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VALIDATION REPORT:

Drinking Water Orthophosphate for Thermo 

Scientific Gallery discrete analyzer

Name of the method: Drinking Water Orthophosphate

Reference: Standard Methods (SM) 4500-P E. Phosphorous /Ascorbic Acid Method. 

EPA approved method under 40 C.F.R. § 141.23 (National Primary 

Drinking Water Regulations, NPDWR, based on SDWA, Safe Drinking 

Water Act).1

Intended use: This paper presents the results of the Alternate Test Procedure (ATP) 

validation study for the Drinking Water Orthophosphate method. Also 

comparison between the method and the EPA Approved Method SM 

4500-P E is presented. 

Revision number: 5

Revision date (mm/dd/yyyy): 02/18/2016

1. Background

1.1 Validation of this automated method covers the determination of orthophosphate (CAS: 14265-

44-2) in drinking water with the Thermo ScientificTM GalleryTM discrete analyzer. 

1.2 The method is based on Standard Method 4500-P E. Ascorbic Acid Method (1999).

1.3 Ammonium molybdate and antimony potassium tartrate react in an acidic medium with 

orthophosphate ion to form an antimony-phospho-molybdate complex. This complex is reduced 

to an intensely blue-colored complex by ascorbic acid. The color is proportional to the 

orthophosphate concentration.

1.4 Only orthophosphate forms a blue color in the test. Polyphosphates (and some organic 

phosphate compounds) may be converted to the orthophosphate form by sulfuric acid 

hydrolysis. Organic phosphate compounds may be converted to the orthophosphate form by 

persulfate digestion. These conversion methods are not covered in this validation.

1.5 The applicable range for this method is from 0.0125 – 0.5 mg PO4-P/L. An extension of the 

range to 5 mg PO4-P/L is achieved when 1:10 automated dilution is configured. The automated 

dilution feature must be confirmed with acceptable analysis of quality control samples by the 

user.
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1.6 The Drinking Water Orthophosphate method for Thermo Scientific Gallery discrete analyzer is 

equally effective in meeting the QC acceptance criteria given in the reference method. More 

detailed information is presented in table 1.

Table 1. Performance of ATP candidate method compared to reference method acceptance criteria.

QC acceptance criteria for SM 4500-P E Performance of DW o-PO4P for 

Gallery analyzer

Minimum detectable concentration: 10 μg 

P/l

Recovery IPR: NA (laboratory specific)

% Recovery CCC: 90 – 110 %

Recovery LFB: Mean ± 3 x SD (n ≥ 20)

Recovery LFM: NA (laboratory specific)

% RSD IPR: NA (laboratory specific)

% RSD LFB: NA (laboratory specific)

% RSD LFM: NA (laboratory specific)

Method Blank: ≤ 1⁄2 x MRL

MDL calculated: 0.00036 mg P/L

MRL: 0.0125 mg P/L

% Recovery IPR: 102 – 103%

% Recovery CCC: 100 – 105%

% Recovery LFB: 102 – 103%

% Recovery LFM: 92 – 98 % 

% RSD IPR: 0.1 – 1.0 %

% RSD CCC: 1.3 – 1.9 %

% RSD LFB: 0.2 – 1.1 %

% RSD LFM: 0.1 – 0.7 %

2. Study implementation

2.1 Study was conducted by Thermo Fisher Scientific - Analyzers & Automation between Nov 5

th

2015 and Feb 12th 2016.

2.2 Analyzers used for the study: Two Thermo Scientific Gallery, automated discrete photometric 

analyzers with wavelength 880 nm filters, operated by two users. Reference method study was 

done with Multiskan GO manual spectrophotometer.

2.3 Reagents used for the study:

Reagents for Gallery: THERMO SCIENTIFIC ORDERING NUMBERS

984366 Phosphate R1, 4 × 20 mL, lot: K509, exp: 2015-11

984368 Phosphate R2, 4 × 20 mL, lot: KA92, exp: 2015-11

Standards for Gallery: THERMO SCIENTIFIC ORDERING NUMBERS

984729 Phosphate standard, 1000 ppm as P (calibration), lot: S2293, exp: 2015-11

984726 Phosphate standard, 1000 ppm as PO4 (2

nd source std), lot: S2122, exp: 2016-01

2.4 Samples were collected to 250 ml laboratory glass bottles from public and private taps and 

preserved refrigerated. No preservatives were added. Sample volumes needed for the analyzer 

are only approx. 0.5 – 4 ml. 

