Lipid classes, separation

This technique was used by Delmas et al. [404] to separate lipid extracts in seawater into various classes. Lipid classes that have been eluted away from the point of application may be burnt off the rod in a partial scan, allowing those lipids remaining near the origin to be developed into the place that has just been simultaneously scanned and reactivated. By analysis of complex mixtures of neutral lipids in this stepwise manner it is possible to be more selective about lipid class separations as well as to be more confident about assigning identities to peaks obtained from a seawater sample. In addition, this approach also reduces the possibility of peak contamination by impurities which would normally coelute with marine lipid classes (e.g., phthalate esters [403]). 

Table D1.6.1 Solvent Systems for Lipid Class Separation on an Iatroscan TLC-FID...

Although use of a constant humidity chamber significantly improves the reproducibility of lipid class separation by Chromarods, minor... 

Christie, W. W., Lipid class separations using high-performance liquid chromatography, in New Trends in Lipid and Lipoprotein Analyses (J. L. Sebedio and E. G Perkins, eds.), AOCS Press, Champaign, IL, 1995, p. 1934. 

High performance and low pressure liquid chromatography (adsorption) Separation of lipid classes, separation of lipids by molecular weight and degree of unsaturation Impractical for most preparative or large-scale processes... 

B. argentifolii Lipid classes separated by high performance TLC using plates coated with silica gel. Developang solvent hexane/ diethyl ether/formic add (80 20 1 v/v/v). Visualization 5% concentrated sulphuric acid in 95% ethanol Buckner et al., 1999... 

For lipid class separation a 10 cm x 4 mm plain silica (3 pm particle size) column is adequate, providing separation across the range from sterol esters and TAGs through to phospholipids and gal-actolipids in 30 min. 

The separation of simple lipid classes from a lipid fraction aims to obtain distinct fractions of sterol esters, triacylglycerols, diacylglycerols, free sterols, free fatty acids, monoacylglycerols, and wax esters. There are numerous methods for lipid class separation, traditionally employing adsorption chromatography with silica gel columns, with increasing use currently of bonded phases such as the nitrile, diol, and polyvinylalcohol phases and of ELSD. These bonded phases give much better reproducibility of retention times than do the usual silica gel columns. Nevertheless, refractive index detection with silica gel columns and isocratic elution is still frequently employed for routine applications. 

In discussing lipid class separations by means of HPLC, it is not possible to use a "recipe" treatment, as the approach will depend largely on the nature of the detection system available to the analyst. For example, this determines the nature of the solvents used in the mobile phase and whether gradient elution is possible. Some relevant separations are therefore described below in terms of specific detectors, as examples of what is possible. 

TLC procedures with silica gel G layers (containing calcium sulfate as binder) have been employed most frequently for lipid class separations. Commonly, the solvent elution system used is hexane- diethyl ether-formic acid (80 20 2 by volume), and this gives the separations shown in Figure 2.6. Cholesterol esters migrate to the solvent front, and they are followed by triacylglycerols, free fatty acids, cholesterol, diacylglycerols, monoacylglycerols and phospholipids (with other polar lipids). For small-scale preparative... 

Lipid class separation and FAMES (Fatty Acid Methyl Esters) preparation... 

Hadioassay of the lipid classes separated by thin-layer chromatography is best carried out using a zonal sean scintillation procedure (Snyder, 1964 Snyder and Kimble, 1965) or autoradiography (Snyder, 1965b). Recommended procedures have been described in detail for both techniques. A zonal scan of a F -triolein preparation before and... 

Some Selected Solvents Used for the Separation of Different Lipid Classes... 

Simple lipids such as CE, WE, EFA, cholesterol, alcohols, ketones, TG, DG, and MG are usually separated on silica gel plates. Depending on the complexity of the lipid material and the variety of lipid classes present in a single sample, either single-or multiple-solvent systems can be used (Figure 12.4a). Although benzene [45] or... 

