Capillary Electrophoresis Dong-Sun Lee/ CAT-Lab / SWU Chapter 33 Capillary Electrophoresis

History Electrophoresis as an analytical tool was introduced by the Swedish chemist Arne Tiselius, first in his doctoral thesis in 1930. For his pioneer work in this field, Tiselius was awarded the Nobel prize in 1948. The first appearance of capillary electrophoresis can be found in 1981 with the publication of an article by J.W. Jorgenson and K.D. Lukacs, working at the University of North Carolina, in Analytical Chemistry, 53 (1981) 1298.

Capillary Electrophoresis Capillary Electrophoresis (CE) is a technique that is utilized to separate complex mixtures of biological and chemical species. The technique employs capillary columns, buffers (with or without additives), and high voltages to perform high resolution separations based on size, shape and charge to mass ratio of organic and inorganic molecules. Both qualitative and quantitative separations are achieved with a number of modes of CE, including free-zone CE, capillary gel electrophoresis, micellar electrokinetic capillary chromatography and isoelectric focusing. A number of detectors are utilized including UV-visible diode array, fluorescence, mass spectrometry, and indirect amperometric. Examples Main component drug assay of pharmaceuticals. Identification of impurities in pharmaceutical and agricultural products. Chiral separations. Separation of DNA and DNA fragments. Separation of nucleotides, nucleosides and bases. Separation of proteins and peptides. Characterization of amine functional and charged polymers.

Capillary electrophoresis (CE) is a technique in which an electrophoretic separation takes place in a narrow-bore fused silica capillary. The capillaries typically used in CE are commercially available at reasonable cost (about $4/meter.) We use capillaries that range from 30 to 50 centimeters in length, 0.150 to 0.375 millimeters in outer diameter, and have a 0.010 to 0.075 millimeter diameter bore. In a CE separation, the capillary is filled with buffer and each end is immersed in a vial of the same buffer. A sample of analyte is injected at one end, either by electrokinesis or by pressure, and a electric field of 100 to 700 volts/centimeter is applied across the capillary. As the analyte mixture migrates through the capillary due to the applied electric field (electrophoresis), differing electrophoretic mobilites drive each of the components into discrete bands. At the other end of the capillary each of the separated analytes is detected and quantified. Electrophoretic mobility is proportional to the charge of the molecule divided by its frictional coefficient. This is approximately equal to the charge to mass ratio of the molecule. So in general any molecules with differering charge to mass ratios can be separated by CE. The diagram below is an attempt to help demonstrate this.

Basic principles of CE The electrophoretic separations of ions The mechanism of separation in electrophoresis is based on the migration of charged particles in an applied electric field. Different particles with different charges and /or sizes migrate with different velocities. The electrostatic force F exerted on an ion i in solution is proportional to the net charge of the ion (qi) and the electric field strength(E, in V/cm) : F = qi E The direction of the force is to the electrode with a charge opposite to that of the ion. Under the influence of the electrostatic force the charged ion is accelerated and starts migrating. Its movement is then opposed by viscosity() of the solution, which increase proportional with the velocity (vi, in cm s–1) of the ion. For a spherical particle(radius, ri) the viscosity is given by the Stokes equation: F = 6  rivi After a very short acceleration time the opposing force (electrostatic and viscous) cancel each other out and the particle then moves with constant velocity through the solution. vi = (qi E) / (6  ri) The electrophoretic mobility (i) has been defined as:  i = vi / E = qi / (6  ri)

 +  +  + q +q  + Anode Cathode Movement of charged particles under the influence of an applied electric filed Electric force (–qE) Viscous drag Cathode Viscous drag Anode q +q  + Electric force (+qE) E Forces acting on charged particles moving in an electric field.

The net effect for the mobility of an ion 1. Mobility is directly proportional to the charge of an ion 2. Mobility is inversely proportional to the viscosity of the solvent 3. Mobility is inversely proportional to the radius of particle (represented by the diffusion coefficient) It is clear that different ions can be separated when they differ either in charge or radius or, better, when their charge/size ratio differs. Wim Kok, Capillary Electrophoresis: Instrumentation and Operation, Chromatographia, Suppl. 51, S9, 2000. Patrick Camilleri, Capillary Electrophoresis-Theory and Practice, CRC, 1993, p66-67

