When an atom in a molecule has four different groups attached to it, there are two possible forms of the molecule which are mirror-images of each other, and non-superimposable. This usually occurs at a carbon atom, but can also occur with others such as nitrogen, phosphorus or sulphur. The two mirror images formed are known as Enantiomers and are chemically almost identical. Hence they can be really hard to separate by HPLC. To achieve resolution it is necessary to design a column with active sites which are accessible to one enantiomer and sterically hindered to the other. It is generally regarded that to achieve separation in this way, a molecule must be attracted to the stationary phase at three different points.

There have been many approaches to this problem, and these are conventionally grouped into five categories:

Type 1: Brush or Pirkle Columns. These columns act by the formation of complexes with the sample, based upon hydrogen bonding, dipole or pi:pi interactions.

Type 2: Cellulose Columns. These columns combine the physical interactions described above with a physical structure which can include or exclude parts of the molecule. Both enantiomers are attracted by the interactions, but one enantiomer is able to approach closer and hence is retained longer on the column. These columns often offer the best chiral resolution, but are very expensive (like £2K!) and have a very long list of Don'ts in the manual, so handle with extreme care.

Type 3: These columns have a chiral enclosure, into which molecules are attracted to form inclusion complexes. Such columns usually contain cyclodextrin (CD), or sometimes crown ethers. Cyclodextrin forms a "flower pot" shaped structure:

There are three sizes available, known as alpha, beta and gamma, and this relates to the number of glucopyranose units used, 6,7 or 8 respectively. It is primarily this type of column which is offered by Shodex.

Type 4: These columns operate by Ligand Exchange, and contain metal complexes. The stationary phase usually comprises an amino acid derivative, and the eluent contains copper ions. These columns are used for the separation of species which form complexes with copper, such as amino acids, or molecules with acid hydroxyl groups and their derivatives.

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Type 5: These columns contain a protein bound to the column, usually Bovine or Human Serum Albumen (BSA, HSA), Acid Glycoprotein (AGP) or Ovomucoid protein (OP). Shodex also offer a BSA-based column.

Most manufacturers specialise in only one or possibly two of these approaches. Each Type is best suited to certain types of sample but there is a lot of overlap in their application. So a sample which can be separated on one Type, can usually be separated on several of the others as well. Which works best for your sample? You decide! Because they work in different ways, methods are not transferrable between separation Types. However, it is usually possible to transfer methods between different manufacturers' columns of the same Type, albeit with a little modification. So there are quite a number of possibilities when confronted with a chiral HPLC problem.

Cyclodextrin columns.

There are three sizes available. We recommend the alpha size for molecules with up to a 6-membered ring, the beta columns for molecules such as naphthalene or bis-phenyl with two rings, and the gamma for larger molecules such as polyaromatics or steroids.

Molecules are attracted to polar OH groups which are found around the wider entrance to the 'flower pot', but the internal region is more non-polar. Hence it is the hydrophobic parts of a molecule which is attracted into this region. However this is a relatively slow process, and hence it is normal for peaks to be broader than expected in other forms of HPLC. However by reducing the flow rate of the eluent, this effect can be minimised. Also, the use of buffered mobile phases can increase the attraction for the eluent to flow into the chamber in the cyclodextrin structure, thus giving more efficient separation.

The interactions at three different parts of the molecule, needed for resolution of chiral molecules, tend to occur at the polar sites on the entrance to the enclosure. Loading capacity is also quite low, so care muct be taken not to overload the column.

Cyclodextrin columns are often favoured because they adopt a reversed phase retention profile, and hence most chromatographers feel at home with the columns. The retention characteristics are however much weaker than for conventional ODS columns, and hence much weaker eluents are required, rarely needing more than 25% acetonitrile as a maximum. This can create a solubility problem for some organic species, which simply will not dissolve in such high water concentrations, and for these molecules, a different column type must be found.