Chromatography Forum

So here are some recent and old articles on IPC of peptides:

Journal of Chromatography A
Vol: 1080, Issue: 1, pp. 49-57, July 1, 2005
The perchlorate anion is more effective than the trifluoroacetate anion as an ion-pairing reagent for reversed-phase chromatography of peptides
Authors: Shibue, M.a; Mant, C.T.a; Hodges, R.S.a(Robert.hodges@uchsc.edu)

Abstract (English): The addition of salts, specifically sodium perchlorate (NaClO4), to mobile phases at acidic pH as ion-pairing reagents for reversed-phase high-performance liquid chromatography (RP-HPLC) has been generally overlooked. To demonstrate the potential of NaClO4 as an effective anionic ion-pairing reagent, we applied RP-HPLC in the presence of 0–100mM sodium chloride (NaCl), sodium trifluoroacetate (NaTFA) or NaClO4 to two mixtures of synthetic 18-residue peptides: a mixture of peptides with the same net positive charge ( 4) and a mixture of four peptides of 1, 2, 3 and 4 net charge. Interestingly, the effect of increasing NaClO4 concentration on increasing peptide retention times and selectivity changes was more dramatic than that of either NaCl or NaTFA, with the order of increasing anion effectiveness being Cl−&ll;TFA−<ClO4−. Such effects were more marked when salt addition was applied to eluents containing 10mM phosphoric acid (H3PO4) compared to 10mM trifluoroacetic acid (TFA) due to the lesser starting anion hydrophobicity of the former mobile phase (containing the phosphate ion) compared to the latter (containing the TFA− ion).

Journal of Chromatography A
Vol: 1080, Issue: 1, pp. 58-67, July 1, 2005
Effect of anionic ion-pairing reagent concentration (1–60mM) on reversed-phase liquid chromatography elution behaviour of peptides
Authors: Shibue, M.a; Mant, C.T.a; Hodges, R.S.a(robert.hodges@uchsc.edu)

Abstract (English): The homologous series of volatile perfluorinated acids—trifluoroacetic acid (TFA), pentafluoropropionic acid (PFPA) and heptafluorobutyric acid (HFBA)—continue to be excellent anionic ion-pairing reagents for reversed-phase high-performance liquid chromatography (RP-HPLC) after more than two decades since their introduction to this field. It was felt that a thorough, step-by-step re-examination of the effects of anionic ion-pairing reagents over a wide concentration range on RP-HPLC peptide elution behaviour is now due, particularly considering the continuing dominance of such reagents for peptide applications. Thus, RP-HPLC was applied over a range of 1–60mM phosphoric acid, TFA, PFPA and HFBA to two mixtures of 18-residue synthetic peptides containing either the same net positive charge ( 4) or varying positive charge ( 1, 2, 3, 4). Peptides with the same charge are resolved very similarly independent of the ion-pairing reagent used, although the overall retention times of the peptides increase with increasing hydrophobicity of the anion: phosphate<TFA−<PFPA−<HFBA−. Peptides of differing charge move at differing rates relative to each other depending on concentration of ion-pairing reagents. All four ion-pairing reagents increased peptide retention time with increasing concentration, albeit to different extents, again based on hydrophobicity of the anion, i.e., the more hydrophobic the anion, the greater the increase in peptide retention time at the same reagent concentration. Interestingly, phosphoric acid produced the best separation of the four-peptide mixture ( 1 to 4 net charge). In addition, concentrations above 10mM HFBA produced a reversal of the elution order of the four peptides ( 1< 2< 3< 4) compared to the elution order produced by the other three reagents over the entire concentration range ( 4< 3< 2< 1).

Journal of Chromatography A
Vol: 1080, Issue: 1, pp. 68-75, July 1, 2005
Effect of anionic ion-pairing reagent hydrophobicity on selectivity of peptide separations by reversed-phase liquid chromatography
Authors: Shibue, M.a; Mant, C.T.a; Hodges, R.S.a(Robert.hodges@uchsc.edu)

Abstract (English): Despite the continuing dominance of trifluoroacetic acid (TFA) as the anionic ion-pairing reagent of choice for peptide separations by reversed-phase high-performance liquid chromatography (RP-HPLC), we believe that a step-by-step approach to re-examining the relative efficacy of TFA compared to other ion-pairing reagents is worthwhile, particularly for the design of separation protocols for complex peptide mixtures, e.g., in proteomics applications. Thus, we applied RP-HPLC in the presence of different concentrations of anionic ion-pairing reagents – phosphoric acid, TFA, pentafluoropropionic acid (PFPA) and heptafluorobutyric acid (HFBA) – to a mixture of three groups of four 10-residue peptides, these groups containing peptides of 1, 3 or 5 net charge. Overall separation of the 12-peptide mixture improved with increasing reagent hydrophobicity (phosphate−<TFA−<PFPA−<HFBA−) and/or concentration of the anion, with reagent hydrophobicity having a considerably more pronounced effect than reagent concentration. HFBA, in particular, achieved an excellent separation at a concentration of just 10mM, whereby the peptides were separated by charged groups ( 1< 3< 5) and hydrophobicity within these groups. There was an essentially equal effect of reagent hydrophobicity and concentration on each positive charge of the peptides, a useful observation for prediction of the effect of varying counterion concentration hydrophobicity and/or concentration during optimization of peptide purification protocols. Peak widths were greater for the more highly charged peptides, although these could be decreased significantly by raising the acid concentration; concomitantly, peptide resolution increased with increasing concentration of ion-pairing reagent.

