Capillary Electrophoresis - Advanced Analytical Chemistry - Lecture Slides, Slides of Analytical Chemistry

Download Capillary Electrophoresis - Advanced Analytical Chemistry - Lecture Slides and more Slides Analytical Chemistry in PDF only on Docsity! 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) docsity.com 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: E=V/d Buffer Buffer Anode Cathode Detector The simplest electrophoretic separations are based on ion charge / size Capillary docsity.com 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 and are fully deprotonated at pH = 9  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 Bulk docsity.com Electroosmosis  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 docsity.com Electroosmotic Flow (EOF) Where: ueo = 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: L VEv eo   docsity.com Controlling Electroosmotic Flow (EOF) EEv eo          4 0 Want to control EOF velocity: 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 docsity.com Electrophoresis and Electroosmosis  Combining the two effects for migration velocity of an ion (also applies to neutrals, but with ep = 0):     L VE eoepeoep    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 docsity.com Electrophoresis and Electroosmosis  A pictorial representation of the combined effect in a capillary, when EO is faster than EP (the common case):     L VE eoepeoep   Figure from R. N. Zare, Stanford docsity.com 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:  Cross-section of a capillary: Figure from R. N. Zare, Stanford uBH / docsity.com 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 = 3 000 V/cm x 100 cm = 3 x 104 V Assuming D = 3 x 10-9 m2/s, and  = 2 x 10-8 m2/Vs, we find that N = 100, 000 theoretical plates. docsity.com 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 docsity.com 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 docsity.com 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 – This approach minimizes the effects of electrophoretic differences between cells and also sweeps away small molecule contaminants – Method detects whole bacterial cells 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. docsity.com 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 (<10 min) – Readily miniaturized 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. docsity.com Capillary zone electrophoresis (CZE, FSCE, or just CE) Capillary gel electrophoresis (CGE) Capillary electrochromatography (CEC) Capillary isoelectric focusing (CIEF) Capillary isotachophoresis (CITP) Micellar electrokinetic capillary chromatography (MEKC) Common Modes of CE in Analytical Chemistry docsity.com Capillary Zone Electrophoresis (CZE), also known as free-solution CE (FSCE), is the simplest form of CE (what we’ve been talking about). The separation mechanism is based on differences in the charge and ionic radius of the analytes. Fundamental to CZE are homogeneity of the buffer solution and constant field strength throughout the length of the capillary. The separation relies principally on the pH controlled dissociation of acidic groups on the solute or the protonation of basic functions on the solute. Capillary Zone Electrophoresis (CZE) Figure from delfin.klte.hu/~agaspar/ce-research.html docsity.com Capillary Gel Electrophoresis (CGE) is the adaptation of traditional gel electrophoresis into the capillary using polymers in solution to create a molecular sieve also known as replaceable physical gel. This allows analytes having similar charge-to-mass ratios to also be resolved by size. This technique is commonly employed in SDS-Gel molecular weight analysis of proteins and in applications of DNA sequencing and genotyping. Capillary Gel Electrophoresis (CGE) docsity.com ● Capillary Electrochromatography (CEC) is a hybrid separation method ● CEC couples the high separation efficiency of CZE with the selectivity of HPLC ● Uses an electric field rather than hydraulic pressure to propel the mobile phase through a packed bed ● Because there is minimal backpressure, it is possible to use small-diameter packings and achieve very high efficiencies ● Its most useful application appears to be in the form of on- line analyte concentration that can be used to concentrate a given sample prior to separation by CZE Capillary Electrochromatography (CEC) docsity.com Capillary Electrochromatography (CEC) R. Dadoo, C.H. Yan, R. N. Zare, D. S. Anex, D. J. Rakestraw,and G. A. Hux, LC-GC International 164-174 (1997).  CEC combines CE and micro-HPLC into one technique: Actual instrument docsity.com Consider a CEC test mixture containing: • The neutral marker thiourea for indication of the electroosmotic flow • Two compounds with very different polarities (#2 and #5) • Two closely related components (#3 and #4) to test resolving power An Example of CEC docsity.com Because the packed length and overall length of these two capillaries are identical, it is possible to make a direct comparison of the performance because the field strength and column bed length are the same. The EOF has decreased dramatically between pH 8 and pH 2.3 with the resulting analysis time increasing from approximately 5 min to over 20 min at the lower pH. Conclusions from the CEC Example docsity.com Electrokinetic Chromatography (EKC): a family of electrophoresis techniques named after electrokinetic phenomena, which include and combine electroosmosis, electrophoresis and chromatography. Examples: • Cyclodextrin-mediated EKC. Here the differential interaction of enantiomers with the cyclodextrins allows for the separation of chiral compounds • Micellar Electrokinetic Capillary Chromatography (next slides) Electrokinetic Capillary Chromatography docsity.com Micellar Electrokinetic Capillary Chromatography (MECC OR MEKC) is a mode of electrokinetic chromatography in which surfactants are added to the buffer solution at concentrations that form micelles. The separation principle of MEKC is based on a differential partition between the micelle and the solvent (a pseudo-stationary phase). This principle can be employed with charged or neutral solutes and may involve stationary or mobile micelles. MEKC has great utility in separating mixtures that contain both ionic and neutral species, and has become valuable in the separation of very hydrophobic pharmaceuticals from their very polar metabolites. Micellar Electrokinetic Capillary Chromatography Analytes travel in here Sodium dodecyl sulfate: polar headgroup, non-polar tails docsity.com