tailieunhanh - Capillary Electrophoresis

LIF (laser-induced fluorescence) is a very popular CE detector – These have ~ attomole sensitivity for fluorescent molecules (. derivatized proteins). Direct absorbance (UV-Vis) can be used for organics. For inorganics, indirect absorbance methods are used instead, where a absorptive buffer (. chromate) is displaced by analyte ions – Detection limits are in the 50-500 ppb range. Alternative methods involving potentiometric and conductometric detection are also used – Potentiometric detection: a broad-spectrum ISE – Conductometric detection: like IC. | 4 30 2012 a W VILLANOVA UNIVERSITY Capillary Electrokinetic Separations Lecture Date April 23rd 2008 Capillary Electrokinetic Separations Outline - Brief review of theory - Capillary zone electrophoresis CZE - Capillary gel electrophoresis CGE - Capillary electrochromatography CEC - Capillary isoelectric focusing CIEF - Capillary isotachophoresis CITP - Micellar electrokinetic capillary chromatography MEKC Reading Skoog et al. - Chapter 30 Capillary Electrophoresis and Electrochromatography Reading Cazes et al. - Chapter 25 Capillary Electrophoresis 1 4 30 2012 What is Capillary Electrophoresis Electrophoresis The differential movement or migration of ions by attraction or repulsion in an electric field Basic Design of Instrumentation Anode Cathode The simplest electrophoretic separations are based on ion charge size Types of Molecules that can be Separated by Capillary Electrophoresis Proteins Peptides Amino acids Nucleic acids RNA and DNA - also analyzed by slab gel electrophoresis Inorganic ions Organic bases Organic acids Whole cells 2 4 30 2012 The Basis of Electrophoretic Separations Migration Velocity Where v migration velocity of charged particle in the potential field cm sec 1 electrophoretic mobility cm2 V 1 sec1 E field strength V cm 1 V applied voltage V L length of capillary cm Electrophoretic mobility . H H Where q charge on ion q viscosity r ion radius - Frictional retarding forces Inside the Capillary The Zeta Potential The inside wall of the capillary is covered by silanol groups SiOH that are deprotonated SiO- at pH 2 SiO- attracts cations to the inside wall of the capillary The distribution of charge at the surface is described by the Stern double-layer model and results in the zeta potential Fused Silica capillary with exposed SiO groups Top figure R. N. Zare Stanford University bottom figure Royal Society of Chemistry s e .Capillary wall Note diffuse layer rich in charges but still mobile Di 1 fuse layer Rigid layer