5] Microcolumn liquid chromatography in biochemical analysis

Abstract

Biochemical methodologies based on various forms of liquid chromatography (LC) have been extremely important to progress in both structural investigations and physicochemical measurements performed on biological macromolecules. The high-performance LC (HPLC) instruments, featuring analytical-scale (4.6-mm i.d.), and preparative columns have now become the standard equipment in biochemical laboratories. While such instrumentation performs admirably in the day-to-day tasks of a biochemist (ranging from isolation work to routine sequencing methods to various other determinations), there is an increasing need to work at the microscale and nanoscale. Miniaturization of chromatographic separations and the associated small-scale manipulations is certainly one way of dealing with the problem of analyzing minute quantities of important biomolecules isolated from complex biological materials. Increasing sophistication of chromatographic detectors, in terms of sensitivity and structural information, provides further incentive for the development of microcolumn separation methods. This chapter discusses the microcolumn LC and its related techniques to provide a perspective on its value in biochemical research. Whereas much developmental work in micro-LC employed separation of small molecules, the primary emphasis of the discussion is on biological macromolecules and their degradation products. After a brief discussion of the microcolumn types and their technology, the instrumental aspects pertinent to the field of biomolecular analysis are described in the chapter. Selected applications further demonstrate certain unique capabilities of microcolumn separation systems.