Thermo Fisher Scientific, A nalyzers & Automation, R&D Industrial diagnostics

Document authors: Marita Jok inen, Annu Suoniemi

- Kähärä , Sari Hartikainen, Katja Laine Corresponding address: marita.jokinen@thermofisher .com

Version 5 Page 1 of 23 

# V ALIDATION 

# R EPORT 

# :

Drinking Water Orthophosphate for Thermo Scientific Gallery discrete analyzer

Name of the method : Drinking Water Orthophosphate

Reference : Standard Meth ods (SM) 4500

- P E. Phosphorous / Ascorbic Acid Method.

EPA a pproved method under 40 C

. F

. R . § 14 1 .23 ( National Primary

Dr inking 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 compariso n between the method and the EPA Approved Method SM

4500

- P E is presented

.

Revision number: 5

Revision date (mm/dd/yyyy) : 02/1 8 /201 6



## 1. Background

1.1 Validation of t his automated method cover s the determination of orthophosphate (CAS: 14265

- 44

- 2 ) in drinking w ater with the Thermo Scientific TM Gallery TM 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

o rthophosphate concentration.

1.4 Only orthophosphate forms a blue color in the test. Polyphosphates (and some organic phosphate compounds) may be conve rted 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 0.0 125

– 0.5 mg PO 4

- P/L. An extension of the range to 5 mg PO 4

- 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

.Thermo Fisher Scientific, A nalyzers & Automation, R&D Industrial diagnostics

Document authors: Marita Jok inen, Annu Suoniemi

- Kähärä , Sari Hartikainen, Katja Laine Corresponding address: marita.jokinen@thermofisher .com

Version 5 Page 2 of 23 1.6 The Drinki ng Water Orthophosphate method for Thermo Scientific Gallery discrete analyzer is equally effective in meeting the QC acceptance criteria given in the reference method. M ore detailed information is presented in table 1.

Table 1

. Performa nce 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

M inimum detectable concentration : 10 μg

P/l

Recovery IPR: NA (laboratory specific)

% Recove ry 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

– 10 3% % 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

- Analyze rs & Automation between Nov 5 th

2015 and Feb 12 th 2016

.

2.2 Analyzers used for the study : T wo T hermo Scientific Gallery, automated discrete photometric analyzers with wavelength 880 nm filters, operated by two users

. Reference method study was

done with Multis kan 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

Standard s for Gallery: THERMO SCIENTIFIC ORDERING NUMBER S 984729 Phospha te standard, 1000 ppm as P (calibration) , lot: S2293, exp: 2015

- 11 984726 Phosp hate standard, 1000 ppm as PO4 ( 2 nd source std ) , lot: S2122 , exp: 2016

- 01

2.4 Samples were collected to 250 ml laboratory glass bo ttles 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), sou rce water: lake

TW

- 04 / Tap water / source water: ground waterThermo Fisher Scientific, A nalyzers & Automation, R&D Industrial diagnostics

Document authors: Marita Jok inen, Annu Suoniemi

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Version 5 Page 3 of 23 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) wer e analyzed to confirm calibration s

. 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. % reco very 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 rea gent blank (LRB). The LFB sample co ncentration 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. T he upper and lower limits for the Prediction Interval of Results (PIR) were

c alculate d and compared to criteria.

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

sam ples 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 sample s (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 th e beginning and end of analysis run and every tenth sample.



## 3. Method Procedure

3.1 Instrument test parame ters are presented in Appendix A

.

3.2 Comparison between Drinking Water Orthophosphate me thod for Galle ry and the reference

method SM 4500

- P E is presented in Appendix B.Thermo Fisher Scientific, A nalyzers & Automation, R&D Industrial diagnostics

Document authors: Marita Jok inen, Annu Suoniemi

- Kähärä , Sari Hartikainen, Katja Laine Corresponding address: marita.jokinen@thermofisher .com

Version 5 Page 4 of 23 

## 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 limi t 5 mg P/L. Dilution limit 0.5 mg P /L.

Sec. dilution 1+9

Calibration Calibration type: 2 nd 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 0.0 3 00 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 10 th , manual

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

Incu bate 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

M ain wavelength 880 nmThermo Fisher Scientific, A nalyzers & Automation, R&D Industrial diagnostics

