(Consensus Reference Water or equivalent) and include ultrapure water blanks in the run (see Note 3).
15. Calculate DON by subtracting DIN from TDN.
3.4 Particulate
Organic Nitrogen
1. Prepare glass fiber filters by placing them in an acid bath (6%
HCl) for 4 h and rinse then thoroughly with plenty of ultrapure
water (see Note 10).
2. Combust the filters and aluminum circles for 4 h at 450
C and
cool to room temperature.
3. Place a filter in the filtration device and apply suction with the
vacuum pump.
4. Pipette a known volume of culture through the filter (see Note
11).
5. Remove the filter with forceps while the suction is on (see Note
12).
6. Place the filter in the Petri dish, and dry it (see Note 13). For
blanks, place pure glass fiber filters in the Petri dishes.
7. Prepare a standard solution by dissolving 1.340 g glycine in
ultrapure water and fill up to a volume of 100 ml. Store the
stock solution in a glass flask in the refrigerator (stable for
1 month).
8. Prepare standards for calibration of the analyzer by adding a
known volume (e.g., 40 μl) glycine stock solution onto pure
glass fiber filters and dry the filters (see Note 13).
9. Place dried filters on the aluminum circles, one filter per circle.
10. Fold with two clean forceps (cleaned with ethanol and quickly
put into the flame of the Bunsen burner).
11. Make into a small ball with the handheld plier.
12. Put the small balls into a 96-well plate (see Note 14).
13. At the time of the measurement, prepare the CN analyzer for
analyses according to the operation manual (see Note 1).
14. Place the aluminum balls in the autosampler of the CN analyzer (see Note 15). Verify proper operation of the instrument
with certified reference material and include blanks in the run
(see Note 3).
4 Notes
1. It is critical to have proper training before running the analyzer.
This will depend on the system that you are using and it is
beyond the scope of this chapter to go into the details of the
operation of the instrument. We have used Aquakem
78
Jaana Koistinen et al.
15. Calculate DON by subtracting DIN from TDN.
3.4 Particulate
Organic Nitrogen
1. Prepare glass fiber filters by placing them in an acid bath (6%
HCl) for 4 h and rinse then thoroughly with plenty of ultrapure
water (see Note 10).
2. Combust the filters and aluminum circles for 4 h at 450
C and
cool to room temperature.
3. Place a filter in the filtration device and apply suction with the
vacuum pump.
4. Pipette a known volume of culture through the filter (see Note
11).
5. Remove the filter with forceps while the suction is on (see Note
12).
6. Place the filter in the Petri dish, and dry it (see Note 13). For
blanks, place pure glass fiber filters in the Petri dishes.
7. Prepare a standard solution by dissolving 1.340 g glycine in
ultrapure water and fill up to a volume of 100 ml. Store the
stock solution in a glass flask in the refrigerator (stable for
1 month).
8. Prepare standards for calibration of the analyzer by adding a
known volume (e.g., 40 μl) glycine stock solution onto pure
glass fiber filters and dry the filters (see Note 13).
9. Place dried filters on the aluminum circles, one filter per circle.
10. Fold with two clean forceps (cleaned with ethanol and quickly
put into the flame of the Bunsen burner).
11. Make into a small ball with the handheld plier.
12. Put the small balls into a 96-well plate (see Note 14).
13. At the time of the measurement, prepare the CN analyzer for
analyses according to the operation manual (see Note 1).
14. Place the aluminum balls in the autosampler of the CN analyzer (see Note 15). Verify proper operation of the instrument
with certified reference material and include blanks in the run
(see Note 3).
4 Notes
1. It is critical to have proper training before running the analyzer.
This will depend on the system that you are using and it is
beyond the scope of this chapter to go into the details of the
operation of the instrument. We have used Aquakem
78
Jaana Koistinen et al.