HPLC triacylglycerol

Chloroform-methanol extracts of Borrelia burgdorferi were used for the identification of lipids and other related components that could help in the diagnosis of Lyme disease [58]. The provitamin D fraction of skin lipids of rats was purified by PTLC and further analyzed by UV, HPLC, GLC, and GC-MS. MS results indicated that this fraction contained a small amount of cholesterol, lathosterol, and two other unknown sterols in addition to 7-dehydrocholesterol [12]. Two fluorescent lipids extracted from bovine brain white matter were isolated by two-step PTLC using silica gel G plates [59]. PTLC has been used for the separation of sterols, free fatty acids, triacylglycerols, and sterol esters in lipids extracted from the pathogenic fungus Fusarium culmorum [60]. 

Silver ion chromatography is a useful technique for separating geometrical isomers of fatty acids (as the methyl ester derivatives) for subsequent analysis by GC. On the other hand, a stable ion-exchange column loaded with silver ions has been developed for HPLC that has proved of value in the simplification of complex mixtures of fatty acids (FAs) of natural origin for subsequent identification by GC-MS and for separating molecular species of triacylglycerols (41). 

Reverse-phase liquid chromatography is now virtually the only method used in the analysis of the TG mixtures. The first paper on TG-HPLC analysis was published in 1975 by Pei et al. (81). Triglycerides were separated on a VYDAC reverse-phase (35 - 44 /xm) column and eluted with methanol-water (9 1). Since Pei et al. first applied RP-HPLC to the separation of triacyl-glycerols, a number of reverse-phase systems have been developed as rapid and efficient resolution of complex triacylglycerol mixtures can be achieved. 

Fig. 29 Separation of triacylglycerols from sunflower seed oil by HPLC with a silver ion column and mass detection. For conditions see text. S = saturated fatty acid M = monounsaturated fatty acid D = di-unsaturated fatty acid.

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.

The first problem in the HPLC analysis of TGs is the identification of the individual TGs. Planner et al. (90) observed that under isocratic conditions the logarithm of the elution volume of a triacylglycerol is directly proportional to the total number of carbon atoms (CN) and inversely proportional to the total number of double bonds (X) in the three fatty acyl chains. This elution behavior is controlled by the equivalent carbon number (ECN) of a triacylglycerol, which may be defined as... 

The major difficulty in the HPLC of triacylglycerols is that of detection. Traditional modes of detection, such as refractive index and ultraviolet absorbance, offer only low sensitivity toward these triacylglycerols. 

Milk fat has presented a particular challenge to analysis in terms of the identification and separation of TGs due to their complex variety of molecular species. The most complex mixtures of natural triacylglycerols require HPLC with gradient elution. 

More recently, butterfat composition has been analyzed by capillary GC on polarizable liquid phases (122,123), reversed-phase high-performance liquid chromatography (124,125), and GC and HPLC with mass spectrometry (MS) (126), as well as by MS-MS (127). These studies have led to an extensive resolution of butterfat triacylglycerols, but the possible presence of small amounts of positional and reverse isomers as well as of species containing two or three short-chain acids per molecule has not been addressed. 

The purpose of their investigation was to determine the relative order of elution of the molecular species of rearranged butterfat triacylglycerols for which the exact quantitative composition can be calculated by statistical considerations. The initial part of the study was concerned with verifying that a random distribution of the fatty acids had indeed been obtained, which was established by detailed GC and HPLC resolution and MS identification of the species. 

Fig. 42 Reversed-phase HPLC profiles of natural (top) and rearranged (bottom) butterfat triacylglycerols as obtained with the light-scattering detector. HPLC conditions Hewlett-Packard Model 1050 liquid chromatograph equipped with a Supelcosil LC-18 column (25 cm X 0.46-cm ID) coupled to a Varex ELSD II light-scattering detector. Solvent linear gradient of 10-90% propanol in acetonitrile at 25°C over a period of 90 min (1 ml/min) recording stopped at 70 min. Peak identification by carbon and double-bond numbers of triacylglycerols.

Reversed-phase HPLC has been used to analyze the oxidation products of triacylglycerols in edible oils. The detection is often based on monitoring the conjugated dienes with an ultraviolet detector (234-235 nm). However, the UV detector provides no information about oxidation products without a conjugated diene structure, e.g., products of oleic acid. Information about these compounds is important when oils with a high oleic acid content are studied. The most common universal detector types—refractive index and flame ionization detectors—are not sensitive enough to detect small amounts of oxidation products. 

