@article{173,
  keywords = {Chromatography, High Pressure Liquid, Chromatography, Reverse-Phase, Formates, Ions, Mass Spectrometry, Protein Conformation, Proteins, Solvents, Trifluoroacetic Acid},
  author = {Balázs Bobály and Eszter Tóth and László Drahos and Ferenc Zsila and Júlia Visy and Jenő Fekete and Károly Vékey},
  title = {Influence of acid-induced conformational variability on protein separation in reversed phase high performance liquid chromatography.},
  abstract = {<p>Influence of acid concentration in the mobile phase on protein separation was studied in a wide concentration range using trifluoroacetic acid (TFA) and formic acid (FA). At low, 0.001-0.01 (v/v%) TFA concentration and appropriate solvent strength proteins elute before the column s dead time. This is explained by the proteins having a structured, but relatively extended conformation in the eluent; and are excluded from the pores of the stationary phase. Above ca. 0.01-0.05 (v/v%) TFA concentration proteins undergo further conformational change, leading to a compact, molten globule-like structure, likely stabilized by ion pairing. Proteins in this conformation enter the pores and are retained on the column. The results suggest a pore exclusion induced separation related to protein conformation. This effect is influenced by the pH and type of acid used, and is likely to involve ion-pair formation. The TFA concentration needed to result in protein folding (and therefore to observe retention on the column) depends on the protein; and therefore can be utilized to improve chromatographic performance. Conformation change was monitored by circular dichroism spectroscopy and mass spectrometry; and it was shown that not only TFA but FA can also induce molten globule formation.</p>},
  year = {2014},
  journal = {J Chromatogr A},
  volume = {1325},
  pages = {155-62},
  month = {2014 Jan 17},
  issn = {1873-3778},
  doi = {10.1016/j.chroma.2013.12.022},
}