Inorganic Nutrients
83
NITROGEN COMPOUNDS
Ammonium Nitrogen
Except under very alkaline conditions (pH> 9.0), most of the ammonia (NH3) in fresh
water exists in the ionic form (NH4 +). Ammonium is an important source of nitrogen
for bacteria, algae, and larger plants in lakes and streams, and, because concentrations
commonly are low, the content of water samples can change quickly and markedly.
Thus, samples of lake and stream water should be analyzed as soon as possible after
collection. If analyses are to be delayed for more than a few hours, samples should be
preserved (see below).
A phenol-hypochlorite method using nitroprusside as a catalyst is recommended for
the measurement of ammonium in water (Solorzano, 1969; Harwood and Kuhn, 1970).
Ammonium reacts with phenol and hypochlorite under alkaline conditions to form
indophenol blue. The color development is proportional to the concentration of
ammonium within a given range (0 to 1000 jlg NH4-N/I). Sensitivity is good, with a
standard deviation under carefully controlled conditions of ± 2 to 5 jlg NH4-NjI.
Sample Storage. Samples should be analyzed within a few hours of collection. If
stored, maintain at 5°C. When hydrogen sulfide is present, acidify to pH 3 and bubble
with an inert gas such as helium until free of the H 2S odor.
Reagents. Distilled water that has been passed through an acidic deionization cation
exchange resin should be used throughout. (A void dust and smoking, as serious
contamination from the air can result.)
Caution: Most of the reagents are caustic and very toxic; therefore, no mouth pi petting.
1. Buffer: Na 3 P0 4 (or K 3 P0 4 ) in a 5% (w/v) solution.
2. Phenol stock: 500 g phenol dissolved in methanol and diluted to 800 ml with
methanol. Store under refrigeration in an amber bottle.
3. 27% NaOH: 270g NaOH pellets dissolved in water, cooled, and diluted to 1 liter.
4. Phenate reagent A: 15 ml of phenol stock and 0.02 g sodium nitroprusside are
diluted to 100 ml with water.
5. Reagent B: Mix equal volumes (15 ml) of sodium hypochlorite (fresh commercial
bleach of 5% chlorine will suffice) and 27% NaOH solution and dilute to 50ml with
distilled water.
6. Store both reagents A and B in amber bottles in a refrigerator; allow to reach room
temperature before using.
7. Ammonium standard: Dissolve 3.819 g of dry NH4Cl in distilled-deionized water
and bring to 1 liter. 1.00ml = 1000 jlg NH4-N. Dilute serially for standards.
Procedures. Using 50-ml graduated cylinders,
1. Dispense 50.0 ml of distilled-deionized water (blank), ammonium standards, and
samples into 50-ml graduated cylinders.
2. Add 2ml phosphate buffer; mix (a vortex mixer works well). Ignore any precipitate
that may form.
3. Add 5 ml of reagent A; mix well.
4. Add 2.5 ml of reagent B; mix well.
5. Allow 1 h for color development (should be stable at room temperature for 24 h).
6. Read the optical density at 630 nm, subtracting the O.D. of the blank, using cells of
appropriate path lengths for the color intensity observed (l-cm path length for high
83
NITROGEN COMPOUNDS
Ammonium Nitrogen
Except under very alkaline conditions (pH> 9.0), most of the ammonia (NH3) in fresh
water exists in the ionic form (NH4 +). Ammonium is an important source of nitrogen
for bacteria, algae, and larger plants in lakes and streams, and, because concentrations
commonly are low, the content of water samples can change quickly and markedly.
Thus, samples of lake and stream water should be analyzed as soon as possible after
collection. If analyses are to be delayed for more than a few hours, samples should be
preserved (see below).
A phenol-hypochlorite method using nitroprusside as a catalyst is recommended for
the measurement of ammonium in water (Solorzano, 1969; Harwood and Kuhn, 1970).
Ammonium reacts with phenol and hypochlorite under alkaline conditions to form
indophenol blue. The color development is proportional to the concentration of
ammonium within a given range (0 to 1000 jlg NH4-N/I). Sensitivity is good, with a
standard deviation under carefully controlled conditions of ± 2 to 5 jlg NH4-NjI.
Sample Storage. Samples should be analyzed within a few hours of collection. If
stored, maintain at 5°C. When hydrogen sulfide is present, acidify to pH 3 and bubble
with an inert gas such as helium until free of the H 2S odor.
Reagents. Distilled water that has been passed through an acidic deionization cation
exchange resin should be used throughout. (A void dust and smoking, as serious
contamination from the air can result.)
Caution: Most of the reagents are caustic and very toxic; therefore, no mouth pi petting.
1. Buffer: Na 3 P0 4 (or K 3 P0 4 ) in a 5% (w/v) solution.
2. Phenol stock: 500 g phenol dissolved in methanol and diluted to 800 ml with
methanol. Store under refrigeration in an amber bottle.
3. 27% NaOH: 270g NaOH pellets dissolved in water, cooled, and diluted to 1 liter.
4. Phenate reagent A: 15 ml of phenol stock and 0.02 g sodium nitroprusside are
diluted to 100 ml with water.
5. Reagent B: Mix equal volumes (15 ml) of sodium hypochlorite (fresh commercial
bleach of 5% chlorine will suffice) and 27% NaOH solution and dilute to 50ml with
distilled water.
6. Store both reagents A and B in amber bottles in a refrigerator; allow to reach room
temperature before using.
7. Ammonium standard: Dissolve 3.819 g of dry NH4Cl in distilled-deionized water
and bring to 1 liter. 1.00ml = 1000 jlg NH4-N. Dilute serially for standards.
Procedures. Using 50-ml graduated cylinders,
1. Dispense 50.0 ml of distilled-deionized water (blank), ammonium standards, and
samples into 50-ml graduated cylinders.
2. Add 2ml phosphate buffer; mix (a vortex mixer works well). Ignore any precipitate
that may form.
3. Add 5 ml of reagent A; mix well.
4. Add 2.5 ml of reagent B; mix well.
5. Allow 1 h for color development (should be stable at room temperature for 24 h).
6. Read the optical density at 630 nm, subtracting the O.D. of the blank, using cells of
appropriate path lengths for the color intensity observed (l-cm path length for high