1 Capillary Electrophoresis

INTRODUCTION

The process of electrophoresis is defined as ‘the differential movement or migration of ions by

attraction or repulsion in an electric field’. In practical terms, a positive (anode) and negative
(cathode) electrode are placed in a solution containing ions. Then, when a voltage is applied
across the electrodes, solute ions of different charge, i.e., anions (negative) and cations (positive),
will move through the solution towards the electrode of opposite charge. Capillary
electrophoresis (CE) is a family of related techniques that employ narrow-bore (20-200 mm i.d.)
capillaries to perform high efficiency separations of both large and small molecules. These
separations are facilitated by the use of high voltages, which may generate electroosmotic and
electrophoretic flow of buffer solutions and ionic species, respectively, within the capillary.

Manufacturing Companies:

• Beckman Coulter (then Beckman Instruments) introduced the first fully automated
capillary electrophoresis system -- the P/ACE 2000 -- in 1989 and has continued to
develop with accepted modifications.

The other major suppliers are:

• Agilent Technologies (formerly Hewlett-Packard Company).

• Bio-Rad Laboratories, Inc.

• Biomidi.

• CVC Microtech

• Lumex

• Sebia Electrophoresis
2 Capillary Electrophoresis

Instrumentation and Construction (Materials and Making):

The basic instrumental configuration for CE is relatively simple. A typical CE instrument uses
the following components to achieve both EOF(Electroosmotic flow) and Electrophoretic
Mobility and therefore separations:

1. Cathode (Negatively Charged Electrode)

2. Anode (Positively Charged Electrode)
3. Power Supply to generate Voltage/Current
4. Catholyte (Buffer Solution at the Cathode End)
5. Anolyte (Buffer Solution at the Anodic End)
6. Capillary (25mm to 100mm ID)
7. A Detection Method(using UV or UV-Vis absorbance, fluorescence detection etc.)
8. Data Acquisition Method

A diagram of a modern instrument, the P/ACE™ 2000 Series, is illustrated below. It is fully
automated instrument. Automation is critical to CE since repeatable operation is required for
precise quantitative analysis.
3 Capillary Electrophoresis

Theory,Working and Principle:

The separation of compounds by capillary electrophoresis is dependent on the differential

migration of analytes in an applied electric field. The electrophoretic migration velocity (up) of
an analyte toward the electrode of opposite charge is

μp is the electrophoretic mobility which is directly proportional to charges on the sample and
inversely to the frictional foeces in the buffer and E is the electric field strength.

Separation Technology
Capillary electrophoresis encompasses a family of related separation techniques that use narrow-
bore fused silica capillaries to separate a complex variety of large and small molecules. High
electric field strengths are used to separate molecules based on differences in charge, size and
hydrophobicity. Sample introduction is accomplished by immersing the end of the capillary into
a sample vial and applying pressure, vacuum or voltage or alternatively gravity injection can also
be used. Depending on the types of capillary and electrolytes used, the technology of CE can be
segmented into several separation techniques.

Main Principle for separation:Electroosmotic flow:

This phenomenon is a consequence of the surface charge on the wall of the capillary. The fused
silica capillaries that are typically used for separations have ionizable silanol groups in contact
with the buffer contained within the capillary. The pI of fused silica is about 1.5. The degree of
ionization is controlled mainly by the pH of the buffer. The negatively-charged wall attracts
positively-charged ions from the buffer, creating an electrical double layer. When a voltage is
4 Capillary Electrophoresis

applied across the capillary, cations in the diffuse portion of the double layer migrate in the
direction of the cathode, carrying water with them. The result is a net flow of buffer solution in
the direction of the negative electrode. This electroosmotic flow can be quite robust, with a linear
velocity around 2 mm/s at pH 9 in 20 mM borate.

The electroosmotic flow (EOF) is defined by

where ∈ is the dielectric constant, ueo is the electroosmotic flow, η is the viscosity of the

buffer, and ζ is the zeta potential measured at the plane of shear close to the liquid-solid
interface.

The electroosmotic flow in capillary electrophoresis is shown in the following diagram:

5 Capillary Electrophoresis

APPLICATIONS

Valuable applications of CE include:

-Genetic analysis

-Analysis of pharmaceuticals (containing nitrogenous bases)

-Pharmaceuticals with chiral centers (enantiomers)

-Counter-ion analysis in drug discovery

-Protein characterization which includes

SDS-Gel Molecular Weight Analysis

Isoelectric Focusing Analysis

Peptide Mapping

-Carbohydrate analysis for the determination of post translational modifications.

REFERENCES
6 Capillary Electrophoresis

• Capillary electrophoresis, Compton, S.W.; Brownlee, R. G., BioTechniques 6, 432-440

(1988)
• High voltage capillary zone electrophoresis: operating parameters effects on
electroendosmotic flows and electrophoretic mobilities, Altria, K. D.; Simpson, C. F.,
Chromatographia 24, 527-532 (1987)
• High-resolution separations based on electrophoresis and electroosmosis, Jorgenson, J.
W.; Lukacs, K. D., J. Chromatography. 218, 209-216 (1981)
• Free-zone electrophoresis in glass capillaries, Jorgenson, J. W.; Lukacs, K. D., ClinIcal.
Chemistry. 27, 1551-1553 (1981)
• Terabe, S.; Otsuka, K.; Ichikawa, K.; Tsuchiya, A.; Ando, T. Anal. Chem. 1984, 56, 111-
113
• Zone electrophoresis in open-tubular glass capillaries, Jorgenson, J. W.;Lukacs, K. D.,
Anal. Chem. 53, 1298-1302 (1981)
• http://bio-animations.blogspot.com/2008/04/capillary-electrophoresis.html
• http://www.beckmancoulter.com/literature/Bioresearch/360643-CEPrimer1.pdf
• http://www.chem.agilent.com/Library/Support/Documents/f01078.pdf

• http://www.promega.com/profiles/602/ProfilesinDNA_602_10.pdf

• http://www.zhdanov.ru/classified-catalogue/manufacturers-and-suppliers/electrophoresis-
equipment-instruments-systems-ie.htm