Created with MindGenius Business 2005Created with MindGenius Business 2005®®

Capillary Electrophoresis Capillary Electrophoresis

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

Separation of analyte ions via differential migration in an electric field, coupled with electro-osmotic flow of mobile phase

Created with MindGenius Business 2005Created with MindGenius Business 2005®®

Capillary Electrophoresis 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 separationBased on interaction of analyte with electric field

Migration velocity v = (µe + µeo) E where µe and µeo are the electrophoretic mobilities of the analyte and buffer, and E is the applied field strength

Created with MindGenius Business 2005Created with MindGenius Business 2005®®

Capillary Electrophoresis 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 buffer pH - 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

Created with MindGenius Business 2005Created with MindGenius Business 2005®®

Capillary Electrophoresis 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

ApplicationsThere 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