CAPILLARY ELECTROPHORESIS

Dr Alireza Shafaati
Associate Professor Pharmaceutical Chemistry, School of Pharmacy, Shaheed Beheshti University of Medical Sciences

1st MNPC- Shiraz May 2005

Capillary electrophoresis (CE) is:

a family of related techniques in which electrophoresis is performed in narrow-bore capillaries, typically 0.025 to 0.100 mm inner diameter.

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Electrophoresis:

The migration of electrically charged particles or ions in solutions due to an applied electric field

In order to overcome problems of convective mixing which were encountered in electrophoretic separations performed in free solutions, various stabilising media have been employed, such as agar, cellulose powder, glass wool, paper, silica gel and acrylamide.

Slab-gel Electrophoresis

An alternative approach to alleviate thermal convection problems in free solution electrophoresis was the use of tubes with small diameters. These small tubes or capillaries dissipate heat better and provide a more uniform thermal cross- section of the sample within the tube.

Capillary Electrophoresis

Low pH

High pH

- H+

Electroosmotic Flow

EOF

Electrophoresis

Electrophoresis + Electroosmosis

EOF

tm (min)

Electrophoresis

0 -

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Electroosmosis

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0

EOF

flow profiles
Hydrodynamic flow Parabolic Resistance to flow at surface More diffusion/broader detection zone

EOF gives plug flow

Minimizes band broadening Narrows detection zone

EOF

flow profiles
Hydrodynamic flow Parabolic Resistance to flow at surface More diffusion/broader detection zone

EOF gives plug flow

Minimizes band broadening Narrows detection zone

Historical Development of CE
1886 Lodge H+ migration in a tube of phenolphthalein Jelly 1937 Tiselius Improved apparatus for moving boundry studies 1967 1981 1984 1989 Hjerten Jorgenson Terabe Free solution electrophoresis in 3 mm tube First successful report on CE Development of MEKC Availability of commercial CE instrument

A model of CE instrument
Capillary cartridge

Detection Compartment

Inlet and oullet vials

Capillary Cartridge

Detection window

C. Capillary electrophoresis
Capillary electrophoresis is formed in fused SiO2 capillary tube long ~ 0.5 m, inner diameter: 25-75 mm.

CE on a chip

Detectors for CE
A wide range of detectors can be link to CE Detection systems commonly used
UV/Vis detectors Fluorescence detector (LIF, laser-induced fluorescence) Mass spectrometry

LIF is the most sensitive of these

Factors Affecting Separations in CE

mep=

Z 6phr
Z = ion charge h = solution viscosity r = ion radius

meo=

ez h
e = dielectric constant z = zeta potential h = solution viscosity

How to control EOF?

Effect of pH on EOF

Modes of CE
Capillary zone electrophoresis (CZE) Micellar electrokinetic chromatography (MEKC) Capillary electrochromatography (CEC) Capillary gel electrophoresis (CGE) Capillary isoelectric focusing (CIEF) Capillary isotachphoresis (CITP)

Capillary Zone Electrophoresis (CZE)

EOF

tm (min)

SDS

Micellar Electrokinetic Chromatography (MEKC)

EOF

Capillary Electrochromatography (CEC)

Capillary Gel Electrophoresis (CGE)

Ep

Capillary Isoelectric Focusing (CIEF)

Low pH buffer

High pH buffer

Capillary Isotachophoresis (CITP)

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Advantages of Capillary Electrophoresis

Extremely high separation efficiency Small sample size required (1-10 ul) Fast separation (1 to 45 min) Easy and predictable selectivity Automation Reproducibility Couple to mass spectrometer

CE Applications:
Proteins, peptides
Purity, heterogeneity Physical properties (MW, pI, peptide mapping) Binding studies Identification (CE-MS) Quantitation Immunoassays

Small Molecules
Pharmaceuticals
Assay Assay (in biologicals) Purity Chiral Separation

Nucleobases, Oligonucleotides
QC (primers, antisense) Fingerprinting PCR Clinical applications

Forensics Environment Foods, additives Ions (organic, inorganic)

Standard Test Mix Separation on an eCAP-SDS 14-200 Capillary

A View of Tehran

Thank You!