I would like to know about this column chromatography material in phytochemistry applications. Can anyone explain how to pack this material and how to load a sample which is non-polar. Also I'd like to know whether it works in reverse phase mode. Thanks in advance.
LH-20 runs as mixed mode. It acts as a GPC (gel permeation chromatography) and also acts (to an extent) as a normal phase column.
Sephadex comes as a dried material and swells when solvent is added. Start with a small amount and add your mobile phase; it will swell, sometimes considerably. The first time I used the stuff, I swelled enough for 10 columns. Let it sit for a couple of hours, decant the fines if desired. I attached a link to the Sephadex instructions at the bottom of this reply; It lists solvents and other useful information.
Sample load is typically 1% to 2% of the volume. Do not let the bed dry out or cracks will form in the column that will reduce resolution. Also, keep the flow rate low. If the flow rate is too high, it causes back pressure which compresses the column, causing more back pressure, more column compression, and the cycle repeats. If using a glass open column, just run the column under gravity flow. If you are running the column on a flash chromatography system (so you can use the UV detector and fraction collector), keep the flow rate down to 1 or 2 mLs per minute unless a large column is being run.
Sephadex LH-20 instructions:
https://www.gelifesciences.com/gehcls_images/GELS/Related%20Content/Files/1314735988470/litdoc56119097AD_20110830235244.pdf
Examples of using a flash system with LH-20:
http://www.isco.com/WebProductFiles/Applications/101/Application_Notes/AN23_Self-pack_Column_Options_for_the_CombiFlash_Rf.pdf
http://www.isco.com/WebProductFiles/Applications/101/Poster_and_Paper_Reprints/Purification_of_Phenolic_Flavonoids.pdf
LH-20 runs as mixed mode. It acts as a GPC (gel permeation chromatography) and also acts (to an extent) as a normal phase column.
Sephadex comes as a dried material and swells when solvent is added. Start with a small amount and add your mobile phase; it will swell, sometimes considerably. The first time I used the stuff, I swelled enough for 10 columns. Let it sit for a couple of hours, decant the fines if desired. I attached a link to the Sephadex instructions at the bottom of this reply; It lists solvents and other useful information.
Sample load is typically 1% to 2% of the volume. Do not let the bed dry out or cracks will form in the column that will reduce resolution. Also, keep the flow rate low. If the flow rate is too high, it causes back pressure which compresses the column, causing more back pressure, more column compression, and the cycle repeats. If using a glass open column, just run the column under gravity flow. If you are running the column on a flash chromatography system (so you can use the UV detector and fraction collector), keep the flow rate down to 1 or 2 mLs per minute unless a large column is being run.
Sephadex LH-20 instructions:
https://www.gelifesciences.com/gehcls_images/GELS/Related%20Content/Files/1314735988470/litdoc56119097AD_20110830235244.pdf
Examples of using a flash system with LH-20:
http://www.isco.com/WebProductFiles/Applications/101/Application_Notes/AN23_Self-pack_Column_Options_for_the_CombiFlash_Rf.pdf
http://www.isco.com/WebProductFiles/Applications/101/Poster_and_Paper_Reprints/Purification_of_Phenolic_Flavonoids.pdf
Thank you for the response. I let the material swell and loaded onto a glass open column with sintered disc. So far so good. The compound of interest that I am after from this particular fraction is of moderate polar which is soluble in methanol but it is no longer in soluble state when water is added. Or is there are any other way to load the sample in this kind of situation?
The best way I know of running this column is by dissolving the sample and loading it carefully on the column (liquid load).
I think that you dissolve your compounds with methanol and load into the column. After that, you can run the column with the mixture of methanol and water.
I'd be careful about loading the column with a large volume of something other than the mobile phase. Sephadex swells to a different extent in different solvents (see the GE reference I posted above- it shows how many mL swollen LH-20 you get from 1 gram dried LH-20 in different solvents). Adding lots of something different than the mobile phase could cause the column to swell, which causes reduced flow rates, or shrink, which causes cracks in the column. The mobile phase (and dissolved sample) will flow preferentially flow through those cracks reducing resolution.
Hi, here's a data file of the Sephadex LH-20 from GE. I hope this could help you in your research. Regards!
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