small scale purification

D.K. dk at no.email.thankstospam.net
Tue Nov 26 22:39:35 EST 2002


In article <as0una$29s$3 at news.Stanford.EDU>, Mark Bowen <mboNOT at slac.stanford.edu> wrote:
>Does anyone have any experience scaling down liquid chromatography?  I 
>am trying to run very small ionic exchange and affinity columns 
>(10-100ul bed volumes), but I am having trouble finding good columns.
> 
>I am using Pierce's 2mL disposable columns, but I would like to get even 
>smaller empty columns to give a taller bed.  Does anyone know a 
>manufacturer of very small (preferably disposable) columns and fittings?

Sure. I've done it many times in various formats. 
Lots of possibilities here (geometries depend a lot on what 
type of chromatography you are doing). 

1. For 150-300 ul, nothing beats reusing Qiagen "pink" mini 
columns (DNA gel purification, etc kits). With cork borer
and a polyethylene filter (taken for example from used 
Qiagen's midi/maxi colums) you can make a top frit so
the bed never gets disturbed during loading/washes. 
3-10 ml syringes fit firmly on top and make great washing 
reservoirs. A piece of narrow silicone/tygon tubing can be 
fit firmly in the colum outlet - inscreasing gravity flow rate
or letting you connect the thing to a pump.

2. 50-500 ul, narrow column. Use 1 ml syringe. Bottom 
filter is made again using cork borer and a polyethylene filter.
Upper frit less needed than for larger columns but too can 
be made easily. 

3. 50 ul and below. Use tips with the very end cut out. Anything
starting with yellow tip through P10 tip to a gel loading tip. A 
small piece of teflon mesh (100 um and below, Spectrum or, 
IIRC, Nalgen) can be fit to make the bottom end. Snag fir using a 
cut out ring from another tip or carefully glue it in place with silicone 
caulk/glue.

4. I've seen (not personal experience) an extreme case of few ul 
columns in gel loading tip clogged with a piece of glass wool. Those 
are quite awkward to deal with. 

If all fails, you can always buy Pharmacia mini FPLC columns
at exorbitant prices :-))

HTH, 

DK



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