Separation mechanism and determination of flavanones with capillary electrophoresis and high-performance liquid chromatography

To probe separation mechanism and determination with capillary zone electrophoresis (CZE) and liquid chromatography (LC), nine compounds with identical flavanone skeleton were studied. Optimum separation of LC was attained with gradient of acetonitrile and 5mM phosphate buffer (pH 6.9). For CE, electrolyte buffer was 4.5mM SDS in 32mM sodium tetraborate buffer (pH 9.2). The distinguishing feature in this work was successful separation of monohydroxyl stereoisomers by CZE. Polarity is generally increased with hydroxyl groups. In a separation mechanism study, polarity would be reduced by intramolecular hydrogen bond between hydroxyl of C5 and carbonyl group of C4. Comparison of the retention results among monohydroxyl flavanones shows polarity with hydroxyl at C6 the least, and that at C4' and C7 nearly equal. Also, elution order of flavones and flavanones would be adverse due to the hydroxyl at C3 in LC. From the numerical value pK(a) of flavanone, the C7-OH is the smallest, and two hydroxyl groups in an adjacent position is always less than the unique one caused by forming a stable 5-membered ring. Investigation of separation mechanism yield only the effect of constituent but also reasonable explanation for contradictory results between Wulf and our laboratory, this due to the hydroxyl at C3.