Cyclodextrins and Capillary Electrophoresis

The answer to the following question was provided by Kevin Altria, LCGC Europe’s “CE Currents” column editor.

Q: What are cyclodextrins and how are they used in capillary electrophoresis?

Cyclodextrins (CDs) are cyclic oligosaccharides composed of D-glucose units that are linked by α(1,4)-glucosidic bonds. The practically important, industrially produced CDs are α-CD, β-CD and γ-CD which differ in the number of glucose units involved (that is, α-CD contains six glucose units, β-CD has seven glucose units and γ-CD has eight glucose units). CDs are generally produced through enzymatic conversion of starch. CDs have the shape of a torus with a hydrophobic interior cavity and a hydrophilic outside. The narrow rim is occupied by the primary hydroxyl groups at C6 while the wider rim contains the secondary hydroxyl groups at C2 and C3. The hydroxyl groups can be chemically modified resulting in a number of derivatives. With regard to their use as chiral selectors in CE the most important difference is the cavity size which is the smallest for α-CD and the largest for γ-CD.

One of the premier applications of capillary electrophoresis (CE) is the resolution of stereoisomers (enantiomers). Enantioseparations by CE are achieved by the addition of a stereochemically pure substance, a so called chiral selector, into the background electrolyte. To achieve a chiral separation the enantiomers must have differing chromatographic interactions with the added chiral selector. The most frequently additives used are cyclodextrins because they are commercially available, UV-transparent and relatively low cost.

There is a large variety of CD derivatives that allow optimization during method development as well as the separation of charged and neutral analytes. Many validated enantioseparation methods have been reported. Chiral CE methods for assessing chiral purity and stability are routinely used in many pharmaceutical companies and have also been included in numerous regulatory submissions. Chiral CE methods have also been included in Pharmacopoeias. For example, there is a chiral test method for epinephrine bitartrate in the USP.

Cyclodextrins are also useful in nonchiral separations. For example, they can be used to separate closely related compounds such as cis- and trans-isomers, which have virtually identical electrophoretic mobilities. These compounds will, however, have differing interactions with the CDs because of their differing shape. For example, a range of closely related impurities of the drug salbutamol were resolved from each other and from the salbutamol enantiomers (1) using a low pH buffer containing 100 mM dimethyl-β-cyclodextrin. A more elaborate buffer of phosphate pH 3.0 containing 12 mM tetrapropyl and 8 mM tetrabutyl ammonium bromide and 20 mM methyl-β-CD was required for the separation of very closely-related Remoxipride achiral impurities from each other (2), a separation will be observed in most cases.

CDs can form complexes with molecules based on their inclusion into the hydrophobic cavity. Secondary interactions may include hydrogen bonding or dipole–dipole interactions with the hydroxyl groups on the CDs, or with other polar substituents of the CDs. In the case of charged CDs ionic interactions will also contribute, or may even dominate, the complexation mechanism. Effectively the CDs are contained in the buffer and their interaction with the analyte either slows or increases movement of the enantiomers. If the interaction is stronger for one enantiomer than the other then a separation will occur.

CDs are naturally chiral molecules, therefore, binding of host enantiomer molecules results in diastereomeric complexes. These complexes differ in properties such as the enantiospecific binding constants, charge or size resulting in an enantioseparation.

(1) M.M. Rogan, D.M. Goodall, and K.D. Altria, Electrophoresis 15, 808–817 (1994).
(2) Stalberg et al., Chromatographia 41, 287–294 (1995).