This document describes a method for measuring reactive phosphorus in water and wastewater samples using an amino acid method. The method involves reacting the sample with molybdate reagent and amino acid reagent to produce a colored complex, then measuring the absorbance. The document provides details on the test procedure, potential interferences, accuracy checking, and method performance.
This document describes a method for measuring reactive phosphorus in water and wastewater samples using an amino acid method. The method involves reacting the sample with molybdate reagent and amino acid reagent to produce a colored complex, then measuring the absorbance. The document provides details on the test procedure, potential interferences, accuracy checking, and method performance.
This document describes a method for measuring reactive phosphorus in water and wastewater samples using an amino acid method. The method involves reacting the sample with molybdate reagent and amino acid reagent to produce a colored complex, then measuring the absorbance. The document provides details on the test procedure, potential interferences, accuracy checking, and method performance.
This document describes a method for measuring reactive phosphorus in water and wastewater samples using an amino acid method. The method involves reacting the sample with molybdate reagent and amino acid reagent to produce a colored complex, then measuring the absorbance. The document provides details on the test procedure, potential interferences, accuracy checking, and method performance.
Scope and application: For water, wastewater and seawater.
1
Adapted from Standard Methods for the Examination of Water and Wastewater.
Test preparation Instrument-specific information Table 1 shows all of the instruments that have the program for this test. The table also shows sample cell and orientation requirements for reagent addition tests, such as powder pillow or bulk reagent tests. To use the table, select an instrument, then read across to find the applicable information for this test. Table 1 Instrument-specific information Instrument
Sample cell orientation
Sample cell
DR 6000
The fill line is to the right.
2495402
DR 3800 DR 2800 DR 2700 DR 1900 DR 5000
The fill line is toward the user.
DR 3900 DR 900
The orientation mark is toward the user.
2401906
Before starting Install the instrument cap on the DR 900 cell holder before ZERO or READ is pushed. For the best results, measure the reagent blank value for each new lot of reagent. Replace the sample with deionized water in the test procedure to determine the reagent blank value. Subtract the reagent blank value from the sample results automatically with the reagent blank adjust option. The contents of one Amino Acid Reagent Powder Pillow can be used as an alternative to the 1 mL of Amino Acid Reagent Solution in the test procedure. Review the Safety Data Sheets (MSDS/SDS) for the chemicals that are used. Use the recommended personal protective equipment. Dispose of reacted solutions according to local, state and federal regulations. Refer to the Safety Data Sheets for disposal information for unused reagents. Refer to the environmental, health and safety staff for your facility and/or local regulatory agencies for further disposal information.
Items to collect Description
Quantity
Amino Acid Reagent
1 mL
Cylinder, 25-mL, graduated mixing
Molybdate Reagent
1 mL
Sample cells (For information about sample cells, adapters or light shields, refer to Instrumentspecific information on page 1.)
Refer to Consumables and replacement items on page 5 for order information.
Sample collection and storage
Collect samples in clean glass or plastic bottles that have been cleaned with 6 N (1:1) hydrochloric acid and rinsed with deionized water. Do not use a detergent that contains phosphate to clean the sample bottles. The phosphate in the detergent will contaminate the sample. Analyze the samples as soon as possible for best results. If immediate analysis is not possible, immediately filter and keep the samples at or below 6 C (43 F) for a maximum of 48 hours. Let the sample temperature increase to room temperature before analysis.
Powder pillow procedure
Start
1. Start program 485 P
React. Amino. For information about sample cells, adapters or light shields, refer to Instrumentspecific information on page 1. Note: Although the program name can be different between instruments, the program number does not change.
mixing cylinder. Invert the mixing cylinder several times to mix. A blue color shows if phosphate is present in the sample.
6. Start the instrument
timer. A 10-minute reaction time starts.
7. Prepare the blank: Fill a
sample cell with 10 mL of untreated sample.
8. When the timer expires,
clean the blank sample cell.
11. Fill a second sample
cell with 10-mL of the prepared sample.
12. Clean the prepared
sample cell.
Prepare the blank while the
timer is counting down.
Zero
9. Insert the blank into the
cell holder.
10. Push ZERO. The
display shows 0.00 mg/L PO43.
Read
13. Insert the prepared
sample into the cell holder.
14. Push READ. Results
show in mg/L PO43.
Interferences Interfering substance
Interference level
Calcium
More than 10,000 mg/L as CaCO3
Chloride
More than 150,000 mg/L Cl
Colored samples
Add 1 mL of 10 N Sulfuric Acid Standard Solution to another 25-mL sample. Use this instead of untreated sample as the blank to zero the instrument. Use a pipet and pipet filler to measure the sulfuric acid standard.
High salt levels (Na+)
May cause low results. To eliminate this interference, dilute the sample until two successive dilutions give about the same result.
Bleaches the blue color. Remove nitrite interference by adding 0.10 g of sulfamic acid to 50 mL sample. Swirl to mix. Use this treated sample in the test procedure.
