days
Laboratory
Analytical sample numberor test day(Factor)
1
2
3
j
･･･
･･･
1
x11
x12
x13
x
･･･
･･･
1j
2
x21
X22
x23
x2j
･･･
･･･
3
x31
X32
x33
x3j
･･･
･･･
･･･
･･･
･･･
･･･
･･･
･･･
･･･
i
xi1
Xi2
xi3
xij
･･･
･･･
･･･
･･･
･･･
･･･
･･･
･･･
･･･
p
xp1
Xp2
xp3
xpj
･･･
･･･

(2)

n
x1n
x2n
x3n
･･･
xin
･･･
xpn

Calculation procedure of reproducibility and repeatability of the results of acollaborative study(2.1) Estimation of true value and variance
In an actual statistical analysis, a true value ( , a true and pure between-laboratory variance(and a true repeatability varianceare unknown. Therefore they are replaced withestimated values obtained from the results of a collaborative study and are expressed as amean (m), a pure between-laboratory variance ( ) and a repeatability variance ( )respectively.
390

(2.2) One-way analysis of varianceExclude ineffective observed values which have clearly objective reasons such as deviationfrom a protocol and malfunction of instruments from the report values by laboratories which
participated in a collaborative study. Further exclude outliers by conducting Cochran’s test
and Grubb’s test. And conduct one-way analysis of variance for the remaining results toobtain the unbiased variance (V) of respective variation factors in Table 2.
Table 2Sum ofsquares

Variation factor

Table of one-way analysis of variance
Degree offreedom

Between-laboratory(L)
Accidental error (e)Comment 1

Comment 2

It is possible to conduct one-way analysis of variance easily using astatistical program or a tool of a spreadsheet program. In this case, it should
be noted that different terminologies may be used (Between-laboratory (L)→ Between-group, Accidental error (e) → Within-group, Unbiased variance→ Mean square, etc.).
Unbiased variance (V) is calculated by (Sum of squares)/ (Degree of
freedom).

(2.3) The calculation of reproducibility and repeatability
The relation of the expectation of variance E (V) of respective factors in Table 2 holds true.
Therefore calculate repeatability variance (sr2) and pure between-laboratory variance ( ) by
the formula (R2.4) and (R2.5), and further calculate reproducibility variance (sR2) by theformula (R2.6) (1) (2).

＋Table 2)
Table 2)
Calculate a repeatability standard deviation ( ) and a reproducibility standard deviation ( )by the formula (R2.7) and (R2.8) using the obtained repeatability variance and reproducibility
variance, and further calculate a repeatability relative standard deviation (RSDr) and a
reproducibility relative standard deviation (RSDR) by the formula (R2.9) and (R2.10) (2) (3).

391Note (1) In case, assume(that is, the pure between-laboratory variance( ) = 0 in the formula (R2.5)) and let the formula (R2.6) form＝
(2) The rounding of a numerical value is not executed in the middle of thecalculation.
(3) The mean and the standard deviation are expressed rounding to the digit of theobserved value. The relative standard deviation is expressed rounding to the firstdecimal place.
(3) Calculation procedure of intermediate precision and repeatability by the replicate
test results on different days.
(3.1) Estimation of a true value and a variance
In an actual statistical analysis, a true value ( , a true test days variance (σ(T)2) and a true
repeatability varianceare unknown. Therefore they are replaced with estimated values
obtained from the replicate test results on different days and are expressed as a mean (m), testdays variance (s(T)2) and repeatability variance ( ) respectively.
(3.2) One-way analysis of variance
Conduct one-way analysis of variance for the replicate test results on different days to obtainthe unbiased variance (V) of respective variation factors in Table 3.
Table 3Sum ofsquares

Variation factor

Table of one-way analysis of variance
Degree offreedom

Test days (T)Accidental error (e)Comment 3

Comment 4

It is possible to conduct one-way analysis of variance easily using astatistical program or a tool of a spreadsheet program. In this case, it should
be noted that different terminologies may be used (Test days (T) →between-group, Accidental error (e) → within-group, Unbiased variance →Mean square, etc.).
Unbiased variance (V) is calculated by (Sum of squares)/ (Degree of
freedom).

(3.3) The calculation of intermediate precision and repeatability
The relation of the expectation of variance E (V) of respective factors in Table 3 holds true.
2
Therefore calculate repeatability variance (sr2) and test days variance () by the formula(R2.11) and (R2.12), and further calculate intermediate variance () by the formula(2) (4)(R2.13).

＋

Table 3)392

Table 3)
Calculate a repeatability standard deviation ( ) and an intermediate standard deviation ()
by the formula (R2.14) and (R2.15) using the obtained estimated values of repeatability
variance and intermediate variance, and further calculate a repeatability relative standard
deviation (RSDr) and an intermediate relative standard deviation (RSDI(T)) by the formula(R2.16) and (R2.17) (2) (3).

