I have been testing a batch of Sigma standard
diacylglycerols on a non-polar GC column as
follows :

Column : CP Sil 5 CB [50m x 0.25mm]
Sample : Sigma 178-9 (dipalmitin, distearin
and diolein [33.33 percent each])
Program: Start at 160 ^C - 320 ^C at 5^ /min
Injector & Detector [FID] both at 280 ^C
(The three compounds are underivatized).

This column gave 3 peaks as I hoped, and with
correct ratios. I assumed the fact that I was
able to resolve distearin and diolein was due
to the length (50m) of the column.

However, when I tried Sigma P3544 (1-Palmitoyl,
3-stearoyl-rac-glycerol), I discovered TWO peaks,
one which coincided with 'dipalmitin' and one
which coincided with 'distearin'.

Running the four diacylglycerol species together
gave me only three peaks, however the peak sizes
seemed to be reasonably similar. It *is* possible
that P3544 was incompletely resolved from either
dipalmitin or distearin, but this seems unlikely.

I considered the possibility that my diacylglycerols
are breaking down (in the injector or column) and
generating free fatty acids and glycerol, and it is
these which I am detecting. From this I would have
expected that the palmitic and stearic peaks would
be larger than oleic - but the similar peak sizes
didn't confirm this one way or the other.

Stearic acid (alone) also has a peak corresponding
with 'distearin', so it would seem that what I am
detecting is fatty acids rather than diacylglycerols.

TLC plates have confirmed that they start off as
diacylglycerols at least, so my questions are :

a) Can underivatized (i.e. unsilylated) diacylglycerols
elute from a non-polar column ?

b) Would a lower injector temperature prevent them
from degrading, or would this just mean that they'd
never get on the column ?

c) If they are breaking down to form fatty acids,
how (if at all) does silylating them help against
this ?

I understand that silylating them blocks the
hydroxyl group and makes them more non-polar
[and thus ideal candidates for resolution on our
CP Sil 88 FAMEs column], but if they fragment
before they get onto the column, it's not much
use... ?

Previously, I have managed to silylate the three
compounds, and they eluted a little later on the
non-polar column (i.e. retained for longer) - but
it now occurs to me that possibly the DAGs were
eluting as fatty acids and that the silylated-DAGs
were converted to silyl-Fatty acids in the injector.

Any thoughts ?
Thanks
Jo Brodie
(lipids moderator)