After we cleaned a RP HPLC column used for proteins with a gradient
of isopropanol/0.1% TFA, we observed something odd (the iPrOH run looked
like a normal blank, i.e., it had some small peaks). The next run, a 5-95%
blank using ACCN/TFA, showed a slow increase in absorbance at 220 and 280
nm. The pressure also slowly increased to about 4X normal. After a long
flush at about 50% ACCN, the pressure and absorbances both slowly declined
to normal values.
The isopropanol was electronic grade and was filtered, but the
bottle was old. Although it is difficult to imagine a mechanism, I can't
think of anything besides the isopropanol wash that was atypical from our
normal operating procedues.
Does anyone have an idea of what happened and how to prevent it?
Obviously, if I use isopropanol again, I will use fresh HPLC grade
material. Thanks for any suggestions.
Chris Halkides
Christopher Halkides
Dept. of Chemistry, UNCW
601 S. College Road
Wilmington, NC 28403-3297
(910) 962-7427