Reversed-phase method development can be a lengthy process which can tie up valuable resources in an analytical laboratory. The process followed and success obtained can vary depending upon experience, resources and available time. Often, a trial and error (or one factor at a time) approach is adopted, where parameters are adjusted and decisions made according to the analytical results obtained for each iterative step. This approach can produce acceptable separations, but it may fail to identify the most suitable method, i.e. the most robust or the most cost effective (e.g. fastest) method.
The adoption of a structured approach to method development is helpful for many reasons: It can lead to the development of better, more robust methods, generate useful retention knowledge for analytes, and has the potential to provide significant savings in both development time and costs. A popular approach is to use screening protocols to systematically explore individual chromatographic parameters (such as column stationary phase, eluent composition, pH etc) and their effects upon retention/separation. Once screening is complete, the most promising combination of conditions can be further optimised, if needed, to produce the final method. This approach is useful, informed and highly recommended. Taking this process further (if required), the screening data can be input to LC retention modelling software to generate retention models and predict analyte retention behaviour at this final optimisation stage. This can be helpful for many industries to improve retention understanding and explore method robustness. Once models have been generated, further method changes can be predicted in silico (e.g. changing gradient ranges/slope) and then experimentally verified with a few injections. The need for further actual experimental work is therefore greatly reduced.
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