loadibility

[jiang295]

for peptide preparative work, if using convectional c18, which form of chromatography will supply higher loadibilility, first formic acid at ph=2.4, or use TFA as ion pair chromatography.

[tom jupille]

Off the top of my head, I don't think there would be an obvious systematic difference. Could go either way.

[jiang295]

Hi tom,
from one of paper(j chromatogr. A, 1075 (2005) 57) I just read, loadibility of peptide will increase with ion pair conc., they argue that ion repulsion is the major cause of overloading. do you think it makes sense.

another question why TFA can form better ion pair than acetic acid? i though acetic anion is more hydrophobic than tfa anion , do you think so ?

Off the top of my head, I don't think there would be an obvious systematic difference. Could go either way.

http://www.sciencedirect.com/science?_o ... 3198f337d9

[tom jupille]

While TFA does seem to act as an ion-pair reagent (more basic peptides become relatively more retained as TFA concentration increases), my guess is that it is more of a secondary interaction rather than the major retention mechanism.

I'm not sure about the relative hydrophobicity of TFA vs acetate (the CF3 moiety is quite hydrophobic), but in any case, there is more than just "ion-pairing" going on. TFA is a chaotrope, so it tends to inhibit secondary coiling of peptides, and it is a stronger acid that acetic, which means that the pH will be lower. Empirically, TFA works better for peptides, but I've never been able to sort out in my own mind exactly what the dominant effects are.

Which is why I could not come up with an "obvious" prediction as to which should have better loadability.

[jiang295]

While TFA does seem to act as an ion-pair reagent (more basic peptides become relatively more retained as TFA concentration increases), my guess is that it is more of a secondary interaction rather than the major retention mechanism.
i agree with that. It is unlikely that TFA can fully cover C18 surface.

I'm not sure about the relative hydrophobicity of TFA vs acetate (the CF3 moiety is quite hydrophobic), but in any case, there is more than just "ion-pairing" going on. TFA is a chaotrope, so it tends to inhibit secondary coiling of peptides, and it is a stronger acid that acetic, which means that the pH will be lower. Empirically, TFA works better for peptides, but I've never been able to sort out in my own mind exactly what the dominant effects are.
CF3CO2 anion is more electron delocated than CH3CO2 anion, probably that is the reason why CF2CO2 is more hydrophobic? I am not sure.


Which is why I could not come up with an "obvious" prediction as to which should have better loadability.

[HW Mueller]

Some time ago the solubility of the two acids were discussed, we never came to an answer if I remember correctly. In any case, if TFA is a ion pairing agent so is acetic acid at higher pH.

[jiang295]

why is solubility important here?

Some time ago the solubility of the two acids were discussed, we never came to an answer if I remember correctly. In any case, if TFA is a ion pairing agent so is acetic acid at higher pH.

[HW Mueller]

Polarity, in relation to retention.