TW-02 / Tap water / source water: lake

TW-03 / Tap water from a ferry (reservoir water), source water: lake

TW-04 / Tap water / source water: ground water
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2.5 Calibrations were done from calibration blank and eight calibrator levels which were 

automatically diluted from a stock calibrator by the analyzer. 

2.6 Quality Control Samples (QCS) were analyzed to confirm calibrations. QCS stock solution was

obtained from a source external to the laboratory and different from the source of calibration 

standards. Two QCS samples of concentrations 0.1 and 0.4 mg/L as P were analyzed as 

duplicates. % recovery and % RSD were calculated.

2.7 Method Detection Limit (MDL) was calculated from seven replicate analysis of low 

concentration laboratory fortified blank sample (LFB) after MDL estimate was calculated from 

ten replicate analysis results of laboratory reagent blank (LRB). The LFB sample concentration 

was approximately five times the MDL estimate. 

2.8 Minimum reporting level (MRL) was confirmed with seven replicate analysis of LFB sample at 

concentration estimated for MRL. MRL was estimated to be at the lowest fortified calibrator 

concentration. The upper and lower limits for the Prediction Interval of Results (PIR) were 

calculated and compared to criteria.

2.9 Initial precision and recovery (IPR) were established with four replicate analyses of three LFB 

samples of different concentrations. % recovery and % RSD were calculated.

2.10 Precision and accuracy from a laboratory fortified blank samples (LFB) were estimated by

analysis of three LFB samples. The samples were run in ten replicates and % recovery and % 

RSD were calculated.

2.11 Precision and accuracy from a laboratory fortified matrix samples (LFM) were estimated by 

analysis of three LFM samples. The samples were run in three replicates and % recovery and % 

RSD were calculated.

2.12 Laboratory Reagent Blanks (LRB) and Continuing Calibration Check samples (CCC) were 

analyzed at intervals: at the beginning and end of analysis run and every tenth sample.

3. Method Procedure

3.1 Instrument test parameters are presented in Appendix A.

3.2 Comparison between Drinking Water Orthophosphate method for Gallery and the reference 

method SM 4500-P E is presented in Appendix B. 
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4. Demonstration Data

4.1 Test parameters and test flow for Gallery

Parameters for Gallery Orthophosphate application:

Limits

Primary calibration range from 0 to 0.5 mg P/L.

Test limit 5 mg P/L.

Dilution limit 0.5 mg P/L.

Sec. dilution 1+9

Calibration

Calibration type: 2nd order

Points/calibrator: duplicate

Acceptance: manual

Calibrator concentrations are shown in Table 1.

QC Profile: PO4P QCS

PO4P 0.1mg/l, conc. 0.1 mg/L, Limits (2 x SD) = ± 2 x 0.0075

PO4P 0.4mg/l, conc. 0.4 mg/L, Limits (2 x SD) = ± 2 x 0.0300

Trigger: With calibration, manual

QC Profile: Ongoing QC

LRB, conc. 0 mg/L, Limits (2 x SD) = ± 2 x 0.0021

PO4P CCC, conc. 0.25 mg/L, Limits (2 x SD) = ± 2 x 0.0125

LFB PO4P 0.1, conc. 0.1 mg/L, Limits (2 x SD) = ± 2 x 0.0075

LFB PO4P 0.4, conc. 0.4 mg/L, Limits (2 x SD) = ± 2 x 0.0300

Trigger: At the start and end of run, every 10th, manual

Testflow

Sample volume 120 μl (dispensed with extra volume 40 μl, extra wash)

Incubate 18 s

End-point blank YES

Reagent 1 Phosphate R1 volume 14 μl (dispensed with extra volume 20 μl)

Incubate 120 s

Reagent 2 Phosphate R2 volume 6 μl (dispensed with extra volume 20 μl)

Incubate 540 s

End-point measurement

Main wavelength 880 nm
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4.2 Method performance data 

4.2.1 Calibration and QCS-samples

Calibration curve was prepared between 0 - 0.5 mg P/L for DW o-PO4P application. 

Calibration was done with automatic dilution from working standard solution of 1.5 mg

P/l and laboratory reagent water. Calibration was verified with QCS-samples (table 3).

Table 2. Example of orthophosphate calibrators, concentrations and responses for DW o-PO4P 

calibration curve.

