4500-P PHOSPHORUS*

4500-P A. Introduction

1. Occurrence

Phosphorus occurs in natural waters and in wastewaters

almost solely as phosphates. These are classified as orthophosphates, condensed phosphates (pyro-, meta-, and other

polyphosphates), and organically bound phosphates. They

occur in solution, in particles or detritus, or in the bodies of

aquatic organisms.

These forms of phosphate arise from a variety of sources.

Small amounts of orthophosphate or certain condensed phosphates are added to some water supplies during treatment. Larger

quantities of the same compounds may be added during laundering or other cleaning, because these materials are major

constituents of many commercial cleaning preparations. Phosphates are used extensively in the treatment of boiler waters.

Orthophosphates applied to agricultural or residential cultivated

land as fertilizers are carried into surface waters with storm

runoff and to a lesser extent with melting snow. Organic phosphates are formed primarily by biological processes. They are

contributed to sewage by body wastes and food residues, and

also may be formed from orthophosphates in biological treatment processes or by receiving-water biota.

Phosphorus is essential to the growth of organisms and can be

the nutrient that limits the primary productivity of a body of

water. In instances where phosphate is a growth-limiting nutrient, the discharge of raw or treated wastewater, agricultural

drainage, or certain industrial wastes to that water may stimulate

the growth of photosynthetic aquatic micro- and macroorganisms in nuisance quantities.

Phosphates also occur in bottom sediments and in biological

sludges, both as precipitated inorganic forms and incorporated

into organic compounds.

2. Definition of Terms

Phosphorus analyses embody two general procedural steps:

(a) conversion of the phosphorus form of interest to dissolved

orthophosphate, and (b) colorimetric determination of dissolved

orthophosphate. The separation of phosphorus into its various

forms is defined analytically but the analytical differentiations

have been selected so they may be used for interpretive purposes.

Filtration through a 0.45-m-pore-diam membrane filter separates dissolved from suspended forms of phosphorus. No claim

is made that filtration through 0.45-m filters is a true separation

of suspended and dissolved forms of phosphorus; it is merely a

convenient and replicable analytical technique designed to make

a gross separation. Prefiltration through a glass fiber filter may be

used to increase the filtration rate.

Phosphates that respond to colorimetric tests without preliminary hydrolysis or oxidative digestion of the sample are termed

“reactive phosphorus.” While reactive phosphorus is largely a

measure of orthophosphate, a small fraction of any condensed

phosphate present usually is hydrolyzed unavoidably in the

procedure. Reactive phosphorus occurs in both dissolved and

suspended forms.

Acid hydrolysis at boiling-water temperature converts dissolved and particulate condensed phosphates to dissolved orthophosphate. The hydrolysis unavoidably releases some phosphate from organic compounds, but this may be reduced to a

minimum by judicious selection of acid strength and hydrolysis

time and temperature. The term “acid-hydrolyzable phosphorus”

is preferred over “condensed phosphate” for this fraction.

The phosphate fractions that are converted to orthophosphate

only by oxidation destruction of the organic matter present are

considered “organic” or “organically bound” phosphorus. The

severity of the oxidation required for this conversion depends on

the form—and to some extent on the amount— of the organic

phosphorus present. Like reactive phosphorus and acid-hydrolyzable phosphorus, organic phosphorus occurs both in the dissolved and suspended fractions.

The total phosphorus, as well as the dissolved and suspended

phosphorus fractions, each may be divided analytically into the

three chemical types that have been described: reactive, acidhydrolyzable, and organic phosphorus. Figure 4500-P:1 shows

the steps for analysis of individual phosphorus fractions. As

indicated, determinations usually are conducted only on the

unfiltered and filtered samples. Suspended fractions generally are

determined by difference; however, they may be determined

directly by digestion of the material retained on a glass-fiber

filter.

3. Selection of Method

a. Digestion methods: Because phosphorus may occur in combination with organic matter, a digestion method to determine

total phosphorus must be able to oxidize organic matter effectively to release phosphorus as orthophosphate. Three digestion

methods are given in 4500-P.B.3, 4, and 5. The perchloric acid

method, the most drastic and time-consuming method, is recommended only for particularly difficult samples, such as sediments. The nitric acid-sulfuric acid method is recommended

for most samples. By far the simplest method is the persulfate

oxidation technique. Persulfate oxidation is coupled with

ultraviolet light for a more efficient digestion in an automated

in-line digestion/determination by flow injection analysis

(4500-P.I).

The persulfate oxidation method in 4500-P.J renders a digestate that can be analyzed for both total nitrogen and total phos-

* Approved by Standard Methods Committee, 1999. Editorial revisions, 2011.

Joint Task Group: 21st Edition—(4500-P.J)—William Nivens (chair), Prem H.

Arora, Lori J. Emery, James G. Poff, Steven C. Schindler; 20th Edition—(4500-

P.G, H, I)—Scott Stieg (chair), Bradford R. Fisher, Owen B. Mathre, Theresa M.

Wright.

1
phorus. This procedure can be used for both parameters because

it occurs over a broad pH range. During the initial stage of the

digestion, sample pH is alkaline (pH12); during the final stage,

sample pH becomes acidic. As a result, nitrogenous compounds

are oxidized to nitrate and phosphorus compounds to orthophosphate.

It is recommended that persulfate oxidation methods be

checked against one or more of the more drastic digestion

techniques and be adopted if identical recoveries are obtained.

b. Colorimetric method: Three methods of orthophosphate

determination are described. Selection depends largely on the

concentration range of orthophosphate. The vanadomolybdophosphoric acid method (C) is most useful for routine analysis

in the range of 1 to 20 mg P/L. The stannous chloride method (D)

or the ascorbic acid method (E) is more suited for the range of

0.01 to 6 mg P/L. An extraction step is recommended for the

lower levels of this range and when interferences must be overcome. Automated versions of the ascorbic acid method (F, G,

Figure 4500-P:1. Steps for analysis of phosphate fractions.

* Direct determination of phosphorus on the membrane filter containing suspended matter will be required where greater precision than that obtained by difference is

desired. Digest filter with HNO3 and follow by perchloric acid. Then perform colorimetry.

† Total phosphorus measurements on highly saline samples may be difficult because of precipitation of large quantities of salt as a result of digestion techniques that

drastically reduce sample volume. For total phosphorus analyses on such samples, directly determine total dissolved phosphorus and total suspended phosphorus and add

the results.

‡ In determination of total dissolved or total suspended reactive phosphorus, anomalous results may be obtained on samples containing large amounts of suspended

sediments. Very often results depend largely on the degree of agitation and mixing to which samples are subjected during analysis because of a time-dependent desorption

of orthophosphate from the suspended particles.

PHOSPHORUS (4500-P)/Introduction

2
and H) also are presented. Careful attention to procedure may

allow application of these methods to very low levels of phosphorus, such as those found in unimpaired fresh-water systems.

Ion chromatography (4110) and capillary ion electrophoresis

(4140) are useful for determination of orthophosphate in undigested samples.

4. Precision and Bias

To aid in method selection, Table 4500-P:I presents the results

of various combinations of digestions, hydrolysis, and colorimetric techniques for three synthetic samples of the following

compositions:

Sample 1: 100 g orthosphosphate phosphorus (PO4

3-P/L),

80 g acid-hydrolyzable phosphate phosphorus/L (sodium hexametaphosphate), 30 g organic phosphorus/L (adenylic acid),

1.5 mg NH3-N/L, 0.5 mg NO3

-N/L, and 400 mg Cl/L.

Sample 2: 600 g PO4

3-P/L, 300 g acid-hydrolyzable

phosphate phosphorus/L (sodium hexametaphosphate), 90 g

organic phosphorus/L (adenylic acid), 0.8 mg NH3-N/L, 5.0 mg

NO3

-N/L, and 400 mg Cl/L.

Sample 3: 7.00 mg PO4

3-P/L, 3.00 g acid-hydrolyzable

phosphate phosphorus/L (sodium hexametaphosphate), 0.230 mg

organic phosphorus/L (adenylic acid), 0.20 mg NH3-N/L, 0.05 mg

NO3

-N/L, and 400 mg Cl/L.

