Effects of chromatography conditions on intact protein separations for top-down proteomics

A chiral stationary phase (CSP) has been prepared by chemically bonding a chiral pseudo-18-crown-6 type host having a 1-phenyl-1,2-cyclohexanediol unit to 3-aminopropyl silica gel. The chiral column was prepared by the slurry-packing method in a stainless steel HPLC column. Normal mobile phases can be used with this CSP in contrast to conventional dynamic coating type CSPs. Enantiomers of 20 out of 30 amino compounds, including 20 amino acids, 2 amino acid methyl esters, 6 amino alcohols, and 2 lipophilic amines, were efficiently separated on columns with this CSP. It is noteworthy that 15 amino compounds out of 30 were separated with better separation factors and shorter retention times compared to the corresponding CSP having pseudo-18-crown-6 with 1-phenyl-1,2-ethanediol as a chiral unit. In view of the correlation between the enantiomer selectivities observed in chromatography and those obtained in gas phase FABMS-EL methods and solution phase titrations, chiral recognition in the host-guest interaction likely contributes to enantiomer separation.