How to eliminate acetic acid in fraction after prep-HPLC ?

[atropine]

I have to use acetic aicd to separate many peaks form main peak.

I couldn't find the method without buffer(ex.acetic acid)

Does anyone have a technique can eliminate acetic acid in fraction after prep-HPLC ?

If it is possilbe, inform me...

Thank you very much in advanced.

[Yury Zelechonok]

Did you try to evaporate your mobile phase?
Acetic acid is volatile and it will evaporate almost completely. Even if acetic acid exist in form of salt with amines it will decompose and evaporate at temperature close to 100C.

[Uwe Neue]

I agree with Yuri. The evaporation of volatile compounds can be accomplished without difficulty usign vacuum together with elevated temperature. You may still end up with your compound in a salt form though. If you need to go beyond this, you will need more complex procedures.

[SaintAlphonzo]

possibly use trifluoroacetic acid as the buffer instead?

much more volatile, you won't have to use nearly any temperature whatsoever with a vacuum...

[Mark]

Possibly another good tefchnique for this would be freeze drying. I have seen lit somewhere (getting old and my memory is going fast so I cannot quote the source) but this should be usable if the analyte is not volatile.

Regards,
Mark

[HW Mueller]

What is your analyte?

[JM]

The most easy way to remove buffers from prep fraction ( desalting ) is to load the fraction on the same column using Deionised water as mobile phase to flush out any buffer or acid . than one can elute the compund of interest by a suitable solvent and evaporate solvent.

hope this will help.

JM

[JA]

The most easy way to remove buffers from prep fraction ( desalting ) is to load the fraction on the same column using Deionised water as mobile phase to flush out any buffer or acid . than one can elute the compund of interest by a suitable solvent and evaporate solvent.

Do you commonly employ this approach? I guess it depends on scale - what if your preparative run is collecting multiple fractions, per run, for your peak of interest..? Unless the job entails little more than a one shot, one tube prep and you can re-inject the entire collected fraction in one go it might be sufficient to just vac down your combined fractions and free base the analyte if it is known to have salted (eg. NMR/elemental). Any stronger base than your analyte should do (we commonly use ~molar aq. NaOH). Stir it up, partition the analyte into an organic solvent, dry, filter and vac it down.

I also like the freeze drying approach, but bear in mind it is typically used for water soluble compounds. If not, you can only guess what form your compound will take as you vac away the organic component of your fractions. Sometimes though, you have to try.. We recently made an attempt on a cpd which I can best describe as forming a glue when vacc'ed down from water/acetonitrile. Sure enough though, it freeze-dried out to give an amorphous solid which was not difficult to handle. The alternative would probably be to just high-vac your fractions.

Unless we know our cpds are thermally stable (and also so in solution) we use the minimum of temperature to recover fractions.. hardly ever going above ~35C for aqueous or buffered mixtures of methanol and acetonitrile.

[JM]

Hi JA,

The desalting of prep fraction is done seperatly from whatever no of fraction you collect. Remove the organic content of the fraction by vac and than load on to column with water wash. The only limitation is that your cmpd should retain in column during water wash.


JM

[JA]

Hi JM,
thank you for illustrating my point - you have to vac the fractions down first I suggested using a separate (and common) technique of 1H NMR to see if the compound is actually an acetate salt before deciding if passing it back down the HPLC is necessary.

There are many other concerns with utilising the HPLC for desalting, mostly related to product scale of which we are not informed. Fair play though