Capillary Electrophoresis Memahami prinsip dasar elektroforesis Memahami berbagai metode yang digunakan dalam elektroforesis Mampu membac elektroforegram yang dihasilkan Mampu merancang suatu analisis menggunakan elektroforesis - 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 Beckman and Coulter - Reading (Skoog et al.) Chapter 30, Capillary Electrophoresis and Electrochromatography - Reading (Cazes et al.)Chapter 25, Capillary Electrophoresis Definition: A separation technique based on the differential transportation of charged species in an electric field through a conductive medium. Primary candidates for CE separation are ions. Can determine the size, shape, and charge of a molecule Different forms of electrophoresis are used for each of these factors independently or in combination. The basic instrumental set-up of CE, consists of a high voltage power supply (0 to 30 kV), a fused silica (SiO2) capillary, two buffer reservoirs, two electrodes, and an on-column detector.Capillary Native Polyacrylimide Gel Electrophoresis (PAGE) SDS-PAGE Slab Paper 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 ProteinsPeptidesAmino acidsNucleic acids (RNA and DNA) - also analyzed by slab gel electrophoresis Inorganic ionsOrganic basesOrganic acidsWhole cells Types of Molecules that can be Separatedby Capillary ElectrophoresisMigration Velocity: Where: v = migration velocity of charged particle in the potential field (cm sec -1) ep = electrophoretic mobility (cm2 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 q = viscosity r = ion radius Frictional retarding forces LVEep ep v = =rqeptq6=- 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 surfaceis 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 - 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 Where: v = electroosomotic mobility co = dielectric constant of a vacuum c = dielectric constant of the buffer , = Zeta potential q = viscosity E = electric field tqc, c40=eo- Net flow becomes 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 veo||.|

\|= =tqc, c40CathodeAnode Electroosmotic flow profile Hydrodynamic flow profile HighPressure LowPressure - driving force (charge along capillary wall) - no pressure drop is encountered - flow velocity is uniform across the capillary Frictional forces at thecolumn walls -cause a pressure drop across the column - Result:electroosmotic flow does not contribute significantly to band broadening like pressure-driven flow in LC and related techniques - A certain solution in a capillary has a electroosmotic mobility of 1.3 x 10-8 m2/Vs at pH 2 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 = 2 At pH = 12 E E veo||.|

\|= =tqc, c40- Want to control EOF velocity: VariableResultNotes Electric FieldProportional change in EOFJoule 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 TemperatureChanges viscosityEasy to control - Combining the two effects for migration velocity of an ion (also applies to neutrals, but with ep = 0): ( ) ( )LVEeo ep eo ep v + = + =- 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 - A pictorial representation of the combined effect in a capillary, when EO is faster than EP (the common case): ( ) ( )LVEeo ep eo ep v + = + =Figure from R. N. Zare, Stanford - 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 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 Cu u B A H + + = /Where A, B, C are constants, and a lower value of H corresponds to a higher separation efficiency - 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: - Cross-section of a capillary: Figure from R. N. Zare, Stanford u B H / =Hydrodynamic injection uses a pressure difference between the two ends of the capillary Vc = APtd4 t 128qLt Vc, calculated volume of injection P,pressure difference d,diameter of the column t, injection time q,viscosity Electrokinetic injection uses a voltage difference between the two ends of the capillary Qi = Vapp( kb/ka)ttr2Ci Q, moles of analyte vapp, velocity t,injection time kb/ka ratio of conductivities (separation buffer and sample) r , capillary radius Cimolar concentration of analyte - 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/Lr2 - For smaller capillaries heat is dissipated due to the large surface area to volume ratio capillary internal surface area= 2t r L capillary internal volume = t r2 L - End result:high potentials can be applied for extremely fast separations (30kV) - 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 - 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. - Single-cell detection of a variety of bacteria - Why is CE a good analytical approach to this problem? Fast analysis times (