Abstract

Chromatography is a widely used analytical technique because of its resolving power, being able to separate sample components from one another prior to measurement. Nonetheless samples encountered in biology present a formidable challenge. They are often very complex, dynamic in nature, contain a diverse set of difficult‐to‐analyse compounds and may require the determination of analytes that are present at very low levels. Proteins, for example, are very large molecules with inherent slow mass transfer that may denature during analysis. While reversed‐phase chromatography may be applicable for the majority of small molecule separations, affinity chromatography, size exclusion chromatography, hydrophobic interaction chromatography and ion exchange chromatography might all need to be investigated before arriving at a suitable analytical method for proteins. In general, the complexity of biological samples is such that mass spectrometric detection in chromatographic methods is now well established and multidimensional chromatographic techniques are increasingly being used for very complex mixtures of small biological molecules such as protein digests.