Orthophosphate

Calibrator

Dilution

1+

Concentration

(mg P/L)

Response

(A)

PO4P-0 0 0.00 -0.0003

PO4P-0 0 0.00 -0.0002

PO4P-St 119 0.0125 0.0044

PO4P-St 119 0.0125 0.0047

PO4P-St 49 0.03 0.0127

PO4P-St 49 0.03 0.0137

PO4P-St 29 0.05 0.0247

PO4P-St 29 0.05 0.0244

PO4P-St 14 0.100 0.0514

PO4P-St 14 0.100 0.0515

PO4P-St 7 0.188 0.1003

PO4P-St 7 0.188 0.0998

PO4P-St 5 0.250 0.1346

PO4P-St 5 0.250 0.1349

PO4P-St 3 0.375 0.1999

PO4P-St 3 0.375 0.1997

PO4P-St 2 0.500 0.2660

PO4P-St 2 0.500 0.2660

Figure 1. Calibration curve for orthophosphate as phosphorous.
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Table 3. Results for orthophosphate QCS-samples after calibration.

Orthophosphate as phosphorous

QCS-sample Duplicate % RSD % Recovery

PO4P 0.1mg/l 0.13 % 96 %

PO4P 0.4mg/l 0.19 % 103 %

4.2.2 Method Detection Limit (MDL)

MDL was established using reagent water (blank) fortified at a concentration five times 

the estimated instrument detection limit (MDLest). The estimate was calculated as three 

times the standard deviation of replicate instrumental measurements of the analyte in 

reagent water. 

MDLest = 3 × (S0)

where S0 = standard deviation of the replicate analyses (n=10) of reagent 

water.

Table 4. Values for determining MDLest for Orthophosphate application.

ample Result (mg/L)

LRB 0.001908

LRB 0.001854

LRB 0.002122

LRB 0.001861

LRB 0.001984

LRB 0.001647

LRB 0.001745

LRB 0.001984

LRB 0.001851

LRB 0.00180

average = 0.00188

n = 10

std. deviation (SD) = 0.00013

MDLest calculated = 0.00040

MDLest assigned= 0.0022
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NOTE! The MDLest had to be assigned higher than the calculated MDLest, because the 

highest individual LRB result of this test was as high as 0.002122 mg/l as P, and criteria 

for LRB sample in this test is, that it should never be above the MDLest.

A laboratory fortified blank sample (LFB) at a concentration which was approximately

five times the MDLest, was analyzed in seven replicates. Each individual result had to 

be within 70 – 130 % of the theoretical value. MDL was calculated as follows:

MDL = (t) × (S)

where t = Student's t-value for a 99 % confidence level and a standard 

deviation estimate with n-1 degrees of freedom [t = 3.14 for 7 

replicates]

S = standard deviation of the replicate analyses

Table 5. Values for determining MDL for DW o-PO4P application. 

Sample Result (mg/L) % Recovery

LFB 0.01 mg/l PO4P 0.00793 79 %

LFB 0.01 mg/l PO4P 0.00791 79 %

LFB 0.01 mg/l PO4P 0.00795 80 %

LFB 0.01 mg/l PO4P 0.00768 77 %

LFB 0.01 mg/l PO4P 0.00796 80 %

LFB 0.01 mg/l PO4P 0.00803 80 %

LFB 0.01 mg/l PO4P 0.00797 80 %

average = 0.007919 79 %

n = 7

std. deviation (SD) = 0.000114

% RSD = 4.5 %

MDL = 0.00036
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4.2.3 Minimum Reporting Level (MRL)

Seven replicate LFBs were analyzed at the proposed MRL concentration.

The mean (Mean) and standard deviation (s) for these replicates were calculated. The

Half Range for the Prediction Interval of Results (HRPIR) was determined using the 

equation:

HRPIR = 3.963s

Where s = standard deviation 

3.963 = constant value for seven replicates

The upper and lower limits for the Prediction Interval of Results (PIR = Mean +HRPIR)

were calculated from the results. 

100 

Fortified Concentration

Mean HR

PIRUpper Limit PIR 100 



Fortified Concentration

Mean HR

PIR Lower Limit PIR

Criteria: The Upper PIR Limit must be ≤ 150 % recovery.

The Lower PIR Limit must be ≥ 50 % recovery

Table 6. Results for MRL confirmation for DW o-PO4P application.

