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 1µM to 200µM. 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.