Tandem columns and mixed-bed columns in high-performance liquid chromatography of proteins

By using a cation- and an anion-exchange column in series, mixtures of acidic and basic proteins were separated in a single chromatographic run with increasing salt gradient at pH 7.0. The serial order of the columns was found to affect the chromatographic results, and the effect was attributed to alteration of the salt gradient profile upon traversing the first ion-exchange column. Single columns, packed with a binary mixture of a cation and an anion exchanger gave similar chromatographic results as the tandem columns and thus offered an alternative approach to the separation of both acidic and basic proteins in a single chromatographic run. A ternary mixed phase was obtained by adding a mildly hydrophobic stationary phase to the mixture of the two ion exchangers. This column could be used with increasing salt gradient as a cation exchanger for the separation of basic proteins, or as an anion exchanger for the separation of acidic proteins. Furthermore, it could be used as a “bipolar” electrostatic-interaction column with increasing salt gradient and as a hydrophobic-interaction column with decreasing salt gradient for the separation of both types of proteins in a single chromatographic run. The constituent stationary phases used in the mixed-bed columns were prepared from the same silica support, i.e., they had the same particle and pore dimensions, density, and pore volume. Besides their obvious advantages in analytical applications, appropriate mixed stationary phases, all having retentive properties for the components to be separated, are expected to be useful also in preparative chromatography to “tailor” column selectivity for a given separation problem without loss of separating capacity.