I used LH-20 resin for separation for small molecule from bacterial biomass. I used MeOH-water for eluting solvent.
How I can equilibrate LH-20 resin before loading the sample onto the column?
Thank Dr. E. Galeano very much for your kind valuable information.
As you know, 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. This will also take care of equilibrating your column
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 Dr. Jack Silver for important information. May you tell me the following:
Firstly I loaded LH20 resin in a large size open column. Before loading LH20 into glass column, I swelled the resin with 50% MeOH (0.1%AcOH) in 3 hrs then I loaded it into open glass column. After loading LH20 resin into column, I used 50% MeOH (0.1% AcOH) for equilibration (about 5 CV) then I loaded my samples (2% of bed volume) and starting eluting my desire compound.Is it ok? or I need to change any thing? Plz suggestion. And how you can prepare your sample?
Dr. Bhuiyan- what you did sounds fine to me. Based on the information I see in this thread thus far, I really wouldn't change anything.
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