Dear Timm,
An informative and useful booklet published by Vydac that
deals mainly with reversed phase purification of peptides states that
the presence of SDS in protein samples seriously degrades separations
of polypeptides. It then adds that the effect is completely
reversible, and that SDS can be removed using a gradient of
acetonitrile in water (presumably with 0.1% TFA also present,
although I would doubt that the TFA is really necessary). They show
data with a C4 column showing that separations after washing away SDS
with an acetonitrile gradient were as good as separations on the same
column before exposure to SDS (even 0.001% SDS in injected samples
had some negative effects on protein separations, so the gradient
apparently at least reduced the level in the eluent below that
concentration). They also cite a couple of references to separations
of hydrophobic proteins having been improved by including detergents.
Although I do not have personal experience with this problem, their
comments lead me to expect that detergent removal should not be too
dificult, as long as the size exclusion column can be used with
organic solvents. Perhaps the manufacturer of the column shows
chromatograms with a test mix that you could at least partially
replicate to test the column after running a gradient to wash away
the detergent. I do not see any serious problems with the
chloroform:MeOH:water mixture, but chlorinated hydrocarbons tend to
be sort of hard on stainless steel components of an HPLC system, and
thus the chloroform should not be left on the system for a prolonged
period of time, if you choose to use it. If the choroform does leach
iron from stainless steel parts of the system onto the column, that
might also be a potential problem, but I am not sure how serious it
is for your columns.
Good luck getting the columns to work.
Dan Brune
>IĄd like to use three TSK-Gel SW ( silica-based ) hplc gel filtration
>columns for protein purification. Anyway, i have been told that these
>columns have been used with detergent ( i don`t have any information
>what kind of ... ). So, what`s best to remove any detergent from the
>columns: gradients of acetonitrile in water or the
>chloroform:MeOH:water (2:5:2) mixture described in an earlier posting (
>might it damage the column ? ) or something else ?
>Second: Is there any easy method ( physical , biotest, spectroscopical )
>to detect trace impurities of detergent in the effluent ?
>
>Any hints are welcome !!!
>
>Thanks, Timm
>
>T Maier
>FU Berlin
>Macromolecular Crystallography
>tmaier@chemie.fu-berlin.de
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