Capillary Electrophoresis

Memahami prinsip dasar elektroforesis

Memahami berbagai metode yang
digunakan dalam elektroforesis
Mampu membac elektroforegram yang
dihasilkan
Mampu merancang suatu analisis
menggunakan elektroforesis
- Outline
Brief review of theory
Capillary zone electrophoresis (CZE)
Capillary gel electrophoresis (CGE)
Capillary electrochromatography (CEC)
Capillary isoelectric focusing (CIEF)
Capillary isotachophoresis (CITP)
Micellar electrokinetic capillary chromatography (MEKC)

- Reading Beckman and Coulter
- Reading (Skoog et al.) Chapter 30, Capillary Electrophoresis and
Electrochromatography
- Reading (Cazes et al.)Chapter 25, Capillary Electrophoresis
Definition: A separation technique based
on the differential transportation of charged
species in an electric field through a
conductive medium.
Primary candidates for CE separation are
ions.

Can determine the size, shape, and charge of
a molecule
Different forms of electrophoresis are used for
each of these factors independently or in
combination.
The basic instrumental set-up of CE, consists
of a high voltage power supply (0 to 30 kV), a
fused silica (SiO2) capillary, two buffer
reservoirs, two electrodes, and an on-column
detector.
Capillary
Native Polyacrylimide Gel Electrophoresis
(PAGE)
SDS-PAGE
Slab
Paper

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
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
Migration Velocity:

Where:
v = migration velocity of charged particle in the potential field (cm sec
-1
)

ep
=

electrophoretic mobility (cm
2
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
q = viscosity
r = ion radius
Frictional retarding forces
L
V
E
ep ep
v = =
r
q
ep
tq

6
=
- 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
- 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
- Net flow occurs as
solvated cations drag
along the solution
Top figure: R. N. Zare (Stanford
University), bottom figure: Royal Society
of Chemistry
Silanols fully
ionized above
pH = 9
Where:
v = electroosomotic mobility
c
o
= dielectric constant of a vacuum

c = dielectric constant of the buffer
, = Zeta potential
q = viscosity
E = electric field
tq
c, c

4
0
=
eo
- Net flow becomes large at higher pH:
A 50 mM pH 8 buffer flows through a 50-cm capillary at 5 cm/min
with 25 kV applied potential (see pg. 781 of Skoog et al.)
- 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:
E E v
eo
|
|
.
|

\
|
= =
tq
c, c

4
0
Cathode Anode
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
- A certain solution in a capillary has a electroosmotic mobility of 1.3 x 10
-8

m
2
/Vs at pH 2 and 8.1 x 10
-8
m
2
/Vs at pH 12. How long will it take a
neutral solute to travel 52 cm from the injector to the detector with 27 kV
applied across the 62 cm long tube?
At pH = 2
At pH = 12
E E v
eo
|
|
.
|

\
|
= =
tq
c, c

4
0
- Want to control EOF velocity:
Variable Result Notes
Electric Field Proportional change in EOF Joule heating may result
Buffer pH
EOF decreased at low pH,
increased at high pH
Best method to control EOF, but may
change charge of analytes
Ionic Strength
Decreases , and EOF with
increasing buffer concentration
High ionic strength means high
current and Joule heating
Organic Modifiers
Decreases , and EOF with
increasing modifier
Complex effects
Surfactant
Adsorbs to capillary wall through
hydrophobic or ionic interactions
Anionic surfactants increase EOF
Cationic surfactants decrease EOF
Neutral hydrophilic
poymer
Adsorbs to capillary wall via
hydrophobic interactions
Decreases EOF by shielding surface
charge, also increases viscosity
Covalent coating
Chemically bonded to capillary
wall
Many possibilities
Temperature Changes viscosity Easy to control
- Combining the two effects for migration velocity of an ion
(also applies to neutrals, but with
ep
= 0):
( ) ( )
L
V
E
eo ep eo ep
v + = + =
- At pH > 2, cations flow to cathode because of positive
contributions from both
ep
and
eo

- At pH > 2, anions flow to anode because of a negative
contribution from
ep
, but can be pulled the other way by a
positive contribution from
eo
(if EOF is strong enough)
- At pH > 2, neutrals flow to the cathode because of
eo
only
Note: neutrals all come out together in basic CE-only separations
- A pictorial representation of the combined effect in a
capillary, when EO is faster than EP (the common case):
( ) ( )
L
V
E
eo ep eo ep
v + = + =
Figure from R. N. Zare, Stanford
- 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
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






Cu u B A H + + = /
Where A, B, C are constants, and a lower
value of H corresponds to a higher
separation efficiency
- 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
u B H / =
Hydrodynamic injection
uses a pressure difference between the two ends of the capillary
V
c
= APtd
4
t
128qL
t

V
c
, calculated volume of injection
P, pressure difference
d, diameter of the column
t, injection time
q, viscosity

Electrokinetic injection
uses a voltage difference between the two ends of the capillary
Q
i
= V
app
( k
b
/k
a
)ttr
2
C
i

Q, moles of analyte
v
app
, velocity
t, injection time
k
b
/k
a
ratio of conductivities (separation buffer and sample)
r , capillary radius
C
i
molar concentration of analyte
- Joule heating is a consequence of the resistance of the
solution to the flow of current
if heat is not sufficiently dissipated from the system the resulting
temperature and density gradients can reduce separation
efficiency
- Heat dissipation is key to CE operation:
Power per unit capillary P/L r
2
- For smaller capillaries heat is dissipated due to the large
surface area to volume ratio
capillary internal surface area = 2t r L
capillary internal volume = t r
2
L

- End result: high potentials can be applied for extremely
fast separations (30kV)
- 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

