ABD 140C HPLC
Gary Hathaway (hathaway@cco.caltech.edu)
Wed, 22 Apr 1998 01:36:40 -0700
At 4:05 PM -0700 4/20/98, Douglas Lamont wrote:
> Dear ABRFers, Would also appreciate any ideas you may have on the
>following problem...? Have following system setup for tryptic mapping
>with online LC/MS 140c HPLC - 10ml syringe pumps, 75ul dynamic
>mixer ABD UV Detector 785A - 2.4ul flowcell Valco low volume
>rheodyne - 20ul loop (PEEK) PEEK tubing to connect everything
>together - 005" TurboChrom Professional S/W to control the pump and
>collect the data. Column - Phenomenex 150x1mm Buffers - low
>TFA (0.02%) in both A and B I ran the above system with a tryptic
>digest sample and compared it with a chromatogram generated using the
>same buffers, column, gradient and sample on a Pharmacia SMART system. The
>Smart system gave a much better chromatogram with sharp well resolved
>peaks. The 140 system gave a poor profile with broad peaks and less
>detail. Any help would be great
> thanks in advance
> Dougie
>Douglas,
Although you do not mention your flowrate, I presume that you are running
at around 50-100 ul/min. I used a 140B system essentially the same as yours
for quite a while until we modified it to a microcapillary HPLC. So if you
are seeing broad peaks, you might try switching the 140c's column to the
Smart HPLC to check to see if the problem follows the column. If it does,
check for headspace on the top of your column. Another source of problems
with the syringe pumps is crossfeeding of A and B buffers during
pressurization. This can let B mix with A and is especially a problem if
you are starting your gradient at 0% B. I start at 5% to make certain that
during equilibration any B that "backed up in the A line will be purged
during the equilibration period (15 minutes). Check to see if you have
leaks at the rear of the pumps and how long it's been since you changed
seals. Also, if you are running ceramic pistons, they sometimes develop
cracks at the end and can wreak havoc with the system. Hope this is helpful.
regards from Pasadena
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Gary M. Hathaway, Director
PPMAL - Protein/Peptide Micro Analytical Laboratory
California Institute of Technology
139-74, Division of Biology
Pasadena, CA 91125
http://www.cco.caltech.edu/~ppmal
email Gary: hathaway@caltech.edu
email facility: ppmal@caltech.edu
phone: lab (818) 395-6388 or office (818) 395-2769
FAX (818) 449-3414
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