Most of my material is retained on the ODS column. It also cannot be washed off the ODS plate (with Rf at below 0.2). The sample is polar and was eluted from D101 resin using Ethanol:Water (gradient elution). I've even tried washing the column out with Acetic acid but still can't get the material out.
What do I do?
What kind of material am I dealing with here?
Hello, do you mean 'material' the compound? Which mobile phase have you used for eluting the compound? Are you trying to purify any compound from plant extracts? Clarify. Better not to use acetic acid for column clean up.
By material, I am referring to my fraction. I extracted alkaloids from my plant, and the non-alkaloid fraction (which I described earlier as "material") is what is giving me a headache--literally. I put it on D101 macroporous resin and eluted with EtOH and water. The sub-fractions were separated further on LH-20, and from then on it has been a real headache. TLC profiles of the fractions from LH-20 are good, but in my attempt to prepare the material for HPLC, I noticed that it is retained on the ODS column and just will not wash out (!). I'm not sure what kind of 'compounds' may be contained in there. (Literature on the plant shows that the non-alkaloid compounds in it are mainly glycosides). I would be glad to receive suggestions on what else to try out.
Dear Paul:
Try to wash/rinse the column by passing solvents with different polarities (starting with polar solvent: water slightly acidified with acetic, ethanol, then isopropanol and finally hexane; then passed again isopropanol and ethanol, finally re-equilibrate the column with the usual ethanol:water mixture).
Compounds like phthalates or glycsides are really hard to desorb from ODS columns.
Best,
Paul:
Have you tried a shorter carbon chain like C8? There should be less hydrophobic interaction with this chain as compared to ODS. I agree with Aurea that you need a less polar solvent but the procedure of going to hexane and then back to aqueous solvents can be time-consuming and you may experience solubility problems in the column if not done in the exact order. Can't you switch from ethanol water to acetonitrile-water. I performed most of my alkaloid separations in ACN-water at an acidic pH. You can also add a small amount of THF to help in elution.
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