5. Sampling and Storage

If dissolved phosphorus forms are to be differentiated, filter

sample immediately after collection. Preserve by freezing at

TABLE 4500-P:I. PRECISION AND BIAS DATA FOR MANUAL PHOSPHORUS METHODS

Method

Phosphorus Concentration

No. of

Laboratories

Relative

Standard

Deviation

%

Relative

Error

%

Orthophosphate

g/L

Polyphosphate

g/L

Total

g/L

Vanadomolybdophosphoric 100 45 75.2 21.6

acid 600 43 19.6 10.8

7000 44 8.6 5.4

Stannous chloride 100 45 25.5 28.7

600 44 14.2 8.0

7000 45 7.6 4.3

Ascorbic acid 100 3 9.1 10.0

600 3 4.0 4.4

7000 3 5.2 4.9

Acid hydrolysis  80 37 106.8 7.4

vanadomolybdophosphoric 300 38 66.5 14.0

acid 3000 37 36.1 23.5

Acid hydrolysis  stannous 80 39 60.1 12.5

chloride 300 36 47.6 21.7

3000 38 37.4 22.8

Persulfate  210 32 55.8 1.6

vanadomolybdophosphoric 990 32 23.9 2.3

acid 10 230 31 6.5 0.3

Sulfuric-nitric acids  210 23 65.6 20.9

vanadomolybdophosphoric 990 22 47.3 0.6

acid 10 230 20 7.0 0.4

Perchloric acid  210 4 33.5 45.2

vanadomolybdophosphoric 990 5 20.3 2.6

acid 10 230 6 11.7 2.2

Persulfate  stannous 210 29 28.1 9.2

chloride 990 30 14.9 12.3

10 230 29 11.5 4.3

Sulfuric-nitric acids  210 20 20.8 1.2

stannous chloride 990 17 8.8 3.2

10 230 19 7.5 0.4

PHOSPHORUS (4500-P)/Introduction

3
or below 10°C. In some cases 40 mg HgCl2/L may be added

to the samples, especially when they are to be stored for long

periods before analysis. CAUTION: HgCl2 is a hazardous

substance; take appropriate precautions in disposal; use

of HgCl2 is not encouraged. Do not add either acid or CHCl3

as a preservative when phosphorus forms are to be determined. If total phosphorus alone is to be determined, add

H2SO4 or HCl to pH2 and cool to 4°C, or freeze without any

additions.

Do not store samples containing low concentrations of

phosphorus in plastic bottles unless kept in a frozen state

because phosphates may be adsorbed onto the walls of plastic

bottles.

Rinse all glass containers with hot dilute HCl, then rinse

several times in reagent water. Never use commercial detergents containing phosphate for cleaning glassware used in

phosphate analysis. More strenuous cleaning techniques may

be used.

6. Bibliography

BLACK, C.A., D.D. EVANS, J.L. WHITE, L.E. ENSMINGER & F.E. CLARK,

eds. 1965. Methods of Soil Analysis, Part 2, Chemical and Microbiological Properties. American Soc. Agronomy, Madison, Wisc.

JENKINS, D. 1965. A study of methods suitable for the analysis and

preservation of phosphorus forms in an estuarine environment.

SERL Rep. No. 65-18, Sanitary Engineering Research Lab., Univ.

California, Berkeley.

LEE, G.F. 1967. Analytical chemistry of plant nutrients. In Proc. Int.

Conf. Eutrophication, Madison, Wisc.

FITZGERALD, G.P. & S.L. FAUST. 1967. Effect of water sample preservation methods on the release of phosphorus from algae. Limnol.

Oceanogr. 12:332.

4500-P B. Sample Preparation

For information on selection of digestion method (4500-

P.B.3–5), see 4500-P.A.3a.

1. Preliminary Filtration

Filter samples for determination of dissolved reactive phosphorus, dissolved acid-hydrolyzable phosphorus, and total dissolved phosphorus through 0.45-m membrane filters. A glass

fiber filter may be used to prefilter hard-to-filter samples.

Wash membrane filters by soaking in distilled water before

use because they may contribute significant amounts of phosphorus to samples containing low concentrations of phosphate.

Use one of two washing techniques: (a) soak 50 filters in 2 L

distilled water for 24 h; (b) soak 50 filters in 2 L distilled water

for 1 h, change distilled water, and soak filters an additional 3 h.

Membrane filters also may be washed by running several

100-mL portions of distilled water through them. This procedure

requires more frequent determination of blank values to ensure

consistency in washing and to evaluate different lots of filters.

2. Preliminary Acid Hydrolysis

The acid-hydrolyzable phosphorus content of the sample is

defined operationally as the difference between reactive phosphorus as measured in the untreated sample and phosphate found

after mild acid hydrolysis. Generally, it includes such condensed

phosphates as pyro-, tripoly-, and higher-molecular-weight species such as hexametaphosphate. In addition, some natural waters contain organic phosphate compounds that are hydrolyzed to

orthophosphate under the test conditions. Polyphosphates generally do not respond to reactive phosphorus tests but can be

hydrolyzed to orthophosphate by boiling with acid.

After hydrolysis, determine reactive phosphorus by a colorimetric method (C, D, or E). Interferences, precision, bias, and

sensitivity will depend on the colorimetric method used.

a. Apparatus:

Autoclave or pressure cooker, capable of operating at 98 to

137 kPa.

b. Reagents:

1) Phenolphthalein indicator aqueous solution.

2) Strong acid solution: Slowly add 300 mL conc H2SO4 to

about 600 mL distilled water. When cool, add 4.0 mL conc

HNO3 and dilute to 1 L.

3) Sodium hydroxide, NaOH, 6N.

c. Procedure: To 100-mL sample or a portion diluted to

100 mL, add 0.05 mL (1 drop) phenolphthalein indicator solution. If a red color develops, add strong acid solution dropwise,

to just discharge the color. Then add 1 mL more.

Boil gently for at least 90 min, adding distilled water to keep

the volume between 25 and 50 mL. Alternatively, heat for 30

min in an autoclave or pressure cooker at 98 to 137 kPa. Cool,

neutralize to a faint pink color with NaOH solution, and restore

to the original 100-mL volume with distilled water.

Prepare a calibration curve by carrying a series of standards

containing orthophosphate (see colorimetric method C, D, or E)

through the hydrolysis step. Do not use orthophosphate standards

without hydrolysis, because the salts added in hydrolysis cause

an increase in the color intensity in some methods.

Determine reactive phosphorus content of treated portions,

using Method C, D, or E. This gives the sum of polyphosphate

and orthophosphate in the sample. To calculate its content of

acid-hydrolyzable phosphorus, determine reactive phosphorus in

a sample portion that has not been hydrolyzed, using the same

colorimetric method as for treated sample, and subtract.

3. Perchloric Acid Digestion

a. Apparatus:

1) Hot plate: A 30-  50-cm heating surface is adequate.

2) Safety shield.

3) Safety goggles.

4) Erlenmeyer flasks, 125-mL, acid-washed and rinsed with

distilled water.

b. Reagents:

1) Nitric acid, HNO3, conc.

PHOSPHORUS (4500-P)/Sample Preparation

4
2) Perchloric acid, HClO4 2H2O, purchased as 70 to 72%

HClO4, reagent-grade.

3) Sodium hydroxide, NaOH, 6N.

4) Methyl orange indicator solution.

5) Phenolphthalein indicator aqueous solution.

c. Procedure: CAUTION: Heated mixtures of HClO4 and

organic matter may explode violently. Avoid this hazard by

taking the following precautions: (a) Do not add HClO4 to a

hot solution that may contain organic matter. (b) Always

initiate digestion of samples containing organic matter with

HNO3. Complete digestion using the mixture of HNO3 and

HClO4. (c) Do not fume with HClO4 in ordinary hoods. Use

hoods especially constructed for HClO4 fuming or a glass

fume eradicator* connected to a water pump. (d) Never let

samples being digested with HClO4 evaporate to dryness.

Measure sample containing the desired amount of phosphorus

(this will be determined by whether Method C, D, or E is to be

used) into a 125-mL erlenmeyer flask. Acidify to methyl orange

with conc HNO3, add another 5 mL conc HNO3, and evaporate

on a steam bath or hot plate to 15 to 20 mL.

Add 10 mL each of conc HNO3 and HClO4 to the 125-mL

conical flask, cooling the flask between additions. Add a few

boiling chips, heat on a hot plate, and evaporate gently until

dense white fumes of HClO4 just appear. If solution is not clear,

cover neck of flask with a watch glass and keep solution barely

boiling until it clears. If necessary, add 10 mL more HNO3 to aid

oxidation.

Cool digested solution and add 1 drop aqueous phenolphthalein

solution. Add 6N NaOH solution until the solution just turns pink.

If necessary, filter neutralized solution and wash filter liberally with

distilled water. Make up to 100 mL with distilled water.

Determine the PO4

3-P content of the treated sample by

Method C, D, or E.

Prepare a calibration curve by carrying a series of standards

containing orthophosphate (see Method C, D, or E) through

digestion step. Do not use orthophosphate standards without

treatment.

4. Sulfuric Acid-Nitric Acid Digestion

a. Apparatus:

1) Digestion rack: An electrically or gas-heated digestion

rack with provision for withdrawal of fumes is recommended.

Digestion racks typical of those used for micro-kjeldahl digestions are suitable.

2) Micro-kjeldahl flasks.

b. Reagents:

1) Sulfuric acid, H2SO4, conc.

2) Nitric acid, HNO3, conc.

3) Phenolphthalein indicator aqueous solution.

4) Sodium hydroxide, NaOH, 1N.

c. Procedure: Into a micro-kjeldahl flask, measure a sample

containing the desired amount of phosphorus (this is determined

by the colorimetric method used). Add 1 mL conc H2SO4 and

5 mL conc HNO3.

Digest to a volume of 1 mL and then continue until solution

becomes colorless to remove HNO3.

Cool and add approximately 20 mL distilled water, 0.05 mL (1

drop) phenolphthalein indicator, and as much 1N NaOH solution

as required to produce a faint pink tinge. Transfer neutralized

solution, filtering if necessary to remove particulate material or

turbidity, into a 100-mL volumetric flask. Add filter washings to

flask and adjust sample volume to 100 mL with distilled water.

Determine phosphorus by Method C, D, or E, for which a

separate calibration curve has been constructed by carrying

standards through the acid digestion procedure.

