Retention mechanism of l-glutamide-derived noncrystalline stationary phases in reversed-phase high-performance liquid chromatography and application for separation of nucleic acid constituents

Abstract

Double alkylated l-glutamide-derived noncrystalline stationary phases Sil-DSG and Sil-DBG have been prepared by coupling N′, N″-dioctadecyl- N-[4-carboxybutanoyl]- l-glutamide (DSG) and N′, N″,-dibutyl- N-[4-carboxybutanoyl]- l-glutamide (DBG) with aminopropylated silica (Sil-APS). TEM observations of DSG and DBG showed that lipid DSG can aggregate in organic solvents (methanol, chloroform, toluene, etc.) and self-assembled nano fibers are observed while such fibrous aggregations are not observed for DBG. The resulting chromatographic data have been provided information about its selective interaction with guest molecules (PAHs) in RP-HPLC. We have observed that the carbonyl groups in Sil-DSG exist in ordered state by forming a condensing thin layer over silica surface while DBG cannot form such an ordered state due to its lower order of short alkyl chain. The ordered carbonyl groups present in Sil-DSG promotes multiple carbonyl π–benzene π interactions with guest PAHs isomers which enhance the selectivity for these compounds. The contribution of π–π interactions was also supported by the substantial effects on the selectivity of benzene and nitrobenzenes. The effect of π-electron containing solvent on the retention behavior of the PAHs was also studied. The selectivity for nucleic acid constituents, i.e. nucleosides and its bases were also evaluated by Sil-DSG and the selectivity for these compounds on Sil-DSG was compared with the selectivity of conventional polymeric ODS phase. It has been found that Sil-DSG provided higher selectivity for nucleic acid constituents than polymeric ODS and that HPLC packing materials can be efficiently employed for routine analysis of these compounds. The effect of methanol content on the separation behavior of nucleosides was also studied.