Differences in preparative loadability between the charged and uncharged forms of ionizable compounds.

Ionizable compounds experience a drastic difference in preparative loadability as a function of pH. It can be shown that the preparative loadability of a compound in the ionic form is by a factor of 20 or more inferior to the loadability of the same compound in the unionized form. In this paper, we demonstrate the reason for this behavior, and show practical applications of the principle.

Role of mass spectrometry in the purification of peptides and proteins.

Experiments were carried out to evaluate the fractionation of proteins and peptides according to mass. Model mixtures were separated by either reversed-phase or ion-exchange chromatography with mass spectrometry-compatible mobile phase additives. Fraction collection was triggered by the mass/charge ratio of each one of the components of the mixture. Chromatography was additionally monitored with a UV-Vis detector in order to compare the new technique with generally accepted in separations. The results indicated that adequate purification is achieved by this new technique. Fraction collection triggered by changes in the mass/charge ratio reduces sample handling and analysis time. This study demonstrates the utility of mass-directed fractionation of peptides and proteins when mass spectrometry-compatible mobile phase additives are used.