column chromatography

Chromatography of lipids using a glass column filled with a suitable material is a common and useful method for fractionation of lipid classes either on an analytical or a semi-preparative scale. While this methodology leads to fractions which need to be later quantified, HPLC allows to separate and quantify lipid compounds in a single process if a convenient detector is present.

Two types of column (or liquid) chromatography are generally used:

procedure which separates lipid mixtures mainly according to their relative polarities

procedure which is based on the importance of ionic groups present in some lipid molecules

This separation is based on differences in the degree of adsorption of lipid components onto a solid and immobilized phase.

HISTORY

Curiously, this industrial and laboratory technique was discovered thanks to a botanist who tried to analyze vegetal pigments which are also coloured lipids. Michael Tswett (1872-1920) is credited with developing and publishing the first concept and technique of chromatography (column chromatography). Tswett was aware that his results possessed significance beyond the mere separation of chlorophyll, xanthophyll, and carotene. The title of one of his papers was "On a new category of adsorption phenomena and their application to biochemical analysis" (Tswett M, Ber Deut Botan Ges 1906, 24, p.316 and 384; Chem Zeutr 1906, 72, 892 and 77, 1286; Chem Ber 1908, 41, 1352). The work of Tswett did not achieve at that time the recognition afforded it today. At that time chemists were not attracted by micro-methods and Tswett was a botanist.
A survey of the life and scientific acitvities of Tswett may be found in LC-GC north America.

Column chromatography was rediscovered and revived by Kuhn in 1931 also in the course of studies on carotenoids (Kuhn R et al., Z Physiol Chem 1931, 197, 141; Ber 1951, 64, 1349). This time this technique received wide attention likely due to the general development of biochemistry which necessitated refinement of the methods of separations. Despite the interest of lipid chemists in the carotenoid substances, the development of chromatographic methods progressed more rapidly with amino acids and carbohydrates than with lipids. Prior to 1950 only a few references to chromatography of fatty substances can be found. In 1936, Thannhauser SJ described the use of an aluminium oxide column to separate phospholipids (J Biol Chem 1936, 116, 527). In 1948, Swain LA described the separation of unsaponifiable lipids into three fractions on an alumina column (Can Chem 1948, 32, 533). The use of silicic acid to separate various lipids was described for the first time in 1952 by Borgstrom B (Acta Physiol Scand 1952, 25, 101).

The retention results in a variety of mechanisms including hydrogen bonding, Van der Waals' forces and also ionic bonding. The solid phase is relatively polar (normal chromatography) and the more polar the lipid, the more strongly is it adsorbed. Thus, the lipids are eluted by increasingly polar solvents. This technique has a low resolution when used at low pressure (Solid Phase Extraction or SPE) but has a high resolution (high performance) when run at high pressure using a stationary phase made of fine particles (HPLC). The former is restricted to the fractionation of complex mixtures into two or three less complex ones, the later being adopted to analyze and quantify purified fractions.

Various procedures were described according to the types of lipids separated and the complexity of the sample studied.

The strategy to be adopted depends on the aim of the study, the available instruments and on the second analysis step which follows the first fractionation step.

1- When all the lipid classes must be studied (non polar and polar lipids), the following procedures are proposed to the analyst:

2- When only neutral lipids must be studied (including fatty acids), several procedures are available: