Lipids chromatogram

Goswami, S. K. KinseUa, J. E. A nondestructive spray reagent for the detection of prostaglandins and other lipids on thin-layer chromatograms. Lipids 1981,16,759-760. 

In practice a few iodine crystals are usually placed on the bottom of a dry, closed trough chamber. After the chamber has become saturated with violet iodine vapor the solvent-free plates are placed in the chamber for 30 s to a few minutes. The iodine vapor condenses on the TLC layers and is enriched in the chromatogram zones. Iodine vapor is a universal detector, there are examples of its application for all types of substances, e.g. amino acids, indoles, alkaloids, steroids, psychoactive substances, lipids (a tabular compilation would be too voluminous to include in this section). 

The detection limit per chromatogram zone is 50—200ng for lipids [11], 200— 400 ng for antioxidants [3] and several ng for ascorbic acid. 

Some substances, e. g. penicillin and pyrazolinone derivatives, are poorly detected by iodine staining with detection limits of 2-4 pg substance per chromatogram zone [6, 7]. The limits for lipids and for opium alkaloids lie with 50-500 ng [8] in the middle nanogram range [9]. 

A review by Bailey and Swain ( ) cited several references which indicated nitrite was responsible for cured meat flavor. These same authors presented chromatograms of volatiles from cured and uncured hams and while the chromatograms were similar, some quantitative differences led to the conclusion that the major difference due to nitrite was its reactivity to retard lipid oxidation. Greene and Price ( ) suggested, however, that sodium chloride was the major factor responsible for cured meat flavor rather than sodium nitrite or an absence of lipid oxidation. It has been concluded from other recent work (2) that nitrite was necessary to produce a typical ham aroma and flavor as well as to retard the development of off-odors and flavors during storage of cooked cured meat. 

Figure 7.2 Partial capillary gas chromatogram displaying the principal lipid solvent soluble constituents of a black tarry deposit on the surface of a Neolithic potsherd from Ergolding Fischergasse, Bavaria, Germany. The sample was trimethylsilylated prior to GC (for further details see Heron et al., 1997). Peak identities (confirmed by GC-MS analysis) 1, lupenone 2, lupeol 3, betulin 4, betulinic acid.

Figure 11.4 Total ion current (TIC) chromatogram of lipid residue extracted from a potsherd of Early Neolithic date (Ecsegfalva, Hungary). Cx y F refer to fatty acids with carbon number (x) and number of unsaturations (y). Cx T refer to triacylglycerols with number of carbon atoms (x). P = plasticizer contamination. (Craig et al., 2007, 354 Figure 18.1, by permission.)...

Fig. 21.15. Partial gas chromatogram of extracted lipids. Peaks Ci4 0 to Ci8 o> saturated straight fatty chain acids (FA) with 14 to 18 carbon atoms C45br and Ci7 r, iso- and anteiso- branched-chain fatty acids Ci8 i FA, mono-unsatu-rated CigtoOH-FA, hydroxylated form derived by bacterial hydration of the original oleic acid. (Reprinted/redrawn from Nature, 432, 35-36, Copyright 2004, Nature Publishing Group, with permission.)...

A representative gas chromatogram with ECD of the analysis of various polar chlorinated pesticides isolated from cod liver oil [179] is shown in Fig. 13. Determination of the polar chlorinated pesticides in cod liver oil required clean up of the lipid matrix with a dimethylformamide/water/hexane liquid-liquid partitioning procedure followed by isolation using a normal-phase LC procedures, and final analysis by GC-ECD [179]. 

A simple method for assessing lipid oxidation is measuring the headspace concentration of hexanal by capillary GLC. Also, the total volatiles appearing in the chromatogram up to hexanal can be taken as oxidation index. The method was applied to determine the amounts of lipid peroxides present in rat liver cells. Enhancement of the hexanal concentration can be achieved on adding ascorbic acid (22), that reduces Fe(ni) present in the matrix to Fe(II), which catalyzes decomposition of hydroperoxides to aldehydes. Significant correlations are found between hexanal concentrations and various oxidation indices, such as TBARS (Section IV.D.2)" . ... 

Develop the chromatogram one- or two-dimensionally using the solvents 1-8. The choice of solvents depends on the lipids to be analyzed and has to be checked experimentally. 

Goswami SK, Frey CF (1974) Separation of bile acids from neutral lipids on thin layer chromatograms. J Chromatogr 100 200-201... 

Ninhydrin to Detect Lipids on TLC Plates Ninhy-drin reacts specifically with primary amines to form a purplish-blue product. A thin-layer chromatogram of rat liver phospholipids is sprayed with ninhydrin, and the color is allowed to develop. Which phospholipids can be detected in this way ... 

