In order to develop a successful preparative HPLC method one first needs to define relevant goals for the specific purification. The most important and common parameters defining the quality of a separation is loading (defined as mcrude/mresin), purity and yield. Purity is usually already defined by regulatory agencies (such as FDA or equivalent) and often includes area percentage of product as well as individual impurities, while yield simply is the ratio of purified product divided by amount loaded onto the column.
Using the terminology described above it seems natural that for a given purification method one cannot increase every parameter individually. For instance, increasing loading will most certainly decrease yield and/or purity. The relationship between these three parameters can be represented by the figure displayed below: By putting the three parameters in the corners of the triangle it becomes even more evident that one cannot perform any purification at the three corners at the same time. Instead, most preparative HPLC purifications operate along one side of the triangle and this is why You as a chromatographic method developer needs to clearly define the purpose of every separation.
Sometimes a combination of HPLC purification methods is needed in a two-step process. In the first step one typically performs the HPLC purification close to C in order to remove more easily removed impurities. Collected fractions of medium purity can then be reintroduced to the column using another method that operates more closely to B. Note that a two-step process always increases both workload and solvent consumption and one always needs to evaluate if working more thoroughly with preparative HPLC method development or upstream purification is a more beneficial alternative.
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