Fig. 44 Reverse-phase HPLC analysis of purified regioselective product triacylglycerols. Sample size 0.5-1.0 mg 5 fi C-18 column (0.49 X 50 cm) 120-min solvent gradient acetonitrile/methylene chloride (70 30 to 40 60, v/v) flow rate, 0.8 ml/min flame ionization detector. Column cleaned with methylene chloride after analysis. L, linoleic Ln, linolenic O, oleic P, palmitic S, stearic.

Fig. 48 Reversed-phase HPLC of autoxidized rapeseed oil triacylglycerols (peroxide value = 393.8 meq/kg). See Fig. 45 for abbreviations and chromatographic conditions. Peaks correspond to both primary and secondary oxidation products of rapeseed oil triacylglycerols.

Triacylglycerols in cocoa butters have been separated by high performance liquid chromatography (HPLC) or gas chromatography (GC). These methods... 

Today, mass spectrometry offers an attractive alternative as a detector to HPLC. Newer techniques for linking HPLC systems with mass spectrometers directly via atmospheric pressure chemical ionization (APCI) and electrospray interfaces should see an expansion of this analytical tool in the analysis of confectionery fats, a field in which it has not yet been applied. Triacylglycerols... 

No comprehensive comparison has been made of the proportions of the free and esterified sterols in cocoa butter with those of fats likely to be used in cocoa butter adulteration. However Gordon and Griffiths (1992) examined the sterol esters of palm kernel oil by isolation with TLC followed by GC and HPLC. They pointed out the problem of co-elution of triacylglycerols with steryl esters with GC. The characterization of esters of triterpene alcohols in CBA fats might well prove useful where the use of fats containing shea is suspected. 

Discriminant analysis has been used in many of the analyses described in this chapter, in particular the classification of cocoa butters by origin and processing from pyrolysis MS data (Radovic et al., 1998), from triacylglycerol profiles obtained by HPLC (Hernandez et al., 1991) and from analysis of volatiles (Pino, 1992). Data from the analysis of mixtures of CBEs with cocoa butter, which model techniques for measuring CBEs in chocolate, have been treated by similar means (Anklam et al., 1996). 

Neri, A., Simonetti, M.S., Cossignani, L. and Damiani, P. (1998) Identification of cocoabutter equivalents added to cocoa butter-I. An approach by fatty acid composition of the triacylglycerol sub-fractions separated by Ag+-HPLC. Z. Lebensm. Unters. Forsch. A., 206(6), 387-392. 

Mottram, H.R. (1999) The Application of HPLC-APCI MS to the Regiospecific Analysis of Triacylglycerols in Edible Oils and Fats. PhD thesis, Department of Chemistry, University of Bristol, UK. Movia, E. and Remoli, S. (1977) Application of enzymic hydrolysis to determine the genuineness of butter., Bollettino dei Chimici dei Laboratori Provinciali, 3, 187-192. 

Two brief, but very definitive, reviews on the HPLC of triacylglycerols (Planner, 1981) and of phospholipids (Porter and Weenan, 1981) are recommended reading. 

Figure 1.4. Reversed-phase HPLC chromatogram of milk fat triacylglycerols (from Robinson and MacGibbon, 1998).

Ruiz-Sala et al. (1996) compared the triacylglycerol composition of the milkfat of sheep, cow and goat by HPLC, using ELSD detection. They... 

Kalo, P., Kemppinen, A., Ollilainen, V., Kuksis, A. 2004. Regiospecific determination of short-chain triacylglycerols in butterfat by normal-phase HPLC with on-line electrospray-tandem mass spectrometry. Lipids, 39, 915-928. 

Triacylglycerols (esp. triolein) Chloroform-methanol extraction, HPLC with detection at 208 nm GC of derivatized or free fatty acids. Bacterial lipase Veeraragavan (1990), Choi and Jeon (1993)... 

Further purification of the total lipid extract from neutral lipids like triacylglycerols is desirable to increase the separation performance and lifetime of reverse-phase HPLC columns. Several authors have utilized a procedure of base-acid wash for this purpose (Evershed et al., 1988 Seitz, 1989 Hakala et al., 2002), where one takes advantage of the phenolic nature of the steryl femlates. Total lipid extract in acetone or... 

Triacylglycerol profiles, determined by high-performance liquid chromatography (HPLC), may also be a tool for the detection of the adulteration of pork by beef fat (44). The presence of 5% or more of pork fat in beef or mutton tallow can be detected and quantified by HPLC analysis of fatty acids in the sn-2 position of the triacylglycerols, because the ratio of 16 0/18 loo9 at this position is about 5.0... 

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