FIGURE 12.4 (A) Diagrammatic representation of the separation of major simple lipid classes on silica gel TLC — solvent system hexane diethylether formic acid (80 20 2) (CE = cholesteryl esters, WE = wax esters, HC = hydrocarbon, EEA = free fatty acids, TG = triacylglycerol, CHO = cholesterol, DG = diacylglycerol, PL = phospholipids and other complex lipids). (B) Diagrammatic representation of the separation of major phospholipids on silica gel TLC — solvent sytem chloroform methanol water (70 30 3) (PA = phosphatidic acid, PE = phosphatidylethanolamine, PS = phosphatidylserine, PC = phosphatidylcholine, SPM = sphingomyelin, LPC = Lysophosphatidylcholine). 

After the extrachon of total lipids from four different genotypes of flax seed (Linum usitassimum) differing markedly in their acyl composihon, PTLC was used for the isolahon of different lipid classes in the neutral lipid frachon [69]. Application of planar chromatographic methods, including PTLC, in the separahon of food lipids has been reviewed with 40 references by Olsson [70]. The polar lipid fraction of niger seed (Guizotia abyssinica Cass.) collected from different regions of Ethiopia could be separated by PTLC on silica gel [71]. 

PTLC was also used for the separation of lipid components in pathogenic bacteria. Mycobacterium avium has a requirement for fatty acids, which can be fulfilled by palmitic or oleic acid, and these fatty acids are then incorporated into triagylglycerols [80]. PTLC was used for the separation of fatty acids and triacylglycerols in the extracts of these bacterial cells to study the lipid classes in the bacterial cells cultured under different growth conditions. 

After the extraction of lipid and nonlipid components from the leaves of mandarin orange Citrus reticulata, the lipid fraction was further separated by PTLC to determine different lipid classes that affect the chemical deterrence of C. reticulata to the leaf cutting ecat Acromyrmex octopinosus. These lipids seem to be less attractive to the ants [81a]. The metabolism of palmitate in the peripheral nerves of normal and Trembler mice was studied, and the polar lipid fraction purified by PTLC was used to determine the fatty acid composition. It was found that the fatty acid composition of the polar fraction was abnormal, correlating with the decreased overall palmitate elongation and severely decreased synthesis of saturated long-chain fatty acids (in mutant nerves) [81b]. 

The partition of different lipids between two immiscible solvents (countercurrent distribution) is useful for crude fractionation of lipid classes with greatly differing polarities. Repeated extractions in a carefully chosen solvent pair increase the effectiveness of the separation but in practice mixtures of lipids are still found in each fraction. A petroleum ether-ethanol-water system can be used to remove polar contaminants (into the alcoholic phase) when interest lies in the subsequent analysis of neutral glycerides, which may be recovered from the ether phase. Carbon... 

Column and thin-layer chromatography (TLC) came into use at about the same time as GLC, with the latter widely accepted because of its speed, ease of use, versatility, resolving power, and, probably most important, ease of visualization. Thin-layer chromatography has been particularly useful in the separation and nondestructive recovery of lipid classes. Tentative identifications can be made by comparison with known compounds, and purity can be checked. Jensen et al. (1961) may well have been the first group to separate milk lipid classes with TLC when they used the technique to obtain diacylglycerols from lipo-lyzed milk lipids. 

Separation of crude lipid extracts into individual lipid classes is difficult and time-consuming. In some cases a crude separation of lipids can be attained by selective solvent extraction. For more extensive purification of lipids, the researcher must turn to chromatography. Chromatographic methods can both resolve a complex lipid mixture into the various classes of lipids and separate the individual components of a single class of lipids. 

Thin-layer chromatography (TLC) on silica gel is well known for its separation power for lipids and related compounds. The flame ionization detector (FID) is a universal analytical instrument that offers high sensitivity and linearity for carbon-containing organic compounds. The combination of TLC and FID led to the wide use of the Iatroscan TLC-FID for the analysis of lipid classes. The adoption of the Iatroscan TLC-FID in both academia and industry has generated sufficient data to indicate that TLC-FID is currently one of the most efficient tools for the quantitation of lipids classes (Ackman et al., 1990 Hammond, 1993). 

Calibration standards are usually mixed together in one solution if they can be separated from each other by Chromarods. If not, individual solutions of the unresolved standards should be prepared separately. Not all of the standards listed in the materials section are necessary for calibration. Selection of calibration standards depends on the lipid-class profile of each particular sample. Dihexadecanoyl lecithin is used to represent the whole group of phospholipids. See Critical Parameters and Troubleshooting for a discussion on calibration standard preparation and FID linearity. 

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