Nitrate Assay Protocol

Nitrate Assay Protocol

Overview

The nitrate assay uses cadmium reduction followed by the Griess test to measure nitrate (NO₃⁻) concentration in aqueous samples. This two-step colorimetric method first reduces nitrate to nitrite, then produces a pink/purple azo dye whose intensity is proportional to nitrate concentration.

Chemistry

Reaction Mechanism

  1. Reduction Step: Nitrate (NO₃⁻) is reduced to nitrite (NO₂⁻) using cadmium metal in acidic conditions

  2. Diazotization: Nitrite reacts with sulfanilamide under acidic conditions to form a diazonium salt

  3. Coupling: The diazonium salt couples with N-(1-naphthyl)-ethylenediamine (NED) to form a pink/purple azo dye

Chemical Equations

Step 1: Cadmium Reduction

NO₃⁻ + Cd + 2H⁺ → NO₂⁻ + Cd²⁺ + H₂O

Step 2: Griess Reaction

NO₂⁻ + Sulfanilamide + H⁺ → Diazonium salt
Diazonium salt + NED → Pink/Purple Azo Dye (λmax = 540 nm, measured at 528 nm)

Specifications

Parameter Value
Detection Method Colorimetric (cadmium reduction + Griess)
Wavelength 528 nm (green LED)
Measurement Range 0-90 ppm NO₃ (can measure 0-40 ppm with API kit)
Reaction Time Minimum 5 minutes
Color Pink/Purple (intensity ∝ concentration)
pH Range Acidic (reagents adjust pH)

Reagents

Required Materials

  1. API Nitrate Test Kit
    • Bottle #1: Cadmium reduction reagent
    • Bottle #2: Griess reagent (sulfanilamide + NED)
    • Important: Shake Bottle #2 vigorously before use (contains suspension)
    • Typical shelf life: Check expiration date
  2. Nitrate Standard
    • 1,000 ppm N stock (equivalent to 4,430 ppm as NO₃)
    • Commercially available or prepare from KNO₃
    • Store in dark at room temperature
  3. Distilled or RO Water
    • For dilutions and blanks
    • Must be nitrate-free

Preparing Calibration Standards

Working Stock (1,000 ppm NO₃)

From 4,430 ppm (1,000 ppm N) standard:

  • Add 4.51 mL of 4,430 ppm standard to a 50 mL tube
  • Add 15.49 mL distilled water
  • Mix thoroughly
  • Final volume: 20 mL at 1,000 ppm NO₃
  • Label and date

Intermediate Stock (250 ppm NO₃)

From 1,000 ppm working stock:

  • Add 6.25 mL of 1,000 ppm stock to a 25 mL volumetric flask
  • Dilute to 25 mL with distilled water
  • Mix thoroughly

Calibration Series

From 250 ppm intermediate stock, prepare:

Standard Concentration (ppm NO₃) 250 ppm Stock Distilled Water Total Volume
Blank 0 0 mL 25 mL 25 mL
S1 5 0.50 mL 24.50 mL 25 mL
S2 10 1.00 mL 24.00 mL 25 mL
S3 20 2.00 mL 23.00 mL 25 mL
S4 40 4.00 mL 21.00 mL 25 mL
S5 60 6.00 mL 19.00 mL 25 mL
S6 80 8.00 mL 17.00 mL 25 mL
S7 90 9.00 mL 16.00 mL 25 mL

Note: Standards should be prepared fresh for calibration. Can be stored for up to 2 weeks in dark bottles at 4°C.

Manual Protocol

Equipment Needed

  • Cuvettes (glass or plastic)
  • Pipettes (accurate to 0.1 mL)
  • Sample bottles with caps (for mixing)
  • Colorimeter with 528 nm capability (green LED)
  • Timer

Step-by-Step Procedure

  1. Sample Preparation
    • Fill sample bottle to 5 mL mark with water sample
    • If sample is turbid, filter or allow to settle
    • Record sample ID and time
  2. Add Reagent #1 (Cadmium Reduction)
    • Add 10 drops of Bottle #1 to sample
    • Cap and invert 3-4 times to mix
    • Solution should remain clear
  3. Prepare Reagent #2
    • SHAKE BOTTLE #2 VIGOROUSLY for 30-60 seconds
    • Reagent contains a suspension that must be mixed
    • Inadequate shaking will result in low readings
  4. Add Reagent #2 (Griess Reagent)
    • Add 10 drops of Bottle #2
    • Shake sample thoroughly (more vigorous than typical)
    • Solution will begin to turn pink/purple
    • START TIMER
  5. Incubation
    • Wait at least 5 minutes for color development
    • Color is stable after 5 minutes
    • Can be read anytime after 5 minutes (no upper time limit like phosphate)
  6. Measurement (After 5+ minutes)
    • Transfer sample to clean cuvette
    • Wipe cuvette exterior
    • Insert into colorimeter
    • Read absorbance at 528 nm (green LED)
    • Record reading
  7. Data Recording
    • Note absorbance value
    • Note time since Reagent #2 addition
    • Compare to calibration curve

