LALI Capillary_Electrophoresis

LALI Capillary_Electrophoresis - Capillary Electrokinetic...

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Capillary Electrokinetic Separations Lecture Date: April 26 th , 2007 1
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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 2
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Electrophoresis: The differential movement or migration of ions by attraction or repulsion in an electric field Anode Cathode Basic Design of Instrumentation : E=V/d Buffer Buffer Anode Cathode Detector The simplest electrophoretic separations are based on ion charge / size 3
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Proteins Peptides Amino acids Nucleic acids (RNA and DNA) - also analyzed by slab gel electrophoresis Inorganic ions Organic bases Organic acids Whole cells Types of Molecules that can be Separated by Capillary Electrophoresis 4
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Migration Velocity : Where: v = migration velocity of charged particle in the potential field (cm sec -1 ) ep = electrophoretic mobility (cm 2 V -1 sec -1) E = field strength (V cm -1 ) V = applied voltage (V) L = length of capillary (cm) Electrophoretic mobility: Where: q = charge on ion = viscosity r = ion radius Frictional retarding forces L V E ep ep r q ep  6 5
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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 Top figure: R. N. Zare (Stanford University), bottom figure: Royal Society of Chemistry Note: diffuse layer rich in + charges but still mobile 6
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It would seem that CE separations would start in the middle and separate ions in two linear directions Another effect called electroosmosis makes CE like batch chromatography Excess cations in the diffuse Stern double- layer flow towards the cathode, exceeding the opposite flow towards the anode Net flow occurs as solvated cations drag along the solution Top figure: R. N. Zare (Stanford University), bottom figure: Royal Society of Chemistry Silanols fully ionized above pH = 9 7
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Where: v = electroosomotic mobility o = dielectric constant of a vacuum = dielectric constant of the buffer = Zeta potential = viscosity E = electric field   4 0 eo Net flow becomes is large at higher pH: – A 50 mM pH 8 buffer flows through a 50-cm capillary at 5 cm/min with 25 kV applied potential (see pg. 781 of Skoog et al.) Key factors that affect electroosmotic mobility: dielectric constant and viscosity of buffer (controls double-layer compression) EOF can be quenched by protection of silanols or low pH Electroosmotic mobility: E E v eo 4 0 8
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LALI Capillary_Electrophoresis - Capillary Electrokinetic...

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