Capillary Electrophoresis docx

4/30/2012 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 4/30/2012 What is Capillary Electrophoresis? Electrophoresis: The differential movement or migration of ions by attraction or repulsion in an electric field Anode Cathode Basic Design of Instrumentation: Anode Cathode The simplest electrophoretic separations are based on ion charge / size Detector Buffer Buffer E=V/d 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 4/30/2012 The Basis of Electrophoretic Separations Migration Velocity:   ep E  ep V L Where: v = migration velocity of charged particle in the potential field (cm sec -1) ep = electrophoretic mobility (cm 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 ep  q 6r 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 > Top figure: R N Zare (Stanford University), bottom figure: Royal Society of Chemistry  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 Note: diffuse layer rich in + charges but still mobile 4/30/2012 Electroosmosis  It would seem that CE separations would start in the middle and separate ions in two linear directions  Another effect called Silanols fully ionized above pH = electroosmosis makes CE like batch chromatography Top figure: R N Zare (Stanford University), bottom figure: Royal Society of Chemistry  Excess cations in the diffuse Stern doublelayer flow towards the cathode, exceeding the opposite flow towards the anode  Net flow occurs as solvated cations drag along the solution Electroosmotic Flow (EOF)  Net flow becomes is large at higher pH: – A 50 mM pH buffer flows through a 50-cm capillary at 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   eo E    4   eo   0 4  E   Where: v = electroosomotic mobility o = dielectric constant of a vacuum  = dielectric constant of the buffer  = Zeta potential  = viscosity E = electric field 4/30/2012 Electroosmotic Flow Profile - driving force (charge along Anode Cathode Electroosmotic flow profile High Pressure Low Pressure capillary wall) - no pressure drop is encountered - flow velocity is uniform across the capillary Frictional forces at the column walls - cause a pressure drop 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  A certain solution in a capillary has a electroosmotic mobility of 1.3 x 10-8 m2/Vs at pH and 8.1 x 10-8 m2/Vs at pH 12 How long will it take a neutral solute to travel 52 cm from the injector to the detector with 27 kV applied across the 62 cm long tube? At pH = At pH = 12 4/30/2012 Controlling Electroosmotic Flow (EOF)     v   eo E    E  Want to control EOF velocity:  4    Variable Result Notes Electric Field Proportional change in EOF Joule heating may result Buffer pH EOF decreased at low pH, increased at high pH Best method to control EOF, but may change charge of analytes Ionic Strength Decreases  and EOF with increasing buffer concentration High ionic strength means high current and Joule heating Organic Modifiers Decreases  and EOF with increasing modifier Complex effects Surfactant Adsorbs to capillary wall through hydrophobic or ionic interactions Anionic surfactants increase EOF Cationic surfactants decrease EOF Neutral hydrophilic poymer Adsorbs to capillary wall via hydrophobic interactions Decreases EOF by shielding surface charge, also increases viscosity Covalent coating Chemically bonded to capillary wall Many possibilities Temperature Changes viscosity Easy to control Electrophoresis and Electroosmosis  Combining the two effects for migration velocity of an ion (also applies to neutrals, but with ep = 0):   ep   eo E   ep  eo  V L  At pH > 2, cations flow to cathode because of positive contributions from both ep and eo  At pH > 2, anions flow to anode because of a negative contribution from ep, but can be pulled the other way by a positive contribution from eo (if EOF is strong enough)  At pH > 2, neutrals flow to the cathode because of eo only – Note: neutrals all come out together in basic CE-only separations 4/30/2012 Electrophoresis and Electroosmosis  A pictorial representation of the combined effect in a capillary, when EO is faster than EP (the common case):   ep   eo E   ep  eo  V L Figure from R N Zare, Stanford The Electropherogram  Detectors are placed at the cathode since under common  conditions, all species are driven in this direction by EOF Detectors