Hallo, with normal column chromatography, using CHCl3/MeOH (1/1-v/v) and also with LH-20 sephadex (elution: MeOH 100% and CHCl3/MeOH 2/8) but unfortunately it s not working. I want to use now HPLC so could anyone give me a suggestion for the best elution?. I guess that i can not use Water because of the precipitation of my product when i put some drop of water.
A little more information is needed prior to a complete answer can being given, simply which two of the glycosphingolipids are you trying to separate, from what matrix are you trying to determine them, what form of detection are using or have you access to ie light scattering or UV-vis or MS or ECd or fluorecence?
From a brief survey of available information the separation is not easy, normal phase chromotography appears to be the best choice and if chloroform methanol is not working then maybe add some acetone to the methanol, gives a slightly different separation compared to methanol alone.
Going for reverse phase HPLC is more difficult due to the polar and nonpolar aspects of these molecules, most researchers have derivatised with benzoyl chloride and gone on from there but its not straight forward and isomers cannot be separated unless separated prior to hplc with TLC.
Personally I would stick with normal phase, for what reason due consider your own attempts unsuccessful, what are you trying and what do you see?
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