Thin layer chromatography lipid analysis

Zaima, N., Goto-Inoue, N., Adachi, K., and Setou, M. 2011. Selective analysis of lipids by thin-layer chromatography blot matrix-assisted laser desorption/ionization imaging mass spectrometry, J. Oleo ScL, 60 93-98. 

Kishimoto, K., Urade, R., Ogawa, T. and Moriyama, T. (2001) Nondestructive quantification of neutral lipids by thin-layer chromatography and laser-fluorescent scanning Suitable methods for "lipidome" analysis. Biochem. Biophys. Res. Commun. 281, 657-662. 

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. 

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preparative applications, but both are losing importance for analytical purposes due to their low resolution and have been replaced by more effective techniques like solid phase, supercritical fluid extraction and counter current chromatography.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. 

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). 

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...

Lipids, relatively nonpolar chemical substances found in plant, bacterial, and animal cells, are among the most ubiquitous of biomolecules. In this experiment, a lipid extract of ground nutmeg will be purified by chromatography on a silica gel column. Analysis of the lipid extract by thin-layer chromatography will provide the classification of the components in the extract. The unknown lipids will be further characterized by saponification and analysis of the fatty acid content by gas chromatography. For an abbreviated experiment, students may be provided samples of natural oils and fats that can be analyzed by saponification and gas chromatography. 

Analysis of the purity of each silica gel column fraction and classification of the unknown lipid can be accomplished by thin-layer chromatography on silica gel plates. On a single plate will be spotted (1) a solution of the crude lipid extract from part A, (2) aliquots from each lipid fraction of the column (or recrystallized lipid), and (3) solutions of standard lipids (listed in the Materials section). On a 20 X 20 cm silica gel plate there is room for nine different analyses. Prepare a 1% solution of the crude lipid from part A in chloroform (10 mg/1 mL). If recrystallized lipid is to be... 

The quality of FAME prepared by the methods described in this unit must be examined by GC analysis. Generally, impurities in the extracted lipid samples are not removed before methylation. If the GC results are not satisfactory due to sample contamination, additional steps may be necessary to clean the sample either before or after methylation. Commonly used techniques for purifying lipid samples are thin-layer chromatography (TLC), solid phase extraction (SPE), and column chromatography. 

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). 

Ratnayake, W.M.N. and Ackman,R.G. 1985. Rapid analysis of Canola gum lipid composition by Iatroscan thin layer chromatography-flame ionization detection. Can. Inst. Food Sci. Technol. J. 18 284-289. 

Low-wavelength UV detection (200-210 nm) is more sensitive and permits the use of gradients but precludes the use of certain common lipid solvents, such as chloroform and acetone, which are opaque in the UV region of interest. With low-wavelength UV detection, the response will also be somewhat dependent on fatty acid composition. For these reasons the mobile phases used in lipid analysis by HPLC may seem rather strange to workers familiar with the Thin Layer Chromatography (TLC) or open column separations. 

PG was purchased from Sigma Chemical Co. (St. Louis, MO) for analysis of drug in the release experiments, a small amount of Relabeled PG (R.P.I. Corp., Mount Pleasanton, IL 50 pCi/pmole) was incorporated into the formulations during preparation. Lipids were obtained from Sigma Chemical Co. (St. Louis, MO) and were checked for purity by thin layer chromatography. Agarose used in the delivery systems was Seaplaque (FMC Corp., Rockland, ME) and the devices were cast on Gelbond (FMC Corp., Marine Colloids Division, Rockland, ME). 

Jee, M.H. and Ritchie, A.S. (1984) Two-dimensional thin-layer chromatography technique for use in lipid analysis. Journal of Chromatogr., 299, 460-464. 

Subsequent to recovery of the total lipids of a cellular preparation as a chloroform-soluble fraction, the total phosphorus content can be determined (see Chapter 3) and then, depending on the amount of lipid phosphorus (or whether the preparation is radiolabeled or not, see below), analytical and/or preparative thin-layer chromatography can be undertaken. In either case, if the experimental protocol is centered on a signal-transduction process, then there may be insufficient material for a phosphorus analysis. In the latter instance, the cellular preparation is prelabeled with 32P or [3H]inositol and the labeled products are located by autoradiography. A preferred type of adsorbent (for thin-layer chromatography) is Merck silica gel 60 (oxalate impregnated). An effective solvent for separation of the phosphatidylinosi-tols and other lipids is chloroform-acetone-methanol-acetic acid-water (80 30 26 24 14, v/v). The approximate / values of cellular phospholipids under these conditions are presented as follows ... 

Salivary lipids should be extracted prior analysis by means of chlorophorm-methanol (2 1) according to Folch s method (40). Thereafter, lipids may be separated and quantified with thin-layer chromatography (9) or with gas-liquid chromatography (23). Salivary lipids may also be fractionated with silicic acid column chromatography. 

Thin-layer chromatography is a powerful tool in the analysis of lipids. It is easy to perform, versatile, and relatively cheap, and it allows for direct quantitative measurements of the separated compounds by means of scanning densitometry. An important feature is that the analyst gets a full picture of the examined sample. 

---