Capillary electrophoresis is a technique that uses narrow bore capillaries to separate charged molecules via electrophoretic mobility. When a voltage is applied, molecules migrate through the capillary at different rates depending on their charge and size. This allows analytes like proteins, nucleic acids, and small molecules to be separated. Key advantages are high efficiency, short analysis times, and low sample volume requirements. Common modes include capillary zone electrophoresis, capillary gel electrophoresis, and micellar electrokinetic capillary chromatography. Applications include analysis in pharmaceuticals and detection of microbial contamination.

1. Capillary electrophoresis
Presented by : k. jayalakshmi
D/o k. kristaiah
1st yr M.pharm, pharmaceutical analysis
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2. Contents
 Introduction
 Principle
 Mechanism
 CE-steps & detectors
 Methods and modes
 Advantages & Disadvantage's
 Applications
 References17-06-2020 2

3. Introduction
Electrophoresis is the motion of dispersed particles relative to a fluid
under the influence of a spatially uniform electric field.
Electrophoresis of positively charged particles is sometimes called
cataphoresis, while electrophoresis of negatively charged particles is
sometimes called anaphoresis.
Principles.
Electrophoresis is a general term that describes the migration and
separation of charged particles (ions) under the influence of an electric
field.
An electrophoretic system consists of two electrodes of opposite charge
(anode, cathode), connected by a conducting medium called an
electrolyte.
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4. TYPES OF ELECTROPHORESIS
 Zone Electrophoresis
 Paper Electrophoresis
 Gel Electrophoresis
 Thin Layer Electrophoresis
 Cellulose acetate Electrophoresis
 Moving Boundary Electrophoresis
 Capillary Electrophoresis
 Isotachophoresis
 Isoelectric Focussing
 Immuno Electrophoresis
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5. What is Capillary Electrophoresis?
Electrophoresis: The differential movement or migration of
ions by attraction or repulsion in an electric field
 Capillary electrophoresis is a family of electrokinetic separation
methods performed in submillimeter diameter capillaries and in
micro- and nanofluidic channels.
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6. principle
Capillary tube is placed between two buffer reservoir, and an
electric field is applied, separation depends on electrophoretic
mobility & electro-osmosis .
▪ Defined volume of analysate is introduced in to the capillary
by replacing one buffer reservoir with sample vial.
▪ Electrophoretic separation is measured by detector.
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7. Using narrow bore tubes, CE removes the Joule heating effect, which
decreases band broadening, giving faster separations than gel.
▪ CE uses tubes 20-100mm diameter and 20-100 cm in length.
▪ CE is used with/without gel. Longitudinal diffusion is the main source
of band-broadening.
▪ Higher electric fields result in high efficiency and narrow peaks (analyte
migrates faster).
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8. Types of Molecules that can be Separated by Capillary
Electrophoresis
 Proteins
 Peptides
 Amino acids
 Nucleic acids (RNA and DNA)
– also analyzed by slab gel electrophoresis
 Inorganic ions
 Organic bases
 Organic acids
 Whole cells
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9. Mechanism
Capillary electrophoresis works on 2 mechanisms
They are
 Electro osmatic flow
Movement/direction of the bulk solution
 Electrophoretic mobility
Separation of charged species
Electro osmatic flow
It is the movement of the separation buffer through the silica capillary
is a results of the existence of a zeta potential at the solvent /silica
interface.
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10. • Net flow becomes is large at higher pH:
• Key factors that affect electroosmotic mobility: dielectric constant and viscosity of
buffer (controls double-layer compression)
• EOF can be quenched by protection of silanols or low pH
• Electro osmotic mobility:
Where:
v = electroosomotic mobility
εo = dielectric constant of a vacuum
ε = dielectric constant of the buffer
ζ = Zeta potential
η = viscosity
E = electric field
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13. Capillary Zone Electrophoresis (CZE), also known as free-solution CE, is the most
standard form of CE. Buffer is flushed through the capillary by pressure, sample is
injected and high voltage is applied. Dependable on the polarity the EOF is towards
the inlet or the outlet.
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14. The Basis of Electrophoretic Separations
Migration Velocity:
Where:
v = migration velocity of charged particle in the potential field
(cm sec -1)
µ ep = electrophoretic mobility (cm2 V-1 sec-1)
E = field strength (V cm -1)
V = applied voltage (V)
L = length of capillary (cm)
Electrophoretic mobility:
Where:
q = charge on ion
η = viscosity
r = ion radius
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15. CE-STEPS
 Electrophoresis is done in buffer filled, narrow bore capillaries
 Each Capillary is about 25-100 µm in internal diameter.
 Small cross sectional area, long length, leading to high resistance, low
currents.
 Vmax =20-100Kv.
 N = 100,000-10,000,000 high resolution.
 When a Voltage is applied to the solution, the molecules move
through the solution towards the electrode of opposite charge
 Depending on the Charge , the molecules move through at different
speeds.
 Thus Separation is Achieved.
 A suitable detector is then used to detect the solute as it comes out
from the end of the Capillary.
 The data Obtained are analyzed by a Computer and represented
Graphically.
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16. Detectors used in capillary electrophoresis
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18. Common Modes of CE in Analytical Chemistry
 Capillary Zone electrophoresis (CZE)
 Capillary gel electrophoresis (CGE)
 Capillary isoelectric focusing (CIEF)

