Overloading Study of Bases Using Polymeric RP-HPLC Columns as an Aid to Rationalization of Overloading on Silica-ODS Phases

Abstract

The separation of ionized bases by reversed-phase liquid chromatography with alkyl silica columns often leads to severely tailed bands that are highly detrimental. Band shape and its dependence on sample mass are notably different when mobile-phase pH is changed, and this behavior has not been previously explained. Ionized silanols present in the stationary phase have been credited with a role in determining peak shape. In the present study, separations on two different polymer columns were compared with those previously obtained on alkyl silica phases. Because silanols are absent from polymer columns, this comparison enabled us to assess the role of silanols in separations on alkyl silica phases and to offer an explanation of why band shape changes with sample size and mobile-phase pH for both polymer and silica-based phases.

Selection of suitable stationary phases and optimum conditions for their application in the separation of basic compounds by reversed‐phase HPLC

“…However, an alternative explanation by the same author was that initially adsorbed charged molecules discourage further adsorption of sample molecules of the same charge ie a mutual repulsion effect [43,44]. In an effort to elucidate the mechanism of overloading, we studied the behaviour of two purely polymeric columns (Hamilton PRP-1 and Asahipak ODP-50) with the bases shown in Figure 1 at low, neutral, and high pH [45]. At low and neutral pH, overloading patterns of these phases were very similar to alkyl-silica phases.…”

Overloading study of basic compounds with a positively charged C18 column in liquid chromatography

“…To avoid the possibility of silanophilic interactions, they studied with a strong acid and found that anionic solutes would also display similar tailing and overloading behaviors with that of bases. This mutual repulsion mechanism was used by McCalley to rationalize the overloading observed on purely polymeric phases [9], which clearly contain no silanols but still showed typical tailing peak shapes as often obtained on silica-based columns. Neue also used this theory to demonstrate the idea that the best loadability is always obtained for the unionized form of the sample, that is to say, basic compounds should be separated at alkaline pH [11].…”

The Use of Ammonium Formate as a Mobile-Phase Modifier for LC-MS/MS Analysis of Tryptic Digests

“…22,23 Basic analytes, including peptides, exhibit poor peak shape and significant peak tailing as a result of repulsion effects between ions of the same charge. 19,24,25 Under RP conditions, including low pH, it has been shown that the overloading of basic peptides occurs at significantly lower sample loads when compared with neutral molecules. [26][27][28] FA/AF has been shown to provide much higher sample load tolerance, similar to TFA, even for basic peptides.…”