Chromatography Forum

Ananda, I didn't mean to sound sarcastic. As long as I've been working with HPLC (>25 years), the "conventional wisdom" has been that RP of peptides involves multiple interactions. Your orginal post implied that the ability to separate these peptides was surprising. The point I was trying to make is that I would be more suprised if you could *not* separate two peptides with such a significant difference.

Or am I oversimplifying?

Tom,

I will back you up with some predicted peptide retention time values.

Below you may find the retention time predicted values of two peptides with 40 and 43 length residues that differ at the three amino acids that Ananda mentioned. Of course I didn't know what are the rest of the sequence so I used another 40 amino acid residue sequence that we have in our database. The retention times are normalized at 100 minutes gradient and these are the predicted retention time you will achieve if you use the separation conditions we describe in the Anal Chem article I mentioned before. Of course our method takes into account sequence information, peptide length and amphipathicity of the peptide so the values might be different as I do not have the actual sequence of the peptides but I think that the elution time difference should be representative.

So, I think that if used the right column, then a water-acetonitrile gradient with TFA-acetic acid as additives and a long gradient should be able to separate them. The supplementatry amino acids contributions in retention time would have made the longer peptide to elute later... (theoretically and with a lot of assumptions as mentioned earlier).

Well, in other words, you seem to have somekind of electrostatic repulsion as it is the only way to explain the reversed selectivity...

TTDVTGVVELQEGVEMVMPGDNVTFTVELIKPIAMEEGLR 74.7
TTDVTGVVELQEGVEMVMPGDNVTFTVELIKPIAMEEGLRITD 76.5

Ananda,

I think your explanation of the basis of separation seems sound. Based on the fact that the separation works with a combination of hydrophobic interactions and electrostatic repulsive interactions but not with a combination of hydrophobic interactions and electrostatic attraction interactions, this suggests that retention differences are additively constructive in your system. That is: hydrophobic interactions are greatest for the pI 4.6 peptide while electrostatic repulsive interactions are least for the pI 4.6 peptide with both effects increasing the retention of the pI 4.6 peptide relative to the pI 4.7 peptide. In the reversed case, TFA will increase electrostatic attraction interactions for the pI 4.7 peptide while the more hydrophobic nature of the pI 4.6 peptide causes an increase in retention, the two effects thereby canceling one another.

Chris

Chris,

Your explnation makes really sense and go in parallel to my rational as well. by the way it is TBA and not TFA. However, I also think it is just the hyrdrophobic interactions and the electrostatic repulsion (charge repluslion) are the parameters for this selectivity in the presence of of TBA.

Kostas

Thanks a lot for your comment and the paper. It is really nice to see there are possibilites of predicting the rT of peptides .

Tom,

No you didnt sound like sarcastic but I thought you overlooked at the problem.
From my experience it is not that easy to separate peptides that are different from 2-3 amino acids. Again it depends on the structure and the pI etc. However, these 2 peptides were not possible to separate with TFA or using any simple RP methods that is why we have to find alternative ways such as TBA and the other method should have tried is IXC which is kind of tedious.

Thanks for your comments again!

Ananda

Ananda,

I know you are using TBA. My expaination was an attempt to explain why TFA doesn't work but TBA does (in an earlier message you reported that you had tried TFA but were unable to accoumplish the separation in this case). I guess the part wasn't sufficiently clear.

Chris

Ananda,

I'm sure I don't have nearly the hands-on experience that you and Tom Jupille do but I remember some papers in the late 80's looking at different combinations of amino acids and finding that, even for exactly the same set of amino acids, retention times varied significantly depending on how they were linked together. I have one as: J. Chrom, 386, 223-228.

Best,
Marc

Marc,

This is the case only for alpha-helical non-amphipathic peptides vs. alpha-helical amphipathic peptides. I do not think that her case qualifies as 40 out of the 43 amino acids are linked at exactly the same way.