slide1 n.
Skip this Video
Loading SlideShow in 5 Seconds..
Capillary Electrokinetic Separations PowerPoint Presentation
Download Presentation
Capillary Electrokinetic Separations

Capillary Electrokinetic Separations

656 Views Download Presentation
Download Presentation

Capillary Electrokinetic Separations

- - - - - - - - - - - - - - - - - - - - - - - - - - - E N D - - - - - - - - - - - - - - - - - - - - - - - - - - -
Presentation Transcript

  1. Capillary Electrokinetic Separations Lecture Date: April 26th, 2007

  2. 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

  3. Anode Cathode Anode Cathode Detector Buffer Buffer E=V/d What is Capillary Electrophoresis? Electrophoresis: The differential movement or migration of ions by attraction or repulsion in an electric field Basic Design of Instrumentation: The simplest electrophoretic separations are based on ion charge / size

  4. 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

  5. Frictional retarding forces The Basis of Electrophoretic Separations Migration 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  = viscosity r = ion radius

  6. 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 • 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

  7. 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

  8. Electroosmotic Flow (EOF) • 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: Where: v = electroosomotic mobility o = dielectric constant of a vacuum  = dielectric constant of the buffer  = Zeta potential  = viscosity E = electric field

  9. Anode Cathode High Pressure Low Pressure Electroosmotic Flow Profile - driving force (charge along capillary wall) - no pressure drop is encountered - flow velocity is uniform across the capillary Electroosmotic flow profile 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

  10. 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 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

  11. Controlling Electroosmotic Flow (EOF) • Want to control EOF velocity:

  12. Electrophoresis and Electroosmosis • Combining the two effects for migration velocity of an ion (also applies to neutrals, but with ep = 0): • 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

  13. Electrophoresis and Electroosmosis • A pictorial representation of the combined effect in a capillary, when EO is faster than EP (the common case): Figure from R. N. Zare, Stanford

  14. 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

  15. 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 Where A, B, C are constants, and a lower value of H corresponds to a higher separation efficiency

  16. 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

  17. 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 D = 3 x 10-9 m2/s , and  = 2 x 10-8 m2/Vs, we find that N = 100, 000 theoretical plates.

  18. 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

  19. 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)

  20. 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

  21. 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.

  22. Detection of Bacterial Contamination with CE • Single-cell detection of a variety of bacteria • 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.

  23. Capillary Electrophoresis: Summary • CE is based on the principles of electrophoresis • The speed of movement or migration of solutes in CE is determined by their charge and size. Small highly charged solutes will migrate more quickly then large less charged solutes. • Bulk movement of solutes is caused by EOF • The speed of EOF can be adjusted by changing the buffer pH • The flow profile of EOF is flat, yielding high separation efficiencies

  24. Advantages and Disadvantages of CE Advantages Offers new selectivity, an alternative to HPLC Easy and predictable selectivity High separation efficiency (105 to 106 theoretical plates) Small sample sizes (1-10 ul) Fast separations (1 to 45 min) Can be automated Quantitation (linear) Easily coupled to MS Different “modes” (to be discussed) Disadvantages Cannot do preparative scale separations Low concentrations and large volumes difficult “Sticky” compounds Species that are difficult to dissolve Reproducibility problems

  25. Common Modes of CE in Analytical Chemistry Capillary Zone electrophoresis (CZE) Capillary gel electrophoresis (CGE) Capillary electrochromatography (CEC) Capillary isoelectric focusing (CIEF) Capillary isotachophoresis (CITP) Micellar electrokinetic capillary chromatography (MEKC)

  26. Capillary Zone Electrophoresis (CZE) 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. Figure from

  27. Capillary Gel Electrophoresis (CGE) 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.

  28. Capillary Isoelectric Focusing (CIEF) Capillary Isoelectric Focusing (CIEF) allows amphoteric molecules, such as proteins, to be separated by electrophoresis in a pH gradient generated between the cathode and anode. A solute will migrate to a point where its net charge is zero. At the solute’s isoelectric point (pI), migration stops and the sample is focused into a tight zone. In CIEF, once a solute has focused at its pI, the zone is mobilized past the detector by either pressure or chemical means. This technique is commonly employed in protein characterization as a mechanism to determine a protein's isoelectric point.

  29. Capillary Isotachophoresis (CITP) Capillary Isotachophoresis (CITP) is a focusing technique based on the migration of the sample components between leading and terminating electrolytes. Solutes having mobilities intermediate to those of the leading and terminating electrolytes stack into sharp, focused zones. Although it is used as a mode of separation, transient ITP has been used primarily as a sample concentration technique.

  30. Capillary Electrochromatography (CEC) • 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

  31. Capillary Electrochromatography (CEC) CEC combines the strengths of two powerful analytical techniques - CE and micro-HPLC.

  32. 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).

  33. An Example of CEC • 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

  34. An Example of CEC This separation is carried out on an ODS stationary phase at pH = 8:

  35. An Example of CEC The separation carried out on an ODS stationary phase at pH = 2.3:

  36. Conclusions from the CEC Example 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.

  37. Electrokinetic Capillary Chromatography Electrokinetic Chromatography (EKC): a family of electrophoresis techniques named after electrokinetic phenomena, which include electroosmosis, electrophoresis and chromatography. A key example of this is seen with cyclodextrin-mediated EKC. Here the differential interaction of enantiomers with the cyclodextrins allows for the separation of chiral compounds. This approach to enantiomer analysis has made a significant impact on the pharmaceutical industry's approach to assessing drugs containing enantiomers.

  38. Micellar Electrokinetic Capillary Chromatography 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. Analytes travel in here Sodium dodecyl sulfate: polar headgroup, non-polar tails

  39. Micellar Electrokinetic Capillary Chromatography • The MEKC surfactants are surface active agents such as soap or synthetic detergents with polar and non-polar regions. • At low concentration, the surfactants are evenly distributed • At high concentration the surfactants form micelles. The most hydrophobic molecules will stay in the hydrophobic region on the surfactant micelle. • Less hydrophobic molecules will partition less strongly into the micelle. • Small polar molecules in the electrolyte move faster than molecules associated with the surfatant micelles. • The voltage causes the negatively charged micelles to flow slower than the bulk flow (endoosmotic flow).

  40. Method Development in CE • Basic guidance, from the Agilent CE system documentation

  41. V P + - New Technology: Electrokinetic Pumping • Voltage controlled, pulseless • No moving parts or seals • Inherently microscale • High pressure generation • Rapid pressure response • Inexpensive