Experiment 9
Ammonium Nitrogen (NH4+-N)
Aim
To measure ammonium nitrogen (NH₄⁺-N) in a given water sample using the Salicylate Method
(Willis et al., 1996).
Methodology
1. Principle
Ammonia occurs naturally in water bodies due to biological degradation of organic matter and
industrial or agricultural runoff. The Salicylate Method involves the reaction of ammonium
compounds with hypochlorite, forming monochloramine, which further reacts with salicylate to
produce 5-aminosalicylate. This compound undergoes oxidation in the presence of sodium
nitroprusside, forming indosalicylate, a blue-colored complex.
• Trisodium phosphate acts as a buffer to maintain pH.
• Citrate is added to complex magnesium and calcium ions to prevent interference.
• The intensity of the green-colored solution is directly proportional to NH₄⁺-N concentration.
• The sample’s absorbance is measured at 685 nm using a spectrophotometer.
2. Apparatus used
• Glass test tubes
• Pipettes
• Spectrophotometer (wavelength: 685 nm)
3. Reagents used
• Salicylate Reagent:
• 32 g sodium salicylate (anhydrous)
• 40 g trisodium phosphate dodecahydrate (Na₃PO₄·12H₂O)
• 0.5 g sodium nitroprusside
• Dissolve in 1 L water
• Hypochlorite Reagent:
• 50 mL bleach (5-5.25% sodium hypochlorite)
• Dilute to 1 L with water
• Stock Ammonium Solution:
• 3.819 g NH₄Cl (anhydrous, dried at 100°C)
• Dissolve in distilled water and dilute to 1000 mL
4. Experimental Procedure
[Link] Preparation:
• Take 0.2 mL of the sample in a test tube.
[Link] Addition:
•Add 4 mL of salicylate reagent and 1 mL of hypochlorite reagent to the test tube.
•Vortex the test tube after adding each reagent.
[Link] Development:
•Allow the solution to develop color for at least 12 minutes (stable for 18 hours).
[Link] Preparation:
•Use 0.2 mL distilled water instead of the sample for the blank.
[Link] Measurement:
•Measure the absorbance of the sample against the blank at 685 nm using a
spectrophotometer.
[Link] Curve Preparation:
•Prepare a set of standard ammonium concentrations using stock solution and
distilled water.
•Plot absorbance vs. concentration to obtain a standard curve (linear up to 20 ppm).
[Link] Calculation:
•Use the standard curve to determine NH₄⁺-N concentration in the sample.
Observation Table
Sr. no. Sample Absorbance
Results & Discussion
Equation and Graph
Comparison with Existing Standards
• Drinking Water (WHO/BIS): <1.5 mg/L
• Surface Water (Natural Rivers/Lakes): <2 mg/L (good), 2-5 mg/L (moderate pollution)
• Wastewater Discharge (CPCB, India): <5 mg/L for inland surface water discharge
• Toxicity Threshold for Aquatic Life: >10 mg/L can cause severe oxygen depletion
The industrial effluent sample (19.8 mg/L) is well above permissible levels and requires biological
nitrification or ammonia stripping before disposal.
Possible Limitations of the Experimental Approach
• Presence of interfering substances (e.g., organic compounds, heavy metals) may affect color
development.
• Variability in reaction time can cause slight fluctuations in absorbance.
• Manual pipetting errors may lead to concentration miscalculations.
Sources of Error
• Improper calibration of the spectrophotometer.
• Sample contamination or residual reagents in glassware.
• Variability in reagent freshness, affecting reaction efficiency.
Precautions
• Ensure accurate pipetting of reagents and samples.
• Use freshly prepared standards for accurate calibration.
• Avoid excessive exposure of samples to air, as ammonia volatilization can alter results.
• Clean spectrophotometer cuvettes thoroughly to prevent contamination.
Environmental Significance
• Drinking Water Quality: High ammonia levels may indicate sewage contamination or poor
water treatment.
• Eutrophication: Excess ammonia in lakes and rivers promotes algal blooms, depleting oxygen
levels and harming aquatic life.
• Industrial Waste Management: High ammonia in effluent water requires proper treatment before
discharge to prevent toxicity.
• Aquatic Toxicity: Ammonia at concentrations above 10 mg/L is toxic to fish and other aquatic
organisms, disrupting ecosystems.