Phosphate Assay Protocol

Phosphate Assay Protocol

Overview

The phosphate assay uses the ascorbic acid method (APHA Standard Method 4500-P E) to measure orthophosphate (PO₄³⁻) concentration in aqueous samples. This colorimetric method produces a blue color whose intensity is proportional to phosphate concentration.

Chemistry

Reaction Mechanism

  1. Complex Formation: Orthophosphate reacts with ammonium molybdate and potassium antimonyl tartrate to form a heteropoly acid (phosphomolybdic acid)

  2. Reduction: Ascorbic acid reduces the phosphomolybdic acid complex to form an intensely blue-colored molybdenum blue compound

  3. Color Development: The blue color intensity is directly proportional to phosphate concentration

Chemical Equation (Simplified)

PO₄³⁻ + Mo⁶⁺ + Sb³⁺ → [PMo₁₂O₄₀]³⁻ (phosphomolybdate complex)
                         ↓ Ascorbic acid
                    Molybdenum Blue (λmax = 880 nm optimal, 625 nm acceptable)

Specifications

Comparison of Test Kits

Parameter API Phosphate Kit Red Sea Phosphate Pro
Detection Method Colorimetric (ascorbic acid) Colorimetric (ascorbic acid)
Optimal Wavelength 880 nm (optimal), 625 nm (API kit uses red LED) 880 nm (IR LED for maximum sensitivity)
Measurement Range 0-30 ppm PO₄ 0-5 ppm PO₄
Optimal Range 0.5-25 ppm 0-1 ppm
Accuracy ±1 ppm ±0.02 ppm
Precision ~0.5 ppm 0.01 ppm
LOD ~0.05 ppm ~0.02 ppm
LOQ ~0.10 ppm ~0.05 ppm
Reaction Time 5-10 minutes Per kit instructions
Color Blue (molybdenum blue) Light blue (molybdenum blue)
Tests per Kit ~150 100
Best Use General aquarium (0-10 ppm) Ultra-low reef (0-1 ppm)

Reagents

Required Materials

  1. API Phosphate Test Kit
    • Bottle #1: Molybdate reagent
    • Bottle #2: Ascorbic acid reagent
    • Typical shelf life: Check expiration date
  2. Phosphate Standard
    • 1,000 ppm PO₄ stock (330 ppm as P)
    • Commercially available or prepare from KH₂PO₄
    • Store in dark at room temperature
  3. Distilled or RO Water
    • For dilutions and blanks
    • Must be phosphate-free

Preparing Calibration Standards

Working Stock (37.5 ppm PO₄)

From 1,000 ppm standard:

  • Add 3.75 mL of 1,000 ppm standard to a 100 mL volumetric flask
  • Dilute to 100 mL with distilled water
  • Mix thoroughly
  • Label and date

Calibration Series

From 37.5 ppm working stock, prepare:

Standard Concentration (ppm PO₄) Working Stock Distilled Water Total Volume
Blank 0 0 mL 25 mL 25 mL
S1 1.0 0.67 mL 24.33 mL 25 mL
S2 2.5 1.67 mL 23.33 mL 25 mL
S3 5.0 3.33 mL 21.67 mL 25 mL
S4 10.0 6.67 mL 18.33 mL 25 mL
S5 15.0 10.0 mL 15.0 mL 25 mL
S6 20.0 13.33 mL 11.67 mL 25 mL
S7 30.0 20.0 mL 5.0 mL 25 mL

Note: Standards should be prepared fresh for calibration. Can be stored for up to 1 week in dark bottles at 4°C.

Red Sea Phosphate Pro Protocol

Overview

The Red Sea Phosphate Pro test kit is designed for ultra-low phosphate measurements in reef aquariums, providing exceptional accuracy (±0.02 ppm) in the 0-1 ppm range where reef systems typically operate.