Document authors: Marita Jok inen, Annu Suoniemi

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Version 5 Page 5 of 23 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 o rthophosphate calibrators , concentrations and responses for DW o

- PO4P

c alibration curve

. Orthophosphate

Calibrator Dilution

1+ Concentration

(mg P /L) Response (A)

PO 4 P

- 0 0 0.00

- 0.0003

PO4 P

- 0 0 0.00

- 0.0002

PO4 P

- 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 o rthophosphate as phosphorous.Thermo Fisher Scientific, A nalyzers & Automation, R&D Industrial diagnostics

Document authors: Marita Jok inen, Annu Suoniemi

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Version 5 Page 6 of 23

Table 3

. Results for o rthophosphate 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 reagen t 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.

MDL est = 3 × (S 0 )

where S 0 = 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

. 0 01861

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

. 0022Thermo Fisher Scientific, A nalyzers & Automation, R&D Industrial diagnostics

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Version 5 Page 7 of 23

NOTE! The MDL est had to be assigned higher than the c alculated MDL est, 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 MDL est

.

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 w as calculated as follows:

MDL = (t) × (S)

where t = Student's t

- value for a 99 % confiden ce 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 Res ult (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 %

av erage = 0

. 007919 79 %

n = 7

std. deviation (SD) = 0

. 000114

% RSD = 4

. 5 %

MDL = 0

. 00036Thermo Fisher Scientific, A nalyzers & Automation, R&D Industrial diagnostics

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Version 5 Page 8 of 23 4.2.3 Minimum Reporting L evel ( MRL )

S even replicate LFBs were analyze d at the proposed MRL concentration.

T he mean ( Mean ) and standard deviation (s) for thes e replicates were calculated

. The Half Range for the Prediction Interval of Results (HR PIR ) was determined using the equation:

HR PIR = 3.963s

Where s = standard deviation

3.963 = constant value for seven replicates

T he u pper and lower limits for th e Prediction Interval of Results (PIR = Mean +HR PIR )

were calculated from the results.

Criteria: The Uppe r 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

HR PIR = 0

. 000921

Upper PIR Limit = 106

. 1 %

Lower PIR Limit = 87

. 7 %

% RSD= 2

. 4 %

Calculate d limits met the criteria and confirmed the MRL to be validated.Thermo Fisher Scientific, A nalyzers & Automation, R&D Industrial diagnostics

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Version 5 Page 9 of 23 4.2.4 Initial Precision and R ecovery (IPR)

F our replicate volumes of reagent water spiked with the analyte of interest (LFB) were analyzed at three different concentrations. % recovery and % RSD w ere calculated.

Res ults 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 %

a verage 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 YESThermo Fisher Scientific, A nalyzers & Automation, R&D Industrial diagnostics

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Version 5 Page 10 of 23 4.2.5 Precision and accuracy from LFB

Precision (%RSD) and accuracy (% Recovery) w ere determined using LFB samples at 0.1, 0.3 and 0.4 mg / L concentrations

. R e s ults are shown in table 8.

Criteria for % recovery: 85

- 115 % , when c is > 2 x MRL a nd

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

avera ge = 0.10257 0.30541 0.40891

n = 10 10 10

std. de v. (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) samp les were prepared by spiking three different tap water samples with ortho phosphate standard solutions to gain spike concentrations of 0.1 and 0.4 mg/L as P for each sample

. The analyte concentration s were high enough to be detected above the original sample and were not less than four times the MDL. The added analyte concentration s 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 uns piked samples by analyzing them and calculating the % Recovery using the next equation. Results for LFM precision and accuracy are shown in table 9.

where C

s = spiked sample concentration

f = spike dilution correction C = sampl e background concentration s = concentration equivalent of analyte added to sample

Note! Because the added spike volume wa s 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.

Crite ria for % recovery: 85

- 115 % , when c is > 2 x MRL and

80

- 120 % , when c is ≤ 2 x MRL

Criteria for % RSD: ±15%Thermo Fisher Scientific, A nalyzers & Automation, R&D Industrial diagnostics

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Version 5 Page 11 of 23

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 10 th
sample, at the beginning and end o f 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

. 003 3 0

. 00 48

% RSD= 1

. 29 % 1

. 87 %

Minimum result: 0

. 249 0

. 252

Maximum result: 0

. 261 0

. 261
Acceptable YES YES

4.3 Reference method performanc e 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.Thermo Fisher Scientific, A nalyzers & Automation, R&D Industrial diagnostics

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Version 5 Page 12 of 23 4.3.3 Same samples and standard stock solutions were use d 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: L aboratory water Sample: V olume 2.5 ml (dispensed manually to test tu be ) Combined reagent: V olume 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 measure d at 880 nm

.