Key Differences from Phosphate Assay

Feature Phosphate Nitrate
Reagent drops 6 drops each 10 drops each
Bottle #2 prep No special prep MUST shake 30-60 sec
Mixing intensity Gentle inversion Vigorous shaking
Timing window 5-10 min (strict) 5+ min (flexible)
Color stability Becomes cloudy after 10 min Stable indefinitely

Automated Protocol (OpenReef System)

System Requirements

  • Calibrated colorimeter at 528 nm (green LED)
  • Peristaltic pumps calibrated for 10-drop equivalent volumes (bidirectional)
  • Bubble mixing via pump reversal (vigorous for nitrate)
  • Reagent bottle bubble mixing for Bottle #2 (to suspend cadmium particles)

Automated Sequence

1. System Cleaning
   └─ Flush with RO water → Waste

2. Sample Loading
   └─ Fill cuvette (5 mL test water)

3. Baseline Measurement
   └─ Zero colorimeter with sample

4. Reagent #2 Preparation
   └─ Bubble mix Reagent #2 bottle (5s reverse, 10s settle)

5. Reagent #1 Addition
   ├─ Pump 10 drops equivalent (~0.50 mL)
   ├─ Clear reagent line
   └─ Bubble mix (3s reverse, 5s settle)

6. Reagent #2 Addition
   ├─ Pump 10 drops equivalent (~0.50 mL)
   ├─ Clear reagent line
   ├─ START TIMER
   └─ Vigorous bubble mix (5s reverse, 10s settle)

7. Incubation
   └─ Wait 5 minutes minimum

8. Measurement
   ├─ Read absorbance @ 528 nm
   └─ Calculate concentration from calibration

9. System Cleaning
   └─ Flush with RO water → Waste

Total Time: ~10 minutes

Critical: Reagent #2 Bottle Mixing

The automated system MUST bubble mix the Reagent #2 bottle before each use:

  • Reverse pump for 5 seconds to create vigorous bubbles
  • Allow 10 seconds for bubbles to suspend cadmium particles
  • Bubble mixing can be repeated between tests if needed
  • Without proper mixing, the cadmium particles settle, causing low readings

Pump Calibration for Reagents

API test kit “drops” must be converted to volume:

  • Measure volume of 10 drops from each reagent bottle
  • Calculate average drop volume
  • Typical: 1 drop ≈ 0.05 mL
  • For 10 drops: ~0.50 mL per reagent
  • Calibrate peristaltic pump to dispense accurate volume

Interpreting Results

Calibration Curve

Plot absorbance (y-axis) vs. concentration (x-axis) for standards:

  • Should be linear in 0-90 ppm range
  • R² > 0.99 for good calibration
  • May show slight non-linearity at high concentrations
  • Use linear regression: y = mx + b
    • y = absorbance
    • x = concentration (ppm NO₃)
    • m = slope (sensitivity)
    • b = y-intercept (blank absorbance)

Calculating Concentration

From measured absorbance (A):

Concentration (ppm NO₃) = (A - b) / m

Where:

  • A = measured absorbance
  • b = y-intercept from calibration
  • m = slope from calibration

Quality Control

Valid Result Criteria:

  • Absorbance within calibrated range
  • Measurement taken after 5 minutes
  • Blank absorbance < 0.050
  • Duplicate samples within 15% RSD (higher tolerance than phosphate)

Duplicate Measurements:

  • Always run samples in duplicate
  • Average the two readings
  • If difference > 15%, run a third replicate

Out of Range:

  • If absorbance > calibration max: dilute sample 1:10 and re-test
  • If absorbance < 0.010: may be below detection limit

Detection Limits

  • Limit of Detection (LOD): ~0.2 ppm NO₃
  • Limit of Quantification (LOQ): ~0.5 ppm NO₃
  • Optimal Range: 1-80 ppm NO₃