Factors affecting electrophoretic mobilities 1. Nature of the charged particles: net charge, size, relative mass, charge-to-size ratio 2. Nature of the electrophoretic system 1) The ionic composition of the electrophoresis buffer 2) The temperature 3) The pH of the electrophoresis buffer 4) The applied voltage 5) The type of support medium: pore size

Principles of electroosmosis When an aqueous solution of electrolytes, as used in electrophoresis, is contact with the wall of the separation capillary, there is a charge separation between the wall and the solution. This can be caused by ionization of the wall material or by specific adsorption of ions from the solution to the wall. With fused silica capillaries the wall is usually negatively charged. Free silanol groups (which has a pKa of 6 to 7) on the surface of the fused silica are deprotonated(at pH> 1.5), leaving negative Si-O– groups. Since the system as a whole must be electrically neutral, the solution in the separation compartment has a net positive charge. This excess of positive ions is located in the solution close to the wall, due to the electrostatic attraction by the negative wall. When a voltage is applied between the ends of the capillary, the electric field exerts a force on the excess of positive charge in the solution close to to the wall. This force drives the solution in the capillary as a whole in the direction of the negative electrode. A constant flow of the solution results when the viscous forces in the thin layer of solution near the wall counteract the electrostatic force. This phenomenon is called electro-osmosis or electro-endoosmosis.

+ – EOF Fixed charges (–) Capillary wall Excess mobile charges (+) The principle of electroosmosis.

(a) Electric double layer created by negatively charged silica surface and nearby cations. (b) Predominance of cations in diffuse part of the double layer produces net electroosmotic flow toward the cathode when an external is applied.

High ectroosmotic mobility Low ectroosmotic mobility Hydrodynamic flow profiles superimposed over the EOF when differences in the electroosmotic mobility exist over the length of the capillary.

Flow profiles for liquids under (a) electroosmotic flow and (b) pressure-induced flow (parabolic velocity profile).

Total electrophoretic mobilty is the vector sum of the electrophoretic mobility of the sample and the effective mobility due to electroosmotic flow.

Types of electrophoretic separations 1) Classical electrophoresis a. Moving boundary electrophoresis b. Zone electrophoresis : paper, cellulose acetate, starch gel, polyacrylamide gel, agarose gel / immuno, rocket / tube, slab, disc, i. Horizontal ii. Vertical c. Steady state electrophoresis i. Isoelectric focusing ii. Isotachophoresis 2) Capillary zone electrophoresis (CZE) 3) Capillary isotachophoresis (CITP) 4) Capillary gel electrophoresis (CGE) 5) Capillary isoelectric focusing (CIEF) 6) Micellar electrokinetic capillary chromatography (MECC) 7) Capillary electrochromatography (CEC)

Initial Final Sample A+B+C Leading electrolyte A+C A A+B+C Moving boundary electrophoresis.

Initial Final Buffer Buffer A+B+C B C A Buffer Buffer Zone electrophoresis.

Initial Final TerminalBuffer Leading Buffer A+B+C TerminalBuffer pH 2 4 6 8 10 Isotachophoesis

Initial Final Buffer Buffer A+B+C pH 2 4 6 8 10 B C A Buffer Buffer Isoelectric focusing.

Moving boundary electrophoresis.

Horizontal electrophoresis.

Isoelectric focussing

Vertical paper electrophoresis.

Tube gel electrophoresis.

Disc electrophoresis.

Electrochromatography In recent years a number of new separation techniques employing high voltages and narrow bore capillaries have been brought to light. These include: Capillary Electrophoresis (CE or CZE) Capillary Gel Electrophoresis (CGE) Micellar Electrokinetic Capillary Chromatography (MECC) The first two techniques in the above list can be viewed as high tech instrumental analogues of the well know slab-gel electrophoresis, whereas MECC makes use of a form of chromatographic partition to achieve the separation of the components. However, the capillary electro- technique which most closely resembles modern HPLC is electrochromatography.

Instrumentation 1) Capillary : 25~75m ID fused silica capillary with a thin outer coating of polyimide 2) Injection volume : 1 l for a 50 cm long, 50 m ID 3) Detector a. Optical absorbance detector b. Laser based absorbance detector c. Refractive index detection d. Thermooptical absorbance e. Fluorescence detector f. Chemiluminescence detector g. Electrochemical detector: Conductivity, Amperometric h. Radioactivity detector I. Hyphenated detection: CE-MS 4) Power supply 5) Buffer and additives

Capillary electrophoresis instrument.