8] Detection in liquid chromatography

Abstract

Publisher Summary Sensitive and selective detection of biological macromolecules is the goal of almost every biotechnology laboratory and chromatography is an important tool in biopolymer characterizations. However, no one detector is capable of completely characterizing an eluting biopolymer from a chromatographic column. The detector is a tool that provides the researcher with different information based on the requirements of the sample. While numerous texts have appeared, dealing with detectors for high-performance liquid chromatography (HPLC) or biopolymer separations, most of these have not specifically emphasized biopolymer detection. This chapter discusses the detection and characterization of components in a biological sample. Each detector provides a different type of information, in terms of spectroscopic, electrochemical, light-scattering properties, molecular weight, etc. The real problem in the unambiguous identification of a given biopolymer lies in the fact that within each class of biopolymers (proteins, nucleic acids, and carbohydrates), properties are similar to one another from a detection point of view. The lack of specificity in spectroscopic and electrochemical detectors is particularly problematic in biotechnology, where minor variants of proteins are frequently the analytes of interest. Therefore, although single-wavelength ultraviolet (UV) absorbance detection is among the most universally applied detection techniques, it often does not provide the selectivity necessary to characterize a biopolymer. If more selectivity is needed, dual-wavelength or linear diode array spectra can be employed. Fluorescence (FL) and electrochemical (EC) detection can also be employed for improved selectivity of appropriate samples. Derivatization can be utilized for improved sensitivity and selectivity. Molecular weights can be determined, with mass spectrometric and light-scattering techniques. Mass spectrometry (MS) can yield further structural information about the biopolymer from fragmentation studies, while low-angle laser light-scattering (LALLS) techniques can give molecular size and may detect aggregation formation. This chapter discusses the UV, FL, EC, and LALLS detectors.