How can fatty acids be fractionated?

This method was modified from the most used lipid fractionation method developed by Kaluzny et al. to separate lipid classes more thoroughly and completely elute phospholipids.  The original method had reported problems of overlapping fractions, in particular, a significant amount of phospholipids were found in elution 1.

To a 500mg Strata^® NH2 cartridge,

Condition- 3 mL chloroform

Equilibrate- 7.5 mL hexane

Load –  20mg sample dissolved in 0.15mL hexane/ chloroform/ methanol (95:3:2)

Elute (I.  Neutral Lipids)- 5 mL chloroform

Elute (II.  Free Fatty Acids)- 5 mL diethyl ether/ acetic acid (98:2)

Elute (III.  Polar Lipids)- 2.5 mL methanol/ chloroform (6:1)

Elute (IV. Polar Lipids)- 2.5 mL 0.05 M sodium acetate in methanol/ chloroform (6:1)

Elution III and IV both contained phospholipids.

1. Ruiz et al. Analytica Chimica Acta 520 (2004) 201–205