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

Abstract A modified C 18 column (Silpr‐2MI‐C18) was prepared using 2‐methylindole and C 18 reagent. The extent of C 18 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 C 18 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 the Silpr‐2MI‐C18 column compared to the commercial C 18 column under RPLC mode. Furthermore, these amino acids were separated on the Silpr‐2MI‐C18 column under HILIC mode. The modified C 18 column was employed to separate amino acids, alkylbenzenes and polycyclic aromatic hydrocarbons 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. Effective retention of biological samples was found on the Silpr‐2MI‐C18 column by comparing the results obtained from the commercial C 18 column.