Sample Result (mg/L) % Recovery

LFB 0.01 mg/l PO4P 0.010088 101 %

LFB 0.01 mg/l PO4P 0.009663 97 %

LFB 0.01 mg/l PO4P 0.009568 96 %

LFB 0.01 mg/l PO4P 0.009378 94 %

LFB 0.01 mg/l PO4P 0.009891 99 %

LFB 0.01 mg/l PO4P 0.009616 96 %

LFB 0.01 mg/l PO4P 0.009605 96 %

average = 0.00969 97 %

n = 7

std. deviation (s) = 0.000232

HRPIR = 0.000921

Upper PIR Limit = 106.1 %

Lower PIR Limit = 87.7 %

% RSD= 2.4 %

Calculated limits met the criteria and confirmed the MRL to be validated.
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4.2.4 Initial Precision and Recovery (IPR)

Four replicate volumes of reagent water spiked with the analyte of interest (LFB) were 

analyzed at three different concentrations. % recovery and % RSD were calculated.

Results are shown in table 7.

Criteria for % recovery: 85 % - 115 %, when c is > 2 x MRL and 

80 % - 120 %, when c is ≤ 2 x MRL 

Criteria for % RSD: ±15% 

Table 7. Results for initial precision and recovery (IPR) for DW o-PO4P application.

Sample Result (mg/L) % Recovery

LFB 0.1 mg/l 0.1027 103 %

LFB 0.1 mg/l 0.1028 103 %

LFB 0.1 mg/l 0.1027 103 %

LFB 0.1 mg/l 0.1028 103 %

average 0.1027 103 %

std.deviation 0.000074

% RSD 0.1 %

Acceptable YES YES

LFB 0.3 mg/l PO4P 0.3056 102 %

LFB 0.3 mg/l PO4P 0.3020 101 %

LFB 0.3 mg/l PO4P 0.3096 103 %

LFB 0.3 mg/l PO4P 0.3051 102 %

average 0.3056 102 %

std.deviation 0.0031

% RSD 1.0 %

Acceptable YES YES

LFB 0.4 mg/l 0.4074 102 %

LFB 0.4 mg/l 0.4083 102 %

LFB 0.4 mg/l 0.4088 102 %

LFB 0.4 mg/l 0.4083 102 %

average 0.4082 102 %

std.deviation 0.00059

% RSD 0.1 %

Acceptable YES YES
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4.2.5 Precision and accuracy from LFB

Precision (%RSD) and accuracy (% Recovery) were determined using LFB samples at 

0.1, 0.3 and 0.4 mg/L concentrations. Results are shown in table 8.

Criteria for % recovery: 85 - 115 %, when c is > 2 x MRL and 

80 - 120 %, when c is ≤ 2 x MRL 

Criteria for % RSD: ±15% 

Table 8. Precision and accuracy data for DW o-PO4P application.

Sample: 

Concentration:

LFB 0.1 mg/l PO4P

0.1 mg P/L

LFB 0.3 mg/l PO4P

0.3 mg P/L

LFB 0.4 mg/l PO4P

0.4 mg P/L

average = 0.10257 0.30541 0.40891

n = 10 10 10

std. dev. (SD) = 0.000209 0.003242 0.000767

% RSD = 0.2 % 1.1 % 0.2 %

Avg. % Recovery = 103 % 102 % 102 %

Acceptable YES YES YES

4.2.6 Precision and accuracy from LFM samples

Laboratory Fortified Matrix (LFM) samples were prepared by spiking three different tap 

water samples with orthophosphate standard solutions to gain spike concentrations of 

0.1 and 0.4 mg/L as P for each sample. The analyte concentrations were high enough to 

be detected above the original sample and were not less than four times the MDL. The 

added analyte concentrations were chosen to be the same as those used for LFB. 

Precision was estimated by analyzing three replicates and calculating the %RSD. 

Accuracy was estimated from LFM and corresponding unspiked samples by analyzing 

them and calculating the % Recovery using the next equation. Results for LFM 

precision and accuracy are shown in table 9.

100 ( )LFMRecovery %, 

 



s

C f C

R

s

where Cs = spiked sample concentration

f = spike dilution correction

C = sample background concentration

s = concentration equivalent of analyte added to sample

Note! Because the added spike volume was less than 1% of the total LFM sample 

volume (0.4 % and 0.12 % of total LFM sample volume), the factor f could be excluded.

Criteria for % recovery: 85 - 115 %, when c is > 2 x MRL and 

80 - 120 %, when c is ≤ 2 x MRL 

Criteria for % RSD: ±15% 
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Table 9. Laboratory Fortified Matrix (LFM) sample results for DW o-PO4 application.

