Novel Click N-benzyl Iminodiacetic Acid And Click 2-(N, N-dimethylamino)-1,3-propanediol For Hydrophilic Interaction Liquid Chromatography

Hydrophilic interaction liquid chromatography (HILIC) is an important chromatographic method for separating polar and hydrophilic compounds.Silica based N-benzyl iminodiacetic acid (IDA) and 2-(N, N-dimethylamino)-1,3-propanediol HILIC stationary phases have been developed for resolving the weaker even without retention of very polar compounds on RP-HPLC. Two stationary phases have been developed by covalently bonding small molecular N-benzyl IDA and N, N-dimethylamino 1,3-propanediol moieties onto silica beads via Cu(Ⅰ) catalyzed Huisgen 1,3-dipolar cycloaddition reaction(CuAAC) between terminal alkynes and organic azido compounds respectively. The key azido intermediates were prepared by transferring the amino groups directly to corresponding azido groups by using an efficient, inexpensive and shelf-stable diazo transfer reagent (imidazole-1-sulfonyl azide hydrochloride) under mild conditions. The structures of two stationary phases were confirmed by FT-IR and elemental analysis, and the structures of all related intermediates were confirmed by NMR, IR and mass spectrum. The data of elemental analysis and IR showed that the N-benzyl IDA and N, N-dimethylamino 1,3-propanediol were successfully immobilized on the surface of silica, the surface coverage were calculated to be 2.23μmol m-2 and 2.82μmol m-2 respectively.The chromatographic performance evaluation of click N-benzyl IDA column was carried out by using nucleosides and bases as probe compounds. The results exhibited that click N-benzyl IDA showed typical HILIC retention characteristics, and it had potential application values in HILIC. Compared with commercially available Atlantis HILIC Silica and ZIC-HILIC column, click N-benzyl IDA demonstrated better abilities in separating hydrophilic and polar compounds such as aromatic acids, natural bases, nucleosides and carbapenems under HILIC mode with higher selectivity and efficiency (the theoretical plate number was up to 44000 plates m-1). The retention mechanism study showed that under HILIC mode, except partitioning interaction, other interactions, such as hydrogen bonding, ion exchange, chelation and electrostatic interactions were also existed on click N-benzyl IDA. In the flow rate of 0.35mL/min, the column displayed the highest column efficiency according to the Van Deemter equation. In addition, click N-benzyl IDA showed good chromatographic stability with relative standard deviation (RSD) of almost all test nucleosides and bases to be less than 0.5%. When it was applied to separate eight amino acids, better selectivity and efficiency were displayed than click lysine and commercial HILIC columns. Under the optimum chromatographic conditions, three vitamins, mixture of lincomycin hydrochloride and clindamycin hydrochloride were well separated on click N-benzyl IDA. Besides, diastereoisomers of phenyl serines could be separated.Click 2-(N, N-dimethylamino)-1,3-propanediol also exhibited good HILIC properties when it was applied in analyzing hydrophilic compounds. Its chromatography retention behavior was studied by investigating the retention of different types of solutes under different buffer concentration and pH values, the experimental results showed that the retention of anions on click 2-(N, N-dimethylamino)-1,3-propanediol depended mostly on HILIC/WAX mechanism, while the chromatographic behavior of cations was controlled by ERLIC mechanism. Furthermore, click 2-(N, N-dimethylamino)-1,3-propanediol was successfully used in separating very polar compounds such as nucleosides and bases, alkaloids, organic acids and antibiotics. The thermodynamic characteristic of click 2-(N, N-dimethylamino)-1,3-propanediol was also studied according to van’t Hoff plot, an exothermic process for transferring analytes from the mobile phase to the stationary phase was observed and there were no change of retention mechanism within the measured temperature range. Besides, click 2-(N, N-dimethylamino)-1,3-propanediol exhibited good reproducibility and stability.