====== Phosphorous determination ======

===== Introduction =====

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.

==== Reagents ====

  * 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 H<sub>2</sub>SO<sub>4</sub>: HClO<sub>4</sub>
  * Reagent 2 (must be made new every time)
    * Sulfuric acid 3 M, (take 1 volume conc H<sub>2</sub>SO<sub>4</sub> 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.

[[chemicals|Full list of chemicals]]

==== Standard ====

  * Potassium dihydrogen phosphate - 4.39 mg contains 1 mg of P\\  take 4.39 g and 100 mL H<sub>2</sub>O and 100 µL contains 1 mg
  * Sample 1 mg/mL

==== Procedure ====

  - 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