Gradient elution mode factors

Fig. 7.6 Options to assess imprinting effects and corresponding response factors, (a) Static mode assessment. (1) Template release. K = partitioning coefficient. (2) Comparison of template binding to a MIP with that to a NIP. IF = imprinting factor. (3) Comparison of binding of template with that of a close analog to the MIP. a = selectivity factor, (b) Flow through SPE mode assessment. Response factors can here be the % recovered template and for gradient elution, the cumulative recovery...

Usually both gradient elution and displacement chromatography offer higher optimum loading factors than isocratic elution, due to the band compression effect of the former two modes. Gradient elution chromatography requires less column efficiency than isocratic elution, ie. shorter columns packed with larger particles can be used which will result in shorter cycle times to the increased flow rate. 

When the chromatographic mode, column type, packing and dimensions have been chosen, the final stage of method development involves solvent optimisation and a choice between isocratic or gradient elution. Many separations can be achieved perfectly satisfactorily under isocratic conditions and are preferred to gradient elution techniques, as these are inconvenient due to the time required to re-equilibrate the column. A measure of the quality of separation is given by the resolution factor which can be expressed as follows ... 

Once the retention volume of a solute is calculated for a particular gradient profile, corresponding bandwidth and resolution can be determined by introducing the appropriate instantaneous retention factor A, at the elution of the peak maximum calculated from the gradient function tpf = /(Tr) and from the retention equation A, = f chromatographic mode and gradient function used [851 ... 

And in this context one last example - assume a component elutes in isocratic mode at a flow rate of 1 ml/min and a given eluent composition in 10 min. When the flow is increased to 2 ml/min, the component elutes after 5 min, because it "goes along with the new speed, the time it spends in the column is reduced by a factor of 2. Suppose now that a component in the gradient mode leaves the... 

In HIC, molecules are bound with a high concentration of salt, usually ammonium or sodium sulfate (1-2 A/) in a buffer (0.02-0.05 M). Elution is attained by a gradient to a lower concentration of salt in the buffer. The pH is controlled and is usually in the range of 6-8, but it is not a critical factor in selectivity. Additives to enhance protein stability are generally compatible with the process. Contrary to its effect on other modes of chromatography, reducing the temperature decreases the retention in HIC due to its being an entropy-driven technique. 

Alkyl-2-acetyl-sn-glycerophosphorylcholine or "platelet-activating factor" (commonly abbreviated to PAF) is present in minute concentrations in platelets and certain other cells, yet exerts profound physiological effects. Its chemistry and biochemistry have been reviewed [353,866]. It is such a polar molecule that HPLC in the adsorption mode is required for its isolation. As the author and others [107,168] have reviewed these methods in some detail. It need only be mentioned here as an example that It elutes between sphingomyelin and lysophosphatidylcholine from a column of silica gel with a gradient of hexane-isopropanol-water in which the water content is increased [110]. No existing method is sufficiently sensitive to demonstrate the... 

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