Hydophylic interaction-reversed phase high performance liquid chromatography application of a recently developed mixed-mode stationary phase

In thise work, a novel multifunctional stationary phases synthesized under another graduate thesis was evaluated as a hydrophilic interaction/reversed-phase mixed-mode stationary phase for high-performance liquid chromatography (HPLC). Successful separation could be achieved by Ile-PBA-Mix column among several aromatic compounds having different polarities under both hydrophilic interaction liquid chromatography (HILIC) and reversed phase (RP) condition. Nucleotides/nucleosides were separated in the HILIC mode. The effects of different separation conditions, such as pH value, mobile phase content, column temperature, buffer concentration and flow rate, on the separation of nucleotides/nucleosides in HILIC mode were investigated. The seven nucleotides/nucleosides were separated within 22 minutes, while six of them were separated within 10 minutes by isocratic elution. To determine the influence of the new multifunctional stationary phase under the RP condition, a number of moderately and weakly polar and nonpolar compounds, such as 10 substituted anilines and eight substituted phenols were separated successfully under the RP condition within 14 and 15 min, respectively. Additionally, nine mixtures of polar/nonpolar test compounds were simultaneously separated within 19 minutes, while seven of them were separated within 12 minutes, under HILIC/RP mixed-mode conditions. Chromatographic parameters, such as the retention factor and peak asymmetry factor, were calculated for all of the analytes, while the theoretical plate number was calculated for analytes separated by isocratic elution. Compared to traditional C18 and commercial HILIC columns, the new stationary phase exhibited both HILIC and RPLC performance, and the scope of analyte separation was thus enlarged.