Sample Result 

avg. n = 3

(mg/L)

Spike. 

mg/L

% 

Recovery

% RSD. 

n =3

Acceptable

TW-02 0.0021

TW-02 +0.1 mg/l PO4P 0.0946 0.10 92 % 0.7 % YES

TW-02 +0.4 mg/l PO4P 0.3906 0.40 97 % 0.1 % YES

TW-03 0.0026

TW-03 +0.1 mg/l PO4P 0.0944 0.10 92 % 0.3 % YES

TW-03 +0.4 mg/l PO4P 0.3943 0.40 98 % 0.2 % YES

TW-04 0.0028

TW-04 +0.1mg/l PO4P 0.0989 0.10 96 % 0.7 % YES

TW-04 +0.4mg/l PO4P 0.3754 0.40 93 % 0.3 % YES

Average = 95 % 0.4 % YES

4.2.7 Continues Calibration Check (CCC)

Continues Calibration Check (CCC) solution was analyzed in intervals of every 10th

sample, at the beginning and end of run. CCC sample concentration was from the mid 

calibration area – 0.25 mg/L. CCC sample result summary is in table 10.

Criteria for CCC % recovery: 90 – 110 %

Table 10. PO4P CCC result summary. Sample: PO4P CCC, concentration 0.25 mg/L.

Analyzer 1 Analyzer 2

Average: 0.254 0.256

% Recovery 102 % 102 %

n = 20 6

std. deviation (SD) = 0.0033 0.0048

% RSD= 1.29 % 1.87 %

Minimum result: 0.249 0.252

Maximum result: 0.261 0.261

Acceptable YES YES

4.3 Reference method performance study

4.3.1 Reference method performance was done with a Multiskan GO spectrophotometer, 

using disposable 1 cm cuvettes and 880 nm wavelength. 

4.3.2 Reagents were done according to the reference method SM4500-P E. 
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4.3.3 Same samples and standard stock solutions were used for reference method results as 

for ATP candidate method. LFM samples were prepared as described in section 4.2.6.

4.3.4 Testflow for reference method, DW o-PO4P manual_1

Instrument Blank: Laboratory water

Sample: Volume 2.5 ml (dispensed manually to test tube)

Combined reagent: Volume 0.4 ml (dispensed manually to test tube)

Mix: Capped test tube was inverted several times to mix

Incubation: Sample-reagent solution was incubated for 15 min.

End-point measurement:

Sample was moved to cuvette and measured at 880 nm.

4.3.5 Reference method performance study results are presented in table 11.

Table 11. Method performance compared to the reference method performance.

DW o-PO4P manual_1

(SM 4500-P E)

DW o-PO4P

Single 

laboratory 

Method 

Performance

MDL: 0.0032 mg P/L

MRL: 0.015 mg P/L

% Recovery IPR: 101 – 102 %

% Recovery CCC: 97 – 104 %

% Recovery LFB: 92 – 104 %

% Recovery LFM: 94 – 101 %

% RSD IPR: 0.4 %

% RSD CCC: 1.49 %

% RSD LFB: 0.29 – 0.96 %

% RSD LFM: 0.24 – 3.0 %

MDL calculated: 0.00036 mg P/L

MRL: 0.0125 mg P/L

% Recovery IPR: 102 – 103%

% Recovery CCC: 100 – 105%

% Recovery LFB: 102 – 103%

% Recovery LFM: 92 – 98 % 

% RSD IPR: 0.1 – 1.0 %

% RSD CCC: 1.3 – 1.9 %

% RSD LFB: 0.2 – 1.1 %

% RSD LFM: 0.1 – 0.7 %

5. Discussion

Comparison of the Drinking Water Orthophosphate method for Thermo Scientific Gallery discrete 

analyzer method to the reference method in table 11 shows the method performance to be in the same 

range as the reference method. MDL, MRL, precision for all quality samples and LFB accuracy, are 

equal or better than in the reference method. Reference method has a slightly better % recovery for 

LFM as the ATP candidate method. The performance is still clearly above the reference value ±15 % 

generally used for drinking water method development. This all considered, the ATP candidate 

method performance is equally good as the reference method performance.

6. Conclusions

The performance results achieved in this validation study shows the method to be a worthy choice for 

analyzing orthophosphate from drinking water. The advantages given by the use of discrete analyzers, 

such as decreasing cost, waste and possibility for human error, makes analysis of drinking water 

samples easier for the laboratories without risking the quality of results.
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APPENDIX A.

Test flow for DW o-PO4P application in the Gallery analyzer.
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APPENDIX B.

