Enhancing enantiomeric and diastereomeric separations: A single chromatograph approach for two-dimensional heart-cut methodology

Abstract

A high-performance chromatograph was adapted and reconfigured to establish a two-dimensional methodology for separations that are not easily achievable using a single enantioselective column. Allethrin, a pyrethroid used widely as a pesticide in domestic environments, was chosen as the model analyte. With three chiral centers, it has eight stereoisomers and is considered one of the more difficult chiral separations. Diastereomeric separation was achieved in the first dimension using a shape-selective stationary phase based on ultrapure porous graphitic carbon (particle diameter, 3 μm) in the reversed phase mode. Advanced asymmetric bidirectional peak functions (half-Gaussian modified Gaussian and exponentially modified Gaussian) were employed to extract overlapping peak areas. The four diastereomeric peaks were heart-cut and sent to the second dimension online using a standard six-port 2-position rotary valve. Enantiomeric separation was attained in the second dimension using an immobilized-cellulose tris(3,5-dichlorophenylcarbamate) column (3 μm) with chromatographic resolutions >2.7 for all enantiomeric pairs. The second dimension method was developed using the information from circular dichroism detection. The baseline separation of seven peaks of allethrin in a single dimension was also demonstrated using (2-hydroxypropyl)-β-cyclodextrin. The enantiomeric purity of a purified formulation of allethrin in a commercial pesticide was validated using the developed 2D approach.