Phosphate Assay

(Chen et al. 1956)

modified by Maitreya Dunham and Cheryl Christianson

December 2005

 

Reagents

 

10% ascorbic acid:

            10g ascorbic acid

            water to 100ml

            store at 4C, good for one month

 

2.5% ammonium molybdate:

            2.5g ammonium molybdate

            water to 100ml

            store at room temperature

 

6N sulfuric acid:

            18ml concentrated acid

            water to 108ml

            store at room temperature

 

Reagent C, prepare fresh each day, keep at 4C until use:

            2 volumes water

1 volume 6N sulfuric acid

1 volume 2.5% ammonium molybdate

1 volume 10% ascorbic acid

 

Assay

 

For each assay, add 1ml reagent C and 1ml sample to a cuvette.  Cover with parafilm and invert several times to mix.

 

For your blank, mix 1ml reagent C and 1ml water. 

 

Also include control samples with known molarities of phosphate ranging from 1ÁM to 200ÁM. These known samples will be used to create a standard curve that plots absorbance vs. molarity. This standard curve will be used to determine the molarities of your unknown samples.

 

Incubate samples for 1.5hrs @ 37C.

 

For each sample, measure absorbance @ 820nm vs. your blank sample.

 

Determine the phosphate concentration in your unknown samples from your standard curve.