STANDARD OPERATING PROCEDURE (SOP)
NITRATE TESTING**
1. Purpose
To provide a standardized procedure for accurate determination of nitrate (NO₃⁻) levels in samples (e.g., water, meat, fertilizers, chemicals, effluents) to ensure compliance with quality and regulatory requirements.
2. Scope
This SOP applies to all technicians and analysts performing nitrate testing in the laboratory using the UV-Visible Spectrophotometric Method or Colorimetric Method (Cadmium Reduction / Griess Reaction).
Responsibilities
Analyst: Perform testing as per SOP and record data accurately.
Supervisor/QA: Verify results, maintain calibration, and ensure compliance.
Lab In-charge: Ensure availability of chemicals, apparatus, and documentation.
Apparatus & Equipment
UV-Visible Spectrophotometer (wavelength ~220 nm or 540 nm depending on method)
Colorimeter (if colorimetric method)
Volumetric flasks (10, 50, 100, 1000 mL)
Pipettes and micropipettes
Test tubes and cuvettes
Analytical balance
. Reagents
For UV-Vis Method:
Nitrate standard solution (1000 mg/L)
Hydrochloric acid
Distilled/deionized water
For Colorimetric (Griess) Method:
Sulfanilamide reagent
N-(1-Naphthyl)ethylenediamine dihydrochloride (NED) reagent
Cadmium reduction column (if applicable)
7. Procedure
A. Sample Preparation
Collect sample in a clean bottle.
Filter if turbid (0.45 µm filter).
Dilute if expected nitrate > calibration range.
B. Calibration Standard Preparation
Prepare a series of nitrate standards (e.g., 0, 1, 2, 5, 10 mg/L).
Measure absorbance to create calibration curve.
C. Nitrate Test Procedure
Method 1: UV-Vis Spectrophotometric (220 nm)
Zero the instrument using distilled water.
Measure absorbance at 220 nm and 275 nm for each sample.
Correct for organic interference:
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Determine nitrate concentration using calibration curve.
Method 2: Colorimetric (Griess Method – 540 nm)
Pipette 10 mL sample into a test tube.
Add 0.4 mL sulfanilamide reagent. Mix well.
Add 0.4 mL NED reagent.
Allow color to develop for 10 minutes (pink color).
Measure absorbance at 540 nm using blank reagent.
Determine nitrate concentration using calibration curve.
8. Calculations
Nitrate (mg/L)
Absorbance
Intercept
Slope
Dilution Factor
Nitrate (mg/L)
Slope
Absorbance−Intercept
)×Dilution Factor
9. Quality Control
Run blank, duplicate sample, and control standard daily.
Acceptable recovery: 90–110%.
Recalibrate instrument weekly or as required.
10. Documentation
Record in:
Raw data sheet
Calibration logbook
Final test report
Instrument maintenance log
Ensure results include:
Sample ID
Date/time
Method used
Analyst name
Final nitrate value (mg/L or ppm)
11. References
AOAC Official Methods
ISO 7890 Nitrate Testing Standards
APHA Standard Methods for Water & Wastewater
Purpose
To validate the analytical method for determining Nitrate (NO₃⁻) in samples (water, food, meat, soil, fertilizer, wastewater) and ensure accuracy, precision, reliability, and regulatory compliance.
2. Scope
This validation applies to all analysts performing nitrate analysis using the selected method in an ISO/IEC 17025 laboratory.
Validation Parameters (As per ISO/IEC 17025 & AOAC Guidelines)
The following characteristics must be evaluated:
✔ 4.1 Linearity & Range
Prepare calibration standards covering expected concentration range (e.g., 0.5 to 20 mg/L).
Plot absorbance vs. concentration.
Acceptable criteria:
R² ≥ 0.995
Linearity residuals < 5%
4.5 Specificity / Selectivity
Check interference from:
nitrite
organic matter
turbidity
chloride / sulfate / bicarbonate
Acceptance:
Interference < 10% of signal
UV method requires correction at 275 nm
✔ 4.6 Robustness
Change small parameters:
Wavelength ±2 nm
Reagent volumes ±10%
Reaction time ±1 min
Acceptance:
Recovery change ≤ ±10%
Absorbance deviation ≤ 5%
✔ 4.7 Measurement Uncertainty (MU)
Include contributions from:
calibration
equipment
analyst variability
standards
repeatability
Calculate expanded uncertainty (k=2).
Typical MU: 5–15% depending on method.
5. Validation Procedure Outline
Prepare fresh calibration standards.
Analyze standards in triplicate.
Analyze spiked samples (L, M, H).
Perform repeatability and reproducibility studies.
Conduct robustness tests.
Calculate LOD, LOQ, R², recovery, RSD, and uncertainty.
Compare results to acceptance criteria.
