Scheme of a CE System

Basics of Capillary Electrophoresis

Acknowledgments: Beckman

Simple Design Polyimide Outer-Coated Capillary Outer 20-150m i.d. 375m o.d. Capillary 20- 150 375 Applied Voltage, 0-30kV 0 Detection On-Line Through Capillary On No Pump

Animation
http://www.shsu.edu/%7Echm_tgc/sounds/pushmovies/CE.gif

CE/Mass Spectrometry
Co-axial
pressure

Sample Injection
bulk movement by pressure

vacuum

bulk movement by EOF plus electrokinesis (differential movement)

electrokinetic

Diagram Courtesy of A.Tomlinson, S. Naylor, L.Benson, Mayo Clinic

+ -

Electrophoretic Mobility

Creation of Electroosmotic Flow

q 6r

q - charge (fixed for strong acids and bases pH dependant for weak acid and bases) 6r - effective ionic volume (N.B. complexation and counterion) - viscosity

Electroosmotic Flow
S - Stern layer (strongly bound) D - Diffuse layer (moves)

Electroosmotic Flow
Pressure (HPLC)
Laminar flow

CAPILLARY WALL

Electroosmotic flow
The gradient of charge from the capillary wall is known as the potential

Plug flow

Mobility

Detection Limits (Mols/inj)

Optical
Absorption Indirect Absorption Laser induced Fluorescence Indirect LIF 10 -16 to 10 -13 10 -14 to 10 -11 10 -20 to 10 -17 10 -17 to 10 -15
10 -17 to 10 -8

e can be used to correct for

any inconsistencies in EOF. It can also be useful for compensating for area changes due to eof shifts. EOF Apparent mobility a Effective mobility e

Mass spectometric

START

General Approaches to Chiral CE

Cavity or Inclusion-Complex Based Chiral Selectors A B C Cyclodextrins Crown Ethers Coiled Polysaccharides

MECHANISM OF SEPARATION
-------------------------------------------------------+A +A

+
+A +A -------------------------------------------------------+A = POSITIVELY CHARGED ANALYTE - - - - = NEUTRAL COATING

= CYCLODEXTRIN

Example of Naproxen
1:10 Mixture of R and S Naproxen

Capillary Gel Electrophoresis

R R,S = 3.75
By Peak Areas

90.1 %
1 1

9.9 %
Data from Andras Guttman

10

11

Gels serve as a molecular sieve for size based separation Biopolymers are run under denaturing conditions Plot of log M.W. vs. migration time(or mobility) shows a linear relationship Generally use electrokinetic injections to protect the gel against extrusion

Time (minutes)

Size Based Separation in Gel

Denatured proteins with constant charge to mass ratio

SDS-CGE of Protein Standards

Longer protein molecules move more slowly through gel