Phosphates, high levels (PO43)
As the concentration of phosphate increases, the color changes from blue to green, then to yellow and finally to brown. The brown color may suggest a concentration as high as 100,000 mg/L PO43. If a color other than blue is formed, dilute the sample and retest.
Sulfide (S2)
Sulfide interferes. For samples with a sulfide concentration less than 5 mg/L, remove sulfide interference as follows: 1. 2. 3.
Add 50 mL of sample to an Erlenmeyer flask.
Add Bromine Water by drops with constant swirling until a permanent yellow color develops. Add Phenol Solution by drops until the yellow color just disappears. Use this treated sample in the test procedure.
Temperature
For best results, sample temperature should be 21 3 C (70 5 F).
Turbidity
May give inconsistent results for two reasons. Some suspended particles may dissolve because of the acid used in the test. Also, desorption of orthophosphate from particles may occur. For highly turbid samples, add 1 mL of 10 N Sulfuric Acid Standard Solution to another 25-mL sample. Use this instead of untreated sample as the blank to zero the instrument. Use a pipet and pipet filler to measure the sulfuric acid standard.
Highly buffered samples or extreme sample pH
Can prevent the correct pH adjustment of the sample by the reagents. Sample pre-treatment may be necessary.
Accuracy check Standard additions method (sample spike) Use the standard additions method (for applicable instruments) to validate the test procedure, reagents and instrument and to find if there is an interference in the sample. Items to collect:
Phosphate 2-mL Ampule Standard, 500-mg/L PO43
Ampule breaker Pipet, TenSette, 0.11.0 mL and tips Mixing cylinders, 25-mL (3)
1. Use the test procedure to measure the concentration of the sample, then keep the (unspiked) sample in the instrument. 2. Go to the Standard Additions option in the instrument menu. 3. Select the values for standard concentration, sample volume and spike volumes. 4. Open the standard solution. 5. Prepare three spiked samples: use the TenSette pipet to add 0.1 mL, 0.2 mL and 0.3 mL of the standard solution, respectively, to three 25-mL portions of fresh sample. Mix well. 6. Use the test procedure to measure the concentration of each of the spiked samples. Start with the smallest sample spike. Measure each of the spiked samples in the instrument. 7. Select Graph to compare the expected results to the actual results. Note: If the actual results are significantly different from the expected results, make sure that the sample volumes and sample spikes are measured accurately. The sample volumes and sample spikes that are used should agree with the selections in the standard additions menu. If the results are not within acceptable limits, the sample may contain an interference.
Use the standard solution method to validate the test procedure, the reagents and the instrument. Items to collect:
10-mg/L Phosphate Standard Solution
1. Use the test procedure to measure the concentration of the standard solution. 2. Compare the expected result to the actual result. Note: The factory calibration can be adjusted slightly with the standard adjust option so that the instrument shows the expected value of the standard solution. The adjusted calibration is then used for all test results. This adjustment can increase the test accuracy when there are slight variations in the reagents or instruments.
Method performance The method performance data that follows was derived from laboratory tests that were measured on a spectrophotometer during ideal test conditions. Users can get different results under different test conditions. Program
Standard
Precision (95% confidence interval)
Sensitivity Concentration change per 0.010 Abs change
485
10.00 mg/L PO43
9.8610.14 mg/L PO43
0.20 mg/L PO43
Summary of method In a highly acidic solution, ammonium molybdate reacts with orthophosphate to form molybdophosphoric acid. This complex is then reduced by the amino acid reagent to yield an intensely colored molybdenum blue compound. The measurement wavelength is 530 nm for spectrophotometers or 520 nm for colorimeters.
Consumables and replacement items
Required reagents Description
Quantity/test
Unit
Item no.
100 tests
2244100
Amino Acid Reagent
1 mL
100 mL MDB
193432
Molybdate Reagent
1 mL
100 mL MDB
223632
Quantity/test
Unit
Item no.
each
189640
Unit
Item no.
946 mL
1420416
Phosphate Standard Solution, 2-mL PourRite Ampule, 500-mg/L PO43
16/pkg
1424220
Wastewater Effluent Standard Solution, Mixed Parameter, for NH3-N, NO3-N, PO43, COD, SO42, TOC
500 mL
2833249
Wastewater Influent Standard Solution, Mixed Parameter, for NH3-N, NO3-N, PO4, COD, SO4, TOC
Phosphate Standard Solution, 50-mg/L, 10-mL Voluette Ampules
16/pkg
17110
Phosphate Standard Solution, 100-mg/L as PO4
100 mL
1436832
Phosphate Standard Solution, 10-mL ampule, 500 mg/L as PO4
16/pkg
1424210
Phosphate Standard Solution, 500-mg/L as PO4
100 mL
1424232
Pipet tips for TenSette Pipet, 0.11.0 mL
Bottle, sampling, with cap, low density polyethylene, 250-mL
Optional standards Description
Ampule Breaker, 10-mL Voluette Ampules
Phosphate Standard Solution, 3-mg/L as PO43 Phosphate Standard Solution, 15-mg/L as PO4 Phosphate Standard Solution, 30-mg/L as PO4
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