Lipid thin layer chromatography

Once several silica gel column fractions have been collected, the eluted fractions must be analyzed for the presence of lipids. Thin-layer chromatography on silica gel plates will be introduced in this experiment for the detection and identification of neutral lipids. 

Padley, F.B., Thin-layer chromatography of lipids, Thin-Layer Chromatography Proceedings Symposium, Rome 1963, Marini-BeHob, G.B., Elsevier, New York, 1964, p. 87. 

Lipid Screening. The problems of lipid analysis in the newborn is difficult because of the fact that most methods for analysis for lipids require substantial amounts of serum, yet a total lipid determination is very important in various types of disease. This problem can be solved by thin-layer chromatography (59). Figure 38 shows a typical pattern obtained when an extract 7rom 10 microliters of serum is subjected to thin-layer chromatography. If these specimens are scanned, and an internal standard is run, one can obtain a rough approximation of the distribution of the various lipids in the serum. This is shown in Figure 39, in which a normal specimen is run in an adult. 

Figure 38, Patterns obtained from the extract of 10 fd of serum for lipid fraction by thin-layer chromatography. In sequence, starting from the bottom, phospholipids, pee cholesterol, cholesterol aniline as an internal standard, triglycerides, and cholesterol esters. The free fatty acids occur between cholesterol and the internal standard and are only barely visible in the print, on the extreme right. They are readily visible, normally, to the eye.

Chedid, A. Haux, P. and Natelson, S. Use of thin layer chromatography on silica gel for serum lipid fractionation and measurement in the routine clinical laboratory. Clin. Chem. (1972), 13, 384 - 390. 

High performance liquid chromatography (HPLC) has been by far the most important method for separating chlorophylls. Open column chromatography and thin layer chromatography are still used for clean-up procedures to isolate and separate carotenoids and other lipids from chlorophylls and for preparative applications, but both are losing importance for analytical purposes due to their low resolution and have been replaced by more effective techniques like solid phase, supercritical fluid extraction and counter current chromatography. The whole analysis should be as brief as possible, since each additional step is a potential source of epimers and allomers. 

Source From Fried, B., Lipids, in Handbook of Thin Layer Chromatography, 3rd ed., Vol. 89, Sherma, J. and Fried, B. Eds., Marcel Dekker, New York, 2003, pp. 99-133. With permission. 

Mangold, H.K., Aliphatic lipid, in Thin-Layer Chromatography, 2nd ed., Stahi, E., Ed., Springer-Verlag, New York, 1969, pp. 363 21. 

The conventional approach to solvent extraction is the batch method. Early work with this method was hampered by the low concentration of the compounds present and the relative insensitivity of the methods of characterization. Thus lipids and hydrocarbons have been separated from seawater by extraction with petroleum ether and ethyl acetate. The fractionation techniques include column and thin-layer chromatography with final characterisation by thin-layer chromatography, infrared, and ultra-violet spectroscopy and gas chromatography. Of these techniques, only gas chromatography is really useful at levels of organic matter present in seawater. With techniques available today such as glass capillary gas chromatography and mass spectrometry, much more information could be extracted from such samples [20]. 

Law S, Wertz PW, Swartzendruber DC, Squier CA (1995) Regional variation in content, composition and organization of porcine epithelial barrier lipids revealed by thin-layer chromatography and transmission electron microscopy. Arch Oral Biol 40 1085-1091... 

Weerheim A, Ponec M (2001) Determination of stratum corneum lipid profile by tape-stripping in combination with high-performance thin-layer chromatography. Arch Dermatol Res 293 191-199. 

The purity of all lipids and anthracyclines exceeded 98% based on thin-layer chromatography (TLC) and/or high-performance liquid chromatography (HPLC) analysis, performed as described by Barenholz and coworkers (38,49,50). 

Fig. C.2 Thin Layer Chromatography. An example of a lipid containing sample developed and stained with Iodine vapors.

Nichols, P. D., Mooney, B. D., and Elliott, N. G. (2001). Unusually high levels of non-saponifiable lipids in fishes escolar and rudderfish identification by gas and thin-layer chromatography. J. Chromatogr. A 936,183-191. 

Thin-layer chromatography (75) and sodium dodecylsulfate-(poly)acrylamide gel electrophoresis (SDS-PAGE) are helpful for analyses of the lipid and protein composition, respectively. Size-exclusion chromatography allows estimation of the size distribution of the (proteo)liposomes and crude fractionation of the material as reviewed in Ref. 76. Accurate determinations of size distributions require analyses by static or dynamic... 

Thin-layer chromatography (TLC) has passed its heyday as an analytical procedure and has been surpassed by more sensitive and automatic methods, but it still has some advantages in the analysis of complex lipids. Despite its decrease in usefulness, because of the simple realization and low-cost apparatus, some examples for TLC are given in this chapter. 

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). 

Lipids can be identified and quantified using thin-layer chromatography (TEC) and gas chromatography (GC) (Galliard, 1968). Extraction of lipids is achieved by homogenizing potato tubers with isopropanol in a blender, followed by a series of filtrations and extractions with chloroform-methanol (2 1). Chloroform is removed by rotary evaporation and the residue is redissolved in benzene-ethanol (4 1). This extract is passed through a DEAE-cellulose column, and the fractions collected are subjected to TEC on 250 p,m layers of silica gel G, using three solvent systems. Fatty acid methyl esters for GC analysis are prepared by transmethylation of the parent lipids, or by diazomethane treatment of the free fatty acids released by acid... 

Fig. 4.5.2 Actual strategies for CDG diagnosis. Initial investigations on CDG patients are routinely carried out by isoelectric focusing (IEF) of serum transferrin. With a CDG type I pattern, subsequent analysis should imply determination of phosphomannomutase (PMM) and phos-phomannose isomerase (PMI) activities. Further studies, like analysis of the lipid-linked- and protein-bound-oligosaccharides, determination of enzyme or sugar transporter activities and molecular biology studies often have to be performed in more specialised laboratories. HPLC High-performance liquid chromatography, TLC thin-layer chromatography...

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. 

Parodi, P. W. 1975. Detection of aceto-diacylglycerols in milk fat lipids by thin-layer chromatography. J. Chromatogr. Ill, 223-226. 

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