By Stavros Kromidas
Along constructing new ones, the optimization of current equipment is a key activity within the HPLC laboratory. And a role that these days should be solved more and more quick and cost-efficiently.This guide presents well-founded tips in greater assembly this problem. across the world popular authors conceal either the final fundamentals and methods in optimization, in addition to the categorical features desirous about such various equipment as RP-HPLC, NP-HPLC, micro- and nano-HPLC, and hyphenated options, comparable to LC/MS. Nor are such themes as column choice and chiral separations left exposed. a few of the contributions current functions utilizing universal optimization software program, akin to DryLab or ChromSword. all the chapters pay attention to the necessities and are written in a essentially orientated variety, whereas their self-contained constitution allows distinct references.Throughout the textual content, the authors provide concrete, sensible advice in addition to pertinent historical past info, including insights into the optimization tools of 7 significant foreign businesses from numerous sectors.The complete is rounded off with real-life stories from skilled clients coming from the various parts of program, specifically the existence sciences, comparable to proteomics or drug improvement.
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Extra info for HPLC Made to Measure: A Practical Handbook for Optimization
Today, however, this distinction is not made everytime; one speaks only about plate number. In the most cases, the theoretical plate number is calculated, but of retained substances. g. e. peak width at the peak base, at 10% or at 50% peak height. Therefore, the comparison of literature values of plate numbers is inherently difficult. α: Separation factor, formerly selectivity factor. α is a measure of the capability of a chromatographic system (chromatographic system: the actual combination of the stationary phase, the mobile phase, and the temperature) to distinguish two given compounds.
The intention is a change of the retention factor k (mostly enhancement), but ideally also of the separation factor α. Otherwise, at constant interaction strength (“chemistry” constant and therefore k and α as well), one attempts to enhance the plate number or in the case of a miniaturization to prevent dilution or to enhance the relative mass sensitivity. Using a trial-anderror procedure, one needs 1–2 weeks. Using a systematic procedure, aided perhaps by an optimization program, the time can be reduced measurably; see Part 4 for chapters on computer-aided optimization.
From a practical point of view, one should think in terms of high flow rates in the course of gradient runs. Increasing the flow while keeping the gradient time constant yields a better resolution because the gradient volume (flow × time) increases. Peak capacity (number of peaks per time unit) and thus resolution itself also increases with increasing gradient volume. Even if resolution is satisfactory, the flow should be increased. For example, if the flow is increased by factor 2 while simultaneously decreasing the gradient time also by a factor of 2 and adapting the gradient accordingly, the resolution will remain the same because the gradient volume remains constant – in only half the time!
HPLC Made to Measure: A Practical Handbook for Optimization by Stavros Kromidas