Comparisons of SM 4500-P E Ascorbic Acid Method for 

Phosphorous and the Gallery DW o-PO4P method

Table 1. The differences and similarities between SM 4500-P E method for phosphorous and the Gallery 

DW o-PO4P method. 

Section SM 4500-P E. Thermo Scientific Gallery 

DW o-PO4 Method

Scope and 

Application

0.01-6.0 mg P/L. Minimum detectable 

concentration approximately 10 μg P/L.

Drinking, ground and surface waters,

and domestic and industrial wastes.

0.0125-0.5 mg P/L. An extension of the 

range to 5 mg PO4-P/L may be achieved 

when a 1:10 automated dilution is 

configured. Method DW o-PO4P was 

tested for drinking water. 

Summary of the 

Method

Ammonium molybdate and potassium 

antimony 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.

Ammonium molybdate and potassium 

antimony 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.

Definitions SI-units SI-units

Interferences Arsenates, hexavalent chromium, nitrite. Arsenates, hexavalent chromium, nitrite,

high iron concentrations.

Safety N/A Only small volumes of sample and 

reagents used. Consult MSDS for 

general safety.

Equipment and 

Supplies

Spectrophotometer with infrared 

phototube for use at 880 nm or filter 

photometer equipped with a red color 

filter.

Thermo Scientific Gallery discrete 

photometric analyzer. Filter for 880 nm.

DECACELL cuvettes.
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Section SM 4500-P E. Thermo Scientific Gallery 

DW o-PO4 Method

Reagents and 

Standards

5 N Sulfuric acid solution

Dilute 70 mL conc H2SO4 to 500 mL

with distilled water.

Ammonium molybdate solution

Dissolve 20 g (NH4)6Mo7O24∙4H2O in 

500 mL distilled water.

Potassium antimonyl 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.

Ascorbic acid

Dissolve 1.76 g ascorbic acid in 100 mL 

distilled water.

Combined reagent

Mix 50 mL 5 N H2SO4. 5 mL potassium

antimonyl tartrate solution. 15 mL 

ammonium molybdate solution and 30 

mL ascorbic acid solution. Mix after 

addition of each reagent. All reagents 

must reach room temperature before 

they are mixed and must be mixed in the 

order given. The solution is stable for 4 

h.

Stock Orthophosphate solution

Dissolve 0.2195 g anhydrous KH2PO4 in 

distilled water and dilute to 1000 mL. 

1.0 mL = 0.05 mg PO4-P.

5 N H2SO4 solution

Slowly add 70 mL of conc. H2SO4 to 

approximately 300 mL of distilled water. 

Cool and dilute to 500 mL.

Ammonium molybdate solution

Dissolve 20 g (NH4)6Mo7O24∙4H2O in 

500 mL distilled water.

Antimony potassium tartrate solution

Dissolve 1.5 g K(SbO)C4H4O6∙1⁄2H2O in 

approximately 250 mL distilled water in 

500 mL volumetric flask. Dilute to 

volume.

Ascorbic acid = Phosphate R2

Dissolve 1.76 g ascorbic acid in 

approximately 80 mL distilled water in 

100 mL volumetric flask. Dilute to 

volume.

Molybdate Reagent = Phosphate R1

Add 15 mL of Ammonium molybdate 

solution to 50 mL of Sulfuric acid 

solution. Add 5 mL of Antimony 

potassium tartrate solution to this 

mixture.

Orthophosphate standard solution 

(stock)

Concentration 1000 mg/L as P. 

Orthophosphate standard solution 

(stock) –secondary source for LFB/LFM

Concentration 1000 mg/L as PO4 

(= 326.2 mg/l as P). 
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Section SM 4500-P E. Thermo Scientific Gallery 

DW o-PO4 Method

Sample 

Collection.

Preservation. and 

Storage

In some cases 40 mg HgCl2/L may be 

added to the samples when they are 

stored for long periods; not encouraged. 

Do not store samples containing low 

concentrations of phosphorous in plastic 

bottles unless kept in a frozen state 

because phosphates may be absorbed 

onto the walls of plastic bottles. Rinse 

all glass containers with hot dilute HCl.

then rinse several times in reagent water.

Sample containers may be of plastic 

material or of Pyrex glass. All bottles 

must be thoroughly cleaned and rinsed 

with distilled water. 

Do not store samples containing low 

concentrations of phosphorous in plastic 

bottles because phosphates may be 

adsorbed to the walls of plastic bottles. 

Samples should be analyzed as soon as 

possible after collection. Samples must 

be cooled and maintained at 4 °C. 