5. Persulfate Digestion Method

a. Apparatus:

1) Hot plate: A 30-  50-cm heating surface is adequate.

2) Autoclave: An autoclave or pressure cooker capable of

developing 98 to 137 kPa may be used in place of a hot plate.

3) Glass scoop, to hold required amounts of persulfate crystals.

b. Reagents:

1) Phenolphthalein indicator aqueous solution.

2) Sulfuric acid solution: Carefully add 300 mL conc H2SO4

to approximately 600 mL distilled water and dilute to 1 L with

distilled water.

3) Ammonium persulfate, (NH4)2S2O8, solid, or potassium

persulfate, K2S2O8, solid.

4) Sodium hydroxide, NaOH, 1N.

c. Procedure: Use 50 mL or a suitable portion of thoroughly

mixed sample. Add 0.05 mL (1 drop) phenolphthalein indicator

solution. If a red color develops, add H2SO4 solution dropwise to

just discharge the color. Then add 1 mL H2SO4 solution and

either 0.4 g solid (NH4)2S2O8 or 0.5 g solid K2S2O8.

Boil gently on a preheated hot plate for 30 to 40 min or until

a final volume of 10 mL is reached. Organophosphorus compounds, such as AMP, may require as much as 1.5 to 2 h for

complete digestion. Cool, dilute to 30 mL with distilled water,

add 0.05 mL (1 drop) phenolphthalein indicator solution, and

neutralize to a faint pink color with NaOH. Alternatively, heat

for 30 min in an autoclave or pressure cooker at 98 to 137 kPa.

Cool, add 0.05 mL (1 drop) phenolphthalein indicator solution,

and neutralize to a faint pink color with NaOH. Make up to

100 mL with distilled water. In some samples a precipitate may

form at this stage, but do not filter. For any subsequent subdividing of the sample, shake well. The precipitate (which is

possibly a calcium phosphate) redissolves under the acid conditions of the colorimetric reactive phosphorus test. Determine

phosphorus by Method C, D, or E, for which a separate calibration curve has been constructed by carrying standards through

the persulfate digestion procedure.

6. Bibliography

LEE, G.F., N.L. CLESCERI & G.P. FITZGERALD. 1965. Studies on the

analysis of phosphates in algal cultures. J. Air Water Pollut. 9:715.

SHANNON, J.E. & G.F. LEE. 1966. Hydrolysis of condensed phosphates in

natural waters. J. Air Water Pollut. 10:735.

GALES, M.E., JR., E.C. JULIAN & R.C. KRONER. 1966. Method for quantitative determination of total phosphorus in water. J. Amer. Water

* GFS Chemical Co., Columbus, OH, or equivalent. Works Assoc. 58:1363.

PHOSPHORUS (4500-P)/Sample Preparation

5
4500-P C. Vanadomolybdophosphoric Acid Colorimetric Method

1. General Discussion

a. Principle: In a dilute orthophosphate solution, ammonium

molybdate reacts under acid conditions to form a heteropoly

acid, molybdophosphoric acid. In the presence of vanadium,

yellow vanadomolybdophosphoric acid is formed. The intensity

of the yellow color is proportional to phosphate concentration.

b. Interference: Positive interference is caused by silica and

arsenate only if the sample is heated. Negative interferences are

caused by arsenate, fluoride, thorium, bismuth, sulfide, thiosulfate, thiocyanate, or excess molybdate. Blue color is caused by

ferrous iron but this does not affect results if ferrous iron concentration is less than 100 mg/L. Sulfide interference may be

removed by oxidation with bromine water. Ions that do not

interfere in concentrations up to 1000 mg/L are Al3, Fe3,

Mg2, Ca2, Ba2, Sr2, Li, Na, K, NH4

, Cd2, Mn2,

Pb2, Hg, Hg2, Sn2, Cu2, Ni2, Ag, U4, Zr4, AsO3

,

Br, CO3

2, ClO4, CN, IO3, SiO44, NO3, NO2, SO42,

SO3

2, pyrophosphate, molybdate, tetraborate, selenate, benzoate, citrate, oxalate, lactate, tartrate, formate, and salicylate. If

HNO3 is used in the test, Cl interferes at 75 mg/L.

c. Minimum detectable concentration: The minimum detectable

concentration is 200 g P/L in 1-cm spectrophotometer cells.

d. Quality control (QC): The QC practices considered to be an

integral part of each method are summarized in Table 4020:I.

2. Apparatus

a. Colorimetric equipment: One of the following is required:

1) Spectrophotometer, for use at 400 to 490 nm.

2) Filter photometer, provided with a blue or violet filter

exhibiting maximum transmittance between 400 and 470 nm.

The wavelength at which color intensity is measured depends

on sensitivity desired, because sensitivity varies tenfold with

wavelengths 400 to 490 nm. Ferric iron causes interference at

low wavelengths, particularly at 400 nm. A wavelength of 470 nm

usually is used. Concentration ranges for different wavelengths are:

P Range

mg/L

Wavelength

nm

1.0–5.0 400

2.0–10 420

4.0–18 470

b. Acid-washed glassware: Use acid-washed glassware for

determining low concentrations of phosphorus. Phosphate contamination is common because of its absorption on glass surfaces. Avoid using commercial detergents containing phosphate.

Clean all glassware with hot dilute HCl and rinse well with

distilled water. Preferably, reserve the glassware only for phosphate determination, and after use, wash and keep filled with

water until needed. If this is done, acid treatment is required only

occasionally.

c. Filtration apparatus and filter paper.*

3. Reagents

a. Phenolphthalein indicator aqueous solution.

b. Hydrochloric acid, HCl, 1  1. H2SO4, HClO4, or HNO3

may be substituted for HCl. The acid concentration in the determination is not critical, but a final sample concentration of 0.5N

is recommended.

c. Activated carbon.† Remove fine particles by rinsing with

distilled water.

d. Vanadate–molybdate reagent:

1) Solution A: Dissolve 25 g ammonium molybdate,

(NH4)6Mo7O24 4H2O, in 300 mL distilled water.

2) Solution B: Dissolve 1.25 g ammonium metavanadate,

NH4VO3, by heating to boiling in 300 mL distilled water. Cool

and add 330 mL conc HCl. Cool Solution B to room temperature, pour Solution A into Solution B, mix, and dilute to 1 L.

e. Standard phosphate solution: Dissolve in distilled water

219.5 mg anhydrous KH2PO4 and dilute to 1000 mL; 1.00 mL 

50.0 g PO4

3-P.

4. Procedure

a. Sample pH adjustment: If sample pH is greater than 10, add

0.05 mL (1 drop) phenolphthalein indicator to 50.0 mL sample and

discharge the red color with 1  1 HCl before diluting to 100 mL.

b. Color removal from sample: Remove excessive color in

sample by shaking about 50 mL with 200 mg activated carbon in

an erlenmeyer flask for 5 min and filter to remove carbon. Check

each batch of carbon for phosphate because some batches produce high reagent blanks.

c. Color development in sample: Place 35 mL or less of

sample, containing 0.05 to 1.0 mg P, in a 50-mL volumetric

flask. Add 10 mL vanadate–molybdate reagent and dilute to the

mark with distilled water. Prepare a blank in which 35 mL

distilled water is substituted for the sample. After 10 min or

more, measure absorbance of sample versus a blank at a wavelength of 400 to 490 nm, depending on sensitivity desired (see ¶

4500-P.C.2a). The color is stable for days, and its intensity is

unaffected by variation in room temperature.

d. Preparation of calibration curve: Prepare a calibration

curve by using suitable volumes of standard phosphate solution

and proceeding as in ¶ c above. When ferric ion is low enough

not to interfere, plot a family of calibration curves of one series

of standard solutions for various wavelengths. This permits a

wide latitude of concentrations in one series of determinations.

Analyze at least one standard with each set of samples.

5. Calculation

mg P/L  mg P(in 50 mL final volume)  1000

mL sample

6. Precision and Bias

See Table 4500-P:I.

* Whatman No. 42, or equivalent. † Darco G60, or equivalent.

PHOSPHORUS (4500-P)/Vanadomolybdophosphoric Acid Colorimetric Method

6
7. Bibliography

KITSON, R.E. & M.G. MELLON. 1944. Colorimetric determination of

phosphorus as molybdovanadophosphoric acid. Ind. Eng. Chem.,

Anal. Ed. 16:379.

BOLTZ, D.F. & M.G. MELLON. 1947. Determination of phosphorus,

germanium, silicon, and arsenic by the heteropoly blue method. Ind.

Eng. Chem., Anal. Ed. 19:873.

GREENBERG, A.E., L.W. WEINBERGER & C.N. SAWYER. 1950. Control of

nitrite interference in colorimetric determination of phosphorus.

Anal. Chem. 22:499.

YOUNG, R.S. & A. GOLLEDGE. 1950. Determination of hexametaphosphate in water after threshold treatment. Ind. Chem. 26:13.

GRISWOLD, B.L., F.L. HUMOLLER & A.R. MCINTYRE. 1951. Inorganic phosphates and phosphate esters in tissue extracts. Anal. Chem. 23:192.

BOLTZ, D.F., ed. 1958. Colorimetric Determination of Nonmetals. Interscience Publishers, New York, N.Y.

