Capillary Electrophoresis (CE) : Amruta L. Suryan

CAPILLARY
ELECTROPHORESIS
(CE)
Presented by –
Amruta L. Suryan
1
CONTENTS:
 Introduction
 Principle
 Related Techniques
 Instrumentation
 Modes of separation
 Efficiency and resolution
 Detection
 Advantages over other techniques
 Disadvantages of capillary Electrophoresis
 Applications of Capillary Electrophoresis
2
INTRODUCTION:
 Electrophoresis
 This technique was first developed by Swedish chemist Arne
Tiselius in 1930s for study of serum proteins
 shares the attributes of both chromatography and electrophoresis
 This technique is able to separate neutral as well as charged
analytes
 Types of sample – Inorganic anions & cations, amino acids,
catecholamine, drugs, vitamins, carbohydrates, peptides,
proteins, nucleic acids, nucleotides, polynucleotide, and other
species.
3
TYPES OF ELECTROPHORESIS:
Electrophoresis is performed in two different formats.
Slab Electrophoresis Capillary Electrophoresis

 Classical/conventional method  Instrumental version of
electrophoresis developed in
1980s
 Typically slow separation  High speed separation
 Labor intensive & difficult to  Simple to automate with high
automate resolution power
 Sample volume – in μl range  Very small sample vol. 0.1-10nL
 Does not yield very precise  Yields precise quantitative
quantitative information information
 Requires staining techniques for  Quantitative detectors similar to
detection HPLC can be used
 Joule heating limits the magnitude  High voltage power supply of
of applied voltage to about 500V
4
PRINCIPLE:
 Separation takes place inside a capillary tube and is based on
“Electrophoretic Mobility”
 It is the velocity with which a particle moves divided by the magnitude
of the electric field is a characteristic number called the
 Migration rates, plate heights and electro-osmotic flow help in
separation
1. Migration Rate (v):

v = μe χ E or v = μe χ V/L
Where , μe = electrophoretic mobility(cm2 v-1 s-1)
E = Field strength (v cm-1)
L = length over which electric field is applied
5
PRINCIPLE: [Contd…]
2. Electro-osmotic flow (EOF) –

 It is a unique feature of CE operation
 Definition: It is the motion of ions in a solvent environment through
very narrow channels, where an applied potential across the channels
cause the ion migration
 Cause of EOF is the electric double layer that develops at silica
solution interface
6
PRINCIPLE: [Contd…]
 EOF leads to bulk solution flow that has flat profile across the tube in
contrast to the laminar(parabolic) pressure driven flow in HPLC
 So it does not lead to band broadening
7
PRINCIPLE:
 The rate of electro-osmotic flow is generally greater than

electrophoretic migration velocities of the individual ions and
effectively becomes mobile phase pump of CE
 Direction of EOF can be reversed by adding cationic surfactants to

buffer
 It is often used to speed up separation of anions
 EOF can be minimized by modifying inside capillary walls with
reagent like Trimethylchlorosilane 8
PRINCIPLE:
3. Plate heights in CE:
 It is calculated by-
N= μe V / 2D
where, N = No. of plates
D = Diffusion coefficient of solute (cm2s-1)
V = applied voltage
μe= electrophoretic mobility
 Contrary to chromatography plate count does not increase with the
length of column
 Resolution increases as plate count increases
 CE normally yields plate counts in range of 1,00,000 to 2,00,000
compared with 5000 to 20,000 plates for HPLC
 Eg: Plate counts of 30,00,000 have been reported for CZE of
dansylated amino acids and about 100,00,000 have been reported for
capillary gel electrophoresis of polynucleotides
9
INSTRUMENTATION
10
INSTRUMENTATION:
 Buffer filled fused silica capillary tubes are used

 Capillary is having 10 to 100μm internal diameter and 30 to 100
cm length
 Buffer reservoir are used
 Outside walls of fused-silica capillary are typically coated with
polyimide for durability, flexibility and stability
 High voltage power supplies of 10 to 25 kV dc are applied
 High voltage electrophoresis compartments are safety interlocked
to protect user
11
SAMPLE INTRODUCTION:
 Most common methods are

 Injections of 5 to 50 nL are common and volumes below 100pL have
been reported
1. Electro-kinetic injection
 A high voltage is applied to cause sample to enter the capillary by
a combination of ionic migration and electro-osmotic flow
 This technique discriminates by injecting larger amount of the
more mobile ions relative to the slower moving ions
2. Pressure injection
 Pressure difference drives the sample solution into the capillary
 This technique does not discriminate because of ion mobility
12
MODES OF SEPARATION:
13
DETECTION:
14
ADVANTAGES OVER OTHER TECHNIQUES:
15
DISADVANTAGES OF CAPILLARY
ELECTROPHORESIS:
16
APPLICATIONS OF CAPILLARY ELECTROPHORESIS:
17

Capillary Electrophoresis (CE) : Amruta L. Suryan

Uploaded by

Copyright:

Available Formats

Capillary Electrophoresis (CE) : Amruta L. Suryan

Uploaded by

Document Information

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Capillary Electrophoresis (CE) : Amruta L. Suryan

Uploaded by

Copyright:

Available Formats

CAPILLARY

Slab Electrophoresis Capillary Electrophoresis

1. Migration Rate (v):

2. Electro-osmotic flow (EOF) –

 The rate of electro-osmotic flow is generally greater than

 Direction of EOF can be reversed by adding cationic surfactants to

 Buffer filled fused silica capillary tubes are used

 Most common methods are

You might also like

Pfad - The Proxy pFad of © 2024 Garber Painting. All rights reserved.