19. Add 2 ml oxidizing reagent solution to the flasks and dissolve
by swirling. Close the flasks and place in the autoclave. Autoclave for 30 min and allow to cool to room temperature.
20. 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).
21. For manual analyses, add 1 ml of reagent I (see step 5) to the
flasks. Mix well by swirling.
22. Add 1 ml of reagent II (see step 6) to the flasks. Mix well by
swirling and allow to react for 10 min.
23. Measure the absorbance of samples in a 5 cm cuvette at 880 nm
(if the absorbance is >0.6, dilute the sample or use 1 cm
cuvette), and calculate the total phosphorus concentration by
the calibration curve (see Note 4).
24. The results provide the total phosphorus for the filtered sample. For determining the dissolved organic phosphorus, subtract the PO 4 concentration (see Sect. 3.1).
3.3 Particulate
Organic Phosphorus
The method is a modified version of Solo ´ rzano and Sharp [5].
1. Prepare glass fiber filters and glass scintillation vials by acid wash
(soak in 6% HCl for 4 h), rinse with ultrapure water, and
combust for at 450
C for 4 h.
2. Prepare sodium sulfate solution (0.17 mol/l) by dissolving
12 g anhydrous Na 2 SO 4 in 500 ml ultrapure water. Store in a
glass bottle at room temperature (stable for several months).
3. Prepare magnesium sulfate solution (0.17 mol/l) by dissolving
42 g MgSO 4 ·7H 2 O in 1 l ultrapure water. Store in a glass bottle
at room temperature (stable for several months).
4. Place the filter in the filtration device, and apply suction with
the vacuum pump.
5. Pipette a known volume of culture through the filter (see Note
5), and for blanks use ultrapure water.
6. Rinse with 4 ml 0.17 mol/l Na 2 SO 4 .
7. Remove the filter with forceps while the suction is on (see Note
6). Rinse the device with ultrapure water between samples.
8. Place the filter in the glass scintillation vial and dry it (see Note
7). For blanks, place pure filters in scintillation vials.
9. Prepare 4.5 mol/l sulfuric acid solution (see Sect. 3.1).
10. Prepare molybdate solution (see Sect. 3.1).
11. Prepare tartrate solution (see Sect. 3.1).
12. Prepare POP reagent I by dissolving 0.7 g C 6 H 8 O 6 in 100 ml
ultrapure water. Store in brown glass bottle in a refrigerator
(usable as long as colorless).
92
Jaana Koistinen et al.
by swirling. Close the flasks and place in the autoclave. Autoclave for 30 min and allow to cool to room temperature.
20. 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).
21. For manual analyses, add 1 ml of reagent I (see step 5) to the
flasks. Mix well by swirling.
22. Add 1 ml of reagent II (see step 6) to the flasks. Mix well by
swirling and allow to react for 10 min.
23. Measure the absorbance of samples in a 5 cm cuvette at 880 nm
(if the absorbance is >0.6, dilute the sample or use 1 cm
cuvette), and calculate the total phosphorus concentration by
the calibration curve (see Note 4).
24. The results provide the total phosphorus for the filtered sample. For determining the dissolved organic phosphorus, subtract the PO 4 concentration (see Sect. 3.1).
3.3 Particulate
Organic Phosphorus
The method is a modified version of Solo ´ rzano and Sharp [5].
1. Prepare glass fiber filters and glass scintillation vials by acid wash
(soak in 6% HCl for 4 h), rinse with ultrapure water, and
combust for at 450
C for 4 h.
2. Prepare sodium sulfate solution (0.17 mol/l) by dissolving
12 g anhydrous Na 2 SO 4 in 500 ml ultrapure water. Store in a
glass bottle at room temperature (stable for several months).
3. Prepare magnesium sulfate solution (0.17 mol/l) by dissolving
42 g MgSO 4 ·7H 2 O in 1 l ultrapure water. Store in a glass bottle
at room temperature (stable for several months).
4. Place the filter in the filtration device, and apply suction with
the vacuum pump.
5. Pipette a known volume of culture through the filter (see Note
5), and for blanks use ultrapure water.
6. Rinse with 4 ml 0.17 mol/l Na 2 SO 4 .
7. Remove the filter with forceps while the suction is on (see Note
6). Rinse the device with ultrapure water between samples.
8. Place the filter in the glass scintillation vial and dry it (see Note
7). For blanks, place pure filters in scintillation vials.
9. Prepare 4.5 mol/l sulfuric acid solution (see Sect. 3.1).
10. Prepare molybdate solution (see Sect. 3.1).
11. Prepare tartrate solution (see Sect. 3.1).
12. Prepare POP reagent I by dissolving 0.7 g C 6 H 8 O 6 in 100 ml
ultrapure water. Store in brown glass bottle in a refrigerator
(usable as long as colorless).
92
Jaana Koistinen et al.