The Lipid Library

SIZE-EXCLUSION CHROMATOGRAPHY OF LIPID POLYMERS

Summary: Recognition that lipid polymers can be present in refined oils, not just thermally abused frying oils, has prompted interest in methods for their isolation and determination. Size-exclusion chromatography has already become established as a standard technique for the analysis of lipid polymers, and a brief introduction to the technique is given.

In a remarkably short time, size-exclusion chromatography (also termed "gel permeation" (GPC) or "gel filtration" chromatography) has become established as a standard technique for the analysis or isolation of lipid polymers. This is an important development, because the presence of such compounds in commercial oils and fats has tended to be ignored - they do not elute from gas chromatography columns and usually migrate with normal lipids with many thin-layer or high-performance liquid chromatography (HPLC) systems.

Excellent reviews of applications of size-exclusion chromatography to lipids has been published by Carmen Dobarganes and Gloria Márquez-Ruiz [1-3], and they are recommended to those who wish to make use of the technique.

Lipid polymers are formed by radical-catalysed reactions of fatty acids in different triacylglycerol molecules. Carbon-carbon bonds can form between the fatty acids, or there may be oxygen (including peroxy) bridges between molecules. Although it has long been recognised that such compounds are formed in appreciable amounts in thermally abused oils, such as those used for frying foods, it is now evident that they can also be present in refined oils, especially fish oils - even those used in health food capsules [4]! Oxidation is a major factor in their formation, but it is probable that the deodorization step in the refining process, in which the oils are heated to high temperatures, contributes substantially to the problem. Potential toxic effects of polymers, and oxidised and cyclic fatty acids in abused frying oils are being studied in a number of laboratories at the moment, and any technique that helps in gathering of reliable data is to be welcomed.

The technique of size exclusion chromatography requires columns packed with a uniform porous material with a defined pore size and compatible with an organic mobile phase appropriate to the solute. If the packing is chosen correctly, small molecules will diffuse into the pores while larger ones will not, so the latter will pass through the column more rapidly and emerge first. The order of elution of solutes is therefore inversely related to the molecular weight. By selecting packing materials with different pore sizes, different groups of compounds can be resolved. Applications to lipids were facilitated by the development of lipophilic gels with sufficient rigidity for HPLC. The most successful ones for lipid applications are based on copolymers of styrene and divinylbenzene with pore sizes in the range 50 to 500Å, and they are sold under trade designations such as Bio-Beads™ SX, Ultrastyragel™, PL-Gel™ and Spherogel™. Spherical particles of 5 to 10 μm in diameter are preferred. Columns of different pore sizes are often used in series to optimize the separations.

The most useful mobile phase is tetrahydrofuran, but dichloromethane and toluene are also used. Refractive index detection has been utilized most often and is probably the best available for the purpose, since the response is linear. However, isocratic elution is required, and this can be a limitation is some circumstances. Evaporative light-scattering detectors are being used increasingly, as they permit the use of gradients, but careful calibration is then necessary. Under optimum conditions, groups of lipids that differ in molecular weight by 10 to 15% should be separable.

There is little doubt that the most important applications of size exclusion chromatography to lipids are for the analysis of triacylglycerol polymers and their oxidation products in thermally abused oils. The reviews cited [1-3] contain all the relevant references. These include two parallel inter-laboratory studies from Europe and the U.S.A., which show that the technique has reached maturity for lipid polymers [5,6].

In the separation shown in Figure 1, two columns in series were required – one containing a 100Å and the other a 500Å gel (a porous, cross-linked styrenedivinylbenzene copolymer (< 10 μm particles)). Tetrahydrofuran (1 ml/min) was the preferred mobile phase, and refractive index detection permitted direct quantification with no need for extensive calibration or correction factors. By this means, oxidized monomer, dimer and polymer fractions can each be separated as well as any hydrolytic products.

By applying a pre-fractionation by means of adsorption column chromatography (such as solid-phase extraction) on silica gel, it is possible to analyse monomeric and dimeric oxidized triacylglycerols separately by size exclusion chromatography [1,7]. It proved possible to distinguish between virgin and refined olive oils by this means, for example [1].

With their usual ingenuity, lipid analysts have found ways to separate lipid classes, such as triacylglycerols, diacylglycerols monoacylglycerols and free fatty acids (as are found in lipolysis mixtures) by size exclusion chromatography as they differ in molecular weight. Adsorption chromatography is probably a more versatile technique for this purpose, however.

A major disadvantage is the high cost of columns for size exclusion chromatography at present, but no doubt this will fall as more analysts begin to use the technique. The packing materials are not as robust as those based on silica gel, so they must be used with care - following the manufacturers' instructions. At least, the instrumental requirements for HPLC/size exclusion chromatography are relatively modest, since isocratic elution is all that is needed and relatively inexpensive detectors can be utilized. Even the mobile-phase can be recycled with care. With automatic injection facilities, a high throughput of samples should be possible in quality control applications.

Size exclusion chromatography with aqueous mobile phases is also proving extremely useful for the separation of lipoprotein classes, but that is another story.

References

1. Dobarganes, C.M. and Márquez-Ruiz, G. Size exclusion chromatography in the analysis of lipids. In: Advances in Lipid Methodology - Two, pp. 113-137 (ed. W.W. Christie, Oily Press, Dundee) (1993).
2. Márquez-Ruiz, G. and Dobarganes, M.C. Analysis of lipid oxidation products by combination of chromatographic techniques. In: New Techniques and Applications in Lipid Analysis, pp. 216-233 (edited by R.E. McDonald and M.M. Mossoba, AOCS Press, Champaign) (1997).
3. Márquez-Ruiz, G. and Dobarganes, M.C. High-performance size-exclusion chromatography for lipid analysis in organic media. In: Lipid Analysis and Lipidomics: New Techniques and Applications, pp. 205-238 (ed: M.M. Mossoba, J.K.G. Kramer, J.T. Brenna and R.E. McDonald, AOCS Press, Champaign, USA) (2006).
4. Shukla, V.K.S. and Perkins, E.G. The presence of oxidative polymeric materials in encapsulated fish oils. Lipids, 26, 23-26 (1991).
5. Beijaars, P.R., van Dijk, R. and Houwen-Claasen, A.A.M. Determination of polymerized triglycerides in frying fats and oils by gel-permeation chromatography: interlaboratory study. J. Assoc. Off. Anal. Chem. Int., 77, 667-671 (1994).
6. Firestone,D. Gel-permeation liquid-chromatographic method for determination of polymerized triglycerides in oils and fats - summary of collaborative study. J. Assoc. Off. Anal. Chem. Int., 77, 957-960 (1994).
7. Hopia, A.I., Lampi, A.-M., Piironen, V.I., Hyvonen, L.E.T. and Koivistoinen, P.E. Application of high-performance size-exclusion chromatography to study the autoxidation of unsaturated triacylglycerols. J. Am. Oil Chem. Soc., 70, 779-784 (1993).

This paper was published first by the author in Lipid Technology, 7, 17-18 (1995), but it has now been substantially updated.

Updated: 28/3/2007 Credits/disclaimer	Scottish Crop Research Institute (and MRS Lipid Analysis Unit), Invergowrie, Dundee (DD2 5DA), Scotland	Right click on the icon to open in a new window