Gas chromatogram of cholesterol and other lipids extracted from bones and derivatized with trimethylsilyl ((CH Si—) groups to increase volatility for chromatography. Bone contains 2 to 50 ng cholesterol/gram of dry bone. 

Since solubility in blood is altered by many factors, including temperature, lipid level, protein, hemoglobin, and hemocrit values, the solubilities of the gases in the samples should be measured individually. This requires only a second equilibration of the original sample with a new aliquot of helium, and from the ratio of the chromatogram peak heights of the two extractions the solubility of the gas concerned can be calculated. 

Typical thin-layer chromatogram of lipids. The solvent system, hexane-diethyl ether-acetic acid (90 10 1). (1) cholesterol, (2) fatty acid, (3) triacylglycerol, (4) fatty acid methyl ester, (5) cholesterol ester, (6) glycerophosphatide, (7) sphingolipid, (8) 1,3- or 1,2-diacylglycerol, (9) 1 - or 2-monoacylglycerol. The Rf for (5), cholesterol ester, is calculated as follows ... 

B 4. How does iodine react to produce the red-brown spots on a developed chromatogram Why do some lipids give darker spots than others ... 

Develop the plate in hexane-diethyl ether-acetic acid (80 20 1) by plac mg the TLC plate in a chamber containing the solvent system, making sun. the edge with the applied samples is down. The solvent level in the cham ber must not be above the application spots on the plate. (Why ) Leave the chromatogram in the chromatography jar until the solvent front rises to about 1 cm from the top of the plate (45 to 60 min). Remove the plate and make a small scratch at the solvent level. Allow the chromatogram to dry (Hood ) and then place it in an iodine chamber for several minutes. Remove the plate and lightly trace, with a pencil or other sharp object, around each red-brown spot. This should be done promptly, as the colors will fade with time. Calculate the mobility of each standard and unknown lipid relative to the solvent front (R() ... 

Figure D1.6.4 latroscan TLC-FID chromatograms of a hydrogenated lipid fraction isolated from...

Figure D1.6.5 Sequential latroscan TLC-FID profiles of the lipid classes extracted from the dorsal white muscle of Atlantic salmon. I, II, and III represent partial chromatograms from the three-stage development of total lipids on aChromarod Sill. The solvent systems were (I) 80 14 1 0.2 (v/v/v/v) hexane/chloroform/isopropanol/formic acid for 55 min (II) acetone for 15 min and (III) 70 30 3 (v/v/v) chloroform/methanol/water for 60 min.

Figure D1.6.6 latroscan TLC-FID chromatograms of (A) a lipid fraction enriched with neutral lipids isolated from cod flesh and stored in ice (B) neutral lipids spiked with authentic 1 -0-palmityl-glyceryl ether dipalmitate (GE), coinciding in position with authentic highly unsaturated acids such as 22 6n-3 (C) hydrogenated neutral lipids spiked with GE. The solvent system was 97 3 1 (v/v/v) hexane/diethyl ether/formic acid for 40 min. Abbreviations O, origin SF, solvent front FFA, free fatty acid PL, phospholipids SE, steryl ester ST, free sterol TG, triglyceride. Reproduced from Ohshima et al. (1987) with permission from AOCS Press.

The sample is now ready for analysis using the procedures described above (see Basic-Protocol and Alternate Protocol). Figure D1.6.6 shows chromatograms of cod muscle lipids before and after hydrogenation. Note the single peaks for steryl esters and free fatty acids in panel C compared to the double peaks in panels A and B. 

Fig. 30 Silver ion high-performance liquid chromatography (Ag-HPLC-FID) with flame ionization detector (FID) analysis of the triacylglycerols of chromatographed Crepis alpina seed oil. Ag-HPLC-FID conditions 0.5-mg sample 5-micron Chromspher Lipids column (Chrompack International, Middelburg, The Netherlands) (4.6 X 250 mm) mobile phase 0.5% acetonitrile in hexane (v/v) flow rate 1.0 ml/min FID. Chromatogram peak triacylglycerol fatty acid abbreviations S, saturated (palmitic and stearic) O, oleic L, linoleic and Cr, crepenynoic fatty acids.

Fig. 6 ELSD chromatogram of a commercial soy lecithin following HPLC on a 5-/tm Ultrasphere Si stationary phase with a gradient of chloroform, methanol, and 28.7% NH4OH. 1 = nonpolar lipids, 2 = PE, 3 = PI, 4 = PC, 5 = PA. (Reproduced from Ref. 73 with the permission of the Journal of High-Resolution Chromatography.)...

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