condensed into a liquid nitrogen-cooled trap during the progress of the reaction, thus
driving the reaction to completion (Scheme 3).
The resulting TG, homogeneous with either EPA or DHA, were afforded in near-
quantitative yields of excellent purity. High-field^1 H- and^13 C-NMR spectroscopy
was found extremely valuable as a probe to monitor the progress of the reac-
tions. It also enabled us to follow the incorporation of EPA and DHA into glycerol
to form the various intermediary acylglycerols participating in the direct esterifica-
tion process, the 1- and 2-MG, 1,2- and 1,3-DG and the TG. The progress of the
reactions is illustrated graphically in Figures 1 and 2, respectively for EPA and
DHA, as based on mol% incorporation of fatty acid equivalents into the acylglycer-
ols. As can be seen in Figures 1 and 2, EPA reacted considerably more rapidly than
DHA. In both cases the reaction mixture was dominated by 1,3-DG during the first
few hours of the reaction. At that stage both 1,2-DG and 1-MG reached a maximum
176 10 Enrichment of Lipids with EPA and DHA by Lipase
Scheme 3. Direct esterification of glycerol with EPA (top) and interesterification of tributyrin with EPA
ethyl ester (bottom) using Candida antarctica lipase.
Figure 1. Progress of the direct esterification of glycerol with EPA.