Kromasil^® notes

Why you can't have your cake and eat it too

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 m_crude/m_resin), 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.

• Click on "?" and then on A, B or C for info
• Side A: Along this side one has sacrificed yield for maximized loading and high purity. This is the area to be close to if the in-house value of the crude from upstream processes is low.
• Side B: In this case the operator sacrifices loading and as direct consequence productivity (kgproduct/h) in order to keep purity and yield high. This is a good choice if the value of the crude coming from upstream processes is high, and if productivity can be kept somewhat low. Due to the low loading, more injections need to be performed in order to purify a given amount of crude sample).
• Side C: At first it may seem intuitively wrong, but sacrificing purity for high loading and yield is not that uncommon in preparative HPLC. However, performing a separation in this area is not a good choice when purifying a substance to the market due to purity restrictions. Instead, operation along this line is performed for quick in-house purification in R&D phase, when a small amount of medium purity sample is needed to perform further studies. Since these separations are usually performed in-house on a small scale, semi-preparative columns (either DAC, pre-packed or spring type) are typically used.
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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.