problems with glycolipid derivatization

Lars Hellgren hellgren at mimer.be.dtu.dk
Fri Mar 31 06:45:59 EST 2000


Dear everyone!

In my lab have we just started to analyse neutral glycosphingolipids on
HPLC, and we use the benzoylchloride derivatization protocol described
in Ullman and McCluer (1987; Methods in enzymology vol 138 117-125) to
per O, N benzoylate the glycolipids (10% beonzoylchloride in pyridine,
37 degrees for 16 hours). We have tried extraction the benzoylated
products with both with hexan and using C18 cartridges as described in
the paper. However, when we try to derivatize a standardmixture
containing cerebroside, lactosylceramide, ceramidetrihexoside and
globoside do we get one very intense peak (cerebrosides), one much
smaller (lactosylceramide) and one which is barely detectable
(ceramidetrihexoside) (Runned on a Luna Silica 5micrometer, 150x4.6 in
different hexan-iso-propanol gradients). We have tested the mixtures on
TLC with other solvent systems with the same result.

I would be very grateful for any suggestion on what might cause this
discrimination of the higher glycosylated lipids.

Thanks in advance!

Lars I. Hellgren
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