Note (4) In case, assume(that is, the test days variance () in the＝formula (R2.12) = 0) and let the formula (R2.13) form

(4) Examples of the calculation of intermediate precision and repeatability by the
replicate test results on different days.
An example of repeatability test results on different days of citric acid-soluble phosphoricacid using sample 1 and sample 2 containing phosphite is shown in Table 4. Conduct one-way
analysis of variance for the test results of respective samples to obtain the unbiased variance(V) of respective variation factors (Table 5).
Examples of the calculation of intermediate precision and repeatability for the sample 1 andsample 2 using the formula (R2.11) to the formula (R2.17) are shown in Table 6-1 and 6-2. In
addition, the results of respective standard deviation are expressed rounding to the digit of theobserved value and the results of the respective relative standard deviations are expressedrounding to the first decimal place.
Table 4
SampleNo

Example of repeatability test results on different days
Test day (factor)
1
2
3
4
5

51.20
52.15
51.00
51.35
51.35
51.45
51.85
51.09
51.28
51.10
5.18
4.90
5.01
5.15
5.14
Sample 2
5.00
5.12
5.06
5.14
5.07
1) The mean is expressed rounding to the digit of the observed value.
Sample 1

Table 5
SampleNo

Table of one-way analysis of variance
Variation factor
Sum
Degreeofofsquares
freedom
Test days (T)1.05706
Sample 1
Accidental error (e) 0.1253
7
Test days (T)0.04786
Sample 2
Accidental error (e) 0.0448
7393

0.176160.01789
0.00797
0.00640(mass fraction (%))
6

7

51.38
51.38
5.13
5.11

51.28
51.43
5.21
5.18

Total
mean
1)

51.38
5.10

Table 6-1
Calculation of intermediate precision and repeatability using the sample 1 replicate test
results on different days1）
Variation factor
Unit
FormulaCalculation
Result2）

Mass fraction
(%)

＝

%

＝

＝

＝

＝

3）

＝
2）

＋

＝

Mass fraction
(%)

＝

＝

%

＝

＝

3）

＋

1) The rounding of a numerical value is not executed in the middle of the calculation.
2) The standard deviation is expressed rounding to the digit of the observed value.
3) The relative standard deviation is expressed rounding to the first decimal place.
Table 6-2
Calculation of intermediate precision and repeatability using the sample 2 replicate test
results on different days1）
Variation factor
Unit
FormulaCalculation
Result2）

Mass fraction
(%)

＝

%

＝

＝

＝

＝

3）

＝
2）

＋

＝

Mass fraction
(%)

＝

＝

%

＝

＝

3）

Footnote: Refer to Table 6-1

394＋Separate sheet: Criteria of trueness and precision in respective concentration levels
The criteria of trueness and precision in respective concentration levels to evaluate test
methods excluding Chromatography are shown in Table 1. The criteria for Chromatography(1)
are shown in Table 2.
The target of trueness (recovery) is generally within the criteria of Table 1 and Table 2. As forthe precision, although the target is recommended to be within the criteria of Table 1 andTable 2, the permissible level is up to 1.5 times as much as the criteria.
Note (1) Gas chromatography, Gas Chromatography/Mass Spectrometry, High
Performance
LiquidChromatography,
High
Performance
LiquidChromatography/Tandem Mass Spectrometry, Ion Chromatography, etc.
Table 1

Criteria of trueness and precision in respective concentration levels (Test methods
excluding Chromatography)

Concentration
level
≧25 %
≧10 %
≧1 %
≧0.1 %≧100 mg/kg≧10 mg/kg≧1 mg/kg≧100 μg/kg≧10 μg/kg<10 μg/kg
Table 2Concentration
level
≧10 %
≧1 %
≧0.1 %≧100 mg/kg≧10 mg/kg≧1 mg/kg≧100 μg/kg≧10 μg/kg<10 μg/kg

Trueness
Recovery(%)
98～10297～10396～10494～10692～10890～11085～11585～11580～12075～125

Reproducibility
relative standard
deviation (%)
2.5
3
4
6
8
11
16
22
22
22

Precision
Intermediate
relative standard
deviation (%)
2
2.5
3.5
4.5
6.5
9
13
18
18
18

Repeatability
relative standard
deviation (%)
1
1.5
2
3
4
6
8
11
11
11

Criteria of trueness and precision in respective concentration levels(Chromatography)
Trueness
Recovery(%)
90～10885～11085～11080～11570～12070～12070～12070～12060～125

Reproducibility
relative standard
deviation (%)
8
8
8
8
11
16
22
22
22

395

Precision
Intermediate
relative standard
deviation (%)
6.5
6.5
6.5
6.5
9
13
18
18
18

Repeatability
relative standard
deviation (%)
4
4
4
4
6
8
11
11
11