Capillary eleCtrophoresis

Sandhya Talla
M.Pharm (Pharmacology)
 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:

Anode Cathode

The simplest electrophoretic

Detector separations are based on ion
charge / size
Buffer Buffer

E=V/d
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
 The Basis of Electrophoretic Separations
V
Migration Velocity: ν = µ ep E = µ ep
L
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)

q
Electrophoretic mobility: µ ep =
6πηr
Where:
q = charge on ion
η = viscosity
r = ion radius Frictional retarding forces
 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
Top figure: R. N. Zare (Stanford
University), bottom figure: Royal Society
of Chemistry

• SiO- attracts cations

to the inside wall of
the capillary

• The distribution of
charge at the surface
is described by the
Stern double-layer
Note: diffuse
model and results in layer rich in +
the zeta potential charges but
still mobile
 Electroosmosis
• It would seem that
CE separations would
start in the middle
and separate ions in
two linear directions
• Another effect called Silanols fully
Top figure: R. N. Zare (Stanford
University), bottom figure: Royal Society
of Chemistry
electroosmosis ionized above
makes CE like batch pH = 9
chromatography
• Excess cations in the
diffuse Stern double-
layer flow towards the
cathode, exceeding
the opposite flow
towards the anode
 Electroosmotic Flow (EOF)
• 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:

 ε 0εζ  Where:
v = µ eo E =   E v = electroosomotic mobility
 4πη  εo = dielectric constant of a vacuum
ε = dielectric constant of the buffer
ε 0εζ ζ = Zeta potential
µ eo = η = viscosity
4πη E = electric field
 Electroosmotic Flow Profile
- driving force (charge along
capillary wall)
Anode Cathode - no pressure drop is
encountered
- flow velocity is uniform across
Electroosmotic flow profile the capillary

Frictional forces at the

High Low column walls - cause a
Pressure Pressure pressure drop across the
column
Hydrodynamic flow profile

• Result: electroosmotic flow does not contribute significantly

to band broadening like pressure-driven flow in LC and
related techniques
 Electrophoresis and Electroosmosis
• Combining the two effects for migration velocity of an ion
(also applies to neutrals, but with µep = 0):

ν = ( µ ep + µ eo ) E = ( µ ep + µ eo )
V
L
• 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

 Electrophoresis and Electroosmosis
• A pictorial representation of the combined effect in a
capillary, when EO is faster than EP (the common case):

ν = ( µ ep + µ eo ) E = ( µ ep + µ eo )
V
L
Figure from R. N. Zare, Stanford
 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
 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

H = A + B / u + Cu
Where A, B, C are constants, and a lower
value of H corresponds to a higher
separation efficiency
 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:

H = B/u

• Cross-section of a capillary:
Figure from R. N. Zare, Stanford
 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)tπr2Ci

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
 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
 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
 Advantages and Disadvantages of CE
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
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)
 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.

Figure from delfin.klte.hu/~agaspar/ce-research.html

 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.
 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.
 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.
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

Analytes travel in here
contain both ionic and neutral species, and has
become valuable in the separation of very
hydrophobic pharmaceuticals from their very polar
Sodium dodecyl sulfate:
metabolites. polar headgroup, non-polar
tails
 Micellar Electrokinetic Capillary Chromatography
• 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.
 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