How to retailn a highly polar compound on RP column

[vkbojja]

Hi,
I am trying to develop a method for highly polar compound. Mol. Wt. 940.
So far I tried Different columns like C8, C18, Amide, Phenyl and hellic columns.
Only on amide column i got good retention but so much tailing.
I tried different ion pairing agents like TFA, TEA, Acetate buffers, Phosphate buffers,
Whatever the case, compound is eluting at dead volume only.
I don’t want to go for Normal phase.
I am expecting suggestions over this forum.
Regars,
vkbojja :

[Gerhard Kratz]

Which HILIC column did you use? You should select a HILIC column with an carbamoyl Group bonding, which has no endcapping. That is one Option. You can try a Zwitter ionic HILIC column to get a) Partition chromatography and b) IEX chromatography effects.
I personally don't like ion pair reagents, but in your case maybe Octan sulfonic acid will help, but be Aware that your C18 column will be modified on the surface, irreversible!!! Take an "old" C18 to test it.
Good luck.

[vahur]

Maybe you can add an apolar group before injection to your polar compound, like FMOC and then try RP column

[HPLCaddict]

It's a bit strange that you don't get retention in reversed phase (C18, C8, Phenyl) AND HILIC (Amide, "HILIC") mode. These two should be orthogonal and usually, if a compound doesn't show any retention in one mode, it should be easily possible in the other one.
No offense please, but did you choose correct conditions for both modes, i.e. high aqueous/low organic in reversed phase and high organic/low aqueous in HILIC?
Concerning the use of ion-pair, it depends on the nature of your compound which reagent you should use. There are anionic (sulfonic acids, SDS,) and cationic (tetraalkylammonium type) regents...

[syedzahidhussain]

I know this might look like a trival answer but I would really reconsider Mobile phase pH. What is the Pka of your analyte? If the pH of the MP is not close enought to the analyte, ionization will occur further increasing the polarity of the analyte and faster elution.

[bisnettrj2]

Have you tried normal phase? Polar column, mobile phase of something like iso-octane-ethanol, IPA-hexane?

[M. Farooq Wahab]

Hi,
I am trying to develop a method for highly polar compound. Mol. Wt. 940.
So far I tried Different columns like C8, C18, Amide, Phenyl and hellic columns.
Only on amide column i got good retention but so much tailing.
I tried different ion pairing agents like TFA, TEA, Acetate buffers, Phosphate buffers,
Whatever the case, compound is eluting at dead volume only.
I don’t want to go for Normal phase.
I am expecting suggestions over this forum.
Regars,
vkbojja :

Have you tried Hypercarb columns? Porous graphitic carbon, surprisingly retains polar compounds very well with a different selectivity, often with good peak shape. Ion pairing agents produce lots of system peaks. Can you share what functional groups are present on the compound?

Regards.

[DJ]

Without divulging too much, it would help tremendously if you could tell us a little more about the chemical nature of this solute.

[vkbojja]

Without divulging too much, it would help tremendously if you could tell us a little more about the chemical nature of this solute.

Soluble only in water and partially soluble in Methanol

[vkbojja]

Which HILIC column did you use? You should select a HILIC column with an carbamoyl Group bonding, which has no endcapping. That is one Option. You can try a Zwitter ionic HILIC column to get a) Partition chromatography and b) IEX chromatography effects.
I personally don't like ion pair reagents, but in your case maybe Octan sulfonic acid will help, but be Aware that your C18 column will be modified on the surface, irreversible!!! Take an "old" C18 to test it.
Good luck.

I dont have much facility about HILIC column. but i will try ion pairing agent whatever you suggested.Thanks

[vkbojja]

It's a bit strange that you don't get retention in reversed phase (C18, C8, Phenyl) AND HILIC (Amide, "HILIC") mode. These two should be orthogonal and usually, if a compound doesn't show any retention in one mode, it should be easily possible in the other one.
No offense please, but did you choose correct conditions for both modes, i.e. high aqueous/low organic in reversed phase and high organic/low aqueous in HILIC?
Concerning the use of ion-pair, it depends on the nature of your compound which reagent you should use. There are anionic (sulfonic acids, SDS,) and cationic (tetraalkylammonium type) regents...

In case of RP i tried all possibilities.But not in HILIC.

[vkbojja]

I know this might look like a trival answer but I would really reconsider Mobile phase pH. What is the Pka of your analyte? If the pH of the MP is not close enought to the analyte, ionization will occur further increasing the polarity of the analyte and faster elution.

Analyte PKa is 3. I am playing with my mobile phases arround that only.

[HPLCaddict]

I know this might look like a trival answer but I would really reconsider Mobile phase pH. What is the Pka of your analyte? If the pH of the MP is not close enought to the analyte, ionization will occur further increasing the polarity of the analyte and faster elution.

Analyte PKa is 3. I am playing with my mobile phases arround that only.

So you have a rather strong acid with an pKa of 3.
Some suggestions:
- Concerning reversed-phase chromatography, at pH-values of 5 and above your acid should be protonated (that is non-ionized) and hopefully hydrophobic enough to be retained on something like C18. Use a high amount of an appropriate buffer and low amount of acetonitrile. For scouting, I'd run something like a gradient from 95% buffer to 50%.
- If you still don't get retention on reversed phase, you might consider ion-pairing. For this, you should use a pH below your analytes pkA (so that it is in an anionic state) an a cationic ion-pairing reagent (some ammonium type stuff e.g. tetrabutylammonium hydrogensulfate).
- For HILIC, I'd also use a buffer below the pkA to get it ionized and run a gradient like 90% acetonitrile to 50% (that's the other way round as in reversed phase!).

I think Vlad is on holiday, otherwise he would have suggested mixed-mode by now . This might be a good option, unfortunately I'm not very experienced with this.

[DJ]

Ion Pair Chromatography

[tom jupille]

Concerning reversed-phase chromatography, at pH-values of 5 and above your acid should be protonated

I think you may have gotten turned around there. An acid will be neutral *below* its pKa. A base will be neutral above its pKa