4.3.5 Refere nce 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

Per formance 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. Discuss i on

Compar ison 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. R eference method has a slightly better % recovery for

LFM as the ATP candidate method . The performance is still clearly above the reference v alue ±15 % generally used for drinking water m ethod 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 ort hophosphate 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 resu lts.Thermo Fisher Scientific, A nalyzers & Automation, R&D Industrial diagnostics

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Version 5 Page 13 of 23 

## APPENDIX A.

Test flow for DW o

- PO4P application in the Gallery analyzer.Thermo Fisher Scientific, A nalyzers & Automation, R&D Industrial diagnostics

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Version 5 Page 14 of 23Thermo Fisher Scientific, A nalyzers & Automation, R&D Industrial diagnostics

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Version 5 Page 15 of 23Thermo Fisher Scientific, A nalyzers & Automation, R&D Industrial diagnostics

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Version 5 Page 16 of 23 

## APPENDIX B.

Comparisons of SM 4500

- P E Ascorbic Acid Method for Phosphorous and the Gallery DW o

- PO4P method

Table 1. The differences an d 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. 0.01 2 5

- 0.5 mg P/L . An extension of the range to 5 mg PO 4 - P/L may be achieved

when a 1:10 automated dilution is configured

. Method DW o

- PO4P was tested for drinking water.

Summar y of the

Method Ammonium m olybdate and potassium

antimony tartrate react in acid medium with orthophosph ate to form a heteropoly acid

– phosphomolybdic acid

– that is reduced to intensely colored molybdenum blue by ascorbic acid. Ammonium m olybdate and potassi um 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 a nd

reagent s used. C onsult MSDS for general safety.

E quipment and Supplies Spectrophotometer with infrared

phototube for use at 880 nm o r filter photometer equipped with a red color

filter. Thermo Scientific Gallery discre t e photometric analyzer

. Filter for 880 nm

. DECACELL cuvettes

.Thermo Fisher Scientific, A nalyzers & Automation, R&D Industrial diagnostics

Document authors: Marita Jok inen, Annu Suoniemi

- Kähärä , Sari Hartikainen, Katja Laine Corresponding address: marita.jokinen@thermofisher .com

Version 5 Page 17 of 23 Section SM 4500

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Reagents and

Standards 5 N Sulfuric acid solution

Dilute 70 mL conc H 2 SO 4 to 5 00 mL

with distilled w ater.

Ammonium molybdate solution

Dissolve 20 g (NH 4 ) 6 Mo 7 O 24 ∙4H 2 O in

5 00 mL distilled water.

P otassium antimonyl tartrate solution

Dissolve 1 .3 715 g K(SbO)C 4 H 4 O 6 ∙1⁄2H 2 O 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 H 2 SO 4

. 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 KH 2 PO 4 in

distilled water and dilute to 1000 mL.

1.0 mL = 0.05 mg PO 4 - P. 5 N H 2 SO 4 solution Slowly add 70 mL of conc

. H 2 SO 4 to approxim ately 300 mL of disti lled water.

Cool and dilute to 500 m

L.

Ammonium molybdate solution

Dissolve 2 0 g (NH 4 ) 6 Mo 7 O 24 ∙4H 2 O in

5 00 mL distilled water.

Antimony potassium tartrate solution

Dissolve 1.5 g K(SbO)C 4 H 4 O 6 ∙1⁄2H 2 O in approximately 2 50 mL distilled wat er in

5 00 mL volumetric flask

. D ilute to

volume.

Ascorbic acid = Phosphate R2 Dissolve 1.76 g ascorbic acid in approximately 80 mL distilled water in

100 mL volumetric flask

. D ilute to

volume.

