5. Prepare reagent II (mix reagent) by mixing 22.5 ml molybdate
solution to 60 ml 4.5 mol/l H 2 SO 4 . Add 2.5 ml tartrate
solution and 35 ml ultrapure water. Store in an amber glass
bottle at room temperature (stable for months).
6. Prepare phosphate standard 310 μg P/ml by dissolving 0.1361 g
potassium dihydrogen phosphate (dried for 2 h at 110
C) in
ultrapure water containing 1 ml 4.5 mol/l H 2 SO 4 , and make up
to 100 ml with ultrapure water. Store in a glass bottle in a refrigerator (stable for months).
7. On the day of analysis, prepare suitable working solutions of
phosphate (0–310 μg P/l) for calibration by diluting the standard stock solution (see step 6) with ultrapure water (e.g.,
0.1 ml in 100 ml ultrapure water for 310 μg P/l solution).
8. Prepare analyzer for analyses according to the operation manual (see Note 1). Dilute the reagents 1:10 for analyzer.
9. Place standards in the autosampler of an analyzer, or measure
the standards manually by a spectrophotometer for a calibration curve and linearity test (see Note 2).
10. For manual analyses, measure 35 ml of each calibration standard and 35 ml of ultrapure water for blanks with graduated
cylinder to reaction flasks.
11. Add 1 ml of reagent I to standards and the blanks. Mix well by
swirling.
12. Add 1 ml of reagent II to standards and the blanks. Mix well by
swirling and allow to react for 10 min.
13. Measure the absorbance of standards and blanks in a 5 cm
cuvette at 880 nm for a calibration curve (see Note 2).
14. Collect the samples in glass bottles or polyethylene bottles and
analyze the samples immediately after collection (within half an
hour). The sample can be stored in a glass bottle in a refrigerator for about 2 h.
15. Place samples in the autosampler of an analyzer, or perform
manual analyses with a spectrophotometer. Verify proper operation of the instrument with certified reference material, and
include ultrapure water blanks in the run (see Note 3).
16. For manual analyses, measure two 35 ml portions of the sample
with a graduated cylinder (sample and reference).
17. Add 1 ml of reagent I to both flasks. Mix well by swirling.
18. Add 1 ml of reagent II to the first flask and 1 ml ultrapure water
to the second flask (the reference). Mix well by swirling and
allow to react for 10 min.
19. Measure the absorbance of samples in a 5 cm cuvette at 880 nm
(if the absorbance is greater than 0.6, dilute the sample or use
smaller cuvette), and calculate the phosphate concentration by
the calibration curve (see Note 4).
90
Jaana Koistinen et al.
solution to 60 ml 4.5 mol/l H 2 SO 4 . Add 2.5 ml tartrate
solution and 35 ml ultrapure water. Store in an amber glass
bottle at room temperature (stable for months).
6. Prepare phosphate standard 310 μg P/ml by dissolving 0.1361 g
potassium dihydrogen phosphate (dried for 2 h at 110
C) in
ultrapure water containing 1 ml 4.5 mol/l H 2 SO 4 , and make up
to 100 ml with ultrapure water. Store in a glass bottle in a refrigerator (stable for months).
7. On the day of analysis, prepare suitable working solutions of
phosphate (0–310 μg P/l) for calibration by diluting the standard stock solution (see step 6) with ultrapure water (e.g.,
0.1 ml in 100 ml ultrapure water for 310 μg P/l solution).
8. Prepare analyzer for analyses according to the operation manual (see Note 1). Dilute the reagents 1:10 for analyzer.
9. Place standards in the autosampler of an analyzer, or measure
the standards manually by a spectrophotometer for a calibration curve and linearity test (see Note 2).
10. For manual analyses, measure 35 ml of each calibration standard and 35 ml of ultrapure water for blanks with graduated
cylinder to reaction flasks.
11. Add 1 ml of reagent I to standards and the blanks. Mix well by
swirling.
12. Add 1 ml of reagent II to standards and the blanks. Mix well by
swirling and allow to react for 10 min.
13. Measure the absorbance of standards and blanks in a 5 cm
cuvette at 880 nm for a calibration curve (see Note 2).
14. Collect the samples in glass bottles or polyethylene bottles and
analyze the samples immediately after collection (within half an
hour). The sample can be stored in a glass bottle in a refrigerator for about 2 h.
15. Place samples in the autosampler of an analyzer, or perform
manual analyses with a spectrophotometer. Verify proper operation of the instrument with certified reference material, and
include ultrapure water blanks in the run (see Note 3).
16. For manual analyses, measure two 35 ml portions of the sample
with a graduated cylinder (sample and reference).
17. Add 1 ml of reagent I to both flasks. Mix well by swirling.
18. Add 1 ml of reagent II to the first flask and 1 ml ultrapure water
to the second flask (the reference). Mix well by swirling and
allow to react for 10 min.
19. Measure the absorbance of samples in a 5 cm cuvette at 880 nm
(if the absorbance is greater than 0.6, dilute the sample or use
smaller cuvette), and calculate the phosphate concentration by
the calibration curve (see Note 4).
90
Jaana Koistinen et al.