Stephen R. Decker
Sdecker at Vines.colostate.edu
Mon Mar 4 23:02:24 EST 1996
I'll assume that you mean the HPX-87H, not 47H. We also use this column extensively
for fermentation analysis. Our parameters are 0.6 mL/min, 0.008N H2SO4 at 65oC with
a 20 ul loop injection. This operates at about 35-40 bar. The higher temp allows
better flow at lower pressure.
We normally do not look for lactate or succinate in our fermentation, although when
we have run lactate standards, they seem to generate split peaks which we never
In general, what you should look for are obvious column problems. Wide gentle peaks
indicate that you column has collapsed and the head space created is causing sample
dilution and mixing prior to entering the column. This problem is notorious in our
lab with the BioRad columns. The first step is to reverse the column and run the
flow backwards at 0.2 mL/min overnight. The return the column to its normal
orientation and try it. If this doesn't work, remove the upstream column end where
the sample enters and visually check to make sure the packing is not below the level
of the frit (still in the fitting). If low, you can simply use a spatula and make a
low conshape of packing using packing from an old column, does not even need to be
the same resin, but should be close. then just screw on the end fitting and get it
running again. I have found that this causes about 90% of our resolution problems.
If you are still having problems, try cleaning the column by reversing it in the flow
and running at 0.2mL/min, 5% Acetonitrile in 0.005N H2SO4 for 4 hours, followed by
30% acetonitrile in 0.005N H2SO4 overnight.
If it is a new column, then call up BioRad and have them replace the column. They
periodically have bad batches of columns and quickly replace the defects.
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