Chromatography Forum

Hi!

I hope to find some input to a sample preparation problem I am facing.

I will try to identify an unknown impurity that we see in the LC-UV method. That method uses tetrabutylammoniumhydrogen sulphate (TBAS) in the mobile phase, so I cannot transfer the method directly to the LC-MS instrument.

I have done fraction collection of the impurity, and injected that in a system without any ion-pairing agents (the primesep 100 column works fine on the main peak). The problem is that the chromatography looks terrible, probably from the excess of TBAS in the sample. There are huge peaks in the chromatogram, so it is hard to see my little 0.1% impurity. Is there a way to remove TBAS from a solution? Or any other good tricks to use?

I can of course inject the sample directly on the Primesep column, but then I have no idea which peak is which. Thanks!

I can't think of anything foolproof, but what about precipitating the sulfate with calcium hydroxide (converting the TBA to the hydroxide form) and then doing a liquid-liquid extraction into an appropriate organic solvent (hopefully the TBA salt of your impurity would be more soluble than TBAH)? I can see a lot of potential problems, including recovery and introduction of additional impurities, but it's the only thing that comes to mind.

Dear Mattias,

You can develop fully MS/ prep compatible method with Primesep by using appropriate column and mobile phase. For mobile phase you can use acetonitrile/water with any of the following modifiers: formic acid, acetic acid, ammonium formate, ammonium acetate or TFA. You can also:
1. Collect fractions on Primesep column with volatile mobile phase, concentrate them and inject in you other column using tetrabutylammoniumhydrogen sulphate Use this fractions as markers to see where compounds are eluting on your regular column.
2. Remove TBAS from fractions by trapping it on anion exchange resin.
3. Use more transparent mobile phase for UV method (sulphuric acid, phosphoric acid, TFA)
4. Let us develop method for this impurity at no charge

What mobile phase you are using for you Primesep runs (solvent ratios, buffers, gradient or isocratic, etc.)?

Contact us if you need help.

Regards,

Vlad

It sound like a good idea to do the fraction collection with the Primesep column, and then inject the concentrated sample into the old system. Why didn't I think of that...

I am almost sure about which peak is which on the Primesep column (the largest impurity).

The Primesep column runs on a gradient (6% ACN in 0.1% TFA to 30% ACN in 0.1% TFA). Separation optimised by Dry-Lab.

Vlad,

A different question (but related to the Primesep 100 column):

How do I clean the column in the best way?

thanks,
/Mattias