Separation, size and charge determination of small molecules using CE in combination with UV area imaging

This article shows how a capillary electrophoresis instrument may be used in conjunction with UV area imaging to determine mobility, diffusion coefficient and charge of components in a mixture of small molecules. The analytes lidocaine, phenylmethanol and benzoate are chosen as representative of small molecules of different charge type: cationic, neutral and anionic respectively in the background electrolyte used, phosphate buffer at pH 7.5. A capillary with three windows is used, with detection at the first window using the Agilent 7100 CE system and at the second and third using the ActiPix D100 UV imaging detector. The separated species are characterised at the first window after short-end injection followed by pressure assisted capillary electrophoresis for 3.5 minutes. The diffusion coefficients and hydrodynamic radii of the molecules are determined from the broadening of the bands between the second and third windows during a 15 minute stage of pressure driven flow. The method allows diffusion coefficients of all species to be measured within a single run, with good repeatability (RSDs better than 2.5%, n=9). Mobilities are found from times to these windows in separate experiments using pressure assisted capillary electrophoresis. Charge follows by combining data on diffusion coefficient and mobility.