Phosphorous determination

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) 1756-1758.

• 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: HClO₄
• 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.

Full list of chemicals

• 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

1. 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
2. Make two blank tubes, two tubes each with 50, 100, 200, and 400 µL standard solution
3. Add 100 µL of fusion mixture to blank, sample and standard and heat for 15-20 minutes. Allow to cool.
4. Add 1 mL water
5. Add 1 mL of reagent 2, shake and leave at 37 °C for 2 h.
6. Read UV-absorbtion at 820 nm
7. Make standard curve and deduce phosphorous content