Affinity chromatography mobile phase

Elution Chromatography The components of the mobile phase supphed to the cohimn ter feed introduction have less affinity for the stationary phase than any of the feed solutes. Under trace conditions, the feed solutes travel through the cohimn as bands or zones at different velocities that depend only on the composition of the mobile phase and the operating temperature and that exit from the cohimn at different times. 

The coupling of supercritical fluid extraction (SEE) with gas chromatography (SEE-GC) provides an excellent example of the application of multidimensional chromatography principles to a sample preparation method. In SEE, the analytical matrix is packed into an extraction vessel and a supercritical fluid, usually carbon dioxide, is passed through it. The analyte matrix may be viewed as the stationary phase, while the supercritical fluid can be viewed as the mobile phase. In order to obtain an effective extraction, the solubility of the analyte in the supercritical fluid mobile phase must be considered, along with its affinity to the matrix stationary phase. The effluent from the extraction is then collected and transferred to a gas chromatograph. In his comprehensive text, Taylor provides an excellent description of the principles and applications of SEE (44), while Pawliszyn presents a description of the supercritical fluid as the mobile phase in his development of a kinetic model for the extraction process (45). 

Affinity chromatography (12) has become an important tool in the isolation of purified fractions of such substances as enzymes. Advantage is taken of specific interactions such as antigen-antibody interactions. One substance of the pair (e.g. antigen) is bonded to a support. When a mixture is passed through the column, the specific interaction retains the corresponding antibody relative to other substances. A change of mobile phase conditions then elutes the pure antibody. This method has a real potential for analysis of specific proteins in body fluids. 

Ion-exchange chromatography (lEC) is used mainly for the separation of ions and easily ionized substances (e.g., substances that form ions by pH manipulation or complexation) in which one of the principal contributions to retention is the electrostatic attraction between mobile phase ions, both sa le and eluent, for immobilized ion centers of opposite charge in the stationary phase. The sample ions are separated based on differences in their relative affinity for the stationary phase ion centers compared to that of the mobile phase counterions in a dynamic exchange system, in which sample ions and eluent ions interact with multiple stationary phase ion centers as they pass through the column. Ion-... 

Some kinds of chromatography require relatively little optimization. In gel permeation chromatography, for example, once the pore size of the support and number of columns is selected, it is only rarely necessary to examine in depth factors such as solvent composition, temperature, and flow rate. Optimization of affinity chromatography is similarly straightforward. In RPLC or IEC, however, retention is a complex and sensitive function of mobile phase composition column type, efficiency, and length flow rate gradient rate and temperature. 

Suresh, V., Gallant, S., and Cramer, S., Immobilized metal affinity chromatography displacer characteristics of traditional mobile phase modifiers, Biotechnol. Prog., 12, 84, 1996. 

Principles and Characteristics Liquid chromatography is the generic name used to describe any chromatographic procedure in which the mobile phase is a liquid. It may be classified according to the mechanism of retention in adsorption, partition, size-exclusion, affinity and ion-exchange (Scheme 4.4). These mechanisms form the basis for the chromatographic modes of... 

Partitioning in chromatography, the physical act of a solnte having different affinities for the stationary and mobile phases. 

This chapter focuses on gas-liquid chromatography, in which compounds in a sample are separated based on vapor pressures and differences in affinity for the stationary phase (a high boiling point liquid) versus the gaseous mobile phase. The time between sample injection and detection of the individual compound eluting from the column is called the retention time. Compounds that have limited solubility in the stationary phase will exit the column quickly as a large proportion will remain in the mobile phase. Compounds with polarity similar to that of the stationary phase will have longer retention times and potentially broader peaks, due to increased interaction with the stationary phase. 

This phenomenon can be exploited for separation and concentration of solutes. If one solute has certain affinity for the micellar entity in solution then, by altering the conditions of the solution to ensure separation of the micellar solution into two phases, it is possible to separate and concentrate the solute in the surfactant-rich phase. This technique is known as cloud point extraction (CPE) or micelle-mediated extraction (ME). The ratio of the concentrations of the solute in the surfactant-rich phase to that in the dilute phase can exceed 500 with phase volume ratios exceeding 20, which indicates the high efficiency of this technique. Moreover, the surfactant-rich phase is compatible with the micellar and aqueous-organic mobile phases in liquid chromatography and thus facilitates the determination of chemical species by different analytical methods [104]. 

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