Hello I am curious about getting properly set up for sephadex LH-20 chromatography.
Can I do this under pressure the same way as with flash chromatography?
Is it the same to set up as with flash chromatography?
You don't want to run much pressure using Sephadex columns. The bed will collapse, creating more back pressure, causing more collapse, until you have no flow. If using an open glass column, you only want to do gravity flow- no air pressure. If you run a pumping system, run at a slow flow rate. If you see any bed compression, back the flow down.
You will need to swell the Sephadex before packing the column. The column swells to a different extent in different solvents (see the information posted by Greg above). This different swelling in different solvents tends to cause purification with isocratic solvent systems, rather than gradients used for flash chromatography..
It is possible to use a flash chromatography system with Sephadex, with a low flow rate if you pack the column yourself. This allows the use of the UV detector and the fraction collector. For an example, see: http://www.isco.com/WebProductFiles/Applications/101/Application_Notes/AN23_Self-pack_Column_Options_for_the_CombiFlash_Rf.pdf
Also- here is a link to the current Sephadex LH-20 manual: https://www.gelifesciences.com/gehcls_images/GELS/Related%20Content/Files/1314735988470/litdoc56119097AD_20110830235244.pdf
You don't want to run much pressure using Sephadex columns. The bed will collapse, creating more back pressure, causing more collapse, until you have no flow. If using an open glass column, you only want to do gravity flow- no air pressure. If you run a pumping system, run at a slow flow rate. If you see any bed compression, back the flow down.
You will need to swell the Sephadex before packing the column. The column swells to a different extent in different solvents (see the information posted by Greg above). This different swelling in different solvents tends to cause purification with isocratic solvent systems, rather than gradients used for flash chromatography..
It is possible to use a flash chromatography system with Sephadex, with a low flow rate if you pack the column yourself. This allows the use of the UV detector and the fraction collector. For an example, see: http://www.isco.com/WebProductFiles/Applications/101/Application_Notes/AN23_Self-pack_Column_Options_for_the_CombiFlash_Rf.pdf
Also- here is a link to the current Sephadex LH-20 manual: https://www.gelifesciences.com/gehcls_images/GELS/Related%20Content/Files/1314735988470/litdoc56119097AD_20110830235244.pdf
Another option you might want to consider is to reverse the direction of your chromatography flow setup, to run it from the bottom of the column to the top. In that case, you might get much higher flow rate even at higher pressure without any danger of compressing the resin under hydrostatic pressure.
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