Analysis triacylglycerol

Animal fats and vegetable oils are triacylglycerols, or triesters, formed from the reaction of glycerol (1,2, 3-propanetriol) with three long-chain fatty acids. One of the methods used to characterize a fat or an oil is a determination of its saponification number. When treated with boiling aqueous KOH, an ester is saponified into the parent alcohol and fatty acids (as carboxylate ions). The saponification number is the number of milligrams of KOH required to saponify 1.000 g of the fat or oil. In a typical analysis, a 2.085-g sample of butter is added to 25.00 ml of 0.5131 M KOH. After saponification is complete, the excess KOH is back titrated with 10.26 ml of0.5000 M HCl. What is the saponification number for this sample of butter ... 

Nova-Pak C18 column in a methanol water chloroform gradient.92 Choline chloride was added to the mobile phase. One review of techniques used in the analysis of triacylglycerols lists over 300 references on separations of the triglyceride fraction of fats using nonaqueous RPLC, aqueous RPLC, argen-tation chromatography, and other chromatographic methods.93... 

Wu LC, Cheng CM (2005) Flow-injection enzymatic analysis for glycerol and triacylglycerol. Anal Biochem 346 234-240... 

Breckenridge, W. C. 1978. Stereospecific analysis of triacylglycerols. In Handbook of Lipid Research, Vol. I Fatty Acids and Glycerides. A. Kuksis (Editor). Plenum Press, New York pp. 197-232. 

As indicated previously, this analysis provides a measure of the mean molecular weight of the fatty acids in a lipid system. It is based on the fact that saponification breaks ester bonds in a lipid system and since fatty acids are attached to the glycerol backbone with an ester bond, SV reflects the number of ester bonds per gram sample. The saponification value indicates the mean molecular weight of the sample s triacylglycerols and when divided by 3 gives the mean molecular weight of the constituent fatty acids. Therefore, the smaller the... 

Transesterification, fatty acid analysis of lipids, 437, 439 Triacetin, lipase assays, 378 Triacylglycerol acylhydrolase, 371, 375, 378. See also Lipases Triacylglycerols, 432 Tributyrin, lipase assays, 378 Trichloroacetic acid (TCA) solubility index for protein hydrolysis, 152 in TBARS determination, 548-550 Trienes, conjugated, determination of, 515-517, 523-524, 526, 528 Trifluoroacetic acid (TFA), for determination of neutral sugars, 721-722, 724-725, 729-730... 

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. 

Figure 29 shows the separation of triacylglycerols from sunflower seed oil. In the analysis of linoleic acid-rich seed oils, well-shaped peaks are obtained, and excellent resolution of all the main fractions is achieved, with species containing linoleic acid being predominant. 

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... 

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. 

As an aid in the identification of such triacylglycerols in butterfat, Marai et al. (128) have investigated the reversed-phase LC-MS behavior of randomized butterfat, which contains the various isomeric triacylglycerols in known and sufficient amounts for analysis. The results show that conventional C, 8 reversed-phase columns would not resol ve the positional and reverse isomers of mixed acid triacylglycerols, but that the resolution of isologous triacylglycerols is retained also when two or three short-chain fatty acids occur per molecule. 

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.

Consequently, a more objective way to measure the habitual intake of milk fat would be the fatty acid composition of adipose tissue. However, this is not routinely performed in larger cohort studies, due to cost and that the procedure is invasive and less tolerated by study participants. Analysis of plasma fatty acid composition is thus a more feasible option for examination to determine dairy intake in the study population. While some groups have separated plasma into its constituent phospholipids and cholesterol esters to analyze serum 15 0 and 17 0 as markers of dairy intake (Smedman et al., 1999), Baylin et al. (2005) found that plasma that was not separated into its constituent cholesteryl ester, phospholipids, and triacylglycerols was still able to reflect habitual dairy intakes comparably to adipose tissue. Thus, whole plasma is an acceptable alternative to fractionated plasma in the absence of adipose tissue for analysis to reflect habitual dairy intakes and may be a cost effective option for consideration when conducting future intervention studies to assess the affect of dairy products on health outcomes. 

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... 

The Padley and Timms (1980) method has been the subject of a collaborative trial which showed the method to be suitable for use by competent laboratories for the analysis of fats that did not contain added milk fat, hazelnut oil or the CBE Calvettta , which has a rather different triacylglycerol composition to other CBEs (FSA, 2001). 

In the analysis of sterols and other components of CBAs, in particular the minor components it must be borne in mind that it is selected fractions of the parent fats obtained by crystallization that are used in confectionery and are potential adulterants. These fat fractions are produced with a view to obtaining desired physical properties, which depend on the triacylglycerol composition, and while they do contain measurable levels of some minor components of varying polarity their composition is likely to vary significantly from that of the parent fat. 

The advent of relatively inexpensive computers has enabled the accumulation and rapid analysis of large sets of data. By this means patterns and trends not always apparent from visual inspection of chromatograms or tables of data can be discriminated by being sorted into recognizable patterns. This approach is essential for some techniques such as pyrolysis where the quantity of data produced would otherwise be overwhelming. Several statistical approaches to exploit the information content of fatty acid and triacylglycerol patterns for the detection and quantification of CBEs in cocoa butter have been reported (Lipp et al., 2001 Simoneau et al., 1999). 

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). 

Takagi, T. and Ando, Y. (1995) Stereospecific analysis of monounsaturated triacylglycerols in cocoa... 

Table 4.7 Triacylglycerol stereospecific analysis of evening primrose and borage oils...

Myher, J.J., Kuksis, A., Gehr, K., Park, P.W. and DiersenSchade, D.A. (1996) Stereospecific analysis of triacylglycerols rich in long-chain polyunsaturated fatty acids. Lipids, 31, 207—215. 

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. 

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