Exercise 6.11 - Quantitative Analysis of Lipid Classes by TLC

LEVEL III

Two methods of lipid analysis are commonly used. The first involves isolation of classes of lipids, followed by Thin Layer Chromatographic separation and quantitation directly on the chromatographic plates. The second method involves the isolation of the components on the TLC plate, followed by conventional quantitative methods.

The lipid classes are divided into:

  1. Neutral lipids (triglycerides)
  2. Polar Lipids (phosphoipids)
  3. Cholesterol

Ordinarily, the neutral lipids are analyzed first, as they are readily separated with one dimensional TLC systems. The polar lipids require two dimensional TLC analysis, and cholesterol needs to be analyzed separately.

The lipids may be chromatographed and measured for the following:

  1. Melting Point
  2. Salkowski Test (color developed with H_2SO_4)
  3. Liebermann-Burchard Test 20 (test for 3-hydroxysteroids using acetic anhydride)
  4. Digitonide Derivative (precipitates cholesterol)
  5. Charring (spraying with an oxidant and heating to carbonize the lipid)

A typical analysis would involve two dimensional chromatography followed by charring. Individual lipids would be identified by the Rf values of the spots on the chromatograms and could be quantitated densitometrically.

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Cell Biology Laboratory Manual
Dr. William H. Heidcamp, Biology Department, Gustavus Adolphus College,
St. Peter, MN 56082 -- cellab@gac.edu