Triacylglycerols analysis, separation

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... 

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. 

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. 

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. 

Prior to the analysis a triacylglycerol internal standard, 1,3-diheptadecanoyl-2,6(Z)-octadecenoyl-glycerol (17 0, 18 1, 17 0), is added to the sample. The sample is then separated into its triacylglycerol components by silver nitrate TLC. The band for /8-SOS (symmetrically disaturated 2-oleoyl-l,3-diacylglycerols) is isolated and the total jS-SOS acylglycerols are converted to methyl esters which are then analysed by GLC. 

Oshima, T., Yoon, H.-S. and Koizumi, C. (1989) Application of selective ion monitoring to the analysis of molecular species of vegetable oil triacylglycerols separated by open-tubular column GLC on a methylphenylsilicone phase at high temperature. Lipids, 24, 535 4. 

An analysis of the positional distribution of fatty acids (as their methyl esters) was done for triacylglycerols in butterfat. Approximately 200 pg of butterfat sample was injected [762]. Excellent separation and peak shapes for a series of individual standards (e.g., C4.o-C20 o. even Ci8 i-C22 i, even Cig 2 and Cig 3) were obtained using a C18 column (ELSD) and a 45-min 20/80 50/50 chloroform/acetonitrile... 

The triacylglycerol (TAG) composition of the seeds of five Amaranthus accessions were determined from the fat residue from a Soxhlet extraction. Two 250 X 4.6mm 30°C C,g columns (RI detector) in series and a 60/40 acetone/ acetonitrile mobile phase were used in the analysis. A lOpL aliquot of a 5% fat extract was injected. The acyl substituents of the TAG ranged from linolenic lino-lenic linoleic to palmitic oleic steric. Squalene was also separated and identified [865]. Elution was complete in 75 min. Pili nut (Canarium ovatum) oil was similarly analyzed. In this case the TAGs ranged up to stearic stearic oleic and elution took 95 min [866]. 

The chapter has examined advantages of TLC, definitions, structure, occurrence, function, sample preparation, sorbents, mobile phases, usual modes of development, and detection procedures for lipids. Although a discussion of 2-D lipid analysis has been provided, mention was not made of the newer technique of multiphase TLC, in which components are separated in two different directions according to different parameters, e.g., conventional silica gel in one direction and reversed phase in the other direction. Ritchie and Jee (143) have used this technique for the analysis of triacylglycerols. 

Figure 4.9 Representative ESI-MS analysis of lipid classes resolved by intrasource separation. Lipid extracts from mouse liver samples were prepared by using a modified procedure of Bligh and Dyer [1]. MS analysis was performed with a TSQ Vantage triple-quadrupole mass spectrometer (Thermo Fisher Scientific, San Jose, CA) equipped with an automated nanospray apparatus (i.e., TriVersa, Advion Bioscience Ltd., Ithaca, NY) and Xcalibur system software. Mass spectra were acqnired directly from the diluted hpid extract in the negative-ion mode (a), after addition of 50 nmol LiOH/mg of protein in the diluted lipid extract and analyzed in the negative-ion mode (h), or the identical hpid solution to that in (b) in the positive-ion mode (c). IS denotes internal standard PC, PE, PG, PI, PS, TAG, NEFA, and CL stand for phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidyUnositoL phosphatidylserine, triacylglycerol, nonesterified fatty acid, and doubly charged cardioUpin, respectively.

Refractive index detectors also have several applications in lipid analysis. They are "universal" detectors, but lack sensitivity, require isocratic elution conditions and are sensitive to minor fluctuations in temperature. Their main value is probably in small-scale preparative applications, say with 1-2 mg of a lipid extract. For example, a refractive index detector was utilized with a column (4.6 x 250 mm) of Ultrasil Si (5 micron silica gel) and isocratic elution with isooctane-tetrahydrofuran-formic acid (90 10 0.5 by volume) to separate most of the common simple lipid classes encountered in animal tissue extracts, such as those of liver [304]. Cholesterol esters, triacylglycerols and cholesterol were each resolved and gave symmetrical peaks. 

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