John,
Four comments come to mind:
(1) From practical experience, replacing the flow cell windows can have a
dramatic effect on the baseline slope and stability. Have you replaced or
cleaned the flow cell windows in the past year? The 6 mAU slope seems a bit
high for the flow cell to be the culprit, but replacing the windows every
year or two is a good idea in any case.
(2) Try using Tim Slattery's "C Solvent" in place of neat acetonitrile in the
"C" position. It does a great job of cleaning the column and keeping it
clean. The solvent is 0.1% TFA, 95% MeCN by volume. Unless your column is
new, it may take several days of sequencing to strip off all of the junk that
contributes to baseline rise. After you try this solvent, you will probably
stop using neat acetonitrile in the "C" position.
(3) The HP/Agilent PTH-B buffer is notorious for the increase in absorbance
with time spent on the HPLC. Historically, "old" high absorbance "B" seemed
to quickly "poison" freshly added "B" with respect to increasing absorbance.
There was a claim a while back that the problem was caused by light exposure,
but covering the "B" bottle locally didn't decrease the baseline rise over
time.
Try putting some fresh "B" in a 50ml culture/centrifuge tube, placing it
on the "B" port and then running a few chromatograms. Alternately, make some
31% IPA (B&J, HPLC-grade, by volume, no buffer), put it in the "B" position
and run a few chromatograms. You will be surprised at how similar the PTH-AA
retention times are to those when using Agilent "B". Since no buffer is
present, you should see a down slope for the first 10 min and may see a
rise/plateau toward the end of the run, depending on your IPA quality.
(4) It's not a perfect fix, but you can add acetone to "A" to balance the A/B
absorbance without significantly disturbing the PTH separation. You may get
a negative peak just before Asp. As a rule-of-thumb, adding 8ul of acetone
to 1L of "A" should increase the "A" baseline absorbance @ 269nm about 1 mAU.
It is a good idea to only add half the calculated amount of acetone and then
test the modified "A" on the HPLC. It is really hard to "undo" adding too
much acetone.
Hope that helps,
Steve Tindall
Argo BioAnalytica Inc.
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