Lecture 10 Modes and Methods in Capillary Electrophoresis © Dr. Rasha Hanafi, GUC Lecture 2, PHCM662-SS2012, 25-02-2012 Lecture 10 Modes and Methods in Capillary Electrophoresis Dr. Rasha Hanafi © Dr. Rasha Hanafi, GUC Lecture 10, SS16 Dr. Rasha Hanafi, 2010-11-07

© Dr. Rasha Hanafi, GUC Lecture 2, PHCM662-SS2012, 25-02-2012 Learning outcomes By the end of this session, the student should be able to: Enumerate advantages of CE. Compare between different modes of CE. Describe injection and detection in CE. Summarize all information acquired about CE to have a global picture about this analytical process. © Dr. Rasha Hanafi, GUC Lecture 10, SS16 Dr. Rasha Hanafi, 2010-11-07

Advantages of capillary electrophoresis Lower instrument costs compared to HPLC, GC (starts at 15,000 $). Small sample size (nL) compared to μL in other separation techniques. Significantly greater speed than HPLC (Voltage applied in CE versus pressure applied in HPLC) High resolution due to the nature of the flow profile (electroosmotic vs. hydrodynamic) and the absence of the A and Cu term parameters in the Van Deemter equation as there is no stationary phase in the capillary. © Dr. Rasha Hanafi, GUC Lecture 10, SS16

Modes of capillary electrophoresis © Dr. Rasha Hanafi, GUC Lecture 2, PHCM662-SS2012, 25-02-2012 Modes of capillary electrophoresis Modes of CE 1st used in “slab electro- -phoresis” CZE Capillary Zone Electrophoresis CIEF Capillary Isoelectric Focusing CITP Capillary Isotachophoresis (what is it? Personal effort) CGE Capillary Gel Electrophoresis © Dr. Rasha Hanafi, GUC Lecture 10, SS16

I. Capillary Zone Electrophoresis CZE © Dr. Rasha Hanafi, GUC Lecture 2, PHCM662-SS2012, 25-02-2012 I. Capillary Zone Electrophoresis CZE 30 anions in 3 min.!! Buffer composition in the capillary: constant Mobility: due to electroosmotic flow, either : 1- to cathode (most common, see lecture 9). Small anions with high mobility and highly negatively charged proteins require very strong electroosmotic flow or otherwise they will not travel toward the cathode. © Dr. Rasha Hanafi, GUC Lecture 10, SS16

I. Capillary Zone Electrophoresis CZE 2- To anode: how? For fast separation of anions, the EOF can be reversed by treating the walls of the capillary with an alkyl ammonium salt (ex: cetyl trimethyl ammonium bromide). NH4+ attaches to the –ve charged surface of the silica, creating a double layer attracted to the anode. N.B.: Neutral compounds would better be derivatized, via formation of borate ions that are readily formed if a borate buffer is used in the capillary. Br- Br- Br- Br- Br- Br- Br- Br- © Dr. Rasha Hanafi, GUC Lecture 10, SS16

I. Capillary Zone Electrophoresis CZE, contd. Usually, the number of theoretical plates (N) achieved in CE= 105!! It is the higher voltage (V) rather than the long capillaries (unlike other types of chromatography) that lead to higher number of plates. N= µapp V Ld /2D Lt Ld: capillary length to the detector ; D: diffusion coefficient; Lt: total capillary length N.B.: Increasing Voltage is restricted to certain values to avoid capillary heating (joule heating) leading to hydrodynamic (parabolic) flow profile. Possible reasons for band broadening: The finite width of the injected band. Occurrence of parabolic flow profile from heating inside the capillary. Adsorption of solute on capillary walls. The finite length of the detection zone. © Dr. Rasha Hanafi, GUC Lecture 10, SS16

II. Capillary Gel Electrophoresis CGE It is a variation of gel electrophoresis which has been a 1ary tool for biochemists in early slab electrophoresis. In CE, the gels are contained inside the capillary. Chemical gels contain cross-linked polymer chains covalently bound deriving their properties from physical entanglement of the polymers. Porous gel polymer matrix are: agarose, methyl cellulose, polyethylene glycol and polyacrylamide (most common, acrylamide is CH2=CH-CO-NH2). The pores of the polymer contain the buffer mixture required for separation. Advantages: Reduce analyte dispersion by convection and diffusion. Provide a medium suitable for handling and detection (in slab electrophoresis). Increasing the amount of cross linking agents results in smaller pore size. Hence, molecular sieving retards analytes based on their sizes (useful in separation of macromolecules, DNA fragments and oligonucleotides that have the same charge but differ in size) © Dr. Rasha Hanafi, GUC Lecture 10, SS16

