Elution competitive

FIGURE 1.5 Typical chromatograms obtained by analytical affinity chromatography on an HSA column during (a) zonal elution competitive binding experiments, in which S-warfarin was the injected analyte and racemic verapamil was a mobile phase additive, and (b) frontal analysis experiments, in which S-verapamil was applied as the target. The concentrations shown represent the amount of (a) racemic verapamil or (b) S-verapamil applied to the column. Source Reproduced with permission from Ref. [91],... 

In its simplest form the competition model assumes the entire adsorbent surface is covered by a monolayer of solute and mobile phase molecules. Under normal chromatographic conditions, the concentration of sample molecules will be small and the adsorbed monolayer will consist mainly of mobile phase molecules. Retention of a solute molecule occurs by displacing a roughly equivalent volume of mobile phase molecules from the monolayer to make the surface accessible to the adsorbed solute aiolecule. For elution of the solute to occur -the above process must be reversible, and can be represented by the equilibrium depicted by equation (4.6)... 

Separations in hydrophobic interaction chromatography have been modeled as a function of the ionic strength of the buffer and of the hydrophobicity of the column, and tested using the elution of lysozyme and ovalbumin from octyl-, butyl- and phenyl-Sepharose phases.2 The theoretical framework used preferential interaction analysis, a theory competitive to solvophobic theory. Solvophobic theory views protein-surface interaction as a two-step process. In this model, the protein appears in a cavity in the water formed above the adsorption site and then adsorbs to the phase, with the free energy change... 

Elution. Incubate the resin for 30 min with rolling in 500 /il Elution Buffer to competitively elute retained proteins. Collect the supernatant after centrifugation at 9300 xg for 5 min at 4° in a microcentrifuge (10,000 rpm in an Eppendorf 5415R). 

In matrix solid-phase dispersion (MSPD) the sample is mixed with a suitable powdered solid-phase until a homogeneous dry, free flowing powder is obtained with the sample dispersed over the entire material. A wide variety of solid-phase materials can be used, but for the non-ionic surfactants usually a reversed-phase C18 type of sorbent is applied. The mixture is subsequently (usually dry) packed into a glass column. Next, the analytes of interest are eluted with a suitable solvent or solvent mixture. The competition between reversed-phase hydrophobic chains in the dispersed solid-phase and the solvents results in separation of lipids from analytes. Separation of analytes and interfering substances can also be achieved if polarity differences are present. The MSPD technique has been proven to be successful for a variety of matrices and a wide range of compounds [43], thanks to its sequential extraction matrices analysed include fish tissues [44,45] as well as other diverse materials [46,47]. 

This sol-gel procedure is an elaboration on well established entrapment methods [29], but with the added advantage of stability and better flow properties. Interestingly, none of the examples presented thus far demonstrate competitive behavior between multiple ligands (i.e. displacement) in the FAC analysis of trimethoprim and pyrimethamine a reversed order of elution based on is described, but this could simply be due to the shift towards an on-rate limited situation for higher affinity compounds, as described earlier. Erosion of dynamic competition between ligands could occur if the sol-gel allows convective mixing of the entrapped protein however the bimodal pore structure of these materials would... 

When the sample introduced into the column is composed of a number of components, the different components of the sample compete for adsorption on the surface of the stationary phase. That competition will affect the individual band profiles and the resulting band shape will depend on several factors, such as the isotherm, the separation factor, the loading factor, the relative concentration of the components, elution order, etc. [1,11],... 

Dunn, B.M. and Chaiken, I.M., Quantitative affinity chromatography. Determination of binding constants by elution with competitive inhibitors, Proc. Natl. Acad. Set U.S.A., 71, 2382-2385, 1974. 

Elution of the bound ligand can be done by competition with the free (unbound) ligand or by partial denaturation of the pro-... 

Competitive desorption (elution) has the advantage of very smooth conditions for protein structure, but the disadvantage of difficult removing of the ligand when further biospecific interactions are intended. 

The isolation of both specific and nonspecific binding proteins on affinity matrices bearing bioactive compounds hinders the identification of drug cellular targets. While solid-phase elution or the competition methods are conventionally used to distinguish between specific and nonspecific receptor-ligand interactions, these approaches are often severely restricted by low ligand solubility and/or slow kinetic dissociation (8). This low solubility of these compounds are not uncommon, since the hydrophobic properties of these compounds are often vital for their bioactivity and/or membrane permeability. 

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