Hardware Requirements

LED Configuration

  • Optimal: 880 nm infrared LED (maximum sensitivity)
  • Alternative: 710-720 nm (95% sensitivity)
  • Not recommended: 625 nm red LED (only 30-40% sensitivity)

Sensor Setup (TSL2591)

  • Use Channel 1 (IR photodiode) for 880 nm LED
  • IR channel has good response in 650-950 nm range
  • Peak sensitivity: 800-850 nm

Preparing Reference Standards (0-1 ppm range)

Stock Solution (100 ppm PO₄)

Materials:

  • Potassium phosphate monobasic (KH₂PO₄, anhydrous, ACS grade)
  • Distilled or RO/DI water
  • 1000 mL volumetric flask
  • Analytical balance (0.0001 g precision)

Preparation:

  1. Weigh 0.1433 g KH₂PO₄ (anhydrous)
    • MW KH₂PO₄ = 136.09 g/mol
    • MW PO₄³⁻ = 94.97 g/mol
    • Conversion: 136.09 / 94.97 = 1.433
  2. Dissolve in ~500 mL distilled water
  3. Dilute to 1000 mL mark
  4. Mix thoroughly (invert 20 times)
  5. Store refrigerated at 4°C (stable 6 months)

Intermediate Solution (10 ppm PO₄) - Recommended

  1. Pipette 10.00 mL of 100 ppm stock
  2. Transfer to 100 mL volumetric flask
  3. Fill to mark with distilled water
  4. Mix thoroughly
  5. Store refrigerated (stable 1 month)

Working Standards (0-1 ppm)

From 10 ppm intermediate solution:

Concentration 10 ppm Stock Distilled Water Total Volume
0.00 ppm 0.0 mL 100 mL 100 mL
0.10 ppm 1.0 mL 99.0 mL 100 mL
0.25 ppm 2.5 mL 97.5 mL 100 mL
0.50 ppm 5.0 mL 95.0 mL 100 mL
0.75 ppm 7.5 mL 92.5 mL 100 mL
1.00 ppm 10.0 mL 90.0 mL 100 mL

Storage: Prepare fresh daily for best accuracy. If stored, refrigerate and use within 1 week.

Calibration Procedure

  1. Blank the colorimeter with distilled water
  2. Measure each standard (3-6 replicates per concentration)
    • Add Red Sea Pro reagents per kit instructions
    • Wait for color development
    • Record absorbance value
  3. Calculate fit coefficients using linear regression
    • Plot concentration (ppm) vs absorbance
    • Should be linear with R² > 0.995
  4. Update calibration file with new coefficients

Expected Performance

With 880 nm LED:

  • Linearity: R² > 0.995
  • Precision: 2-5% RSD
  • Sensitivity: ~3x better than 625 nm LED
  • Detection limit: 0.02 ppm PO₄

Software Configuration

Modify src/light_sensor.py

Change to use IR channel for 880 nm LED:

self.channel = 1  # Use IR channel (1) for 880nm LED

Update calibrations.json

{
  "Phosphate Red Sea Pro": {
    "units": "ppm",
    "led": "880",
    "fit_type": "polynomial",
    "fit_coef": [
      3.521845,
      -0.012456
    ],
    "range": {
      "min": 0.00,
      "max": 0.65
    }
  }
}

Note: fit_coef values shown are examples. Perform calibration to determine actual coefficients for your system.

Quality Control

Method Blank:

  • Distilled water processed with reagents
  • Should read 0.00 ± 0.01 ppm

Continuing Calibration Verification (CCV):

  • Run 0.50 ppm standard every 10 samples
  • Should be within ±10% of expected value

Duplicate Samples:

  • Run 1 duplicate per 10 samples
  • RPD < 10%

Commercial Reference Standards (Alternative)

If preparing standards is impractical:

Hanna Phosphate Standards:

  • HI774-11: 0.00 ppm (blank)
  • HI774-12: 0.25 ppm
  • HI774-13: 0.50 ppm

NIST Traceable Standards:

