Mechanism of Liquid Chromatographic Resolution of Chiral Compounds

The resolution of chiral compounds in liquid chromatography can be divided into three controllable parts: mobile phase composition, chiral stationary phase type and instrument operating conditions. The above three parts all contribute to the resolution of chiral compounds. In general, the selectivity of the chiral stationary phase takes precedence over the composition of the mobile phase, which takes precedence over the instrument operating conditions.

There are many principle models of chiral separation, but the most accepted is the three-point interaction model (Fig. 1). To achieve chiral compound resolution, it is necessary to simultaneously exert force in three directions of force, and at least one direction of the force is three-dimensional or enantioselective. There are many types of these forces, including electrostatic interactions, dipole-dipole interactions, inclusion interactions, hydrophobic interactions, π-π interactions, and hydrogen bonding interactions.

The three-point interaction modelFig. 1. The three-point interaction model (Protein Sci. 2002, 11(6): 1330-9).

1. Inclusion Interactions

The chiral stationary phase of inclusion interaction can form chiral selective cavities with certain spatial structure, such as cellulose derivatives, starch derivatives, cyclodextrins and their derivatives, and macrolide antibiotics. The benzene or naphthalene ring of aromatic compounds and other five-, six- or heterocyclic rings can be contained in the helix of polysaccharide derivatives or the cavity of cyclodextrin and its derivatives during separation, acting as a directional force.

Structure and deriving points of cyclodextrin.Fig. 2. Structure and deriving points of cyclodextrin.

Fig 2 shows the structure of cyclodextrins and their derivatization sites. The oxygen atom containing lone pair electrons in the cyclodextrin cavity can have electrostatic interaction with the polar part of the compound to be separated. Therefore, when the alpha or beta position of the chiral carbon atom has a halogen-substituted aromatic group, a chiral stationary phase such as cyclodextrin and its derivatives can be used. In addition, inclusion interactions are also more common in polysaccharide derivative chiral stationary phases, including amylose and cellulose derivatives. The inclusion interaction of polysaccharide derivatives for chiral compounds are not closed ring cavities like cyclodextrins, but helical cavities formed by certain monosaccharide units. Generally, amylose has a more uniform helical structure than cellulose. Therefore, the chiral fixation of amylose derivatives is stronger than that of cellulose. In addition, the strength of the inclusion interaction is also related to the mobile phase used, the setting of instrument conditions, and the type of compound.

2. Hydrophobic Interactions

Hydrophobic interactions are another major force in the resolution of enantiomers on chiral stationary phases, and are generally the most common in reversed-phase liquid chromatography. When there are benzene ring, carbonyl group and hydroxyl group near the chiral carbon, the hydrophobic interaction of the benzene ring and the weak hydrogen bonding of the other two groups in reversed-phase liquid chromatography may be the basis for the chiral resolution of the compound. In general, when the separated enantiomers contain larger hydrophobic groups in their structure, it is more appropriate to try reversed-phase liquid chromatography.

3. π-π Interactions

Aromatic benzene can be considered to be composed of three sigma bonds and three π bonds, and can also be considered as a large π bond ring structure formed by three sigma bonds and one 6 electron. When the hydrogen of the benzene ring is replaced by an electron-withdrawing or electron-donating group, the corresponding π acid or π base can be formed ( Fig. 3).

π-π stacking of the two aromatic ringsFig. 3. π-π stacking of the two aromatic rings (Molecular Catalysis. 2020, 498: 111242).

The hydrogen on the benzene ring is substituted by electron-donating group such as methyl to form the corresponding π base, and replaced by electron-withdrawing groups such as nitro to form the corresponding π-acid. The π-π stacking electrostatic effect between π acids and π bases is the main force type for the resolution of various chiral chromatographic columns, including the most widely used polysaccharide derivatives.

4. Hydrogen Bond Interactions

Hydrogen bond interactions are very common forces, including N-H and O-H (Fig. 4). The bond energies of different types of hydrogen bonds and hydrogen bonds formed between different groups are also different. Hydrogen bond interaction mainly occurs between ketones, esters, carboxylic acids, amides and amines with the amino groups of amines and hydroxyl groups of alcohols, which is one of the main mechanisms of chiral separation by normal phase chromatography.

Schematic diagram of hydrogen bonding.Fig. 4. Schematic diagram of hydrogen bonding.

Enantiomers have the same chemical and physical properties and the same chromatographic retention behavior in achiral environment. Certain properties differ between enantiomers in the chiral environment, which is the basis for the resolution of chiral compounds. The separation of relative enantiomers using chiral fixation is the most commonly used method in the laboratory, and there are many types of forces that play a separation role. In addition, which force plays a decisive role in the separation is related to the type of chiral stationary phase, the composition of the mobile phase, the type and amount of additives, and the characteristics of the compounds to be analyzed.

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