Journal of Chromatography A
Vol: 660, Issue: 1-2, pp. 17-23, February 4, 1994

Explanation of the selectivity differences between reversed-phase ion-pair chromatographic systems containing trifluoroacetate or heptafluorobutyrate as pairing ion
Author: Patthy, Miklósa

Abstract (English): The well documented selectivity differences found between reversed-phase ion-pair chromatographic systems containing trifluoroacetate or heptafluorobutyrate as pairing ion were explained after determining sorption isotherms for trifluoroacetate and heptafluorobutyrate on Nucleosil 100-5 C18 from a solution similar to the eluents used for the separation of transmitteramines and peptides. Based on the isotherms and retention data obtained with reversed-phase, ion-exchange and reversed-phase ion-pair chromatographic systems, it is proposed that the selectivity differences between the systems studied are caused by the fact that trifluoroacetate and heptafluorobutyrate are not interchangeable in terms of their surface concentrations at the practical eluent concentrations of the pairing ions concerned.

And as I had to dig in my biblio to find these, I found the old articles that had investigated all the different fundamentals parameters which probably cover everything else...

These are the Vigh and co-workers studies which published 5 different articles with the general title "Studies in reversed phase ion pair chromatography" and then a second title with the parameters investigated. If I am not wrong, there are pieces taken out from there for the book "Practical HPLC development"...

Also, if I am not wrong, the first article published for peptide separation using different perfluorocarboxylic acids is from Bennett et al. J. Liq. Chromatography, 3, 1353-1365 (1980) with title: The use of perfluorinated carboxylic acids in the reversed phase HPLC of peptides

Thanks for the bibliographic work.

No problem,

Actually I meant to put the references of Vigh and co-workers work but I forgot:

So here they are:
Studies in reversed phase ion pair chromatography:
I. Adsorption isotherms of tetraalkylammonium ion pair reagents on lichrosorbRP18 in methanol water elutents J. Chromatogr. 260, 1983, 337
II. Retention of positive and neagetive ions and neutral solutes in tetrabutylammonium bormide containg methonal water elutents on lichrosorb RP-18 J. Chromatogr. 265, 1983, 182
III. The effect of counter ion concentration J. Chromatogr. 291, 1984, 91
IV. The role of the chain-length of the pairing ion. J. Chromatogr. 303, 1984, 29
V. Simultaneous effects of the eluent concentration of the inorganic counter ion and the surface concentration of the pairing ion J. Chromatogr. 395 1987, 503

And as a bonus from the same author:
Ratinalization of the selection of the type of the organic modifier(s) for selectivity optimization in reversed phase ion pair chromatogrpahy J. Chromatogr. 485, 1989, 403

Kostas-yes, thanks for this useful literature survey.

I still think Mark's work is valuable because he has roughly evaluated in the same study with the same instrument (I guess) quite a range of inorganic and organic additives enabling a direct chromatographic comparison. Other workers have e.g. concentrated on organic ion pair additives because they were interested in MS work, and have also e.g. concentrated on the mass spec response with the different organic additives. These studies are valuable. However, the chromatographic performance is not entirely understood-for example the comparison of peak shapes with the different additives. Oh, I think C. Huber has published some papers which hint at differences in peak shape, but I do not think a systematic investigation of such phenomena was a major objective of his studies either.

Kostas, the rt predictability you mention is just one of the reasons why I feel sorry for our poor doctoral student: The "easy" stuff is done. He would have to see whether he can predict what would be the best concentration of water, organic, other modifyers, the pH, the temperature, the stationary phase, flow rate. Already I can´t fathom all the material in this chain.....

Although there is some academic interest on doing so, and unless you are doing targeted peptide analysis, considering any possible combination might not be of much value (not to mention very challenging if you are considering a broad spectrum of peptide structures).

For example you might have hard time to convince people to go to other than TFA mobile phase additives, which also means acidic vs. basic pH etc. Also such a study will also be based I guess to isocratic separations which again are useful for limited amount of compounds or purification purposes.

The new forthcoming method I developed is based on emperical data, from a huge database we have in house. Specifically the study use data from >12000 LC-MS-MS runs, with >345,000 confidently identified unique peptides. The filtering was done out of 4 billion non-unique peptide identifications. Sophisticated allignement algorithms implies that we can use the model for other similar separations prediction, all you need is a LC-MS-MS run from that other separation (with several hundrend peptide identifications). -The alignement results are preliminary so I can put exact numbers on this...-

Do you have a doctoral student working on these stuff?

The most recent doctoral student has been trying to find out whether a radio-I labeled monoclonal antibody can be used to locate, maybe even destroy, colon cancer and metastases (it didn´t work well enough). I am mentioning this to underline that to someone in such an application oriented position it would indeed be exceedingly important that those in more basic research would not play the "True Believer" (Eric Hoffer), but instead go on with unbiased (and unequivocal) studies.
In the case of TFA it would be very important to know whether it´s uniqueness is allowing for separations which are not practical otherwise, and why. So, I am not really trying to convice anybody not to use TFA, but rather am trying to convince that more studies are needed.

Well! I don't know Hans...

It looks to me that there is only one bias here, and that it is yours against the TFA . After all, you said it yourself that TFA in your hands was only trouble (one of your previous posts in this tread).

Also, if one goes through the pubications or even through the study that Mark did, someone (openminded) would at least conclude that TFA seems to have a competitive edge vs. other mobile phase additives when comes to peptide separation.

However, your post were always, "yes but..." or "if I interpret the results correctly we could do the same with something else..." or implying that basic research in this field is biased...

PS: I am having a tough day at work so I feel confrotentional

Yes, my experience has produced a certain bias and uncertainty, that´s the reason I am watching discussions on TFA closely, it just happens that I have not seen any unequivocal evidence. Humans are not so helpless as not to know when some things are "nailed down".

Good to know others have bad days also, hopefully these are not too many!