Published Online: 15 SEP 2006
Copyright © 2000 John Wiley & Sons, Ltd. All rights reserved.
Encyclopedia of Analytical Chemistry
How to Cite
Huber, C. G. 2006. Biopolymer Chromatography. Encyclopedia of Analytical Chemistry.
- Published Online: 15 SEP 2006
Biopolymers are biological macromolecules including peptides, proteins, nucleic acids, and polysaccharides whose physical and chemical properties differ considerably from those of small molecules. Biopolymer chromatography is a branch of liquid chromatography which is adapted to the specific requirements of biopolymers in order to obtain rapid and high-resolution chromatographic separations of these molecules. Because of the low diffusion coefficients of biopolymers, special stationary phase configurations and surface modifications are necessary to permit sufficiently fast mass transfer kinetics between the mobile and stationary phase. Exploiting different molecular properties of the biopolymers, such as size, charge, hydrophilicity, hydrophobicity, the ability to form complexes with metal ions, or biological function, the most popular liquid chromatographic modes for biopolymer separation are size-exclusion, ion-exchange, normal-phase, reversed-phase, ion-pair reversed-phase, metal-interaction, and affinity chromatography.
Because of their large size and the multitude of different functional groups present in biopolymers, more than one mechanism may be responsible for interaction with the stationary phase, resulting in enhanced selectivity due to mixed-mode interactions. Adsorption at multiple sites results in very steep elution isotherms as a function of the mobile phase solvent strength. Therefore, elution with a gradient of increasing solvent strength is usually applied in biopolymer chromatography. The choice of a suitable chromatographic environment is of special importance ifthe native confirmation of a biopolymer, which is essential for its biological activity, is to be preserved for subsequent experiments. Generally, conditions that come close to the physiological environment are most appropriate.
The most important detection techniques for biopolymers are ultraviolet/visible absorbance detection, fluorescence detection, electrochemical detection, mass spectrometry, and low-angle laser light scattering photometry. Miniaturization and multidimensional separation together with mass spectrometric detection allow the routine separation and identification of femtomoleattomole amounts of hundreds to thousands of different biopolymers contained in complex mixtures of biological origin.