similar to those used in LC, typically UV absorption, fluorescence, and MS – Sensitive detectors are needed for small concentrations in CE  The general layout of an electropherogram: Figure from Royal Society of Chemistry 4/30/2012 CE Theory The unprecedented resolution of CE is a consequence of the its extremely high efficiency Van Deemter Equation: relates the plate height H to the velocity of the carrier gas or liquid H  A  B / u  Cu Where A, B, C are constants, and a lower value of H corresponds to a higher separation efficiency CE Theory  In CE, a very narrow open-tubular capillary is used – No A term (multipath) because tube is open – No C term (mass transfer) because there is no stationary phase – Only the B term (longitudinal diffusion) remains: H  B/u  Cross-section of a capillary: Figure from R N Zare, Stanford 4/30/2012 Number of theoretical plates N in CZE N = L/H H = B/v = 2D/v v =  E = V/L Therefore, N = L/[2D/(V/L)] = V/2D The resolution is INDEPENDENT of the length of the column! Moreover, for V = 000 V/cm x 100 cm = x 104 V D = x 10-9 m2/s , and  = x 10-8 m2/Vs, we find that N = 100, 000 theoretical plates Sample Injection in CE Hydrodynamic injection uses a pressure difference between the two ends of the capillary Vc = Pd4 t 128Lt Vc, calculated volume of injection P, pressure difference d, diameter of the column t, injection time , viscosity Electrokinetic injection uses a voltage difference between the two ends of the capillary Qi = Vapp( kb/ka)tr2Ci Q, moles of analyte vapp, velocity t, injection time kb/ka ratio of conductivities (separation buffer and sample) r , capillary radius Ci molar concentration of analyte 4/30/2012 Capillary Electrophoresis: Detectors     LIF (laser-induced fluorescence) is a very popular CE detector – These have ~0.01 attomole sensitivity for fluorescent molecules (e.g derivatized proteins) Direct absorbance (UV-Vis) can be used for organics For inorganics, indirect absorbance methods are used instead, where a absorptive buffer (e.g 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 J Tanyanyiwa, S Leuthardt, P C Hauser, Conductimetric and potentiometric detection in conventional and microchip capillary electrophoresis, Electrophoresis 2002, 23, 3659–3666 Joule Heating  Joule heating is a consequence of the resistance of the solution to the flow of current – if heat is not sufficiently dissipated from the system the resulting temperature and density gradients can reduce separation efficiency  Heat dissipation is key to CE operation: – Power per unit capillary P/L  r2  For smaller capillaries heat is dissipated due to the large surface area to volume ratio – capillary internal surface area = 2 r L – capillary internal volume =  r2 L  End result: high potentials can be applied for extremely fast separations (30kV) 10 4/30/2012 Capillary Electrophoresis: Applications  Applications (within analytical chemistry) are broad: – For example, CE has been heavily studied within the pharmaceutical industry as an alternative to LC in various situations  We will look at just one example: detecting bacterial/microbial contamination quickly using CE – Current methods require several days Direct innoculation (USP) requires a sample to be placed in a bacterial growth medium for several days, during which it is checked under a microscope for growth or by turbidity measurements – False positives are common (simply by exposure to air) – Techniques like ELISA, PCR, hybridization are specific to certain microorganisms Detection of Bacterial Contamination with CE  Method – A dilute cationic surfactant buffer is used to sweep microorganisms out of the sample zone and a small plug of “blocking agent” negates the cells’ mobility and induces aggregation – Method detects whole bacterial cellls Lantz, A W.; Bao, Y.; Armstrong, D W., “Single-Cell Detection: Test of Microbial Contamination Using Capillary Electrophoresis”, Anal Chem 2007, ASAP Article Rodriguez, M A.; Lantz, A W.; Armstrong, D W., “Capillary Electrophoretic Method for the Detection of Bacterial Contamination”, Anal Chem 2006, 78, 4759-4767 11 4/30/2012 Detection of Bacterial Contamination with CE  The electropherograms show single-cell detection of a variety of bacteria with good S/N  Why is CE a good analytical approach to this problem? – Fast analysis times (