Lipids preparative layer chromatography

Chapter 12 Application of Preparative Layer Chromatography to Lipids.299... 

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preparative applications, but both are losing importance for analytical purposes due to their low resolution and have been replaced by more effective techniques like solid phase, supercritical fluid extraction and counter current chromatography.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

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... 

Each column fraction is analyzed for lipid material by spotting on a 5 X 10 cm silica gel thin-layer plate and exposing it to iodine vapor as follows. Prepare seven tapered capillary tubes and use these to place a spot of each solution on the TLC plate. Put at least 10 capillary applications from a single fraction on a spot. Your final plate should then have seven spots, one for each fraction. Set the plate in an iodine chamber and allow it to remain for about 15 minutes or until some spots are yellow or red-brown. The presence of lipid in a fraction is indicated by the red-brown color. Retain all the column fractions that appear to have lipid. Each of these fractions will be analyzed in part B by thin-layer 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. 

Figure 1 shows the scheme for the preparation of purified lipid A from endotoxin. S. typhimurium G30/C21 was extracted by the method of Galanos t aK (24) and submitted to one of two different conditions of hydrolysis (a) 0.1 N HC1 [in methanol-water (1 1, v/v)], 100 °C, 45 min, to yield the crude monophosphoryl lipid A (nontoxic), and (b) 0.02 M sodium acetate, pH 4.5, 100 °C for 30 min (two cycles) to yield the crude diphosphoryl lipid A (toxic). The 0.1 N HC1 hydrolysis product was fractionated on a Sephadex LH-20 column (23). Each of these fractions was then separated by preparative thin layer chromatography (TLC) on silica gel H (500 ym), with the solvent system chloroform-methanol-waterconcentrated ammonium hydroxide (50 25 4 2, v/v) as previously described (23) to yield TLC fractions 1-7 and 1-9 respectively. 

In brief [43, 44], primary neurons were prepared from the cerebellum of 6-day-old mice and the cells were treated with different concentrations of the target compounds for 24 h. Radiolabeled 3-[ 14C] serine or [14C]galactose was added to the culture medium and incorporation into newly synthesized sphingolipids was analyzed after labeling for 24 h. Lipids were extracted, separated by thin-layer chromatography, and visualized with a phosphoimager. Radioactivity found in the selected lipids is expressed in relation to untreated cells. 

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). 

The lipids isolated from a cell preparation are separated on thin-layer chromatography, and the desired fractions are recovered by the extraction procedure described earlier in this chapter. In this instance, interest is centered on the phosphatidylcholine fraction, and it can be subjected to the protocol outlined in Scheme A (Figure 4-24). 

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 ... 

In an interesting experimental protocol, Silvestro et al. (1993) utilized HPLC-mass spectrometry with an ion spray (electrospray) interface for determination of PAF and lysoPAF in human PMN (neutrophils). Both unstimulated and stimulated (with complement-activated zymosan) cells were used as starting material. The total lipids were isolated in the usual way, and the PAF was isolated and purified by a combination of thin-layer chromatography, HPLC, and silica chromatography. This final PAF preparation was subjected to a bioassay with the inclusion of 3H 16 0 PAF to monitor recoveries. 

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