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Chiral Separations by High-Performance Liquid Chromatography

Liquid Chromatography

  1. Timothy J. Ward,
  2. Karen D. Ward

Published Online: 17 DEC 2012

DOI: 10.1002/9780470027318.a5905.pub2

Encyclopedia of Analytical Chemistry

Encyclopedia of Analytical Chemistry

How to Cite

Ward, T. J. and Ward, K. D. 2012. Chiral Separations by High-Performance Liquid Chromatography . Encyclopedia of Analytical Chemistry. .

Author Information

  1. Millsaps College, Jackson, MS, USA

Publication History

  1. Published Online: 17 DEC 2012

Abstract

The word ‘chiral’ is derived from the Greek word ‘cheir’, which means hand. Chiral molecules are molecules that are related to each other in the same way that a left hand is related to a right hand. These molecules are mirror images of each other and are nonsuperimposable. Chiral separations traditionally have been considered among the most difficult of all separations because enantiomers have identical chemical and physical properties in an achiral environment. In this article, we focus on techniques used in high-performance liquid chromatography (HPLC). Most chiral separations by HPLC are accomplished via direct resolution using a chiral stationary phase (CSP). In this technique, a chiral resolving agent is bound or immobilized to an appropriate support to make a CSP, and the enantiomers are resolved by the formation of temporary diastereomeric complexes between the analyte and the CSP.

Various types of CSPs have been developed, including polysaccharide-based, cyclodextrin-based, cyclofructan-based, macrocyclic antibiotic-based, protein-based, Pirkle-type, and ligand-exchange CSPs. The most commercially common CSPs, polysaccharide-based phases, consist of derivatized cellulose and amylose coated on silica gel and a new generation of polysaccharide-based phases, which is immobilized through covalent bonding to a silica gel. Generally, analytes separated using the cyclodextrins require the formation of an inclusion complex between the analyte and the cyclodextrin. Separation is most favorable when the analyte also interacts with the mouth of the cyclodextrin molecule via hydrogen bonding to the functional groups present. The cyclofructan-based CSPs are the newest development in chiral HPLC columns and are now available commercially. These bonded CSPs show excellent enantioselectivity toward all types of primary amines as well as other varieties of compounds. Numerous analytes have been separated using the macrocyclic antibiotic-based CSPs, which can also be derivatized to alter their selectivity. Protein-based CSPs comprise a number of commercially available columns. The protein-based CSPs resemble the bonded macrocyclic antibiotic CSPs in many ways; however, the macrocyclic antibiotic CSPs are more stable and have greater capacities than the protein-based CSPs. The Pirkle-type CSPs typically use nonpolar organic mobile phases and often require derivatization of the analyte to add a π-acid or π-basic moiety, or both, to achieve separation. Ligand-exchange phases are used with aqueous buffer mobile phases in which enantiomers are separated based on the differences in their charge and ionization constants. Limitations of ligand-exchange separations are that only ionized analytes can be resolved using this technique and the copper-salt-containing mobile phases used absorb in the ultraviolet (UV) region, decreasing detection sensitivity.