32
May/June 2013
Capillary Electrophoresis: an Attractive Technique for Chiral Separations
Cari E Sänger – van de Griend 1,2 , Ylva Hedeland 2 , Curt Pettersson 2
1 Kantisto BV, Callenburglaan 22, 3742 MV Baarn, the Netherlands 2
Uppsala University, Department of Medicinal Chemistry, Division of Analytical Pharmaceutical Chemistry, Biomedical Centre PO Box 574, SE-751 23 Uppsala, Sweden
Capillary Electrophoresis (CE) separates compounds that differ in charge to hydrodynamic size ratio and is an excellent technique for the analysis of polar compounds. The technique is particularly applicable to chiral separations. Chiral CE separation is achieved by adding a chiral selector to the so called background electrolyte. The enantiomers then form fast, reversible equilibria with the selector. In this paper a simple method development strategy for basic, acidic and neutral compounds is presented and illustrated with examples and common pitfalls. Some important good working practices (background electrolyte buffer recipes, temperature, corrected peak area, injection, polyimide coating removal from capillary ends) are highlighted, so that a good chiral separation can be developed into a robust analytical method.
the United States Pharmacopeial Convention (USP) [[3]
, [1] ]. Figure 1: Achiral and chiral separation of local anaesthetics. Reprinted with permission from [[27]and [10]]. Introduction
Capillary Electrophoresis has been used in the pharmaceutical industry since the first commercially available instruments appeared on the market in the late 1980s. After a decline in use during the first decade of this
century, a recent revival has been observed [1]
, not least because of the increasing
prevalence of large biological molecules as drugs. Electrophoresis has traditionally been applied mainly to proteins and nucleic acids (DNA, RNA) but CE is applicable over a wide range of analytes and anything from small inorganic ions to cell organelles and even complete cells and viruses have been analysed with CE. It is being used to complement or to replace traditional gel electrophoresis and chromatographic techniques. Typical successful applications
for small molecule are chiral as well as achiral purity determinations of drug substance and products as well as the determination of drug counter ions or small ions such as metal ions, organic acids etc. Since CE works quite well for protein analysis it has recently gained an increased interest within the biotech industry. The therapeutic proteins that are drug candidates need additional characterisation during drug discovery and development compared to traditional small molecules. Furthermore, CE methods are being used for the quality control analysis of approved biotechnological drugs. That CE has successfully gained a position in the pharmaceutical and biotech industry is demonstrated by the general chapters and monographs in pharmacopoeias e.g., the European Pharmacopoeia (
Ph.Eur.) [[2]
, [1] ] and
Since electrophoresis separates analytes that differ in their charge to their hydrodynamic size ratio in an applied electric field, a charge on the analyte is a prerequisite for electrophoretic mobility. Either the analyte needs to have one or more charged functional group(s) or need to form a covalent bond or a reversible complexes with either any of the background electrolyte (BGE) components or an additive (e.g., a chiral selector or a micelle). The use of additives has led to several sub techniques, such as chiral CE. It is in the field of chiral separations that CE has had its major impact in small molecule analysis. Chiral CE has the advantage of promoting highly efficient separations at a reasonable cost. In comparison, chiral gas chromatography shows effective enantioseparations but may often include a time-consuming derivatisation step. Furthermore, GC is only suitable for volatile and thermo-stabile compounds and consequently is less used today. Even though liquid chromatography (LC) and supercritical fluid chromatography (SFC) have a general high success rate for chiral separations, chiral CE is often better suited for rapid screening of optimal conditions since the enantiomeric separation is generally faster in CE and there is no need for a long equilibration time when switching to a different chiral BGE. Moreover, CE has been used to screening of selectors that later have been successfully applied as chiral
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