To determine the content of phosphorous in biological matter one
can use the colour which is formed through reduction of a
phospho-molybdate complex. A 10% solution of ascorbic acid is used
for the reduction. The suitable range is 1-8 µg of phosphorous.
This procedure is adapted from Chen, Toribara and Warner, Anal. Chem. 28 (1956)
- Heating facilities (ca. 200 °C)
- Reagent 1 (stable at room temperature for months)
- Sulfuric acid, 95-98%
- Perchloric acid, 70%
- Fusion mixture 3:2 v/v of H2SO4
- Reagent 2 (must be made new every time)
- Sulfuric acid 3 M, (take 1 volume conc H2SO4
and 5 volumes of water), use 3 mL
- Water 6 mL
- Ammonium molybdate tetrahydrate - 2.5% 3 mL, take 75 mg
- Ascorbic acid - 10% 3 mL, take 300 mg (This can be stored at +4 °C for at least four weeks).
Add to reagent 2 just before use.
- Potassium dihydrogen phosphate - 4.39 mg contains 1 mg of P
take 4.39 g and 100 mL H2O
and 100 µL contains 1 mg
- Sample 1 mg/mL
- Dry your sample to constant weight preferably, use at least a couple
of mg for dissolution. Normally overnight drying is sufficient.
Make a solution ca 1 mg/mL and take volume corresponding to 2-8 µg
- Make two blank tubes, two tubes each with 50, 100, 200, and 400 µL standard solution
- Add 100 µL of fusion mixture to blank,
sample and standard and heat for 15-20 minutes. Allow to cool.
- Add 1 mL water.
- Add 1 mL of reagent 2, shake and leave at 37 °C for 2 h.
- Read UV-absorbtion at 820 nm
- Make standard curve and deduce phosphorous content
Chen, Toribara and Warner, Anal. Chem. 28 (1956) 1756-1758.