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Determination of Total Phosphorus


 

Equipment

Apparatus:

  • Hewlett-Packard 8452A Diode Array Spectrophotometer
  • Hewlett-Packard HP UV/VIS Chemstation Software
  • Compatible Computer
  • HELLMA UV Quartz Sample Cell (QS 1.000)
  • Kimwipes
  • 30 ml Disposable Glass Tubes, alternatively 16 x 125mm tubes with screw cap closures
  • Volumetric Flask (1 L & two 100 ml)
  • Twelve marbles, if using 30ml disposable tubes

     

Reagents:

  • Deionized water
  • Conc. H2SO4 (Mallinckrodt), cat# 2876
  • Ammonium molybdate(VI) tetrahydrate (Aldrich cat. no 22,123-6)
  • L-Ascorbic acid (Aldrich cat. no. 25,556-4)
  • 0.65 mM Phosphorus standard solution (Sigma cat. no. 661-9)
  • Hydrogen peroxide (Fisher cat. no. H323-500)


Procedure

Prepare the solutions

  • 8.9 N H2SO4 solution.Slowly add 247 ml of conc. H2SO4 to 753 ml of deionized water, allow the heat to dissipate from the 1L volumetric flask, and mix the solution well. Store in a sealed container at room temperature for up to 1 year.
  • 10% Ascorbic acid solution. Place 10 g of ascorbic acid into a 100 ml volumetric flask. Add 50 ml and mix the solution well. Dilute to 100 ml with deionized water. Store in an amber screw-cap bottle at 4°C for up to 1 month.
  • 2.5% Ammonium molybdate(VI) tetrahydrate solution. Place 2.5 g of ammonium molybdate(VI) tetrahydrate into a 100 ml volumetric flask. Add 50 ml and mix the solution well. Dilute to 100 ml with deionized water. Store in an amber screw-cap bottle at 4°C for up to 1 month.

Prepare the sample tubes

  • Place sample (0.1 µmoles phosphorus) into the bottom of each tube. Gently remove any solvent from the tubes with N2.

Prepare the 5 standard tubes

  • Place the following quantities of phosphorus standard into six separate tubes: 0 µmoles (0µl) blank, 0.0325 µmoles (50 µl), 0.065 µmoles (100 µl), 0.114 µmoles (175 µl), 0.163 µmoles (250 µl), and 0.228 µmoles(350 µl).

Digestion of organic sample to inorganic phosphate

  • The samples, six standards should be labeled and placed in a vinyl-coated test tube rack. Add 0.45 ml H2SO4 to each of the standard tubes and sample tubes.
  • Heat all tubes in an aluminum block in the hood at 200-215°C for 25 minutes. Important: temperature must be above 200°C.
  • Remove tubes from the block and allow them to cool 5 minutes before adding 150 µl H2O2 to the bottom of all tubes.
  • Replace the tubes in the block and continue to heat for an additional 30 minutes. The samples should be colorless at this point (if any brown color persists, add 50 µl of H2O2 to all cooled tubes and continue heating tubes for 15 minutes). Cool tubes to ambient temperature.
  • Add 3.9 ml deionized water to all tubes.
  • Add 0.5 ml ammonium molybdate(VI) tetrahydrate solution to all tubes and vortex each tube 5 times.
  • Add 0.5 ml ascorbic acid solution to all tubes and vortex each tube 5 times.
  • Cap each tube with a screw cap or put a marble on each tube to prevent evaporation. Heat all tubes @ 100°C for 7 minutes.
  • Cool the tubes to ambient temperature.

Spectrophotometric analysis of samples

  • Zero the spectrophotometer using the 0 µmoles standard (reagent blank).
  • Determine the absorbance of each of the five standards at 820 nm.
  • Determine the absorbance of each of the samples at 820 nm.
  • Generate a calibration curve using the standards and determine the concentration of phosphorus in the samples.

References

  • Chen, Toribara, and Warner (1956) Anal. Chem. 28:1756-1758.
  • Fiske & Subbarow (1925) J. Biol. Chem. 66:374-389.