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Capillary Electrophoresis

Capillary Electrophoresis. Separation of analyte ions via differential migration in an electric field, coupled with electro-osmotic flow of mobile phase. Advantages Only needs nL sample High speed and resolution, virtually no band broadening Instrumentation

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Capillary Electrophoresis

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  1. Capillary Electrophoresis Separation of analyte ions via differential migration in an electric field, coupled with electro-osmotic flow of mobile phase Advantages • Only needs nL sample • High speed and resolution, virtually no band broadening Instrumentation • Capillary tube (10 - 100mM internal diam., 40-100cm long) • Two buffer reservoirs, with platinum electrodes • DC potential (20-30 kV) applied along capillary • Sample introduced one end, detector at other • Direct of potential depends on charge (+/-) of analyte

  2. Capillary Electrophoresis Mobile Phase • Commonly phosphate or borate buffer (20-100 mM) • pH and Ionic strength must be controlled • Can add detergents to transport neutral molecules in a micelle (MEKC) Stationary Phase • No stationary phase for true CE • Newer developments introducing a stationary phase combine CE and HPLC to give electrochromatography Principles of separation Based on interaction of analyte with electric field Migration velocity v = (µe + µeo) E where µeand µeoare the electrophoretic mobilities of the analyte and buffer, and E is the applied field strength

  3. Capillary Electrophoresis Retention and Resolution: dependent on • Charge / size ratio is primary separation factor • Charge gives v  and thus RT  • Size gives v  and thus RT  • Interaction with buffer ions / molecules • pH and ionic strength of buffer affects ionisation of analyte – and thus RT • Applied field • Column Length • Diffusion Optimisation: may involve • bufferpH - alter charge status of analyte • buffer ionic strength - change capacity to produce electro-osmotic flow • organic modifiers: influence ionic strength, and can “capture” analytes selectively to affect charge/size ratio

  4. Capillary Electrophoresis Detection: normally similar to LC detectors but • Peak area is dependent on rate of movement through the detector • Peak area not independent of retention time • Mostly “on-column”, i.e. capillary forms flow cell • Short path length gives reduced sensitivity • Indirect methods may be needed to increase sensitivity Applications There are a variety of named techniques, each suitable for different analytical problems. You may see mentioned: • Capillary zone electrophoresis • Capillary gel electrophoresis • Capillary isotachophoresis • Capillary isoelectric focusing • Capillary electrochromatography • Micellar electrokinetic capillary chromatography

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