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

Reference:

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