Chiral chromatography and its application to the pharmaceutical industry

Terms used in the chiral chromatography:

1) Chiral stationary phase: A stationary phase which incorporates a chiral selector. If not a constituent of the stationary phase as a whole, the chiral selector can be chemically bonded to (chiral bonded stationary phase)or immobilized onto the surface of a solid support or column wall (chiral coated stationary phase),or simply dissolved in the liquid stationary phase.

2) Chiral selector: The chiral component of the separation system capable of interacting enantioselectively with the enantiomers to be separated.

3) Chiral additive: The chiral selector which has been added as a component of a mobile phase or electrophoretic medium.

4) Chiral mobile phase: A mobile phase containing a chiral selector

The most effective separation techniques that can be used for the analysis of enantiomers are chromatography and electrophoresis. However, separation can only be achieved by using chiral agents in the separation process.

Two substances can only be separated if their standard energy of distribution differ, which means that their standard enthalpies and/or their standard entropies of distribution also differ.

For any chiral separation the stationary phase is chosen such that the spatial arrangement of its composite atoms results in the probability or proximity of interaction differing significantly between the two enantiomers to be separated.

Separation of chiral compounds typically is performed using by capillary electrophoresis and chromatographic techniques such as HPLC, GC, and SFC.

The Mechanism of Solute Retention:

The retention of a solute is directly related to the volume of stationary phase available and this is rarely the same as the total amount of stationary phase present in the column.

The available stationary phase is limited by the porosity of the support (only those molecules small enough to enter the pores can interact with the stationary phase) and the chiral characteristics (spatial arrangement) of the stationary phase.

Only those molecules having the appropriate spatial arrangement will achieve close interaction with the stationary phase molecules or surface.

Conversely, those solute molecules of opposite chirality will be partially hindered from close interaction with the stationary phase or surface, and experience reduced interaction.

Retention depends on (1) The magnitude of the interactive forces between the molecule and the two phases, (2) The change in the random nature of the solute molecule when it transfers from one phase

to the other(3) Theavailability of the stationary phase, which will depend on the exclusion properties of the

support and the chiral nature of the surface.

Gas Chromatography Chiral Stationary Phases:

Chiral Polysiloxane Stationary Phases Chiral Metal Chelating Stationary Phases Cyclodextrin Chiral Stationary Phases:

1. In the case of the cyclodextrins, the chiral selectivity will vary with the type of cyclodextrin and the type of solute derivative that is used.

2. Due to the structure of the cyclodextrins, retention can result from interactions arising from inclusion or exclusion of the solute from the natural cavities contained by the molecule.

3. The best phase system is usually determined by experiment and will not only be influenced by the stationary phase selected, but also the type of solute derivative that is employed.

Liquid Chromatography chiral stationary phases:

There are five general types of LC chiral stationary phases. They are:

1. Protein based phases bonded to silica, 2. Small molecular weight chiral compounds bonded to silica (the Pirkle phases), 3. Cellulose and amylose polymers type phases that are coated onto silica gel, 4. Macrocyclic glycopeptides that are bonded to silica and 5. Cyclodextrin based materials that are also bonded to silica.

There are several protein type stationary phases, a1-acid glycoprotein, cellobiohydralase and human serum albumin being examples.

These stationary phases are used with mobile phases with a high water content (mostly > 90%) and thus separations are largely by dispersive interactions.

There are quite a large number of small chiral molecules that are used as Pirkle stationary phases, some of the more common being the substituted urea carbamates. These types of stationary phases can be operated either in the reversed phase mode or the normal mode.

The cellulose or amylose polymers are derivatized in order to link groups such as carbamates or benzoates to the free hydroxylgroups.

The cyclodextrin phases can be made form a, b or g cyclodextrins and offer a large number of chiral centers and, as result of their unique structure, contain a number of substantially deep inclusion centers.

These inclusion centers allow selective intrusion by appropriately sized molecules and thus can play an important part in chiral selectivity.

Applications

1) Indirect Enantiomeric Resolution:

It involves the coupling of the enantiomers with an auxiliary chiral reagent to convert them into diastereomers. The diastereomers can then be separated by any achiral separation technique.

2) Direct Enantiomeric Resolutions: Direct enantiomeric resolutions are only feasible in chromatographic systems which

contain an appropriate chiral selector. The chiral selector can be incorporated into the stationary phase that is chiral stationary

phase or be permanently bonded to or coated onto the surface of the column packing material, chiral bonded and chiral coated stationary phases.

3) Enantioselective chromatographya) On chiral stationary phases In the case of chiral stationary phases, the enantiomer that forms the more stable association

with the chiral selector will be the more strongly retained species of the racemate. The enantioselectivity of the chiral chromatographic system is then expressed as the ratio of the

retention factors of the two enantiomers.b) On achiral stationary phases

Enantioselective chromatography can also be performed on achiral chromatographic columns using the required chiral selector as a chiral mobile phase or a chiral mobile phase additive.

A chiral mobile phase reduces the retention of the solute enantiomer which forms a stronger association with the chiral selector.

The effective enantioselectivity of the chromatographic system will therefore be proportional to the ratio of the enantioselectivities of the association processes in the stationary and mobile phases.

These differ in their thermodynamic stability, provided that at least three active points of the selector participate in the interaction with corresponding sites of the solute molecule. This three-point interaction rule is generally valid for enantioselective chromatography.

Enantioselective chromatography and capillary electrophoresis are extensively employed in the analysis of the enantiomeric composition (enantiomeric excess, optical purity) of chiral compounds.

Other Applications:

Chiral gas chromatography is one of the most frequently used techniques for the analysis of essential oils.

It is used for the analysis of food and beverages, flavors, perfumes and medicinal oils. Due to the importance of chirality in the physiological response of many drugs, chiral gas

chromatography is routinely used in drug analysis. The technique can be applied to the analysis of all types of spatial isomers, not merely those

with chiral carbon atoms. Chiral gas chromatography also has a wide field of application in research, in environmental

studies and environmental monitoring, in biochemistry and biotechnology, in forensic science and in all types of quality control.

Use of chiral chromatography for rapid compound screening and compound purification, will allow rapid elimination of racemic compounds with undesirable characteristics.

Preparative and semi-preparative separations for pre-clinical and early clinical development will continue to be the fast growing area.

The single biggest application would be the analytical separation and preparative purification of chiral pharmaceuticals