Question

how does the Ascorbic Acid Method (SPR) work?

Answer 1

Ammonium molybdate and potassium antimonyl tartrate react in acid medium with orthophosphate to form a heteropoly acid - phosphomolybdic acid - that is reduced to intensely colored molybdenum blue by ascorbic acid. This method for measuring phosphate is based on APHA Standard Method 4500-P E.

Reagents and Equipment

Assembled colorimeter

Cuvettes

5 mL screw capped tubes

Sulfuric acid, (14% v/v), Labchem, Cat # LC256002

Potassium antimonyl tartrate solution, Labchem, Cat # LC187201

Ammonium molybdate solution (4%), Labchem, Cat # LC112101

Ascorbic acid, Labchem, Cat # LC115309

1mL adjustable micropipette

Distilled water. Optional, for diluting water sample

Colorimeter set-up

Wavelength: 625 nm. Use Red led (colorimeter RGB board).

Phosphate standard data: download a data file or prepare your own phosphate standard data

Before measuring samples, calibrate the colorimeter against water or a distilled water sample developed with ascorbic acid reagent as described below.

Method

Step 1: Prepare fresh ascorbic acid reagent

1. 25 mL sulfuric acid solution;
2. 2.5 mL potassium antimonyl tartrate solution;
3. 7.5 mL ammonium molybdate solution;
4. 15 mL ascorbic acid solution (prepared in step 1

• Mix the ascorbic acid reagent. The reagent is stable for 4 hours.

Step 2: Sample testing

• Fill an empty sample bottle to the 5 mL line with your water sample
• Add 800 µL of test reagent solution, cap and invert several times to mix
• Note the time since adding the reagent to your water sample. When it has been at least 5-10 minutes, pour some of your sample into a cuvette, place in the colorimeter and click measure.The program will return the phosphate concentration in ppm.