The FA composition of SCO was 4.2 mol% myristic acid (MA), 2.5 mol% penta-
decanoic acid, 46.3 mol% palmitic acid (PA), 1.3 mol% stearic acid (SA),
10.2 % DPA, and 35.5 mol% DHA. The TAG fraction of the reaction mixture
was subjected to silver-ion HPLC. In the spectrophotometric detection at 206 nm,
the sensitivity of each TAG species depends mainly on its double bond number.
Therefore, the detector shows very weak responses for all saturated TAG species.
The detector can only estimate the ratio of the desired TAG to their positional iso-
mers that contain the same number of double bonds because the detector’s response
is assumed to have the same value for each isomer. The ratio of the positional isomers
can be estimated from the corresponding peak areas. Most major peaks, amounting to
15–17 in number, could be identified. The desired sTAGs, C 8 (DHA)C 8 and
C 8 (DPA)C 8 could be separated from their positional isomer, C 8 C 8 (DHA) [and
(DHA)C 8 C 8 ], C 8 C 8 (DPA) [and (DPA)C 8 C 8 ], respectively.
9.2.5 Chiral HPLC
Silver-ion HPLC cannot separate stereoisomers, i.e. AAB and BAA types of sTAG.
In order to determine the enantiomeric excess of the chiral diacid sTAGs, a stereo-
specific analysis of the product must be carried out. The basis of the stereospecific
analysis of TAG is the generation of the enantiomeric MAGs and DAGs from the
parent TAG by Grignard reagent. These are then converted while maintaining their
chiral information into diastereomeric derivatives that allow measurement of fluor-
156 9 Lipase-Catalyzed Synthesis of Structured Triacylglycerols
Table 3.Peak identification for the transesterification reaction between DHAEE and tricaprylin by silver-
ion HPLC.
Peak
no.
Chemical species Possible
isomersMolecular
weightNumber of
double bonds1 Tricaprylin, caprylic acid ethyl ester
2 Caprylic acid, dicaprylin
3 Dioctanoyldocosahexaenoylglycerol C 8 (DHA)C 8 655 6
4 Dioctanoyldocosahexaenoylglycerol (DHA)C 8 C 8 ,
C 8 C 8 (DHA)
655 65 Doicosahexaenoic acid ethyl ester (DHAEE) 357 6
6 Docosahexaenoyloctanoyl diacylglycerol (DHA)C 8 (OH),
C 8 (DHA)(OH)
(OH)(DHA)C 8 ,
(OH)C 8 (DHA)
(DHA)(OH)C 8 ,
C 8 (OH)(DHA)
503 67 Docosahexaenoic acid (DHA) 329 6
8 Didocosahexanoyloctanoyl triacylglycerol (DHA)C 8 (DHA),
(DHA)(DHA)C 8 ,
C 8 (DHA)(DHA)
839 12