Basic HPLC Theory and Definitions: Retention, Thermodynamics, Selectivity, Zone Spreading, Kinetics, and Resolution

Liquid chromatography is a very important separation method used in practically all chemistry fields. For many decades, it has played a key role in academic and industrial laboratories where it is used to analyze or purify components from complex mixtures. For example, it is used to separate proteins/drugs from impurities and to analyze drugs and endogenous components in biological materials. Most breakthroughs in biochemical and pharmaceutical sciences would probably not have been possible without chromatography. Chromatography is generally considered to have been developed in the early twentieth century by the Russian botanist Tswett. He found that he could separate components from plant extracts by flushing a sample with organic solvents through a glass tube packed with an inorganic adsorbent. Distinct bands of various colors evolved and migrated at different rates down the column. The bands corresponding to the different plant pigments could be collected at the outlet at the bottom of the tube. Tswett chose to call his technique “chromatography,” which means “color writing” in Greek. The name has been kept for historical reasons, although it is not very descriptive of the method in general. His publications had, however, little impact, and the technique fell into oblivion for several decades. Chromatography is based on the partitioning of solutes between two phases and is, therefore, related to simple liquid–liquid extraction. In chromatography, however, one phase (the mobile phase) is in constant movement relative to the other one (the stationary phase). The sample molecules are partitioned between the phases; those in the stationary phase are retained, whereas those in the mobile phase move. The interaction between the solutes and the stationary phase is most often based on adsorption. During a chromatographic separation, a solute normally partitions between the phases many thousand times. The basis of separation is that different kinds of molecules on average spend different amount of time in the stationary phase. Due to the large number of partitioning steps, chromatography has enormous resolving power and can separate mixtures of components with very similar physical properties. In the most common format, called column chromatography, the stationary phase is a highly porous solid material packed inside a cylindrical column (steel or glass), whereas the mobile phase is a liquid, a gas, or a supercritical fluid. If a successful separation has been 1