| Composition of reagent 1: |
- Sodium salicylate 120 g/L
- Sodium citrate 120 g/L
- Sodium nitroprusside * 2 H20 0.925 g/L
|
| Composition of reagent 2: |
- Sodium hydroxide 32 g/L
- Dichloroisocyanuric acid Sodium salt * 2 H2O 2 g/L
|
- Centrifuge 1 mL of culture in an Eppendorf tube at 14’000 for 12 min.
- Dilute the supernatant with dest. water to reach an ammonium concentration in a measurement range of 0 to 50 mg/L NH4+ (3 mM). (i.e. 50x – 100x dilution in case of E. coli HCD fermentation at late stage)
- Mix 100 μL sample with 200 μL reagent 1 and 200 μL reagent 2 in a 4.5 mL photometric cell with 1 cm thickness. (intensification of green colour with increasing ammonium content)
- After 15 minutes reaction, the mixture is diluted with 3 mL distilled water and measure absorption photometrically at 658 nm.
External standard calibration is used to determine NH4+ concentration in samples. NH4Cl or (NH4)2SO4 can be used standard references preparation (std 1, 5 mg/L; std 2, 15 mg/L; std 3, 30 mg/L; std 4, 50 mg/L).
| Ammonium dihydrogen phosphate |
|
| µM NH4+ |
Absorption at 658nm |
| 43.47 |
0.0267 |
| 130.4 |
0.0768 |
| 260.08 |
0.1548 |
| 434.67 |
0.2634 |
