An Introduction to Chiral Chromatography

The International Union of Pure and Applied Chemists defines chirality as ‘the geometric property of a rigid object of being non-superimposable on its mirror image.’ Another term used when discussing chirality is handedness, and the simplest example is our hands — left and right mirror images of each other. In chemistry, chiral molecules are called enantiomers.

Enantiomers

Enantiomers are chiral molecules that are mirror images of one another that are non-superimposable. This means that the molecules cannot be orientated on top of one another and give the same molecule. Chiral molecules with one or more stereocenters can be enantiomers. In many biological molecules chirality is conferred due to a carbon atom being bonded to four different functional groups. The carbon atom is then known as a chiral centre; chiral molecules can contain one or more chiral centres.

Enantiomers are termed optical isomers because they rotate plane polarized light in different directions. If the enantiomer rotates the light clockwise, it is known as dextrorotary and are denoted as (d) or (+) isomers. If the light is rotated anticlockwise they are known as levorotary (l) or (-). Another way of denoting an enantiomer is using the configuration of functional groups around the chiral centre. These are known as the Cahn-Ingold-Prelog rules, and use the priority of the functional groups to establish an order. A chiral centre can be denoted as R or S. The configuration, R or S is independent of the optical activity, consequently we can have (S)-(+)-lactic acid and (R)-(-)-lactic acid.

Why do the differences matter?

Some of the properties of enantiomers show no differences, for example, the densities and melting points are the same. But some properties do differ; particularly toxicity and biological activity which can be critical. Often during chemical synthesis, if the molecules produced have chiral centres, the reactions result in the formation of an equivalent mixture of a pair of enantiomers known as a racemate mixture, which is optically inactive.

When both enantiomers are present, problems can sometimes result. The drug thalidomide is racemic. One enantiomer is effective against morning sickness and the other enantiomer causes tetraplegia. The drug naproxen has two enantiomers; one is used to treat arthritis and the other causes liver toxicity. So it is vital that the correct enantiomer is used and separation of racemic mixtures is critical in some biological applications.

Chiral Chromatography

One of the methods of separating racemates is to use chiral chromatography. GC, SFC and HPLC can be used, although SFC or HPLC are the preferred separation methods for biological compounds due to their thermal sensitivity.

Enantiomeric separations can only be carried out in chromatography systems that contain a chiral selector. The selector is usually linked with the stationary phase and the effectiveness of separation is then due to the enantiomer selectivity of the stationary phase. The factors affecting an SFC separation are discussed in The Effect of Temperature, Pressure and Co-Solvent on a Chiral Supercritical Fluid Chromatography Separation.