Chromatography Forum

Hi!
I have hydrophlic fraction from tamarind plant extract. When i ran HPLC with 5% ACN or even water only in C18 column everythign comes out in void volume..I ordered hilic columns from waters Atlantis Analytical HILIC Silica 3 um 2.1x50 mmm
but i am not getting good peak shape or better separation..lots of compds still comming out within 1 or 2 min. I have use 2% ammoniam acetate buffer for best separation i could get..what are my other choices..for buffer and column...your suggestions are higly appreciated.

Thanks

First, what is your sample dissolved in when you inject it? I would recommend starting with at least 85% ACN in the mobile phase. The sample should then contain at least 75% ACN, and preferably 85%. Second: Atlantis HILIC consists of uncoated silica. If your sample solvent composition meets the above criteria, then the next thing to try is a column with a thick, well-hydrated coating attached to the silica. Examples: Tosoh's Amide-80 or PolyLC's PolyHYDROXYETHYL A. Both of them have been used successfully for HILIC of polar small molecules in plant extracts.

What type of compounds are you working with? MWs?

I might also add: Don't uncritically use TFA in the mobile phase just because that's done in RPC. Chaotropes like TFA promote retention in RPC but have the opposite effect in HILIC. For best retention, use something like 10-15 mM of a buffered salt, like ammonium format, pH 3.5, or ammonium acetate, pH 4.5.

Adding to Andy Alpert's excelent comments, if your hplc does partial loop injections, make sure that the additional solvent filling the loop is not water or methanol. Anything with -OH groups is likely to be strongly eluting in hilic.

Hilic also depends on pH

Do also try asking your Waters rep. Column manufacturers often have some useful tips.

thanks for all you suggestion..i will try suggested conditions

Yes. I did not invent the mode, though. It's been used since 1975 for analysis of sugars on amino columns. Not everyone understood how the columns were working, though, or that it was a general-purpose mode. My 1990 paper merely brought it all together and filled in the gaps with some applications data of my own for classes of compounds not run before in this manner, just to prove my points about the mechanism.
Incidentally, the term "HILIC" is an abbreviation, not an acronym. The acronym for Hydrophilic Interaction Chromatography would be HIC, but that term had already been appropriated to stand for Hydrophobic Interaction Chromatography. After some consideration, I settled on HILIC as an expression that would be easy to pronounce.

Cool. I really find it fascinating that this forum has people who are considered some of the foremost minds in chromatography posting answers - users like Uwe Neue and yourself, seasoned vets like Bruce Hamilton and HW Mueller and danko who've worked in the liquid chromatography field for years, who find the time to post answers to newbies like me and others that post here, who must be asking questions the vets have seen time and time again over their long careers. It's humbling, but helpful, and appreciated.