Analysis of phosphate using molybdate blue colorimetry.

Principle Phosphate and molybdate ions form a complex that turns blue upon
reduction by ascorbic acid.

Solutions A H2SO4 2.5 mol L‐1. Top up 25.5 g 96 % H2SO4 to 100 mL water.
B Dissolve 10 g ammonium heptdamolybdate tetrahydrate in water,
top up to 250 mL.
C Dissolve 0.28 g potassium antimonyltartrate hydrate in water, top
up to 100 mL.
D Dissolve 0.88 g ascorbic acid in water, top up to 50 mL.

Staining reagents SR1 (for samples containing no H2SO4)

Mix 10 mL A
3 mL B
1 mL C
6 mL D

SR2 (for containing 0.25 mol L‐1 H2SO4)

Mix 10 mL water
3 mL B
1 mL C

Shelf life Reagents A, B and C can be stored at room temperature for several
months. Reagents D, SR1 and SR2 have to be made daily.

Detection and LOD ABS 0.002 1

quantification limit LOQ ABS 0.006 1 (≈ 10 µg P L‐1)

Standards This method is linear in the range 10 – 500 (sometimes 750/1000) µg P L‐1.
Be sure to calculate your standards correctly, the concentration on our
bought standard stock solution is 1000 mg L‐1 PO43‐.

Staining non‐acidic Add 0.2 mL SR1 to 1 mL sample. Let react for 15‐20 min.
samples

Staining 0.25 mol Add 0.14 mL SR2 to 1 mL sample, add 0.06 mL D (ascorbic acid). Let react
L‐1 H2SO4 samples for 15‐20 min.

1
These values were determined by Christine Seilerbeck and Jakob Santner in May
2013.

Prepared by Jakob Santner 10 December 2013

Analysis Measure on a UV/VIS spectrophotometer at a wavelength of 881 nm.
Make sure you know how to use the instrument and that you understand
how the calibration blanks are used. This is different for one‐ and two‐
channel instruments, the one we use (on the 2nd floor) is a two‐channel
one.

Note The formation of the colour complex requires an acid matrix of 0.25 mol
L‐1 H2SO4. The colour and its intensity are very sensitive to the matrix. If
sample conservation is required before analysis, it can be done by
bringing the sample to 0.25 mol L‐1 H2SO4. If strongly acidic samples (e.g.
digests) are to be measured the samples have to be diluted before
analysis.

Do not  change the concentration of the reactants.

 change the H2SO4 concentration.
 confuse calibration and sample blank.

as this will lead to erroneous results.

Attention Non‐acid washed, reusable labware may contain high loads of P

depending on what it was used for (e.g. nutrient solutions, standard
stocks,…). Therefore make sure your stuff is properly cleaned.

New, single‐use lab plastics are usually free of P, so there is no necessity

to acid‐wash them. On rare occasions we had contaminations, so if
persistant errors occur (after checking for other issues) that might be the
reason.

References Murphy J and Riley J P 1962, A modified single solution method for the
determination of phosphate in natural waters. Anal. Chim. Acta 27,
31‐36.

Prepared by Jakob Santner 10 December 2013