AMERICAN WATER WORKS ASSOCIATION. 1958. Committee report. Determination of orthophosphate, hydrolyzable phosphate, and total

phosphate in surface waters. J. Amer. Water Works Assoc. 50:1563.

JACKSON, M.L. 1958. Soil Chemical Analysis. Prentice-Hall, Englewood

Cliffs, N.J.

ABBOT, D.C., G.E. EMSDEN & J.R. HARRIS. 1963. A method for determining orthophosphate in water. Analyst 88:814.

PROFT, G. 1964. Determination of total phosphorus in water and wastewater as molybdovanadophosphoric acid. Limnologica 2:407.

4500-P D. Stannous Chloride Method

1. General Discussion

a. Principle: Molybdophosphoric acid is formed and reduced

by stannous chloride to intensely colored molybdenum blue.

This method is more sensitive than Method C and makes feasible

measurements down to 7 g P/L by use of increased light path

length. Below 100 g P/L an extraction step may increase

reliability and lessen interference.

b. Interference: See 4500-P.C.1b.

c. Minimum detectable concentration: The minimum detectable

concentration is about 3 g P/L. The sensitivity at 0.3010 absorbance is about 10 g P/L for an absorbance change of 0.009.

d. Quality control (QC): The QC practices considered to be an

integral part of each method are summarized in Table 4020:I.

2. Apparatus

The same apparatus is required as for 4500-P.C, except that a

pipetting bulb is required for the extraction step. Set spectrophotometer at 625 nm in the measurement of benzene-isobutanol extracts and at 690 nm for aqueous solutions. If the instrument is not

equipped to read at 690 nm, use a wavelength of 650 nm for

aqueous solutions, with somewhat reduced sensitivity and precision.

3. Reagents

a. Phenolphthalein indicator aqueous solution.

b. Strong-acid solution: Prepare as directed in 4500-P.B.2b2).

c. Ammonium molybdate reagent I: Dissolve 25 g

(NH4)6Mo7O24 4H2O in 175 mL distilled water. Cautiously

add 280 mL conc H2SO4 to 400 mL distilled water. Cool, add

molybdate solution, and dilute to 1 L.

d. Stannous chloride reagent I: Dissolve 2.5 g fresh

SnCl2 2H2O in 100 mL glycerol. Heat in a water bath and stir

with a glass rod to hasten dissolution. This reagent is stable and

requires neither preservatives nor special storage.

e. Standard phosphate solution: Prepare as directed in 4500-

P.C.3e.

f. Reagents for extraction:

1) Benzene–isobutanol solvent: Mix equal volumes of benzene

and isobutyl alcohol. (CAUTION: This solvent is highly flammable.)

2) Ammonium molybdate reagent II: Dissolve 40.1 g

(NH4)6Mo7O24 4H2O in approximately 500 mL distilled water.

Slowly add 396 mL ammonium molybdate reagent I. Cool and dilute

to 1 L.

3) Alcoholic sulfuric acid solution: Cautiously add 20 mL

conc H2SO4 to 980 mL methyl alcohol with continuous mixing.

4) Dilute stannous chloride reagent II: Mix 8 mL stannous

chloride reagent I with 50 mL glycerol. This reagent is stable for

at least 6 months.

4. Procedure

a. Preliminary sample treatment: To 100 mL sample containing not

more than 200 g P and free from color and turbidity, add 0.05 mL (1

drop) phenolphthalein indicator. If sample turns pink, add strong acid

solution dropwise to discharge the color. If more than 0.25 mL (5

drops) is required, take a smaller sample and dilute to 100 mL with

distilled water after first discharging the pink color with acid.

b. Color development: Add, with thorough mixing after each

addition, 4.0 mL molybdate reagent I and 0.5 mL (10 drops)

stannous chloride reagent I. Rate of color development and

intensity of color depend on temperature of the final solution,

each 1°C increase producing about 1% increase in color. Hence,

hold samples, standards, and reagents within 2°C of one another

and in the temperature range between 20 and 30°C.

c. Color measurement: After 10 min, but before 12 min, using

the same specific interval for all determinations, measure color

photometrically at 690 nm and compare with a calibration curve,

using a distilled water blank. Light path lengths suitable for

various concentration ranges are as follows:

Approximate

P Range

mg/L

Light Path

cm

0.3–2 0.5

0.1–1 2

0.007–0.2 10

Always run a blank on reagents and distilled water. Because

the color at first develops progressively and later fades, maintain

equal timing conditions for samples and standards. Prepare at

PHOSPHORUS (4500-P)/Stannous Chloride Method

7
least one standard with each set of samples or once each day that

tests are made. The calibration curve may deviate from a straight

line at the upper concentrations of the 0.3- to 2.0-mg/L range.

d. Extraction: When increased sensitivity is desired or interferences

must be overcome, extract phosphate as follows: Pipet a 40-mL sample, or one diluted to that volume, into a 125-mL separatory funnel.

Add 50.0 mL benzene-isobutanol solvent and 15.0 mL molybdate

reagent II. Close funnel at once and shake vigorously for exactly 15 s.

If condensed phosphate is present, any delay will increase its conversion to orthophosphate. Remove stopper and withdraw 25.0 mL of

separated organic layer, using a pipet with safety bulb. Transfer to a

50-mL volumetric flask, add 15 to 16 mL alcoholic H2SO4 solution,

swirl, add 0.50 mL (10 drops) dilute stannous chloride reagent II, swirl,

and dilute to the mark with alcoholic H2SO4. Mix thoroughly. After 10

min, but before 30 min, read against the blank at 625 nm. Prepare blank

by carrying 40 mL distilled water through the same procedure used for

the sample. Read phosphate concentration from a calibration curve

prepared by taking known phosphate standards through the same procedure used for samples.

5. Calculation

Calculate as follows:

a. Direct procedure:

mg P/L 

mg P (in approximately 104.5 mL

final volume)  1000

mL sample

b. Extraction procedure:

mg P/L 

mg P (in 50 mL

final volume)  1000

mL sample

6. Precision and Bias

See Table 4500-P:I.

4500-P E. Ascorbic Acid Method

1. General Discussion

a. Principle: Ammonium molybdate and antimony potassium

tartrate react in acid medium with orthophosphate to form a

heteropoly acid—phosphomolybdic acid—that is reduced to intensely colored molybdenum blue by ascorbic acid.

b. Interference: Arsenates react with the molybdate reagent to

produce a blue color similar to that formed with phosphate.

Concentrations as low as 0.1 mg As/L interfere with the phosphate determination. Hexavalent chromium and NO2

 interfere

to give results about 3% low at concentrations of 1 mg/L and 10

to 15% low at 10 mg/L. Sulfide (Na2S) and silicate do not

interfere at concentrations of 1.0 and 10 mg/L.

c. Minimum detectable concentration: Approximately 10 g

P/L. P ranges are as follows:

Approximate

P Range

mg/L

Light Path

cm

0.30–2.0 0.5

0.15–1.30 1.0

0.01–0.25 5.0

d. Quality control (QC): The QC practices considered to be an

integral part of each method are summarized in Table 4020:I.

2. Apparatus

a. Colorimetric equipment: One of the following is required:

1) Spectrophotometer, with infrared phototube for use at

880 nm, providing a light path of 2.5 cm or longer.

2) Filter photometer, equipped with a red color filter and a

light path of 0.5 cm or longer.

b. Acid-washed glassware: See 4500-P.C.2b.

3. Reagents

a. Sulfuric acid, H2SO4, 5N: Dilute 70 mL conc H2SO4 to

500 mL with distilled water.

b. Antimony potassium tartrate solution: Dissolve 1.3715 g

K(SbO)C4H4O6 1⁄2H2O in 400 mL distilled water in a 500-mL

volumetric flask and dilute to volume. Store in a glass-stoppered

bottle.

c. Ammonium molybdate solution: Dissolve 20 g

(NH4)6Mo7O24 4H2O in 500 mL distilled water. Store in a

glass-stoppered bottle.

d. Ascorbic acid, 0.1M: Dissolve 1.76 g ascorbic acid in 100 mL

distilled water. The solution is stable for about 1 week at 4°C.

e. Combined reagent: Mix the above reagents in the following proportions for 100 mL of the combined reagent:

50 mL 5N H2SO4, 5 mL antimony potassium tartrate solution,

15 mL ammonium molybdate solution, and 30 mL ascorbic

acid solution. Mix after addition of each reagent. Let all

reagents reach room temperature before they are mixed and

mix in the order given. If turbidity forms in the combined

reagent, shake and let stand for a few minutes until turbidity

disappears before proceeding. The reagent is stable for 4 h.

f. Stock phosphate solution: See 4500-P.C.3e.

g. Standard phosphate solution: Dilute 50.0 mL stock phosphate

solution to 1000 mL with distilled water; 1.00 mL  2.50 g P.

4. Procedure

a. Treatment of sample: Pipet 50.0 mL sample into a clean, dry

test tube or 125-mL erlenmeyer flask. Add 0.05 mL (1 drop)

phenolphthalein indicator. If a red color develops add 5N H2SO4

solution dropwise to just discharge the color. Add 8.0 mL

combined reagent and mix thoroughly. After at least 10 min but

PHOSPHORUS (4500-P)/Ascorbic Acid Method

8
no more than 30 min, measure absorbance of each sample at

880 nm, using reagent blank as the reference solution.

b. Correction for turbidity or interfering color: Natural color

of water generally does not interfere at the high wavelength used.