  • Available from scientific suppliers
  • Certified accuracy
  • Typical: 0.1, 0.5, 1.0, 5.0 ppm PO₄

Troubleshooting Red Sea Pro

Issue Cause Solution
Readings negative Calibration intercept too high Re-calibrate with fresh standards
Poor repeatability (>10% RSD) Inconsistent mixing/timing Control temperature, use timer
Non-linear curve Reagent issues, LED saturation Fresh reagents, reduce integration time
Drift over time Reagent degradation Re-calibrate monthly
Doesn’t match comparator Different timing Ensure exact same reaction time

Best Practices

  • Always blank before measurements
  • Control temperature (20-25°C)
  • Follow exact reaction timing
  • Filter turbid samples
  • Store reagents cool and dark
  • Check expiration dates
  • Re-calibrate monthly

Manual Protocol (API Kit)

Equipment Needed

  • Cuvettes (glass or plastic)
  • Pipettes (accurate to 0.1 mL)
  • Sample bottles with caps (for mixing)
  • Colorimeter with 625 nm capability
  • 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 (Molybdate)
    • Add 6 drops of Bottle #1 to sample
    • Cap and invert 3-4 times to mix
    • Solution should be clear
  3. Add Reagent #2 (Ascorbic Acid)
    • Add 6 drops of Bottle #2
    • START TIMER immediately
    • Cap and invert 3-4 times to mix thoroughly
    • Solution will begin to turn blue
  4. Incubation
    • Wait 5-10 minutes for color development
    • Color develops progressively
    • CRITICAL: Do not exceed 10 minutes
  5. Measurement Window (5-10 minutes after Reagent #2)
    • Transfer sample to clean cuvette
    • Wipe cuvette exterior
    • Insert into colorimeter
    • Read absorbance at 625 nm
    • Record reading
  6. Data Recording
    • Note absorbance value
    • Note exact time since Reagent #2 addition
    • Compare to calibration curve

Critical Timing Warning

⚠️ IMPORTANT: After approximately 10 minutes, the solution becomes cloudy and you will not get an accurate reading. The measurement MUST be taken within the 5-10 minute window.

Automated Protocol (OpenReef System)

System Requirements

  • Calibrated colorimeter at 625 nm
  • Peristaltic pumps calibrated for 6-drop equivalent volumes (bidirectional)
  • Bubble mixing via pump reversal
  • Temperature control (optional but recommended)

Automated Sequence

The OpenReef system automates the entire protocol:

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 #1 Addition
   ├─ Pump 6 drops equivalent
   ├─ Clear reagent line
   └─ Bubble mix (3s reverse, 5s settle)

5. Reagent #2 Addition
   ├─ Pump 6 drops equivalent
   ├─ Clear reagent line
   ├─ START TIMER
   └─ Bubble mix (3s reverse, 5s settle)

6. Incubation
   └─ Wait 7.5 minutes (mid-point of 5-10 min window)

7. Measurement
   ├─ Read absorbance @ 625 nm
   └─ Calculate concentration from calibration

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

Total Time: ~12 minutes

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 6 drops: ~0.30 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 0-30 ppm range
  • R² > 0.99 for good calibration
  • Use linear regression: y = mx + b
    • y = absorbance
    • x = concentration (ppm PO₄)
    • m = slope (sensitivity)
    • b = y-intercept (blank absorbance)

Calculating Concentration

From measured absorbance (A):

Concentration (ppm PO₄) = (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 at 5-10 minutes
  • Blank absorbance < 0.050
  • Duplicate samples within 10% RSD

Out of Range:

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

Detection Limits

  • Limit of Detection (LOD): ~0.05 ppm PO₄
  • Limit of Quantification (LOQ): ~0.10 ppm PO₄
  • Optimal Range: 0.5-25 ppm PO₄

