Hello all;
Many thanks for all the input, posted and Emailed. It really helps to have
you propose possibilities in a puzzling situation like this so that they
can be eliminated one by one. For those interested, I think there is a
light at the end of the tunnel...read on.
I'm sure that there is more than one problem going on. Surprise, surprise.
By pure fluke I happened to catch one of them yesterday while watching my
cycles for clues, yet again. After the R3 TFA (metered, liquid pulse in the
473) is added, there is a long incubation time to allow for cleavage and
then, at step 57 in my cycle, the block is filled with S2 ethyl acetate and
then flushed out to waste, presumably to get rid of any residual TFA
hanging around in the block. The time on this "blk 1 flsh" step is 10 sec
and has been since day one, August 23, 1991. For some reason (?) 10 sec is
no longer sufficient and I was getting a quick shot of ethyl acetate
delivered to the reaction cartridge at the start of the next step, a "dry
cart". It makes sense that the hydrophobes would be more affected. It
happened in a heartbeat and is a valuable (and expensive) lesson to me that
when optimizing, I can't snooze during the "boring" parts of the cycle when
I'm not expecting anything signifigant to happen! I'm sure we all pay rapt
attention when there's a drydown or tranfer step in progress but then go do
some paperwork. No more.
As for the lifespan of the columns, that is still a mystery. Some of the
responses I've gotten indicate that this is more or less normal and other
people think I should return the columns. At nearly six hundred bucks
(CAN$), I'd like them to last at least as long as they did in the good ol'
days.
Thanks so much for all the help!
Sandy Kielland
Victoria Protein Microchemistry Centre
Department of Biochemistry and Microbiology
University of Victoria
Victoria B.C. CANADA
phone: (250) 7218884
FAX (250) 7218855
Email Kielland@UVic.ca
Webpage http://www.proteincentre.com
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