Last time when I done a Sephadex lh20 for Alternaria sp. metabolites. I found in my 1st bottle there are some small molecules near 197 m/z according to the LCMS (further work I know them are tenuazonic acid), then the large molecules come out in following bottles. So I got confused about this. Please give me a reason, thanks.
Sephadex LH20 is different from ordinary Sephadex gel filtration resins. It acts more like a hydrophobic interaction chromatography resin due to chemical modification. Instead of separating molecules on the basis of size (with larger molecules eluting earlier), it separates mainly on the basis of hydrophobicity, with more polar molecules eluting earlier.
The order of elution alone is not a adequate description of the elution pattern. One should use the fractions of a column volume. As you know, small molecules are retained on a size exclusion column longer than the large ones. If, in your case, small molecules were eluted at approximately one column volume, then the column worked properly. Probably, your large molecules were very hydrophobic and were retained on the column by forces of different nature.
If the small molecules were eluted substantially earlier than one column volume, you have a problem either with the sorbent or the packing of your column.
I hope this helps.
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