IUBio

Problems due protein accumulation on a RP-18 column.

Peter pxpst2 at unixs.cis.pitt.edu
Thu Mar 11 09:09:26 EST 1999


In article <36E791AA.3C9F3036 at bibo.lb.tu-berlin.de>, Klaus Throm
<lewr at bibo.lb.tu-berlin.de> wrote:

> For quantifying amino acids, I use a OctaDeacetylSulphate 2 column.
> I´ve got protein accumulation due an inadequate precolumn.
> For purging the column, i´ve got two protocols:
> 
> 
>      60% isopropanol, 40% 4M urea
>      60% isopropanol, 40% 4M acetic acid
> 
> 
> Does anybody have any recommendations about other protocols or
> deviations like thiourea?
> 
First, It is best to use repackable precolumns.  You can buy bulk packing
from the manufacture.  Alternatively you can get premade guard columns.
Second,  It is generally best to stick to the washing protocols that is
provided by the manufacture.  They know more about the chemistry of their
columns better than anyone else.  With this said, I find that one of the
best techniques for cleaning a column is to reverse the flow and run over
night at a slow flow rate(<0.2 ml/min).  Normally it is the frits on the
inlet side that get clogged and increase the back pressure.  If you are
seeing spikes in your absorbance, then you do not have a dirty column but
rather you have air in the column.  If this is the case the column is
destroyed for any analytical use.  The biggest problem with this back wash
technique is that the void space that sometimes develops on the front end
of the column may cause the packing to resettle and thus channels may form
and you will lose resolution(plate count decreases)  therefore, you have
to recalibrate the column following the back wash.  Once again, it is
smart to consult the column manufacture and ask if they have a "zero dead
volume" when the column is packed.   One note about the isopropanol, is
that it will greatly inhibit the resolvation of the proteins and make it
harder to remove.  Also, I would be very apprehensive about using the
Acetic acid.  Most columns will be severely damaged if the pH is outside
of the range of 2-8.5.  This is due to the "special" chemistry" of a
bonded phase to a siliceous support.  Generally, I use 8M urea in Water
only to clean very dirty columns but If you are preparing your samples
properly(filter thru .5um and pass over precolumn) these problems rarely
arise.
If you give me more specifics like who made your column, what the pressure
and the sive of the particles ,I might be able to give you more specific
advice.

Hope this helps

-- 
Peter



" Don't you eat that yellow snow
     Watch out where the huskies go"
                                    FZ



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