Re: HPLC: 1 mm columns
Gary Hathaway (hathaway@cco.caltech.edu)
Wed, 13 May 1998 01:31:26 -0700
At 11:06 AM -0700 5/12/98, Mark Lively wrote:
>ABRF colleagues,
>
> I would like to know if it is possible to use an Applied
>Biosystems Model 140B Solvent Delivery system to run 1 mm
>i.d. columns.
Hello from Pasadena,
Mark,
I use the 140B HPLC pumps and the 785 detector. Currently I run 0.5mm
columns as preparative HPLC for proteolytic digests and off-line collection
(MALDI-TOF MS followed by Edman degradation). Since this column runs best
with a flowrate of 10ul/min. I use a precolumn splitter from LC Packings
and their capillary flowcell which I recall has a volume of 35 nl and a
pathlength of 8 mm(?). If you split precolumn, there is little reason to
remove the 75ul mixer as your main buffer flowrate will be in the 50-100
ul/min range. I get very good reproductibility for peptide digests and
detect at 200nm. For the 1 mm column, you don't need to use a splitter as
the optimal flowrate is near to 50ul/min. However, you will find that the
75ul mixer may give unsatisfactory gradient delays. I tried (at ABI's
suggestion) removing the mixer from the system and just routed the flow
directly from the 140's mixing T. This worked very well with good
reproducibility, but again for peptide digests. I tried another company's
mixing T, but got much worse results and so went back to ABI's version. You
may want to replace some of the delivery lines with 0.005" peek (65 micron
I.D.) to minimize dead volumes. We reconfigured our column heater and
detector to minimize runs and drilled a hole in the bottom of the 785 to
get flow going in an upward direction (to help clear potential bubbles) and
added a short 50 micron I.D. fused silica capillary delivery line to
minimize post flowcell delays and mixing. I added a pair of solvent bottles
with helium overlay and sparging to complete the modifications. I hope this
helps. If you need further details, you can contact me directly.
regards
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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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