Chromatography Forum

The peptides were purified by using 0.1%TFA/water as buffer A and 0.1%TFA/acetonitrile as buffer B on a reverse phase C18 HPLC column. I am looking for an easy way of removing Triluoroacetic acid from the lyophilized peptide and to make an acatate salt of the peptide. When I tried SPE method-I am loosing 50% of the peptide. Is there any other technique that can used to exchange TFA salt- acetate salt and with minimum loss.

Thanks
vv

Maybe one can improve the SPE method. Please describe what you have done.

From your description, I assume that you tried to use a C18 or C8 SPE to get ride of TFA, but also lost peptides during the washing step. Apparantly, C18 may not be a good option for retaning peptides for this purpose.

I would suggest you to try other available phases. One phase, called SDB (styene DivinylBenze ??), could work.

Maybe one can improve the SPE method. Please describe what you have done.

I used STRATA GIGA Tubes from Phenomenex for replacing TFA with acatic acid. (The purification of the peptide was done by C18RP-HPLC). I dissolved teh peptide in DI water and the column was conditioned with 100ml of 5%acetic acid in acatonitrile then equilibrated with 100 ml of water, then peptide was loaded, then washed with 200ml of water and fractions were collected , then peptide was eluted with 5%acetic acid in acetonitrile and collected fractions were lyophilized.

The simplest thing to try is to add acetic acid to all steps that use water at this moment. Dissolve the peptide in water with 5% acetic acid. Wash the SPE with acetonitrile with 5% acetic acid, then with water with 5% acetic acid. Then load your sample, and then wash with water with 5% acetic acid and elute with acetonitrile with 5% acetic acid. If this does not work we will need to think about a better scheme.

The simplest thing to try is to add acetic acid to all steps that use water at this moment. Dissolve the peptide in water with 5% acetic acid. Wash the SPE with acetonitrile with 5% acetic acid, then with water with 5% acetic acid. Then load your sample, and then wash with water with 5% acetic acid and elute with acetonitrile with 5% acetic acid. If this does not work we will need to think about a better scheme.

Thank you, I am concerned about the recovery too. How to improve the recovery?

Have you considered a strong cation exchange SPE? You can elute with 1M ammonium acetate at pH 4.5. Lyophilization will remove the excess ammonium salt.

Have you considered a strong cation exchange SPE? You can elute with 1M ammonium acetate at pH 4.5. Lyophilization will remove the excess ammonium salt.

No, I haven't. I am thinking of Dowex beads too. Do you have the protocol for this ion exchange SPE?

Thank you
Vasanti

I suggested the improvements to your method to see if your recovery improves. I suspect that you loose some of your analyte, because you did not control the pH in the intermediate steps. I also suggest that you collect all the fractions to see where the lost part of your peptide ends up. If one knows where the problem is, one can make improvements to the method.

There are possibly some other methods that could be worth exploring. You could potentially work with an Oasis ion exchanger, but let us try to improve this method first.

If the peptide is not too small you may use ultrafiltration or SEC, possibly saving one lyophilization to boot.

I've had success using Phenomenex Strata X-C using the following procedure:

Condition: 1 column volume of methanal
Load: Sample (HPLC fraction containing basic analyte + TFA)
Wash: 2 column volumes of methanol
Elute: 2 column volumes of 5% ammonia in methanol

I'm am cheating a bit. This was from an application note they sent me a while ago. It worked great for me!

Yes ....
That is the method for basic analytes developed by my group and copied later by Phenomenex. I guess I should be flattered ...