For highly colored or turbid waters, prepare a blank by adding all

reagents except ascorbic acid and antimony potassium tartrate to

the sample. Subtract blank absorbance from absorbance of each

sample.

c. Preparation of calibration curve: Prepare individual calibration curves from a series of six standards within the phosphate ranges indicated in 4500-P.E.1c. Use a distilled water

blank with the combined reagent to make photometric readings

for the calibration curve. Plot absorbance vs. phosphate concentration to give a straight line passing through the origin. Test at

least one phosphate standard with each set of samples.

5. Calculation

mg P/L 

mg P (in approximately 58 mL

final volume)  1000

mL sample

6. Precision and Bias

The precision and bias values given in Table 4500-P:I are for

a single-solution procedure given in the 13th Edition. The present procedure differs in reagent-to-sample ratios, no addition of

solvent, and acidity conditions. It is superior in precision and

bias to the previous technique in the analysis of both distilled

water and river water at the 228-g P/L level (Table 4500-P:II).

7. References

1. EDWARDS, G.P., A.H. MOLOF & R.W. SCHNEEMAN. 1965. Determination of orthophosphate in fresh and saline waters. J. Amer. Water

Works Assoc. 57:917.

2. MURPHY, J. & J. RILEY. 1962. A modified single solution method for the

determination of phosphate in natural waters. Anal. Chim. Acta 27:31.

8. Bibliography

SLETTEN, O. & C.M. BACH. 1961. Modified stannous chloride reagent

for orthophosphate determination. J. Amer. Water Works Assoc.

53:1031.

STRICKLAND, J.D.H. & T.R. PARSONS. 1965. A Manual of Sea Water

Analysis, 2nd ed. Fisheries Research Board of Canada, Ottawa.

4500-P F. Automated Ascorbic Acid Reduction Method

1. General Discussion

a. Principle: Ammonium molybdate and antimony potassium

tartrate react with orthophosphate in an acid medium to form an

antimony–phosphomolybdate complex, which, on reduction

with ascorbic acid, yields an intense blue color suitable for

photometric measurement.

b. Interferences: As much as 50 mg Fe3/L, 10 mg Cu/L, and

10 mg SiO2/L can be tolerated. High silica concentrations cause

positive interference.

In terms of phosphorus, the results are high by 0.005, 0.015, and

0.025 mg/L for silica concentrations of 20, 50, and 100 mg/L,

respectively. Salt concentrations up to 20% (w/v) cause an error of

less than 1%. Arsenate (AsO4

3) is a positive interference.

Eliminate interference from NO2

 and S2 by adding an excess

of bromine water or a saturated potassium permanganate (KMnO4)

solution. Remove interfering turbidity by filtration before analysis.

Filter samples for total or total hydrolyzable phosphorus only after

digestion. Sample color that absorbs in the photometric range used

for analysis also will interfere. See also 4500-P.E.1b.

c. Application: Orthophosphate can be determined in potable,

surface, and saline waters as well as domestic and industrial

wastewaters over a range of 0.001 to 10.0 mg P/L when photometric measurements are made at 650 to 660 or 880 nm in a 15-

or 50-mm tubular flow cell. Determine higher concentrations by

diluting sample. Although the automated test is designed for

orthophosphate only, other phosphorus compounds can be converted to this reactive form by various sample pretreatments

described in 4500-P.B.1, 2, and 5.

d. Quality control (QC): The QC practices considered to

be an integral part of each method are summarized in Table

4020:I.

2. Apparatus

a. Automated analytical equipment: An example of the continuous-flow analytical instrument consists of the interchangeTABLE 4500-P:II. COMPARISON OF PRECISION AND BIAS OF ASCORBIC ACID METHODS

Ascorbic Acid

Method

Phosphorus

Concentration,

Dissolved

Orthophosphate

g/L

No. of

Laboratories

Relative

Standard

Deviation

%

Relative

Error

%

Distilled

Water

River

Water

Distilled

Water

River

Water

13th Edition1 228 8 3.87 2.17 4.01 2.08

Current method2 228 8 3.03 1.75 2.38 1.39

PHOSPHORUS (4500-P)/Automated Ascorbic Acid Reduction Method

9
able components shown in Figure 4500-P:2. A flow cell of 15 or

50 mm and a filter of 650 to 660 or 880 nm may be used.

b. Hot plate or autoclave.

c. Acid-washed glassware: See 4500-P.C.2b.

3. Reagents

a. Antimony potassium tartrate solution: Dissolve 0.3 g

K(SbO)C4H4O6 1⁄2H2O in approximately 50 mL distilled water

and dilute to 100 mL. Store at 4°C in a dark, glass-stoppered bottle.

b. Ammonium molybdate solution: Dissolve 4 g

(NH4)6Mo7O24 4H2O in 100 mL distilled water. Store in a

plastic bottle at 4°C.

c. Ascorbic acid solution: See 4500-P.E.3d.

d. Combined reagent: See 4500-P.E.3e.

e. Dilute sulfuric acid solution: Slowly add 140 mL conc

H2SO4 to 600 mL distilled water. When cool, dilute to 1 L.

f. Ammonium persulfate, (NH4)2S2O8, crystalline.

g. Phenolphthalein indicator aqueous solution.

h. Stock phosphate solution: Dissolve 439.3 mg anhydrous

KH2PO4, dried for 1 h at 105°C, in distilled water and dilute to

1000 mL; 1.00 mL  100 g P.

i. Intermediate phosphate solution: Dilute 100.0 mL stock

phosphate solution to 1000 mL with distilled water; 1.00 mL 

10.0 g P.

j. Standard phosphate solutions: Prepare a suitable series of

standards by diluting appropriate volumes of intermediate phosphate solution.

4. Procedure

Set up manifold as shown in Figure 4500-P:2 and follow the

general procedure described by the manufacturer.

Add 0.05 mL (1 drop) phenolphthalein indicator solution to

approximately 50 mL sample. If a red color develops, add H2SO4

(4500-P.F.3e) dropwise to just discharge the color.

5. Calculation

Prepare standard curves by plotting response of standards

processed through the manifold against P concentration in standards. Compute sample P concentration by comparing sample

response with standard curve.

6. Precision and Bias

Six samples were analyzed in a single laboratory in septuplicate. At an average PO4

3 concentration of 0.340 mg/L, the

average deviation was 0.015 mg/L. The coefficient of variation

was 6.2%. In two samples with added PO4

3, recoveries were 89

and 96%.

7. Bibliography

HENRIKSEN, A. 1966. An automatic method for determining orthophosphate in sewage and highly polluted waters. Analyst 91:652.

LOBRING, L.B. & R.L. BOOTH. 1973. Evaluation of the AutoAnalyzer II; A

progress report. In Advances in Automated Analysis: 1972 Technicon

International Congress. Vol. 8, p. 7. Mediad, Inc., Tarrytown, N.Y.

U.S. ENVIRONMENTAL PROTECTION AGENCY. 1979. Methods for Chemical

Analysis of Water and Wastes. EPA-600/4-79-020, National Environmental Research Center, Cincinnati, Ohio.

U.S. ENVIRONMENTAL PROTECTION AGENCY. MDQARL Method Study 4,

Automated Methods. National Environmental Research Center,

Cincinnati, Ohio.

4500-P G. Flow Injection Analysis for Orthophosphate

1. General Discussion

a. Principle: The orthophosphate ion (PO4

3) reacts with

ammonium molybdate and antimony potassium tartrate under

acidic conditions to form a complex. This complex is reduced

with ascorbic acid to form a blue complex that absorbs light at

880 nm. The absorbance is proportional to the concentration of

orthophosphate in the sample.

Also see 4500-P.A, B, and F, and Section 4130, Flow Injection

Analysis (FIA).

b. Interferences: Remove large or fibrous particulates by filtering sample through glass wool. Guard against contamination

from reagents, water, glassware, and the sample preservation

process.

Silica forms a pale blue complex that also absorbs at 880 nm.

This interference is generally insignificant because a silica conFigure 4500-P:2. Phosphate manifold for automated analytical system.

PHOSPHORUS (4500-P)/Flow Injection Analysis for Orthophosphate

10
centration of approximately 30 mg/L would be required to produce a 0.005 mg P/L positive error in orthophosphate.

Concentrations of ferric iron greater than 50 mg/L cause a

negative error due to competition with the complex for the

reducing agent ascorbic acid. Treat samples high in iron with

sodium bisulfite to eliminate this interference, as well as the

interference due to arsenates.

Glassware contamination is a problem in low-level phosphorus

determinations. Wash glassware with hot dilute HCl and rinse with

reagent water. Commercial detergents are rarely needed but, if they

are used, use special phosphate-free preparations.

Also see 4500-P.F.

c. Quality control (QC): The QC practices considered to be an

integral part of each method are summarized in Table 4020:I.

2. Apparatus

Flow injection analysis equipment consisting of:

a. FIA injection valve with sample loop or equivalent.

b. Multichannel proportioning pump.

c. FIA manifold (Figure 4500-P:3) with tubing heater and

flow cell. Relative flow rates only are shown in Figure 4500-P:3.

