Capillary Electrophoresis

4/30/2012 Capillary Electrokinetic Separations Lecture Date: April 23rd, 2008 Capillary Electrokinetic Separations  Outline – Brief reviewof 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 differentialmovement or migration of ions by attraction or repulsion in an electric field Anode Cathode Basic Design of Instrumentation: Anode Cathode Detector Buffer Buffer E=V/d The simplestelectrophoretic separations are based on ion charge/ size Types of Molecules that can be Separated by Capillary Electrophoresis Proteins Peptides Amino acids Nucleic acids (RNAand DNA) - also analyzed by slab gel electrophoresis Inorganic ions Organic bases Organic acids Wholecells 2 4/30/2012 The Basis of Electrophoretic Separations Migration Velocity:  = epE = ep V Where: v = migrationvelocity of charged particlein the potential field (cm sec -1) ep =electrophoretic mobility (cm2 V1 sec-1) E = field strength (V cm -1) V = applied voltage(V) L = length of capillary (cm) Electrophoretic mobility: ep = 6πr Where: q = charge on ion  = viscosity r = ion radius Frictionalretarding forces Inside the Capillary:  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 The Zeta Potential Top figure:R. N. Zare (Stanford University),bottomfigure: RoyalSociety ofChemistry Note: diffuse layer rich in + charges but still mobile 3 4/30/2012  It would seem that CE separations would start in the middle and separate ions in two linear directions  Another effect called electroosmosis makesCE like batch chromatography  Excess cations in the diffuseStern double-layer flowtowards the cathode, exceeding the opposite flow towards the anode  Net flow occurs as solvatedcations drag along the solution Electroosmosis Silanols fully ionized above pH = 9 Top figure:R. N. Zare (Stanford University),bottomfigure: RoyalSociety ofChemistry Electroosmotic Flow (EOF)  Net flow becomes is large at higher pH: – A50 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: v = eoE =0 E 0 eo 4π Where: v = electroosomotic mobility  = dielectric constant of a vacuum  = dielectric constant of the buffer  = Zeta potential  = viscosity E = electric field 4 4/30/2012 Electroosmotic Flow Profile Anode Cathode Electroosmotic flowprofile - drivingforce (charge along capillarywall) - no pressure drop is encountered - flow velocity is uniformacross thecapillary High Pressure Low Pressure Frictionalforces at the columnwalls - cause a pressuredrop across the column Hydrodynamic flow profile  Result: electroosmotic flow does not contribute significantly to band broadening like pressure-driven flow in LC and related techniques Example Calculation of EOF at Two pH Values  Acertain solution in a capillary has a electroosmotic mobility of 1.3 x 10-8 m2/Vsat pH 2 and 8.1 x 10-8 m2/Vsat pH 12. How long will it take a neutral solute to travel52 cm fromthe injector to the detector with 27 kV applied across the 62 cm long tube? At pH = 2 At pH = 12 5 ... - tailieumienphi.vn