54 August / September 2016

Chromatography Today Help Desk Important Considerations for Repeatable HPLC Assays of Chiral Compounds

The manufacturing process of many of the active pharmaceutical ingredients that are used in prescription drugs today may result in racemic mixtures of the compound being synthesised. In some cases, the efficacy and toxicity of the different mirror forms of the drug will be nominally the same and so the consequences of not having a single enantiomer pure drug are mild. However, there many cases where this is not true. Probably the most discussed drug that falls into this category is thalidomide and the birth defects associated with the application of this substance. The drug was prescribed in Canada, Europe, Australia, and parts of Asia in the late 1950’s-1960’s to pregnant women in their first trimester to treat nausea and vomiting caused by morning sickness. After the introduction of thalidomide as a pharmaceutical, there was a huge increase in birth defects, especially in Europe and Canada, caused by an enantiomeric impurity. It is estimated that as many as 10,000 children were born with major malformations due to their mothers taking this drug during pregnancy. The thalidomide molecule is a racemic glutamic acid analogue, consisting of S- and R+ enantiomers that interconvert under physiological conditions. The S- form potently inhibits release of tumour necrosis factor from peripheral mononuclear blood cells, whereas the R+ form seems to act as a sedative, probably mediated by sleep receptors in the forebrain, the different forms of the molecule are shown in Figure 1.

Check Valves Single pump heads

One obvious cause of retention time shift is that the pump is not delivering the correct flow rate. This is easily assessed, by either connecting a flowmeter or by using a stop watch and a measuring cylinder / syringe (the latter is for low flow rates, with the needle attached to the outlet tubing from the LC system). If the pump has been identified as the cause of the retention time shift then resolving the issue needs to assess several criteria, which will depend on the type of pump that is being used. For isocratic and low pressure mixing pumps, there is a single pump head and the pump seals and check valves on this pump should be checked.

Single pump heads may include two pistons which will drive the flow under pressure through the LC system. There are several arrangements for pistons, either reciprocating so that one pump head is delivering flow whilst the other is refilling the solvent chamber or flow-through, where there is a primary and a secondary pump in line. The primary pump pumps liquid through the secondary pump which delivers flow whilst the primary pump refills the solvent chamber. This design differs from the dual reciprocating pump where each piston delivers flow independently to a mixing tee.

Dual pump heads Figure 1. The two enantiomers of thalidomide The two enantiomers of thalidomide

Quality procedures to ensure that the compound has only one form of an enantiomer are very dependent on retention times. If there is a shift in the retention time, there is a strong probability that the concentration of the wrong compound may be determined. For some separations, the separation factor is large enough to ensure that this is not a problem, however the nature of a chiral separation is that peaks elute close together.

This raises the question of what causes retention time shift? Unfortunately, there is no one solution, as there can be several factors. This article will look at causes of a shift in retention time. Although in this case it is applied to the analysis of enantiomers, it is very relevant to all analytes.

A high pressure binary pump will utilise two pumps that mix together at a tee. With this physical arrangement, inaccuracies in the flow rate can be attributed to either or both of the pump heads. One common issues associated with older style pump heads is that the pump head delivering the organic solvent does not contribute its share to the overall flow of the mobile phase. There are a range of materials that can be used for check valves (the most common being sapphire, ruby or ceramic) and selection of the correct check valve material may overcome this issue. As a temporary fix it is possible to clean the check valve in a water methanol mixture to allow the pump head to function appropriately. A better approach is to employ a ceramic check valve that will work better in high acetonitrile composition mobile phases. The use of mechanically assisted check valves will also eliminate the issue. The cause of this is explained in detail by an excellent article Dolan [1].

Compressibility Factor

Solvents compressed under pressure will have differing degrees of compressibility. This is something that will typically have to be programmed into the pump delivery system. Compressibility is the fractional change in volume per unit increase in pressure. Water has a compressibility factor of 46.4 x 10-6

, therefore, for each atmosphere increase in pressure, the volume of water would decrease 46.4 parts per million. The compressibility


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