Characterization of High‐Pressure Liquid Chromatography Columns using Chromatographic Methods

Abstract

A great variety of columns for liquid chromatography (LC) are available in dimensions ranging from industrial scale to micro‐bore, nano‐bore, and capillary size, and on‐chip columns. The columns may be used in various liquid chromatography modes or in capillary electrochromatography, depending on the support materials and stationary phase chemistry. Every year many new column types are introduced on the market, with improved selectivity and efficiency, long lifetime, and mobile phase compatibility, intended for general use, for liquid chromatography/mass spectrometry (LC/MS) applications, proteomic research, or for the analysis of other specific sample types. Considerable improvement in pH, high‐temperature, and high‐pressure stability of new column types, together with advances in the instrumentation, enabled introduction of capillary, high‐temperature, and ultra‐high‐pressure HPLC into routine practice. Even though reversed‐phase mode is still by the most widely used in contemporary LC, applications of other separation modes (such as ion, normal‐phase, or high‐interaction liquid chromatography (HILC)) have become more frequent recently, because of unique separation selectivity for certain sample types. Characterization of column quality is not a simple task, because a number of factors should be taken into account, that affect the selectivity, efficiency and resolution of sample separation and the reproducibility of chromatographic data. These include the type of the support, the arrangement and density of the stationary phase on the adsorbent surface, the homogeneity of the chromatographic bed, etc. Various physicochemical techniques are used for characterization of the properties of column packings however, most of them are suitable for bulk materials only and cannot be directly applied for commercial columns without damaging them. Not to destroy the columns, often precious and expensive, practicing chromatographers can apply chromatographic methods to characterize columns and evaluate their analytical suitability under real‐life conditions, where the intermolecular interactions between the analytes, the stationary phase, and the mobile phases affect the retention. The present review reports various chromatographic tests and strategies available for column evaluation.