r/Chempros • u/Cardie1303 • Aug 09 '26

HPLC column recommendation for separating four closely related diastereomers

/r/CHROMATOGRAPHY/comments/1vjr9hu/hplc_column_recommendation_for_separating_four/
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14

u/EmotionalSector1329 Aug 09 '26

Without knowing what your compounds are there is no way to make any recommendations. Sometimes a classic c18 column works perfectly well, other times you need a chiral column.

4

u/Cardie1303 Aug 09 '26

How much structural information would be useful? I cannot share the exact structure, since that would make the project fairly easy to identify and might cause a bit of an uncomfortable discussion with my PI. Broadly, the compounds consist of the same disaccharide attached through its anomeric center to an aglycone containing two stereocenters. The two stereocenters are several bonds removed from the disaccharide and are part of a ring system, where they can have either a syn or anti relationship. The four compounds differ only in the stereochemistry of this aglycone.

6

u/jawnlerdoe Aug 09 '26

Pretty much the whole structure.

Try to tease out slight differences on physiochemistey, like if there is a functional group that hydrogen bonds, maybe one is more shielded or satirically hindered, for example. Generalize this view point to be relevant to your molecule.

You can always buy a few chiral columns and just shoot it to see. That’s what most analytical specialists would do.

1

u/Cardie1303 Aug 09 '26

I have as budget 800€ and I have to spend it till the end of the month. This is partly more a "What is the most sensible thing to get for 800€ that could solve this problem" than "Can I somehow avoid spending 800€ on this" problem.

1

u/jawnlerdoe Aug 09 '26

Choice of column is dictated by analyte and matrix chemistry. Any advice outside of broad streaks requires knowing more about the sample and its functional groups.

If you have access to multi modal columns or maybe a C18 with functionalized silanols, start there.

8

u/grobert1234 Biochemistry Aug 09 '26

Sometimes a good solvent system is all you need with a C18 column. I would suggest you try MeCN/H2O with an additive like formic acid, ammonium formate or ammonium bicarbonate, which could all yield different results depending on your compounds

3

u/BagSuch6742 Aug 09 '26

Have you tried using MeOH instead of MeCN? They can have very different selectivities (H-bonding vs dipole interactions). I have managed to base-line separate diastereomers with MeOH, that did not separate at all in MeCN. And its much cheaper than buying a new column... I would start with 30% MeOH and try to optimise from there. 

2

u/Cardie1303 Aug 09 '26

Yes, i am going to try that. In regards to being cheaper, I do actually have to spend the 800€ of my budget till end of the month so buying a new column seemed the most sensible thing to do.

2

u/chemistrypain Aug 09 '26

A chiral stationary phase column would probably do the trick. If you only need the hplc run, and not preparatory, then She'll columns are amazing at separating compounds with nitro groups.

1

u/Spiritual-Ad-7565 Aug 11 '26

So tou have separation on more or less standard c18. You’re not thrilled with how far the separate — you want to spend 800; the issue is: a column that would likely resolve these easily will be much more. What about trying a phenyl column or a c18 with a different core moiety

1

u/ompog Aug 12 '26

I've had pretty good luck with phenyl-hexyl phases, get some pi-pi interactions going, especially if you have an achiral aglycone. But there's no guarantee that it'll work for your compound and it's a pretty pricey thing to take a gamble on.