Tubing volumes are given as an example only; they may be

scaled down proportionally. Use manifold tubing of an inert

material, such as TFE.

d. Absorbance detector, 880 nm, 10-nm bandpass.

e. Injection valve control and data acquisition system.

3. Reagents

Use reagent water (10 megohm) to prepare carrier and all solutions. To prevent bubble formation, degas carrier and buffer with

helium. Pass He at 140 kPa (20 psi) through a helium degassing tube.

Bubble He through 1 L solution for 1 min. As an alternative to

preparing reagents by weight/weight, use weight/volume.

a. Stock ammonium molybdate solution: To a tared 1-L container add 40.0 g ammonium molybdate tetrahydrate

[(NH4)6Mo7O24 4H2O] and 983 g water. Mix with a magnetic

stirrer for at least 4 h. Store in plastic and refrigerate.

b. Stock antimony potassium tartrate solution: To a 1-L dark,

tared, container add 3.0 g antimony potassium tartrate (potassium

antimonyl tartrate hemihydrate), K(SbO)C4H4O6 1⁄2H2O, and

995 g water. Mix with a magnetic stirrer until dissolved. Store in a

dark bottle and refrigerate.

c. Working molybdate color reagent: To a tared 1-L container

add 680 g water, then add 64.4 g conc sulfuric acid. CAUTION:

This solution becomes very hot! Swirl to mix. When mixture

can be handled comfortably, add 213 g stock ammonium molybdate solution (¶ a above) and 72.0 g stock antimony potassium tartrate solution (¶ b above). Shake and degas with helium.

TABLE 4500-P:III. RESULTS OF SINGLE-LABORATORY STUDIES WITH

SELECTED MATRICES

Matrix

Sample/Blank

Designation

Known

Addition

mg P/L

Recovery

%

Relative

Standard

Deviation

%

Wastewater

treatment

Reference

sample*

— 101 —

plant Blank† 0.05 96 —

influent 0.1 95 —

Site A‡§ 0 — 0.7

0.05 98 —

0.1 101 —

Site B‡§ 0 — 5

0.05 75 —

0.1 91 —

Site C‡§ 0 — 0.6

0.05 88 —

0.1 97 —

Wastewater

treatment

Reference

sample*

— 100 —

plant Blank† 0.05 96 —

effluent 0.1 96 —

Site A‡ 0 — 0.7

0.05 94 —

0.1 96 —

Site B‡ 0 — 0.3

0.05 94 —

0.1 99 —

Site C‡ 0 — 0.5

0.05 109 —

0.1 107 —

Landfill

leachate

Reference

sample*

— 98 —

Blank† 0.05 94 —

0.1 95 —

Site A‡# 0 — 0.9

0.05 105 —

0.1 106 —

Site B‡# 0 — 6.7

0.05 89 —

0.1 94 —

Site C‡# 0 — 0.9

0.05 110 —

0.1 109 —

* U.S. EPA QC sample, 0.109 mg P/L.

† Determined in duplicate.

‡ Samples without known additions determined four times; samples with known

additions determined in duplicate.

§ Sample dilutions: A - 5-fold; B - 100-fold; C - 10-fold. Typical relative

difference between duplicates 0.5%.

 Sample dilutions: A - 5-fold; B - 20-fold; C - 10-fold. Typical relative difference

between duplicates 0.3%.

# Sample dilutions: A - 20-fold; B - 10-fold; C - 20-fold. Typical relative

difference between duplicates 1%.

Figure 4500-P:3. FIA orthophosphate manifold.

PHOSPHORUS (4500-P)/Flow Injection Analysis for Orthophosphate

11
d. Ascorbic acid solution: To a tared 1-L container, add 60.0 g

granular ascorbic acid and 975 g water. Stir or shake until dissolved.

Degas this reagent with helium, then add 1.0 g dodecyl sulfate,

CH3(CH2)11OSO3Na, stirring gently to mix. Prepare fresh weekly.

e. Stock orthophosphate standard, 25.00 mg P/L: In a 1-L

volumetric flask, dissolve 0.1099 g primary standard grade anhydrous potassium phosphate monobasic (KH2PO4) that has

been dried for 1 h at 105°C in about 800 mL water. Dilute to

mark with water and invert to mix.

f. Standard orthophosphate solutions: Prepare orthophosphate standards in desired concentration range, using stock standard (¶ 3e) and diluting with water.

4. Procedure

Set up a manifold equivalent to that in Figure 4500-P:3 and follow

method supplied by manufacturer or laboratory standard operating

procedure.

5. Calculations

Prepare standard curves by plotting absorbance of standards

processed through the manifold versus orthophosphate concentration. The calibration curve is linear.

6. Precision and Bias

a. Recovery and relative standard deviation: Table 4500-P:III

gives results of single-laboratory studies.

b. MDL: A 700-L sample loop was used in the method described

above. Using a published MDL method,1 analysts ran 21 replicates of

a 5.0-g P/L standard. These gave a mean of 5.26 g P/L, a standard

deviation of 0.264 g P/L, and MDL of 0.67 g P/L.

7. Reference

1. U.S. ENVIRONMENTAL PROTECTION AGENCY. 1984. Definition and procedure for the determination of method detection limits. Appendix B

to 40 CFR 136 Rev. 1.11 amended June 30, 1986. 49 CFR 43430.

4500-P H. Manual Digestion and Flow Injection Analysis for Total Phosphorus

1. General Discussion

a. Principle: Polyphosphates are converted to the orthophosphate form by a sulfuric acid digestion and organic phosphorus

is converted to orthophosphate by a persulfate digestion. When

the resulting solution is injected onto the manifold, the orthophosphate ion (PO4

3) reacts with ammonium molybdate and

antimony potassium tartrate under acidic conditions to form a

complex. This complex is reduced with ascorbic acid to form a blue

complex that absorbs light at 880 nm. The absorbance is proportional to the concentration of total phosphorus in the sample.

See 4500-P.A for a discussion of the various forms of phosphorus found in waters and wastewaters, 4500-P.B for a discussion of sample preparation and digestion, and Section 4130,

Flow Injection Analysis (FIA).

b. Interferences: See 4500-P.G.1b.

c. Quality control (QC): The QC practices considered to be an

integral part of each method are summarized in Table 4020:I.

2. Apparatus

Digestion and flow injection analysis equipment consisting of:

a. Hotplate or autoclave.

b. FIA injection valve with sample loop or equivalent.

c. Multichannel proportioning pump.

d. FIA manifold (Figure 4500-P:4) with tubing heater and

flow cell. Relative flow rates only are shown in Figure 4500-P:4.

Tubing volumes are given as an example only; they may be

scaled down proportionally. Use manifold tubing of an inert

material, such as TFE.

3. Reagents

Use reagent water (10 megohm) for all solutions. To prevent

bubble formation, degas carrier and buffer with helium. Pass He

at 140 kPa (20 psi) through a helium degassing tube. Bubble He

through 1 L solution for 1 min. As an alternative to preparing

reagents by weight/weight, use weight/volume.

Prepare reagents listed in 4500-P.G.3a, b, d, e, and f, and in

addition:

a. Sulfuric acid carrier, H2SO4, 0.13M: To a tared 1-L container add 993 g water, then add 13.3 g conc H2SO4. Shake

carefully to mix. Degas daily. Prepare fresh weekly.

b. Molybdate color reagent: To a tared 1-L container add

694 g water, then add 38.4 g conc H2SO4. CAUTION: The

solution becomes very hot! Swirl to mix. When mixture can be

handled comfortably, add 72.0 g stock antimony potassium

tartrate (4500-P.G.3b) and 213 g stock ammonium molybdate

(4500-P.G.3a). Shake to mix, and degas.

4. Procedure

See 4500-P.B.4 or 5 for digestion procedures. Carry both

standards and samples through the digestion. The resulting solutions should be about 0.13M in sulfuric acid to match the

concentration of the carrier. If the solutions differ more than 10%

from this concentration, adjust concentration of carrier’s sulfuric

acid to match that of digested samples.

Figure 4500-P:4. FIA total phosphorus manifold.

PHOSPHORUS (4500-P)/Manual Digestion and FIA for Total Phosphorus

12
Set up a manifold equivalent to that in Figure 4500-P:4 and

analyze digested samples and standards by following method supplied by manufacturer or laboratory’s standard operating procedure.

Use quality control protocols outlined in Section 4020.

5. Calculations

Prepare standard curves by plotting absorbance of standards

processed through the manifold versus phosphorus concentration. The calibration curve is linear.

6. Precision and Bias

a. MDL: A 780-L sample loop was used in the method

described above. Using a published MDL method,1 analysts ran

21 replicates of a 3.5-g P/L standard. These gave a mean of

3.53 g P/L, a standard deviation of 0.82 g P/L, and MDL of

2.0 g P/L. The MDL is limited mainly by the precision of the

digestion.

b. Precision study: Ten injections of a 100.0-g P/L standard

gave a percent relative standard deviation of 0.3%.

7. Reference

1. U.S. ENVIRONMENTAL PROTECTION AGENCY. 1984. Definition and

procedure for the determination of method detection limits. Appendix B to 40 CFR 136 Rev. 1.11 amended June 30, 1986.

