Rate constants Affinity chromatography

Walters, R.R., Practical approaches to the measurement of rate constants by affinity chromatography, in Analytical Affinity Chromatography, Chaiken, I.M., Ed., CRC Press, Boca Raton, FL, 1987, 1986, Chap. 3. 

The approach of implementing a biological assay as a postcolumn reaction detection system after liquid chromatography can not only be applied to antibody-based assays (immunoassays) but also to assays employing other affinity interactions with high association and low dissociation rate constants, such as receptors. Information obtained from such a detection system not only provides quantitative results but also indicates the biological activity of the detected compound. 

The importance of polysaccharides as support media in the field of affinity chromatography continues, as numerous reviews have indicated. Quantitative uses of affinity chromatography in purification, functional characterization, determination of rate constants, and measurement of macromolecule-ligand equilibrium binding have been discussed. Improvement of affinity chromatographic separations may be achieved by use of mixed solvents at carefully controlled concentrations and at temperatures in the normal to sub-zero range. Results of a theoretical study of the concentration dependence of elution volume for particular ligands have been presented. ... 

An alternative approach for equilibrium constant measurements in affinity chromatography is to use frontal analysis. This method is sometimes known as frontal affinity chromatography FAC). In this technique, a solution containing a known concentration of the analyte is continuously applied to an affinity column at a fixed flow rate (Fig. 2). As the analyte binds to the immobilized ligand, the ligand... 

Moore, R.M. Walters, R.R. Peak-decay method for the measurement of dissociation rate constants by high-performance affinity chromatography. J. Chromatogr. 

A simple procedure reported for the isolation of jack-bean a-D-mannosidase was based on affinity chromatography on agarose-immobilized benzidine. The influence of substituents on the hydrolysis of substituted phenyl a-D-mannopyranosides by a-D-mannosidase from Medicago sativa seeds has been investigated. As indicated by structure-activity relations, the electronic effect of the substituent has an influence on the rate of formation of the intermediate D-mannosyl-enzyme complex. This effect depends not only on the nature of the substituent, but also on its position meta or para) and on the temperature of the experiment. Hammett-type linear free energy relationships show that the reaction constant p changes its sign at ca. 27 °C. Substrates... 

A mixture of analytes is introduced as a solution onto the stationary phase and the mobile phase sweeps the components through it. Each analyte equilibrates or partitions between the phases in a different manner, primarily because they exhibit a different affinity for the stationary phase. Thus, each analyte has a specific equihbrium constant, and will have a different migration rate. This causes the analytes to be retained for different periods of time on the stationary phase, and to elute at different rates from the column. Each component of a mixture will be associated with a specific retention time. This type of chromatography is termed elution chromatography. 

A description of the principles of paper chromatography is not within the scope of this summary. Suffice it to say that the compounds to be separated have varying degrees of affinity for the cellulose fibers of the paper and differential solubilities in the solvent system used hence, different compounds will travel at different rates with relation to the rate of travel of the solvent. The ratio between the rates of travel of the solvent and the particular compoimd in question is constant under standard conditions and is designated the R/ of that compound ... 

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