Quality Control 4020-Quality Assurance/Quality 

Control.5

Additional QC procedures can and 

should be used. Some regulatory

programs may require additional QC or 

have alternative acceptance limits.

Quality control (QC) program

Formal protocol required for the 

laboratory using this method.
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Section SM 4500-P E. Thermo Scientific Gallery 

DW o-PO4 Method

Initial

Demonstration of 

Capability

Initial demonstration of capability 

(IDC):

MDL calculation in SM 1020-B.4.: min. 

7 replicates of MDL sample, 1-5 x MDL 

estimate conc. analyzed in 3 days and 

ideally by different users. Recoveries 

should be 50 – 150% and %RSD ±20%. 

MDL=3.14s, where s= standard 

deviation (n=7).

Done for each matrix separately and

checked annually at minimum.

Define operational range (upper and 

lower limits). Minimum reporting level

is set at or above the smallest calibrator, 

and verified with QC sample at 1 to 2 

times the MRL conc. Result is 

acceptable if precision and accuracy 

meet laboratory method requirements.

Quality Control Samples (QCS):

Externally created blind samples that 

must reach laboratory acceptance criteria 

are analyzed annually at minimum. 

Accuracy study with each calibration. 

Criteria: ±15 % Recovery

Initial precision and recovery : Min. 4 

replicate volumes of reagent water 

spiked with analyte (LFB). Compare to 

the given recovery limits or calculate 

limits: ±5.84 x SD (n = min. 20).

The initial demonstration 

of laboratory capability 

Calibration curve: min. 5 calibration 

standards and a blank; the correlation 

coefficient shall be equal to or greater 

than 0.995

Method Detection Limit (MDL) in 

accordance with the procedure at 40 

CFR part 136. Appendix B. This 

protocol is very similar to SM 1020-B.4.

Minimum Reporting Level (MRL) —

The minimum reporting level is 

estimated at the level of lowest fortified 

calibrator. MRL is established to this 

concentration by confirming that the 

upper and lower PIR limits fulfill the 

acceptance criteria (50- 150 % 

Recovery).

External QC samples (QCS)

Externally created blind samples that 

must reach laboratory acceptance criteria 

are analyzed with every calibration, 

annually at minimum, e.g. Proficiency 

tests. Criteria: ±15 % Recovery

Initial precision and recovery (IPR): 4 

replicate volumes of reagent water 

spiked with analyte (LFB). 

% recovery: 85 - 115 % or 80 - 120 % 

when c is ≤ 2 x MRL, %RSD: ±15%.
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Section SM 4500-P E. Thermo Scientific Gallery 

DW o-PO4 Method

Assessing 

Laboratory 

Performance

The instrument calibration with min. 3

calibration standards and a blank for 

linear and 5 calibration standards and a 

blank for second-order; the correlation 

coefficient ≥ 0.995 and the backcalculated values of individual 

calibrators should be within 90 – 110 % 

of the true value (except at lower 

operational limit). 

Calibration verification. The value of the 

instrument check standard should fall 

between 90 and 110 % of the true value. 

Frequency: every 10th and at the end. 

Method Blank

Frequency: daily, every 20th. 

Result < 1/2 x MRL. 

Laboratory Fortified Blank (LFB): 

Must reach laboratory acceptance 

criteria.

Sample Duplicates

Frequency: daily, every 20th. Must reach 

laboratory acceptance criteria.

LFM and LFMD

Frequency: daily, every 20th. Must reach 

laboratory acceptance criteria.

Calibration curve: min. 5 calibration 

standards and a blank; the correlation 

coefficient shall be equal to or greater 

than 0.995. 

Continuing Calibration Check (CCC)

sample: 90 - 110 % Frequency: At the 

beginning, every 10th, at beginning and 

end of run.

Method Blank

Frequency: daily, every 10

th

. 

Result < 1/3 x MRL. 

Laboratory Fortified Blank (LFB): 

Frequency: daily, two different 

concentration samples for every batch of 

max. 10 samples.

Criteria: ±15 % Recovery or must reach 

laboratory acceptance criteria.

Laboratory Fortified Matrix Sample

(LFM/LFMD): 

Frequency: daily, one LFM sample as 

duplicate for every batch of max. 10 

samples.

85 - 115 % or 80 - 120 % when c is ≤ 2 

x MRL, RSD: ±20%.

Calibration and 

Standardization

Prepare individual calibration curves 

from a series of six standards within the 

phosphate ranges 0.01-0.25 mg P/L. Plot 

absorbance vs. phosphate concentration

to give a straight line passing through 

origin.