Troubleshooting

Issue Cause Solution
No color development Expired reagents Replace API kit
  Bottle #2 not shaken Shake vigorously 30-60 sec
  Wrong reagent order Ensure Bottle #1 before #2
Very weak color Bottle #2 settled Shake Bottle #2 before EVERY use
  Insufficient mixing Shake sample vigorously
  Low nitrate Normal - within sensitivity
Inconsistent results Bottle #2 not mixed properly Always shake Bottle #2
  Temperature variation Control temperature (20-25°C)
  Dirty cuvettes Clean with dilute HCl, rinse well
Results lower than expected Cadmium particles settled Shake Bottle #2 longer
  Reagent degradation Replace Bottle #2
Color fades over time Normal for very low concentrations Read within 30 min
Precipitate in reagent bottle Cadmium particles (normal) Shake vigorously before use
High blank readings Contaminated distilled water Use fresh nitrate-free water
  Reagent contamination Prepare fresh blank

Interferences

Positive Interferences (False High)

  • Nitrite (NO₂⁻): measured directly by Griess reaction (adds to reading)
  • High chloride (> 1,000 ppm): can interfere with cadmium reduction
  • Iron (Fe²⁺): can interfere with color development

Negative Interferences (False Low)

  • High sulfide: inhibits Griess reaction
  • EDTA or other chelators: bind cadmium, prevent reduction

Mitigation

  • For nitrite interference: measure nitrite separately and subtract
  • Dilute high-salinity samples (1:10 or 1:100)
  • Remove sulfide by purging or oxidation before analysis

Safety

Reagent Hazards

Bottle #1 (Cadmium Reduction):

  • Contains cadmium metal (toxic heavy metal)
  • Wear gloves and eye protection
  • Avoid ingestion and inhalation
  • Handle in well-ventilated area

Bottle #2 (Griess Reagent):

  • Contains sulfanilamide and NED
  • Mild irritant
  • Avoid skin contact

Waste Disposal

⚠️ IMPORTANT: This test uses cadmium, which is a toxic heavy metal.

  • DO NOT dispose of waste down the drain
  • Collect all waste in a designated cadmium waste container
  • Dispose as hazardous chemical waste
  • Follow local environmental regulations
  • Contact your institution’s EH&S or local hazardous waste facility

Cadmium Exposure Prevention

  • Wear nitrile gloves when handling reagents
  • Work in well-ventilated area
  • Wash hands thoroughly after testing
  • Do not eat, drink, or smoke while performing assay
  • Clean work surfaces after use

References

  1. APHA Standard Method 4500-NO₃⁻ B: Cadmium Reduction Method
  2. Griess, P. (1879) Bemerkungen zu der Abhandlung der HH. Weselsky und Benedikt
  3. API Nitrate Test Kit Instructions
  4. IoRodeo Nitrate Protocol: https://sites.google.com/iorodeo.com/biorodeo/nitrate

Appendix: Conversion Factors

Nitrate Units

Different reporting conventions:

NO₃⁻ (as NO₃) ÷ 4.427 = N (as N)
N (as N) × 4.427 = NO₃⁻ (as NO₃)

Example:

  • 44.3 ppm NO₃ = 10.0 ppm N
  • 10.0 ppm N = 44.3 ppm NO₃

Common Reporting Formats

Unit Conversion
ppm NO₃ As measured
ppm NO₃-N Divide by 4.427
mg/L NO₃ Same as ppm NO₃
mg/L N Divide by 4.427
mM NO₃ Divide by 62.0

Standard Preparation

To prepare 1,000 ppm N (4,430 ppm NO₃) from KNO₃:

1,000 ppm N = 7,218 mg KNO₃ per liter

Calculation:
MW(KNO₃) = 101.1 g/mol
MW(N) = 14.0 g/mol

(1,000 mg N/L) × (101.1/14.0) = 7,218 mg KNO₃/L

Procedure:

  1. Dissolve 7.218 g KNO₃ in ~800 mL distilled water
  2. Add 2 mL chloroform (preservative, optional)
  3. Dilute to 1 liter
  4. Store in dark bottle, stable for 6 months

Comparison: Nitrate vs Nitrite

The Griess test actually measures nitrite (NO₂⁻), not nitrate directly. The cadmium reduction step converts nitrate to nitrite.

Species Measured By Notes
NO₃⁻ (Nitrate) Cadmium reduction + Griess Total test (this protocol)
NO₂⁻ (Nitrite) Griess only (skip Bottle #1) Direct measurement

To measure nitrite only:

  • Skip Bottle #1
  • Add only Bottle #2
  • Result is nitrite concentration

To calculate nitrate only:

  • Run test with both bottles (total = NO₃ + NO₂)
  • Run test with only Bottle #2 (nitrite only)
  • Nitrate = Total - Nitrite