Хиральная колоночная хроматография: принципы и типы стационарных фаз.

Chiral column chromatography is a variant of column chromatography that is employed for the separation of chiral compounds, i. e. enantiomers, in mixtures such as racemates or related compounds. The chiral stationary phase (CSP) is made of a support, usually silica based, on which a chiral reagent or a macromolecule with numerous chiral centers is bonded or immobilized. The chiral stationary phase can be prepared by attaching a chiral compound to the surface of an achiral support such as silica gel. For example, one class of the most commonly used chiral stationary phases both in liquid chromatography and supercritical fluid chromatography is based on oligosaccharides such as Amylose Cellulose or Cyclodextrin (in particular with β cyclodextrin, a seven sugar ring molecule) immobilized on silica gel. The principle can be also applied to the fabrication of Monolithic HPLC columns or Gas Chromatography columns. or Supercritical Fluid Chromatography columns.

The chiral stationary phase, CSP, can interact differently with two enantiomers, by a process known as chiral recognition. Chiral recognition depends on various interactions such as hydrogen bonding, π π interaction, dipole stacking, inclusion complexation, steric, hydrophobic and electrostatic interaction, charge transfer interactions, ionic interactions etc, between the analyte and the CSP, to form in situ transient diastereomeric complexes. Most of the types of stationary phases can be classified as Pirkle type (Brush type), Protein based, Cyclodextrins based, Polymer based carbohydrates (polysaccharide based CSPs), Macrocyclic antibiotic, Chiral crown ethers, imprinted polymers, etc.

A protein based chiral stationary phase is based on silica gel, on which a protein is immobilized or bonded. The protein is based on many chiral centers, therefore the mechanism of chiral interaction between the protein and the analytes involves many interactions, such as hydrophobic and electrostatic interactions, hydrogen bonding and charge transfer interactions, which may contribute to chiral recognition. Hydrophobic interactions between the protein and the analyte are affected by percent organic in the mobile phase. As the organic content increases, retention on protein based columns decreases.

Cyclodextrin (CD) chiral stationary phase is produced by partial degradation of starch by the enzyme cyclodextrin glycosyltransferase, followed by enzymatic coupling of the glucose units, forming a toroidal structure. CDs are cyclic oligosaccharides consisting of six (α CDs), seven (β CDs) and eight (γ CDs) glucopyranose units. The chiral recognition mechanism is based on a sort of inclusion complexation. Complexation involves the interaction of the hydrophobic portion of an analyte enantiomer with the non polar interior of the cavity, while the polar functional groups can form a hydrogen bond with the polar hydroxyl chiral cavity space. The most important factor that determines whether the analyte molecule will fit into the cyclodextrin cavity is its size. The α CD consists of 30 stereo selective centers, β CD consists of 35 stereo selective centers and γ CD consists of 40 stereo selective centers. When the hydrophobic portion of the analyte is larger or smaller than the toroid's cavity size, inclusion will not occur.

Macrocyclic chiral stationary phases consist of a silica support, on which macrocyclic antibiotic molecules are bonded. of conducting a systematic screening of various CSP's by advanced column switching devices, trying automatically and systematically various mobile phase combinations, effectively employing a trial and error strategy.