Prepare a series of at least five standards 

covering the desired range and a blank

for a calibration curve. The Gallery 

automatically plots instrument response 

against standard concentration.
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Version 5 Page 21 of 23

Section SM 4500-P E. Thermo Scientific Gallery 

DW o-PO4 Method

Procedure 1. 50 mL sample

2. 8.0 mL combined reagent

3. Incubate 10-30 minutes

4. Absorbance measurement at 880 nm

1. 120 μL sample/standard.

2. Incubate 18 sec.

3. Blank measurement.

4. Add 14 μL Phosphate R1 solution.

5. Incubate 120 sec.

6. Add 6 μL Phosphate R2 solution.

7. Incubate 9 minutes at 37 °C.

8. Measure absorbance at 880 nm.

Data Analysis 

and Calculation

Convert the photometer readings 

obtained with the sample to mg P/L by 

use of the calibration curve. 

The Gallery computes sample 

concentrations as mg P/L by comparing 

sample response with the standard curve.

Single laboratory 

Method 

Performance

MDL: 0.0032 mg P/L

MRL: 0.015 mg P/L

% Recovery IPR: 101 – 102 %

% Recovery CCC: 97 – 104 %

% Recovery LFB: 92 – 104 %

% Recovery LFM: 94 – 101 %

% RSD IPR: 0.4 %

% RSD CCC: 1.49 %

% RSD LFB: 0.29 – 0.96 %

% RSD LFM: 0.24 – 3.0 %

MDL calculated: 0.00036 mg P/L

MRL: 0.0125 mg P/L

% Recovery IPR: 102 – 103%

% Recovery CCC: 100 – 105%

% Recovery LFB: 102 – 103%

% Recovery LFM: 92 – 98 % 

% RSD IPR: 0.1 – 1.0 %

% RSD CCC: 1.3 – 1.9 %

% RSD LFB: 0.2 – 1.1 %

% RSD LFM: 0.1 – 0.7 %

Pollution 

Prevention

N/A Small reagent volumes reduce the 

amount of reagent wastes considered 

dangerous for the environment, 

compared to manual or FIA methods.

1Waste 

Management

N/A Excess reagents, samples and method 

process wastes should be characterized 

and disposed of in an acceptable

manner.
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Section SM 4500-P E. Thermo Scientific Gallery 

DW o-PO4 Method

References Edwards. Molof & Schneeman. 1965. 

Determination of orthophosphate in 

fresh and saline waters. J. Amer. Water 

Works Assoc. 57:917.

Murphy & Riley. 1962. A modified 

single solution method for the 

determination of phosphate in natural 

waters. Anal. Chim. Acta 27:31.

4500-P E. PHOSPHOROUS/Ascorbic 

acid method. Standard Methods for the 

Examination of Water and Wastewater, 

22nd Edition, American Public Health 

Association (APHA), 2012. 

Code of Federal Regulations 40 § 

141.23. Inorganic chemical sampling 

and analytical requirements.

Code of Federal Regulations 40 § 136, 

Appendix B — Definition and 

Procedure for the Determination of the 

Method Detection Limit – Revision 

1.11. 

Protocol for the Evaluation of Alternate 

Test Procedures for Organic and 

Inorganic Analytes in Drinking Water, 

EPA Office of Waters, February 2015.

4020 Quality Assurance/Quality 

Control. Standard Methods for the 

Examination of Water and Wastewater, 

22nd Edition, American Public Health 

Association (APHA), 2012.

1020-B.4 Method Detection Level 

Determination and Application. 

Standard Methods for the Examination 

of Water and Wastewater, 22nd Edition, 

American Public Health Association 

(APHA), 2012.
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Version 5 Page 23 of 23

Table 2. Final ratios in the cuvette.

SM 4500-P E Thermo Scientific Gallery 

DW o-PO4 Method

Final ratios

Combined reagent without Ascorbic acid (70 

mL) : ascorbic acid in Combined reagent (30 mL)

1 : 0.43

Sample : Combined reagent

1 : 0.16

Sample : Comb without Ascorbic acid

1 : 0.112

Sample : Ascorbic acid

1 : 0.05

Final ratios

Phosphate R1 : Phosphate R2 (Ascorbic acid)

1 : 0.43

Sample : R1+R2

1 : 0.17

Sample : Phosphate R1

1 : 0.117

Sample : Phosphate R2 (Ascorbic acid)

1 : 0.05