Troubleshooting

Issue Cause Solution
No color development Expired reagents Replace API kit
  Wrong reagent order Ensure Bottle #1 before #2
  Insufficient mixing Ensure adequate bubble mixing after each reagent
Weak/pale color Low phosphate Normal - within sensitivity
  Reagent degradation Check expiration, replace if old
Solution cloudy before 10 min Contamination Use clean cuvettes/bottles
  Particulates in sample Filter sample
Solution cloudy after 10 min Normal reaction progress Re-run test, read at 5-10 min
Color too dark to read Concentration too high Dilute sample (1:10 or 1:100)
Inconsistent results Temperature variation Control temperature (20-25°C)
  Timing inconsistency Use automated timer
  Dirty cuvettes Clean with dilute HCl, rinse well
Negative concentration Blank contamination Prepare fresh blank
  Calibration error Re-calibrate system

Interferences

Positive Interferences (False High)

  • Silicate (> 10 ppm): can form similar blue complex
  • Arsenate: reacts identically to phosphate
  • High turbidity: scatters light, increases absorbance

Negative Interferences (False Low)

  • High sulfide: reduces color development
  • High iron (> 100 ppm): can precipitate phosphate

Mitigation

  • Filter turbid samples
  • Use sample dilution if interferences suspected
  • For silicate interference: Use 880 nm IR LED instead of 625 nm
    • 880 nm is optimal wavelength for molybdenum blue method
    • Provides 3x better sensitivity than 625 nm
    • Reduces silicate interference
    • Required for ultra-low range (0-1 ppm) measurements

Safety

Reagent Hazards

Bottle #1 (Molybdate):

  • Contains sulfuric acid (corrosive)
  • Wear gloves and eye protection
  • Avoid skin contact

Bottle #2 (Ascorbic Acid):

  • Generally safe
  • Mild irritant

Waste Disposal

  • Small volumes can be neutralized and disposed of via drain
  • Larger volumes: collect and dispose as chemical waste
  • Follow local regulations

References

Standards and Methods

  1. APHA Standard Method 4500-P E: Standard Methods for the Examination of Water and Wastewater
  2. Murphy, J. and Riley, J.P. (1962) A modified single solution method for the determination of phosphate in natural waters
  3. EPA Method 365.3: Phosphorous, All Forms (Colorimetric, Ascorbic Acid)

Test Kit Documentation

  1. API Phosphate Test Kit Instructions
  2. Red Sea Phosphate Pro Manual: https://g1.redseafish.com/support/manuals/products/phosphate-pro/
  3. IoRodeo Phosphate Protocol: https://sites.google.com/iorodeo.com/biorodeo/phosphate

Sensor Documentation

  1. TSL2591 High Dynamic Range Digital Light Sensor Datasheet: https://cdn-shop.adafruit.com/datasheets/TSL25911_Datasheet_EN_v1.pdf
  2. AMS TSL25911 Product Page: https://ams-osram.com/products/sensor-solutions/ambient-light-color-spectral-proximity-sensors/ams-tsl25911-ambient-light-sensor

Appendix: Conversion Factors

Phosphate Units

Different reporting conventions:

PO₄³⁻ (as PO₄) ÷ 3.066 = P (as P)
P (as P) × 3.066 = PO₄³⁻ (as PO₄)

Example:

  • 1.0 ppm PO₄ = 0.33 ppm P
  • 0.33 ppm P = 1.0 ppm PO₄

Standard Preparation

To prepare 1,000 ppm PO₄ from KH₂PO₄:

1,000 ppm PO₄ = 1,433 mg KH₂PO₄ per liter

Calculation:
MW(KH₂PO₄) = 136.09 g/mol
MW(PO₄) = 94.97 g/mol

(1,000 mg PO₄/L) × (136.09/94.97) = 1,433 mg KH₂PO₄/L

Procedure:

  1. Dissolve 1.433 g KH₂PO₄ in ~800 mL distilled water
  2. Add 5 mL concentrated H₂SO₄ (preservative)
  3. Dilute to 1 liter
  4. Store in dark bottle, stable for 6 months