Front Matter

9.2.2 Chemical degradation using Grignard reagent

This method uses allyl magnesium bromide to partially deacylate the TAG. From the

resulting mixture, four different classes of partial acylglycerols (2-MAG, 1,2-DAG,

2,3-DAG and 1,3-DAG) and unreacted TAG are isolated on a boric acid-impregnated

thin-layer chromatography (TLC) plate. The FA composition of each acylglycerol is

then analyzed as described in Section 9.2.1. In the chemical degradation analysis,

analytical errors may occur readily, and the method time-consuming and labor-

intensive.

9.2.3^13 C-NMR

This technique is very useful and precise for the analysis of the positional distribution

of PUFA in TAG, but requires an expensive instrument, and also rather large amounts

of samples (>5 mg) which should be as pure as possible.

9.2.4 Silver-ion HPLC

None of the methods described in Sections 92.1–9.2.3 tell us what types of mole-

cular species of TAG are present. Unless each molecular species is separated, only

the positional distributions of FAs are determined. In order to determine the com-

position of a sample and its positional distribution of PUFA, the sample must be

separated and purified into each molecular species using a chromatographic techni-

que, after which the purified species must be analyzed by enzymatic, chemical or

NMR methods. Accordingly, an easy, simple, and accurate method is needed to

determine simultaneously both the molecular species composition of sTAG and

the positional distribution of FAs of the reaction mixture.

Silver-ion chromatography is a technique that utilizes the property of silver ions to

form reversible polar complexes with double bonds in organic molecules such as

unsaturated lipids. This technique enables the separation of unsaturated species ac-

cording to the number, geometric configuration and position of the double bond.

Some researchers have demonstrated the separation of positional isomers of TAG

that contain FAs having the same number of double bonds (Christie, 1988; Jef-

frey, 1991; Adlof, 1995; Dobson et al., 1995). However, no separation of isomeric

TAG containing PUFAwas reported. In the author’s laboratory, we have succeeded in

the separation of (EPA)C 8 C 8 [and/or C 8 C 8 (EPA)] and C 8 (EPA)C 8 by using 2–pro-

panol as a modifier in hexane-acetonitrile-based mobile phase for silver-ion HPLC

(Han et al., 1999a; Iwasaki et al., 1999). In silver-ion HPLC with spectrophotometric

detection at 206 nm, a hexane-acetonitrile-based mobile phase is generally used.

However, the mobile phase has a solubility limit of acetonitrile in hexane (Ad-

lof, 1994). 2-Propanol can serve as a third solvent, ensuring good solubility of acet-

onitrile in hexane. Figure 1A and B show the silver-ion HPLC charts of reaction

mixtures produced by a 1,3-specific lipase (LipozymeTM) and a non-specific lipase

152 9 Lipase-Catalyzed Synthesis of Structured Triacylglycerols