Preparation and evaluation a mixed-mode stationary phase with C18 and 2-methylindole for HPLC.

Abstract

A modified C18 column (Silpr-2MI-C18) was prepared using 2-methylindole and C18 reagent. The extent of C18 hydrocarbon chain, conjugative rings and anion exchange site provided multiple retention mechanisms, including reversed phase liquid chromatography (RPLC), π-π interaction, hydrophilic interaction liquid chromatography (HILIC) and anion exchange chromatography (AEC). The separation of protected amino acids was investigated on the commercial C18 and Silpr-2MI-C18 columns, while the chromatographic conditions, including methanol content and pH of the mobile phase, were studied. The separation arrangement of the hydrophilic amino acids was different on Silpr-2MI-C18 column compared to the commercial C18 column under RPLC mode. Furthermore, these amino acids were separated on the Silpr-2MI-C18 column under HILIC mode. The modified C18 column was employed to separate amino acids, alkylbenzenes, polycyclic aromatic hydrocarbons (PAHs) under RPLC mode and inorganic anion under AEC mode. The results confirm that this new stationary phase of RPLC/HILIC/AEC has multiple interactions with different analytes. The effective retention of biological sample was found on the Silpr-2MI-C18 column by comparing the results obtained from the commercial C18 column.