49 CFR 43430.

4500-P I. In-line UV/Persulfate Digestion and Flow Injection Analysis for Total Phosphorus

1. General Discussion

a. Principle: Organic phosphorus is converted in-line to orthophosphate by heat, ultraviolet radiation, and persulfate digestion. At the same time, inorganic polyphosphates are converted

to orthophosphate by in-line sulfuric acid digestion. The digestion processes occur before sample injection. A portion of the

digested sample is then injected and its orthophosphate concentration determined by the flow injection method described in

4500-P.H.1.

See 4500-P.A for a discussion of the various forms of phosphorus found in waters and wastewaters, Section 4500-P.B for a

discussion of sample preparation and digestion, and Section

4130, Flow Injection Analysis (FIA).

b. Interferences: See 4500-P.G.1b.

c. Quality control (QC): The QC practices considered to be an

integral part of each method are summarized in Table 4020:I.

2. Apparatus

Flow injection analysis equipment consisting of:

a. FIA injection valve with sample loop or equivalent.

b. Multichannel proportioning pump.

c. FIA manifold (Figure 4500-P:5) with tubing heater; in-line

ultraviolet digestion fluidics, including a debubbler consisting of

a gas-permeable TFE membrane and its holder; and flow cell.

Relative flow rates only are shown in Figure 4500-P:5. Tubing

volumes are given as an example only; they may be scaled down

proportionally. Use manifold tubing of an inert material, such as

TFE. The block marked “UV” should consist of TFE tubing

irradiated by a mercury discharge ultraviolet lamp emitting radiation at 254 nm.

d. Absorbance detector, 880 nm, 10-nm bandpass.

e. Injection valve control and data acquisition system.

3. Reagents

Use reagent water (10 megohm) for all solutions. To prevent

bubble formation, degas carrier and all reagents with helium.

Pass He at 140 kPa (20 psi) through a helium degassing tube.

Bubble He through 1 L solution for 1 min. As an alternative to

preparing reagents by weight/weight, use weight/volume.

a. Digestion reagent 1: To a tared 1-L container, add 893.5 g

water, then slowly add 196.0 g sulfuric acid, H2SO4. CAUTION:

This solution becomes very hot! Prepare weekly. Degas before

using.

b. Digestion reagent 2: To a tared 1-L container, add 1000 g

water, then add 26 g potassium persulfate, K2S4O8. Mix with a

magnetic stirrer until dissolved. Prepare weekly. Degas before

using.

c. Sulfuric acid carrier, 0.71M: To a tared 1-L container,

slowly add 70 g H2SO4 to 962 g water. Add 5 g sodium chloride,

NaCl. Let cool, then degas with helium. Add 1.0 g sodium

dodecyl sulfate. Invert to mix. Prepare weekly.

d. Stock ammonium molybdate: To a tared 1-L container add

40.0 g ammonium molybdate tetrahydrate, (NH4)6Mo7O24 4H2O,

and 983 g water. Mix with a magnetic stirrer for at least 4 h. The

solution can be stored in plastic for up to 2 months if refrigerated.

e. Stock antimony potassium tartrate: To a 1-L dark, plastic,

tared container add 3.0 g antimony potassium tartrate (potassium

Figure 4500-P:5. FIA in-line total phosphorus manifold.

PHOSPHORUS (4500-P)/In-line UV/Persulfate Digestion & FIA for Total P

13
antimonyl tartrate trihydrate), C8H4K2O12Sb2 3H2O, and 995 g

water. Mix with a magnetic stirrer until dissolved. The solution

can be stored in a dark plastic container for up to 2 months if

refrigerated.

f. Molybdate color reagent: To a tared 1-L container add

715 g water, then 213 g stock ammonium molybdate (¶ d above)

and 72.0 g stock antimony potassium tartrate (¶ e above). Add

and dissolve 22.8 g sodium hydroxide, NaOH. Shake and degas

with helium. Prepare weekly.

g. Ascorbic acid: To a tared 1-L container add 70.0 g ascorbic

acid and 975 g water. Mix with a magnetic stirrer until dissolved.

Degas with helium. Add 1.0 g sodium dodecyl sulfate. Mix with

a magnetic stirrer. Prepare fresh every 2 d.

h. Stock orthophosphate standard, 1000 mg P/L: In a 1-L

volumetric flask dissolve 4.396 g primary standard grade anhydrous potassium phosphate monobasic, KH2PO4 (dried for 1 h at

105°C), in about 800 mL water. Dilute to mark with water and

invert to mix. Prepare monthly.

i. Standard solutions: Prepare orthophosphate standards in

desired concentration range, using stock orthophosphate standards (¶ h above), and diluting with water. If the samples are

preserved with sulfuric acid, ensure that stock standard and

diluted standards solutions are of the same concentration.

4. Procedure

Set up a manifold equivalent to that in Figure 4500-P:5 and

follow method supplied by manufacturer or laboratory’s standard operating procedure. Use quality control procedures described in Section 4020.

5. Calculations

Prepare standard curves by plotting absorbance of standards

processed through manifold versus phosphorus concentration.

The calibration curve is linear.

Verify digestion efficiency by determining tripolyphosphate

and trimethylphosphate standards at regular intervals. In the concentration range of the method, the recovery of either of these

compounds should be 95%.

6. Precision and Bias

a. MDL: A 390-L sample loop was used in the method

described above. Using a published MDL method,1 analysts ran

21 replicates of a 0.10-mg P/L orthophosphate standard. These

gave a mean of 0.10 mg P/L, a standard deviation of 0.003 mg

P/L, and MDL of 0.007 mg P/L.

b. Precision of recovery study: Ten injections of a 10.0-mg P/L

trimethylphosphate standard gave a mean percent recovery of

98% and a percent relative standard deviation of 0.8%.

c. Recovery of total phosphorus: Two organic and two inorganic complex phosphorus compounds were determined in triplicate at three concentrations. The results are shown in Table

4500-P:IV.

d. Comparison of in-line digestion with manual digestion

method: Samples from a wastewater treatment plant influent and

TABLE 4500-P:IV. RECOVERIES OF TOTAL PHOSPHORUS

Compound

Known

Concentration

mg P/L

Mean

Concentration

Recovered

mg P/L

Recovery

%

Relative

Standard

Deviation

%

Sodium 10 8.99 90.3 0.5

pyrophosphate 2 1.81 90.2 0.6

0.2 0.19 93.4 1.0

Phenylphosphate 10 10.10 101.5 0.2

2 2.12 105.0 5.6

0.2 0.20 101.3 1.0

Trimethylphosphate 10 8.99 90.3 0.2

2 1.86 92.7 0.3

0.2 0.18 95.3 1.1

Sodium 10 10.61 106.7 1.0

tripolyphosphate 2 2.14 106.6 0.2

0.2 0.22 108.9 0.9

TABLE 4500-P:V. COMPARISON OF MANUAL AND IN-LINE TOTAL

PHOSPHORUS METHODS

Samples

Concentration

by Manual

Persulfate

Digestion

Method

mg P/L

Concentration

by In-Line

Digestion

Method

mg P/L

Relative

Difference

%

Influent (I2) 5.93 5.52 6.9

Influent (I3) 5.03 4.50 10.5

Influent (I5) 2.14 2.11 1.4

Influent (I6) 1.88 1.71 9.0

Effluent (E1) 3.42 2.87 16.1

Effluent (E2) 3.62 3.55 1.9

Effluent (E3) 3.26 3.34 2.4

Effluent (E4) 8.36 8.16 2.4

Effluent (E5) 0.65 0.71 9.2

Effluent (E6) 0.74 0.81 9.5

Phenylphosphate 1.95 1.91 2.1

Trimethylphosphate 1.87 1.80 3.7

Sodium pyrophosphate 1.90 1.73 8.9

Sodium tripolyphosphate 1.84 1.73 6.0

Figure 4500-P:6. Correlation between manual and in-line total phosphorus methods.

PHOSPHORUS (4500-P)/In-line UV/Persulfate Digestion & FIA for Total P

14
effluent and total phosphorus samples at 2.0 mg P/L were determined in duplicate with both manual persulfate digestion followed by the method in 4500-P.H and in-line digestion method.

Table 4500-P:V gives the results of this comparison, and Figure

4500-P:6 shows the correlation between manual and in-line total

phosphorus methods.

7. Reference

1. U.S. ENVIRONMENTAL PROTECTION AGENCY. 1984. Definition and

procedure for the determination of method detection limits. Appendix B to 40 CFR 136 Rev. 1.11 amended June 30, 1986.

49 CFR 43430.

4500-P J. Persulfate Method for Simultaneous Determination of Total Nitrogen and Total

Phosphorus

1. General Discussion

a. Principle: The oxidation of nitrogenous compounds for

determining total nitrogen must occur in an alkaline medium.

Conversely, the oxidation of phosphorus compounds for determining total phosphorus must occur under acidic conditions.

Methods determining total nitrogen have used a persulfatesodium hydroxide system to oxidize nitrogenous compounds to

nitrate. Accordingly, methods determining total phosphorus have

used persulfate in an acidic medium.

During the initial stage of the digestion, sample pH is alkaline

(pH12). In the final stage of the digestion, the sodium hydroxide is

consumed, causing sample pH to become acidic (pH2). By means of

this broad pH range, the method allows for the oxidation of both

nitrogen and phosphorus compounds. The digested sample is analyzed

for nitrate and orthophosphate, yielding total nitrogen and total phosphorus results.

b. Selection of nitrate/orthophosphate measurement methods:

Using a dual-channel autoanalyzer that performs nitrate-nitrite

by the cadmium reduction method and orthophosphate by the

ascorbic acid reduction method, total nitrogen and total phosphorus can be measured simultaneously. Alternatively, other

methods for orthophosphate and nitrate can be used.

