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HPLC Separation of Phospholipids

Separation of Phospholipids by HPLC

Apparatus

  • Hewlett Packard 1050 Pump Station
  • Sedex 55 Evaporative Light Scattering Detector
  • Hewlett Packard 2-D ChemStation
  • 500 Fl & 10 ml Syringes (Hamilton Co.)
  • Astec Diol 5 mm diol bonded silica normal phase Spherical Column, 250 X 4.6 mm ,
  • Advanced Separation Technology (ASTEC), cat. # 51080 Whippany, NJ.
  • Sonicator
  • Small Glass Test Tubes
  • 1 Liter Graduated Cylinder
  • 1 Liter Erlenmeyer Flask

Reagents

  • Chloroform (Mallinckrodt)
  • Methanol (B&J)
  • Ammonium Hydroxide (Mallinckrodt)
  • Deionized Water

Procedure

  1. Prepare Mobile Phase A.
    1. Mix 800 ml chloroform, 195 ml methanol, and 5 ml ammonium hydroxide in a 1 liter graduated cylinder.
    2. Mix well by inverting cylinder approximately 20 times.
    3. Transfer solvent mixture to a 1 liter erlenmeyer flask and sonicate for 10-15 minutes to remove air bubbles.
  2. Prepare Mobile Phase B.
    1. Mix 600 ml chloroform, 340 ml methanol, 50 ml deionized water, and 5 ml ammonium hydroxide in a 1 liter graduated cylinder.
    2. Mix well by inverting cylinder approximately 20 times.
    3. Transfer solvent mixture to a 1 liter erlenmeyer flask and sonicate for 10-15 minutes to remove air bubbles.
  3. Sample Preparation.
    1. Dissolve sample in chloroform:methanol:deionized water (73:23:3 v/v/v) at a concentration of 2mg/ml.
    2. Inject 200µl into a 20µl loop and load on column; elute with gradient.
  4. Gradient.
    1. 0-14 min: 100% A to 100% B linear gradient
    2. 14-25 min: hold 100% B
    3. 25-30 min: 100% B to 100% A linear gradient
    4. 30-45 min: hold 100% A to regenerate
  5. Flow Rate. 1ml/minute

References

  • Becart et al. (1990) Journal of High Resolution Chromatography, 13:126-129.