Capillary electrophoresis is a separation technique that uses charged molecules' differential migration in response to an applied electric field. Key components include a capillary, buffers, and detectors. Molecules are separated based on their charge and size. There are several modes, including capillary zone electrophoresis which separates based on charge and size, and micellar electrokinetic capillary chromatography which uses micelles to separate charged and neutral molecules. Capillary electrophoresis provides high resolution, efficiency, and versatility in analyzing various molecules like proteins, nucleic acids, and inorganic ions.

1. Capillary eleCtrophoresis
Sandhya Talla
M.Pharm (Pharmacology)

2. What is Capillary Electrophoresis?
Electrophoresis: The differential movement or migration
of ions by attraction or repulsion in an electric field
Anode
Cathode
Basic Design of Instrumentation:
E=V/d
Buffer
Buffer
Anode Cathode
Detector
The simplest electrophoretic
separations are based on ion
charge / size

3. Proteins
Peptides
Amino acids
Nucleic acids (RNA and DNA)
- also analyzed by slab gel electrophoresis
Inorganic ions
Organic bases
Organic acids
Whole cells
Types of Molecules that can be Separated
by Capillary Electrophoresis

4. The Basis of Electrophoretic Separations
Migration Velocity:
Where:
v = migration velocity of charged particle in the potential field (cm sec -1
)
µep = 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 Frictional retarding forces
L
V
E epep µµν ==
r
q
ep
πη
µ
6
=

5. Inside the Capillary: The Zeta Potential
• The inside wall of the
capillary is covered
by silanol groups
(SiOH) that are
deprotonated (SiO-
)
at pH > 2
• SiO-
attracts cations
to the inside wall of
the capillary
• The distribution of
charge at the surface
is described by the
Stern double-layer
model and results in
the zeta potential
Top figure: R. N. Zare (Stanford
University), bottom figure: Royal Society
of Chemistry
Note: diffuse
layer rich in +
charges but
still mobile

6. Electroosmosis
• It would seem that
CE separations would
start in the middle
and separate ions in
two linear directions
• Another effect called
electroosmosis
makes CE like batch
chromatography
• Excess cations in the
diffuse Stern double-
layer flow towards the
cathode, exceeding
the opposite flow
towards the anode
Top figure: R. N. Zare (Stanford
University), bottom figure: Royal Society
of Chemistry
Silanols fully
ionized above
pH = 9

7. Electroosmotic Flow (EOF)
Where:
v = electroosomotic mobility
εo = dielectric constant of a vacuum
ε = dielectric constant of the buffer
ζ = Zeta potential
η = viscosity
E = electric fieldπη
εζε
µ
4
0
=eo
• 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
• Electroosmotic mobility:
EEv eo 





==
πη
εζε
µ
4
0

8. Electroosmotic Flow Profile
CathodeAnode
Electroosmotic flow profile
Hydrodynamic flow profile
High
Pressure
Low
Pressure
- driving force (charge along
capillary wall)
- no pressure drop is
encountered
- flow velocity is uniform across
the capillary
Frictional forces at the
column walls - cause a
pressure drop across the
column
• Result: electroosmotic flow does not contribute significantly
to band broadening like pressure-driven flow in LC and
related techniques

9. Electrophoresis and Electroosmosis
• Combining the two effects for migration velocity of an ion
(also applies to neutrals, but with µep = 0):
( ) ( )
L
V
E eoepeoep µµµµν +=+=
• At pH > 2, cations flow to cathode because of positive
contributions from both µep and µeo
• At pH > 2, anions flow to anode because of a negative
contribution from µep, but can be pulled the other way by a
positive contribution from µeo (if EOF is strong enough)
• At pH > 2, neutrals flow to the cathode because of µeo only

10. Electrophoresis and Electroosmosis
• A pictorial representation of the combined effect in a
capillary, when EO is faster than EP (the common case):
( ) ( )
L
V
E eoepeoep µµµµν +=+=
Figure from R. N. Zare, Stanford

11. The Electropherogram
• Detectors are placed at the cathode since under common
conditions, all species are driven in this direction by EOF
• Detectors similar to those used in LC, typically UV
absorption, fluorescence, and MS
– Sensitive detectors are needed for small concentrations in CE
• The general layout of an electropherogram:
Figure from Royal Society of Chemistry

12. CE Theory
The unprecedented resolution of CE is a consequence of
the its extremely high efficiency
Van Deemter Equation:
relates the plate height H to the velocity of the carrier gas
or liquid
CuuBAH ++= /
Where A, B, C are constants, and a lower
value of H corresponds to a higher
separation efficiency

13. CE Theory
• In CE, a very narrow open-tubular capillary is used
– No A term (multipath) because tube is open
– No C term (mass transfer) because there is no stationary phase
– Only the B term (longitudinal diffusion) remains:
• Cross-section of a capillary:
Figure from R. N. Zare, Stanford
uBH /=

14. Sample Injection in CE
Hydrodynamic injection
uses a pressure difference between the two ends of the capillary
Vc = ∆Pπd4
t
128ηLt
Vc, calculated volume of injection
P, pressure difference
d, diameter of the column
t, injection time
η, viscosity
Electrokinetic injection
uses a voltage difference between the two ends of the capillary
Qi = Vapp( kb/ka)tπr2
Ci
Q, moles of analyte
vapp, velocity
t, injection time
kb/ka ratio of conductivities (separation buffer and sample)
r , capillary radius
Ci molar concentration of analyte

15. Capillary Electrophoresis: Detectors
• LIF (laser-induced fluorescence) is a very popular CE
detector
– These have ~0.01 attomole sensitivity for fluorescent
molecules (e.g. derivatized proteins)
• Direct absorbance (UV-Vis) can be used for organics
• For inorganics, indirect absorbance methods are used
instead, where a absorptive buffer (e.g. chromate) is
displaced by analyte ions
– Detection limits are in the 50-500 ppb range
• Alternative methods involving potentiometric and
conductometric detection are also used
– Potentiometric detection
– Conductometric detection
J. Tanyanyiwa, S. Leuthardt, P. C. Hauser, Conductimetric and potentiometric detection in
conventional and microchip capillary electrophoresis, Electrophoresis 2002, 23, 3659–3666

16. Capillary Electrophoresis: 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 innoculation (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

17. Advantages
Offers new selectivity, an alternative to HPLC
Easy and predictable selectivity
High separation efficiency (105
to 106
theoretical plates)
Small sample sizes (1-10 ul)
Fast separations (1 to 45 min)
Can be automated
Quantitation (linear)
Easily coupled to MS
Disadvantages
Cannot do preparative scale separations
“Sticky” compounds
Species that are difficult to dissolve
Reproducibility problems
Advantages and Disadvantages of CE

18. Capillary Zone electrophoresis (CZE)
Capillary gel electrophoresis (CGE)
Capillary isoelectric focusing (CIEF)
Capillary isotachophoresis (CITP)
Micellar electrokinetic capillary chromatography (MEKC)
Common Modes of CE in Analytical Chemistry

19. 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.
Capillary Zone Electrophoresis (CZE)
Figure from delfin.klte.hu/~agaspar/ce-research.html

20. 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.
Capillary Gel Electrophoresis (CGE)

21. 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.
Capillary Isoelectric Focusing (CIEF)

22. 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.
Capillary Isotachophoresis (CITP)

23. 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 Electrokinetic Capillary Chromatography
Analytes travel in here
Sodium dodecyl sulfate:
polar headgroup, non-polar
tails

24. • 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.
Micellar Electrokinetic Capillary Chromatography

25. 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