- We will look at just one example: 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
- Method
A dilute cationic surfactant buffer
is used to sweep
microorganisms out of the
sample zone and a small plug of
blocking agent negates the
cells mobility and induces
aggregation

Method detects whole bacterial
cellls
Lantz, A. W.; Bao, Y.; Armstrong, D. W., Single-Cell Detection: Test of Microbial Contamination Using Capillary Electrophoresis, Anal. Chem. 2007, ASAP Article.
Rodriguez, M. A.; Lantz, A. W.; Armstrong, D. W., Capillary Electrophoretic Method for the Detection of Bacterial Contamination, Anal. Chem. 2006, 78, 4759-4767.
- Single-cell detection of a
variety of bacteria

- Why is CE a good
analytical approach to this
problem?
Fast analysis times (<10
min)
Readily miniaturized



Lantz, A. W.; Bao, Y.; Armstrong, D. W., Single-Cell Detection: Test of Microbial Contamination Using Capillary Electrophoresis, Anal. Chem. 2007, ASAP Article.
Rodriguez, M. A.; Lantz, A. W.; Armstrong, D. W., Capillary Electrophoretic Method for the Detection of Bacterial Contamination, Anal. Chem. 2006, 78, 4759-4767.
CE is based on the principles of electrophoresis
The speed of movement or migration of solutes in
CE is determined by their charge and size. Small
highly charged solutes will migrate more quickly
then large less charged solutes.
Bulk movement of solutes is caused by EOF
The speed of EOF can be adjusted by changing the
buffer pH
The flow profile of EOF is flat, yielding high
separation efficiencies

Advantages
Offers new selectivity, an alternative to HPLC
Easy and predictable selectivity
High separation efficiency (10
5
to 10
6
theoretical plates)
Small sample sizes (1-10 ul)
Fast separations (1 to 45 min)
Can be automated
Quantitation (linear)
Easily coupled to MS
Different modes (to be discussed)

Disadvantages
Cannot do preparative scale separations
Low concentrations and large volumes difficult
Sticky compounds
Species that are difficult to dissolve
Reproducibility problems

Advantages and Disadvantages of CE

Capillary Zone electrophoresis (CZE)
Capillary gel electrophoresis (CGE)
Capillary electrochromatography (CEC)
Capillary isoelectric focusing (CIEF)
Capillary isotachophoresis (CITP)
Micellar electrokinetic capillary chromatography (MEKC)

Common Modes of CE in Analytical Chemistry
Capillary Zone Electrophoresis
(CZE), also known as free-solution CE
(FSCE), is the simplest form of CE
(what weve 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.

The separation relies principally on the
pH controlled dissociation of acidic
groups on the solute or the protonation
of basic functions on the solute.
Capillary Zone Electrophoresis (CZE)
Figure from delfin.klte.hu/~agaspar/ce-research.html
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 SDS-Gel molecular weight
analysis of proteins and in applications of DNA sequencing and
genotyping.

Capillary Gel Electrophoresis (CGE)
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
solutes 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)
Capillary Isotachophoresis (CITP) is a focusing technique based on
the migration of the sample components between leading and
terminating electrolytes.

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)
Capillary Electrochromatography (CEC) is a hybrid
separation method
CEC couples the high separation efficiency of CZE with
the selectivity of HPLC
Uses an electric field rather than hydraulic pressure to
propel the mobile phase through a packed bed
Because there is minimal backpressure, it is possible to
use small-diameter packings and achieve very high
efficiencies
Its most useful application appears to be in the form of on-
line analyte concentration that can be used to concentrate
a given sample prior to separation by CZE
Capillary Electrochromatography (CEC)
CEC combines the strengths of two powerful
analytical techniques - CE and micro-HPLC.
Capillary Electrochromatography (CEC)
R. Dadoo, C.H. Yan,
R. N. Zare, D. S.
Anex, D. J.
Rakestraw,and G. A.
Hux, LC-GC
International 164-
174 (1997).
Capillary Electrochromatography (CEC)
Consider a CEC test mixture containing:
The neutral marker thiourea for indication of the electroosmotic flow
Two compounds with very different polarities (#2 and #5)
Two closely related components (#3 and #4) to test resolving power
An Example of CEC
An Example of CEC
This separation is carried out on an ODS stationary phase at pH = 8:
An Example of CEC
The separation carried out on an ODS stationary phase at pH = 2.3:
Because the packed length and overall length of these two
capillaries are identical, it is possible to make a direct comparison of
the performance because the field strength and column bed length
are the same.
The EOF has decreased dramatically between pH 8 and pH 2.3 with
the resulting analysis time increasing from approximately 5 min to
over 20 min at the lower pH.
Conclusions from the CEC Example
Electrokinetic Chromatography (EKC): a family of electrophoresis
techniques named after electrokinetic phenomena, which include
electroosmosis, electrophoresis and chromatography.

A key example of this is seen with cyclodextrin-mediated EKC. Here the
differential interaction of enantiomers with the cyclodextrins allows for
the separation of chiral compounds.

This approach to enantiomer analysis has made a significant impact on
the pharmaceutical industry's approach to assessing drugs containing
enantiomers.

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 Electrokinetic Capillary Chromatography
Analytes travel in here
Sodium dodecyl sulfate:
polar headgroup, non-polar
tails
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.
The voltage causes the negatively
charged micelles to flow slower than
the bulk flow (endoosmotic flow).
Micellar Electrokinetic Capillary Chromatography
- Basic guidance,
from the Agilent
CE system
documentation
New Technology: Electrokinetic Pumping
P
V

+ -
Voltage controlled, pulseless
No moving parts or seals
Inherently microscale
High pressure generation
Rapid pressure response
Inexpensive

V
d
V
k
P
P P
2
max
32c, c,
= =