Molybdate Reagent = Phosphate R1

Add 15 mL of Ammoniu m molybd ate
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

Concen tration 1000 mg/L as PO4 (= 326.2 mg/l as P)

.Thermo Fisher Scientific, A nalyzers & Automation, R&D Industrial diagnostics

Document authors: Marita Jok inen, Annu Suoniemi

- Kähärä , Sari Hartikainen, Katja Laine Corresponding address: marita.jokinen@thermofisher .com

Version 5 Page 18 of 23 Section SM 4500

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Sample

Collection

. Preservation

. and Storage In some cases 40 mg HgCl 2 /L may be

added to the samples when they are stored for long periods; not encouraged. Do not store samples containing low

concentration s 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 p lastic 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 bott les. 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

sho u ld 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.Thermo Fisher Scientific, A nalyzers & Automation, R&D Industrial diagnostics

Document authors: Marita Jok inen, Annu Suoniemi

- Kähärä , Sari Hartikainen, Katja Laine Corresponding address: marita.jokinen@thermofisher .com

Version 5 Page 19 of 23 Section SM 4500

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Initial

Demonstration of Capability Initial demonstration of capability

(IDC):

MDL calculati on 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

m eet 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 p recision 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 R eporting L evel (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).

E xternal 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

rep licate volumes of reagent water spiked with analyte (LFB). % recovery: 85

- 115 % or 80

- 120 % when c is ≤ 2 x MRL, %RSD: ±15 %.Thermo Fisher Scientific, A nalyzers & Automation, R&D Industrial diagnostics

Document authors: Marita Jok inen, Annu Suoniemi

- Kähärä , Sari Hartikainen, Katja Laine Corresponding address: marita.jokinen@thermofisher .com

Version 5 Page 20 of 23 Section SM 4500

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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 back

calculated 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 10 th and at the end

.

Method Blank Frequency: daily, every 20 th

. Result < 1/2 x MRL.

Laboratory Fortified Blank (LFB):

Must reach laborator y acceptance
criteria.

Sample Duplicates Frequency: daily, every 20 th . Must reach laboratory acceptance criteria.

LFM and LFMD Frequency: daily, every 20 th . 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 10 th , at beginning and

end of run

.

M ethod Blank Frequency: daily, every 1 0 th

. Res ult < 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 /L FMD ) : 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 wit hin 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.Thermo Fisher Scientific, A nalyzers & Automation, R&D Industrial diagnostics

Document authors: Marita Jok inen, Annu Suoniemi

- Kähärä , Sari Hartikainen, Katja Laine Corresponding address: marita.jokinen@thermofisher .com

Version 5 Page 21 of 23 Section SM 4500

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Procedure 1. 50 mL sample

2. 8.0 mL c ombined rea gent
3. Incubate 10

- 30 minutes

4 . Absorba nce measurement at 880 nm 1. 120 μL sample/standard.

2. Incubate 18 sec

. 3. B lank 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: 0.0 032 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

M RL: 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 S mall reag ent volumes reduce the

amount of reagent wastes considered dangerous for the environment

, compared to manual or FIA methods.

1 Waste Management N/A Excess reagents

, samples and method process wastes should be characterized and disposed of in an acceptable

manner.Thermo Fisher Scientific, A nalyzers & Automation, R&D Industrial diagnostics

Document authors: Marita Jok inen, Annu Suoniemi

- Kähärä , Sari Hartikainen, Katja Laine Corresponding address: marita.jokinen@thermofisher .com

Version 5 Page 22 of 23 Section SM 4500

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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

wa ters. 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 chem ical 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 Orga nic 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.Thermo Fisher Scientific, A nalyzers & Automation, R&D Industrial diagnostics

Document authors: Marita Jok inen, Annu Suoniemi

- Kähärä , Sari Hartikainen, Katja Laine Corresponding address: marita.jokinen@thermofisher .com

Version 5 Page 23 of 23 Table 2. Final ratios in the cuvette.

SM 4500

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Final ratios

Combined reagent without A scorbic acid (70 mL) : ascorbic acid in Combined reagent (30 mL)

1 : 0.43

Sample : Combined reagent

1 : 0.16

Sample : Comb without A scorbic acid

1 : 0.112

Sample : Ascorbic acid

1 : 0. 05 Final ratios

Phosphate R1 : Phosphate R2 (A scorbic acid)

1 : 0.43

Sample : R1+R2

1 : 0.17

Sample : Phosphate R1

1 : 0.117

Sample : Phosphate R2 (A scorbic acid)

1 : 0.05