Thin-layer chromatography, lipid classes

Chen, X., and B. R. T. Simoneit, Epicuticular Waxes from Vascular Plants and Particles in the Lower Troposphere Analysis of Lipid Classes by Iatroscan Thin-Layer Chromatography with Flame Ionization Detection, . /. Atmos. Chem., 18, 17-31 (1994). 

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. 

Many methods exist to separate triglycerides into fractions on the basis of their degree of unsaturation. These include fractional crystallization from solvents, and separation by column and thin layer chromatography (TLC). The classes of triglycerides may then be studied and after methylation the fatty acid content determined by GLC. Yet even this is not the whole story since the position of fatty acids on the triglyceride molecule may uniquely affect the physical and biological properties of the lipid. 

D1.6 Quantitation of Lipid Classes by Thin-Layer Chromatography with Flame Ionization Detection... 

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). 

An important (and early) objective of this chapter was to illustrate an approach to evaluation of the lipid classes present in a mammalian cell, such as the human platelet. Thus, armed with the information presented above, it is now feasible to start to explore this problem. As emphasized earlier, TLC can provide significant information on the types (or classes) of lipids present in a lipid extract analytical and/or structural technique to elucidate the chemical nature of the individual groups of compounds. A brief examination of the results that can be obtained in two types of thin-layer chromatography follows. 

Early studies employed thin layer chromatography (TLC) in the separation, identification, and quantification of individual classes of lipids. These techniques have been optimized for different classes of glycerol phospholipids, neutral hpids, sphin-golipids, and fatty acids. Methods and general references to TLC can be found in the Lipid Library http //www.lipidlibrary.co. 

The lipid fraction of the supernatants is obtained through a Folch extraction, and the lipid classes are separated by analytical thin layer chromatography. The amount of activity associated with each separated lipid moiety is then quantified. Treatment of line-10 cells with antibody plus... 

The fatty acid composition of lipids is usually analyzed by gas chromatography following transesterification into methyl esters. Unmodified lipids can be analyzed by HPLC or by soft chemical ionization mass spectrometry. In the course of sample preparation it is often necessary to separate the various membrane fractions (plasma membrane, thylakoid, microsomal, mitochondrial, etc.) by sophisticated gradient centrifugations, as well as the individual lipid classes within a membrane fraction, usually by thin-layer chromatography (TLC). 

Bergen, B.J., J.G. Quinn and C.C. Parrish. Quality assurance study of marine lipid-class determination using Chromarod-Iatroscan thin layer chromatography-flame ionization detector. Environ. Toxicol. Chem. 19 2189-2197, 2000. 

Thin-layer chromatography remains one of the main methods for class fractionation and speciation of lipids [23,24] and is used increasingly to determine the botanical origin, potency, and flavor potential of herbs and spices [25-27]. In the pharmaceutical industry, it is used for the analysis of complex and dirty samples with poor detection characteristics and for stability and content uniformity testing [28-31]. It continues to be widely used in the standardization of plant materials used as traditional and modem medicines. In addition, it retains an historic link with the characterization of dyes and inks and the control of impurities in industrial chemicals. 

The polarity of a lipid affects its volatility, solubility, and nonspecific binding to other polar compounds. Lipids are often functionally classified as neutral or polar on the basis of their mobility on thin-layer chromatography using a neutral or polar solvent system. Neutral lipids such as wax esters, steryl esters, ether lipids, and TAG are chemically neutral while other neutral lipids, such as free FA, fatty alcohols, and monoacylglycerols, are actually slightly polar due to the presence of hydroxyl or carboxyl groups. Table 3.8 lists the relative polarities of some common lipid classes. 

FIGURE 9.6 Thin-layer chromatography-flame ionization detection (TLC-FID) chromatograms of lipid extracts from the deep-sea flsh, orange roughy Hoplostethus atlanticus) (a) ovary, (b) muscle, and (c) liver. In this example, lipid classes were separated by a single development in a solvent system (mobile phase) of hexane diethyl etheriacetic acid (60 17 0.2, v/v/v) with the rod (stationary phase) fully scanned. See Section 9.4.2. 

The biological activity of total, polar and neutral lipids, as well as purified fractions of each lipid class after thin layer chromatography separations (Nasopoulou et al., 2007) was studied against washed rabbit platelets according to the method of Demopoulos et al. (1979)... 

High-performance silica is used in high-performance thin-layer chromatography (HPTLC). HPTLC differs from normal TLC in that the size of the absorbent (usually silica) is only 5 pm, with a narrow distribution. This enables HPTLC to give better separations compared with TLC, which uses a standard silica, and, moreover, HPTLC requires a smaller sample size and has a lower detection limit compared with conventional TLC. HPTLC plates of varying sizes are commercially available and of late have found considerable applications in the field of lipids. Weins and Hauck (1995), in their survey of TLC, conclude that the use of HPTLC plates increased by 30% over the period 1993-95. An excellent application of HPTLC is illustrated in Fig. 1.1 for the separation of neutral and complex lipids. Yao and Rastetter (1985) have achieved separation of more than 20 lipid classes of tissue lipids on HPTLC plates using four developing solvents. 

Indrasena, W. M., Parrish, C. C., Ackman, R. G. et al. (1990) Separation of lipid classes and carotenoids in Atlantic salmon feeds by thin layer chromatography with latroscan flame ionization detection. Bulletin of Aquaculture Association of Canada, 4, 36-40. 

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