Separation of coumarins by subcritical water chromatography

In this study consist of two parts. In the first part of the study, we investigated the potential application of SBWC technique for separation of coumarin, vanillin, and ethyl vanillin using new design chromatography system. Chromatographic separations were performed on five columns including Waters XTerra MS C18, Zorbax RRHD Eclipse Plus, Zorbax SB-Phenyl, Zorbax SB-C18, and Waters XBridge C18 columns. Retention time of all three solutes was shortened with increasing water temperature. While separations on Zorbax SB-Phenyl and Zorbax SB-C18 columns are good, Waters XBridge C18 column yielded the best separation among the five commercial columns tested. Separation of coumarin, vanillin, and ethyl vanillin has been carried out using SBWC at temperatures ranging from 100 to 200 oC with both constant and variable flow rates. The temperature effect on retention factor was examined using van't Hoff equation. In the second part of the study, was synthesized the poly (HEMA-MAH) (PHEMAH) poly (hydroxyethyl methacrylate-N-methacryloyl-(L-histidine)-methylester) microspheres to be used as the column filling material (stationary phase) in subcritical water chromatography and their stability and durability tests were carried out at elevated temperature. These molecules can exhibit stability and durability at elevated temperature. Separation of coumarin, vanillin, ethyl vanillin, 6-methyl coumarin and 7-methyl coumarin have been carried out with the synthesised this column filling material at temperatures ranging from 125 to 200 °C. Methanol/ Water mixture (%2-97 and %5-94) were used as mobile phase in this process. The best separation was observed at % 5-94 methanol/water ratio at high temperature.