Imaging Peptide and Protein Chirality via Amino Acid Analysis by Chiral × Chiral Two-Dimensional Correlation Liquid Chromatography.

The present contribution illustrates the utilization of a chiral × chiral two-dimensional liquid chromatography (2DLC) setup with tert-butylcarbamoyl quinine chiral stationary phase (CSP) in the first dimension (1D) and tert-butylcarbamoyl quinidine CSP in the second dimension (2D) to analyze FMOC-derivatized d and l amino acids from peptide hydrolysates. Hereby, in the 1D and 2D chiral separation dimensions factors such as selector and immobilization chemistry of the CSPs, mobile phase, temperature, column hardware dimensions, stationary phase supports, particle type and packing were identical. Orthogonality between 1D and 2D CSPs was solely based on their stereochemistry, i.e. their opposite configurations in two chiral centers of the selector molecules, which results in inversion of enantiomer elution orders in the two dimensions. Using Coreshell CSPs for fast chromatography allowed 2D-flow rates which were 60 times faster than the 1D-flow rates to enable online comprehensive two-dimensional chromatography (LC × LC). Due to very similar chemoselectivity, yet opposite elution orders of corresponding enantiomers in 1D and 2D, characteristic 2D-elution patterns for achiral and chiral components can be generated. Peaks of achiral components and impurities are lined up on the diagonal line in the 2D separation space (contour plot) and thereby removed from the chromatographic space of the target enantiomers avoiding overlaps with potential interferences. Corresponding enantiomers provide cross peaks on the 2D chromatogram. Moreover, enantioselectivity of both single CSPs is combined to result in an enhanced overall 2D enantioselectivity. The concept is illustrated for the therapeutic peptides gramicidin and bacitracin. Since all amino acids give a consistent elution order as FMOC-derivatives, all enantiomers of the same configuration are either above or below the diagonal line allowing straightforward imaging of the configuration of the amino acids in peptides by the 2D chromatogram.