I am doing chromatography of Ethyl Acetate Fraction in ods column . Initially I used 80% meoh in water . Then I decreased the polarity gradually to 90% and 100% meoh. However, some part of the loaded sample remained at the top. So I used acetone 40% in Meoh so that I could elute all the remaining samples. then i found some white particles in my fractions. It may be the resin leaked form the column. Is it right to use acetone in ods column? What is the best way to clean the column to remove the residual sample?
I think from my best knowledge of ODS columne that you should make your mobile phase more acidic by by adding approximately 0.1 % phosphoric acid or acetic acid.
@Baraa Siyo thank you for your response.......but i have further queries......if we use acidic solvent then i think it would be difficult to remove the acid from the recovered fraction....or is there other method to remove the acid in a simple way
Actually , I hadnot such problem before because I never use ODS column but I have been read a little bite about it before. But I think in such case you should keep in mind which solvent you used during discussion your result. Good Luck
If ethyl acetate is still present in your ODS column, I would not use an elution solvent with any aqueous content since ethyl acetate and water are immicible. If you want to load your extract in ethyl acetate and do not plan on completely removing it, I would suggest using MeOH/Acetone or MeOH/Ethyl acetate for elution. Acetone may damage an ODS column eventually, but most bonded phases are rugged enough to withstand a fair amount.
Based on the solvent you are loading your sample onto the column with and the proportion of MeOH required for elution, it may be worth looking at a less hydrophobic stationary phase, such as NH2 for future samples.
At 80% methanol in water, there should be reasonable solubility of ethyl acetate, at least from an analytical injection, maybe even more concentrated.
You can run acetone through a C18 column as a mobile phase; what you were running is called "non-aqueous reverse phase" and I've used the techniques to purify lycopenes.
I'm wondering if the column is old. Buffers left in columns can hydrolyze the C18 off the column over time, but the hydrolyzed C18 sticks to the C18 still bonded to the silica until washed out with a sufficiently non-polar solvent.
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