2. Apparatus

Clean all glassware with HCl before use.

a. Autoclave, capable of achieving a temperature of 120°C for

a minimum of 120 min.

b. Glass culture tubes, 13-mm-OD 100-mm-long with autoclavable caps.

c. Autopipettor, capable of pipetting a 6.0-mL portion.

d. Repeating pipettor, capable of pipetting 1.25-mL portion.

e. Erlenmeyer flask, 3000-mL.

f. Aluminum foil.

g. Automated continuous-flow instrument system for nitrate

and phosphate determination: The suggested analytical instruments are described in Sections 4500-NO3

.F.2 and 4500-

P.F.2a.

3. Reagents

a. Deionized water, high-quality, free of phosphorus and nitrogen

compounds. Prepare by ion-exchange or distillation methods as directed in 4500-NH3.B.3a and 4500-NO3

.B.3a.

b. Sodium hydroxide, 3N: Dissolve 120 g low-nitrogen NaOH

in 800 mL deionized water in a 1000-mL volumetric flask. Cool

and dilute to volume.

c. Oxidizing reagent: Dissolve 64 g low-nitrogen (0.001%

N) potassium persulfate, K2S2O8, in 500 mL deionized water.

Use low heat if necessary. Add 80 mL 3N NaOH, prepared from

low-nitrogen sodium hydroxide, and dilute to 1000 mL. Store in

a brown bottle at room temperature.

d. All of the reagents listed for determining nitrate  nitrite as

indicated in Section 4500-NO3

.F.3.

e. All of the reagents listed for determining phosphate as

indicated in Section 4500-P.F.3.

f. Nicotinic acid p-toluenesulfonate stock and working standards: Dry nicotinic acid p-toluenesulfonate in an oven at 105°C

for 24 h. Dissolve 2.1084 g in deionized water and dilute to 100 mL;

1 mL  1 mg N. To prepare a working standard, dilute 2.0 mL

stock solution to 1000 mL; 1 mL  2 g N.

g. Adenosine triphosphate stock and working standards: Dissolve 0.6514 g adenosine triphosphate in deionized water and

dilute to 1000 mL; 1 mL  0.1 mg P. To prepare a working

standard, dilute 20.0 mL stock solution to 1000 mL; 1 mL 

2 g P. To prepare a low-range working standard, dilute 1.0 mL

stock solution to 1000 mL; 1 mL  0.1 g P.

4. Procedure

a. Calibration curve: Prepare a minimum of five standards over the

desired calibration ranges using a stock calibration standard containing

both nitrate and orthophosphate. Treat standards in the same manner as

samples. Include blanks in calibration curves.

b. Sample preparation: If necessary, dilute sample with deionized

water so expected nitrogen and phosphorus concentrations fall within

the range of the calibration standards. Samples preserved with acid

cannot be analyzed by this digestion method.

c. Digestion check standards: Analyze quality-control standards containing organic nitrogen and phosphorus on each analytical run (see 4500-P.J.3f and g for suggested standards and preparation procedures). These standards provide reference checks on the

calibration and test the efficiency of the digestion.

d. Digestion: Pipet 6.0 mL of sample or standard into the culture

tubes. Add 1.25 mL oxidizing reagent to each tube using a repeating

pipettor. Cover the tubes with loose-fitting plastic caps. Prepare an

autoanalyzer wash water in an erlenmeyer flask by adding oxidation

reagent to deionized water in the same proportion as was added to

the samples. Cover flask with foil. Autoclave samples and wash

water for 55 min at 120°C. Cool to room temperature. Add 0.05 mL

of 3N NaOH to each tube before proceeding to nitrate  nitrite and

phosphate analyses. Shake to mix. Add same proportion of 3N

NaOH to digested wash water.

PHOSPHORUS (4500-P)/Persulfate Method for Simultaneous Determination of Total N and Total P

15
e. Final nitrate  nitrite measurement: Use the automated

cadmium reduction method for the determination of nitrate-nitrite

after digestion. See Section 4500-NO3

.F. Other nitrate analysis

methods may be applicable; however, precision and bias data do not

exist for these methods on this matrix at this time.

f. Final phosphate measurement: Use the automated ascorbic

acid reduction method for the determination of phosphate after

digestion. See Section 4500-P.F. Other phosphate analysis methods may be applicable; however, precision and bias data do not

exist for these methods at this time.

5. Calculation

Prepare nitrogen and phosphorus standard curves by plotting

the instrument response of standards against standard concentrations. Compute the nitrogen and phosphorus concentrations by

comparing the sample response with the standard curve. Where

necessary, multiply sample concentration by the appropriate

dilution factor to determine final concentration.

6. Quality Control

The QC practices considered to be an integral part of each

method are summarized in Table 4020:I. Use protocols specified

in Section 4020 to verify performance. These include daily use

of reagent blanks, laboratory-fortified blanks, and known additions. Regulatory analysis may require additional quality control

procedures.

7. Precision and Bias

a. Total nitrogen:

1) Nitrogen digestion check standards—Four different organic nitrogen standards (2 mg N/L) were analyzed by a single

laboratory on three separate analytical runs yielding the following results:

Nitrogen Compound

Mean

mg N/L

Recovery

of N

%

Standard

Deviation

mg N/L

Relative

Standard

Deviation

%

Urea 2.03 102 0.021 1.0

Ammonium p-toluenesulfonate 1.93 97 0.037 1.9

Glycine p-toluenesulfonate 1.94 97 0.034 1.8

Nicotinic acid p-toluenesulfonate 1.86 93 0.044 2.4

2) Mixed ammonia-nitrate solution—A mixed ammonianitrate sample (0.55 mg N/L) was analyzed nine times, yielding

the following results:

Nitrogen Compounds

Mean

mg/L

Recovery

of N

%

Standard

Deviation

mg/L

Relative

Standard

Deviation

%

Ammonium sulfate & potassium

nitrate 0.557 101 0.012 2.1

3) Samples—A single laboratory analyzed five samples in

quadruplicate on three separate occasions. Samples analyzed for

total nitrogen included surface water and diluted wastewater

samples. At an average concentration of 15.0 mg N/L, the

average standard deviation was 0.143 mg/L.

b. Phosphorus:

1) Adenosine triphosphate solutions—Two concentrations (2

mg P/L and 100 g P/L) adenosine triphosphate were analyzed

by a single laboratory on two separate analytical runs yielding

the following results:

Phosphorus Compound

Mean

mg P/L

Recovery

of P

%

Adenosine triphosphate, 2 mg P/L 2.05 103

Adenosine triphosphate, 0.100 g/L 0.103 103

2) Samples—A single laboratory analyzed five samples in

quadruplicate on three separate occasions. Samples were analyzed for total phosphorus over two different calibration ranges.

Surface water and diluted wastewater samples were analyzed

using a low calibration range [0 to 250 g P/L, method

detection level (MDL)  2 g P/L]. At an average concentration

of 1670 g P/L, the average standard deviation was 29.4 g/L.

Surface water and undiluted wastewater samples were analyzed

using a high calibration range (0 to 6 mg P/L, MDL  0.05 mg

P/L). At an average concentration of 1.97 mg P/L, the average

standard deviation was 0.028 mg/L.

8. Bibliography

KOROLEFF, F. 1972. Determination of total nitrogen in natural waters

by means of persulfate oxidation. In S.R. Carlberg, ed. New

Baltic Montreal. ICES Cooperative Res. Rep., Ser. A, No. 29,

p. 73.

KOROLEFF, F. 1977. Simultaneous persulphate oxidation of phosphorus

and nitrogen compounds in water. In K. Grasshoff, Report of the

Baltic Intercalibration Workshop. Annex. Interim Commission for

the Protection of the Environment of the Baltic Sea, p. 52.

NYDAHL, F. 1978. On the peroxodisulphate oxidation of total nitrogen in

waters to nitrate. Water Res. 12: 1123.

U.S. ENVIRONMENTAL PROTECTION AGENCY. 1979. Method 365.1 in

Methods for Chemical Analysis of Water and Wastes. EPA

600/4-79/020, U.S. Environmental Protection Agency, Cincinnati, Ohio.

VALDERRAMA, J.C. 1981. The simultaneous analysis of total nitrogen and

total phosphorus in natural waters. Marine Chemistry 10:109.

U.S. ENVIRONMENTAL PROTECTION AGENCY. 1987. Handbook of Methods

for Acid Deposition Studies: Laboratory Analyses for Surface Water Chemistry. EPA 600/4-87/026, U.S. Environmental Protection

Agency, Washington, D.C., p. 1811.

AMEEL, J.J., R.P. AXLER & C.J. OWEN. 1993. Persulfate Digestion for

Determination of Total Nitrogen and Phosphorus in Low Nutrient

Waters. Center for Water and the Environmental, Natural Resources Research Inst. Amer. Environ. Lab. Oct 1993:2.

PHOSPHORUS (4500-P)/Persulfate Method for Simultaneous Determination of Total N and Total P

16