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Spectral properties of some ion-pairing reagents commonly used in reversed-phase high-performance liquid chromatography of proteins and peptides in acetonitrile gradient systems

1986; Elsevier BV; Volume: 361; Linguagem: Inglês

10.1016/s0021-9673(01)86906-5

1873-3778

Günther Winkler, Peter Briza, Christian Künz,

Chromatography in Natural Products

Abstract Electronic spectra of some anionic anc cationic ion-pairing reagents in aqueous solution and in solvent mixtures with various contents of acetonitrile were recorded between 200 and 300 nm, and optimum conditions for highly sensitive detection using linear solvent gradients in reversed-phase high-performance liquid chromatography were proposed. Butane-, hexane and octanesulphonic acids showed no remarkable absorbance and recorded baseline drifts are therefore mainly caused by the increasing contents of acetonitrile. For trifuloroacetic acid (TFA) and heptafluorobutyric acid (HFB), the baseline trace of a linear acetonitrile gradient system is the result of a discontinuous change from the π → π* band of the anion in aqueous solution to the n → π* transition of the undissociated acid in the organic solvent and therefore depends strongly on the wavelength used. For both acids, there exists an optimal recording point below 220 nm (TFA: 215 nm, HFB: 219 nm). Cationic hydrophobic ion-pairing reagents such as alkylated amines are weakly protonated at a higher pH and show relatively strong absorbance below 250 nm. The ionization is further reduced by the addition of acetonitrile, leading to an extreme baseline drift. At low pH, however, complete ionization is achieved even at high contents of organic solvent, but ion-pairing with the carboxylic group of peptides is then impossible. In contrast to the amines, the tetraalkylammonium cation is a relatively strong Lewis acid and the pH dependency as well as the organic solvent dependency of the electronic spectra is therefore less marked. Nevertheless, in the case of tetrabutylammonium phosphate the baseline drift shows a minimum at pH 5 and therefore this pH is highly recommended for sensitive detection using acetonitrile gradients.