7 Chromatography: Separation Techniques in Biology
W John Lough and Mark Carlile
School of Pharmacy and Pharmaceutical Sciences, University of Sunderland, Chester Road, Sunderland, SR1 3SD, UK
7.1 Introduction to Chromatographic Separation
Chromatography is a term that is used to describe a group of separation techniques that utilise the distribution of molecules between a stationary phase and a mobile phase [1]. Separation is achieved by exploitation of a molecule's physicochemical properties that promote it to interact with the stationary phase or stay within the mobile phase. Interactions with the stationary phase promote a slower passage through the separation system. It is this distribution and the modulation of the distribution between these phases that makes chromatography a very powerful and versatile separation technique. Different types of molecule have different distribution constants and hence come off the column at different times. Thus, they are separated from one another before being measured. Importantly, this means that quantitative determinations based on chromatography can be optimised to give good specificity. In an analytical method validation to demonstrate that an analytical method is fit for its intended purpose, accuracy, precision, linearity, limit of quantitation and limit of detection may be evaluated, but it is the test for specificity that is often the most challenging of the validation tests [2]. Therein lies perhaps the main reason ...