III. Capillary IsoElectric Focusing CIEF CIEF is commonly used to separate zwitterionic compounds (amphiprotic) whose charge depends on the functional groups attached to the main chain and the surrounding pH of the environment (peptides).   An amphiprotic compound is a specie that in solution is capable of donating or accepting a proton. Most common example: amino acids! When glycine is dissolved in water, 3 important equilibria operate: N.B.: The isoelectric point (pI) is the pH at which the amino acid is neutral, i.e. the zwitterion form is dominant. At this point, the amino acid does not migrate in an electric field. At a pH < pI, the molecule is positive, and when the pH > pI it is negative .  © Dr. Rasha Hanafi, GUC Lecture 10, SS16

III. Capillary IsoElectric Focusing CIEF, contd. © Dr. Rasha Hanafi, GUC Lecture 2, PHCM662-SS2012, 25-02-2012 III. Capillary IsoElectric Focusing CIEF, contd. NaOH and H3PO4 in each of the reservoirs what happens when potential is applied? H+ moves to cathode and OH- moves to anode which creates regions of different pH all along the capillary (pH gradient). H+ OH- NaOH H3PO4 © Dr. Rasha Hanafi, GUC Lecture 10, SS16

III. Capillary IsoElectric Focusing CIEF, contd. Strategy: Because the charge changes with pH, a pH gradient can be used to separate molecules in a mixture. The anodic end of the capillary sits in acidic solution (low pH), while the cathodic end sits in basic solution (high pH). During a CIEF separation, the capillary is filled with the sample in solution and typically no EOF is used (EOF is removed by using a coated capillary to hide the active silanol groups or any interacting groups). When the voltage is applied, the ions will migrate to a region where they become neutral (pH=pI). Compounds of equal isoelectric points are “focused” into sharp segments and remain in their specific zone, which allows for their distinct detection. © Dr. Rasha Hanafi, GUC Lecture 10, SS16

III. Capillary IsoElectric Focusing CIEF, contd. If the analyte is –ve, it migrates to anode, progressively to lower pH regions leading to continuous protonation giving it less –ve charge until net charge = 0 (isoelectric point) where it stops moving. Since isoelectric points are different for different analytes, separation takes place with sharp boundaries. To move the focused uncharged bands at the end of the experiment, pressure is applied to force them out. © Dr. Rasha Hanafi, GUC Lecture 10, SS16

Injection in CE Electrokinetic Pressure © Dr. Rasha Hanafi, GUC Lecture 2, PHCM662-SS2012, 25-02-2012 Injection in CE Electrokinetic An electric field is applied to drive the sample into the capillary. One end of the capillary and its electrode are removed from the buffer and put in the sample cup. A potential is applied causing the sample to enter the capillary by ? ;-) and ? ;-).. Disadv.: Because each analyte has a different mobility, in quantitative analysis, the injected sample does not have the same composition as the original sample, i.e. unintentional injection of larger amounts of highly mobile ions relative to slower moving ions usually takes place. Adv: most useful in capillary gel electrophoresis in which the liquid in the capillary is too viscous for hydrodynamic injection. Pressure A pressure difference between the 2 ends of the capillary is used to drive the sample into it (vacuum at the detector end or pressure at the sample end). Disadv: can not be used in gel–filled capillaries © Dr. Rasha Hanafi, GUC Lecture 10, SS16

Detection in CE On column detection. © Dr. Rasha Hanafi, GUC Lecture 2, PHCM662-SS2012, 25-02-2012 Detection in CE On column detection. The detector cell: A small section of the protective polyimide coating is removed from the exterior of the capillary by burning, dissolution or scraping. Path length (b) = only 50-100 μm ! Possible detectors: UV-VIS Absorbance. Fluorescence. ESI Mass spectrometry. Electrochemical (conductometry and amperometry). Indirect detection??? Smart  (ex: indirect detection of Cl- isotopes in presence of UV-absorbing anion chromate), how?? © Dr. Rasha Hanafi, GUC Lecture 10, SS16

How to improve Detection sensitivity? b b 1. The bubble cell 2. Right angle bend Limitation: band broadening  successive peaks must be separated by 3 mm at least or they will overlap. 3. Silver coating on a section of the capillary allows multiple internal reflection. © Dr. Rasha Hanafi, GUC Lecture 10, SS16

CE versus LC Advantages: Higher resolution. Low waste production. Generally simpler equipment. Drawbacks: Higher Limit of Detection (LOD). Run-to-run irreproducibility of migration times. Insolubility of some analytes in common electrolyte solns. Inability to scale up to a preparative separation. Although liquid chromatography is 2 decades more mature, CE is preferred for some forensic ex: analysis of alkaloids in opium and heroin. © Dr. Rasha Hanafi, GUC Lecture 10, SS16

References Textbook: Principles of instrumental analysis, Skoog et al., 5th edition, chapter 30, pp. 782 to 795. Textbook: Textbook: Quantitative Chemical Analysis, Harris, chapter 26. http://bio-animations.blogspot.com/2008/04/capillary-electrophoresis.html © Dr. Rasha Hanafi, GUC Lecture 10, SS16