 Capillary isotachophoresis (CITP)
 Micellar electrokinetic capillary chromatography (MEKC)
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19. Capillary Zone Electrophoresis (CZE)
 Capillary Zone Electrophoresis (CZE), also known as free-solution CE
(FSCE), is the simplest form of CE (what we’ve been talking about).
 The separation mechanism is based on differences in the charge and
ionic radius of the analytes.
 Fundamental to CZE are homogeneity of the buffer solution and
constant field strength throughout the length of the capillary.
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20. Capillary Gel Electrophoresis (CGE)
 Capillary Gel Electrophoresis (CGE) is the adaptation of traditional gel
electrophoresis into the capillary using polymers in solution to create
a molecular sieve also known as replaceable physical gel.
 This allows analytes having similar charge-to-mass ratios to also be
resolved by size.
 This technique is commonly employed in Gel molecular weight
analysis of proteins and in applications of DNA sequencing and
genotyping.
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21. Capillary Isoelectric Focusing (CIEF)
 Capillary Isoelectric Focusing (CIEF) allows amphoteric molecules, such
as proteins, to be separated by electrophoresis in a pH gradient
generated between the cathode and anode.
 A solute will migrate to a point where its net charge is zero. At the
solute’s isoelectric point (pI), migration stops and the sample is focused
into a tight zone.
 In CIEF, once a solute has focused at its pI, the zone is mobilized past
the detector by either pressure or chemical means.
 This technique is commonly employed in protein characterization as a
mechanism to determine a protein's isoelectric point.
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22. Capillary Isotachophoresis (CITP)
 Capillary Isotachophoresis (CITP) is a focusing technique based on the
migration of the sample components between leading and terminating
electrolytes.
 (isotach = same speed)
 Solutes having mobilities intermediate to those of the leading and
terminating electrolytes stack into sharp, focused zones.
 Although it is used as a mode of separation, transient ITP has been used
primarily as a sample concentration technique.
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23. Micellar Electrokinetic Capillary
Chromatography
 Micellar Electrokinetic Capillary Chromatography (MECC OR MEKC) is a
mode of electrokinetic chromatography in which surfactants are added to
the buffer solution at concentrations that form micelles.
 The separation principle of MEKC is based on a differential partition
between the micelle and the solvent (a pseudo-stationary phase).
 This principle can be employed with charged or neutral solutes and may
involve stationary or mobile micelles.
 MEKC has great utility in separating mixtures that contain both ionic and
neutral species, and has become valuable in the separation of very
hydrophobic pharmaceuticals from their very polar metabolites.
 Micellar
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24. Micellar Electrokinetic Capillary Chromatography
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25.  The MEKC surfactants are surface active agents such as soap or synthetic
detergents with polar and non-polar regions.
 At low concentration, the surfactants are evenly distributed
 At high concentration the surfactants form micelles.
 The most hydrophobic molecules will stay in the hydrophobic region on the
surfactant micelle.
 Less hydrophobic molecules will partition less strongly into the micelle.
 Small polar molecules in the electrolyte move faster than molecules
associated with the surfatant micelles.
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27. Advantages and Disadvantages of CE
 Simple
 Automated
 High efficiency of separation
 Short analysis time
 Low sample volume
 Ease of operation
 Ability to separate both charged and non-charged molecules
 Different mechanisms for selectivity
 Low cost
 Use aqueous rather organic solvents hence environment friendly
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28. Disadvantages
Disadvantages Cannot do preparative scale separations “Sticky”
compounds Species that are difficult to dissolve Reproducibility problems
 Aged , improperly stored blood samples – degradation products
 Abnormal Hb – use other means of identification
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29. Applications
• Applications (within analytical chemistry) are broad: – For example, CE
has been heavily studied within the pharmaceutical industry as an
alternative to LC in various situations
• detecting bacterial/microbial contamination quickly using CE – Current
methods require several days.
• Direct inoculation (USP) requires a sample to be placed in a bacterial
growth medium for several days, during which it is checked under a
microscope for growth or by turbidity measurements
 False positives are common (simply by exposure to air)
 Techniques like ELISA, PCR, hybridization are specific to certain
microorganisms
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30. References
1.Watson G.David,pharmaceutical analysis,2nd edi.2005,Churchill
Livingstone,Pno.333-353.
2.Frank A. Settle,Handbook of Instrumental Techniques for Analytical
chemistry,1st edi.,2004,Pearson education,Pno.165.
3.http://www.ceandcec.com/presentation.htm
4.http://www.hbc.ukans.edu/CBAR/Electrochrom.htm
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