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Capillary Electrokineti c Separations
Lecture Date: April 23
rd
, 2008
Capillary Electrokineti c Separations
- 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 (Skoog et al.)
Chapter 30, Capillary Electrophoresis and Electrochromatography
- Reading (Cazes et al.)
Chapter 25, Capillary Electrophoresis
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What is Capillary Electrophoresi s?
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 Electrophoresi s
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The Basis of Electrophoreti c Separations
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
=viscosity
r =ion radius Frictional retarding forces
L
V
E
ep ep
= =
r
q
ep

6
=
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
Topfigure: R. N. Zare(Stanford
University),bottomfigure: Royal Society
of Chemistry
Note: diffuse
layer rich in +
charges but
still mobile
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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
- Net flow occurs as
solvated cations drag
along the solution
Topfigure: R. N. Zare(Stanford
University),bottomfigure: Royal Society
of Chemistry
Silanols fully
ionized above
pH =9
Electroosmotic Flow (EOF)
Where:
v =electroosomotic mobility
c
o
=dielectric constant of a vacuum
c =dielectric constant of the buffer
, =Zeta potential
q =viscosity
E =electric field

4
0
=
eo
- Net flow becomes is 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
|
|
.
|

\
|
= =

4
0
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Electroosmotic Flow Profile
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
Example Calculation of EOF at Two pH Values
- 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
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Controlling Electroosmotic Flow (EOF)
E E v
eo
|
|
.
|

\
|
= =

4
0
- Want to control EOF velocity:
Variable Result Notes
Electric Field Proportional change in EOF J oule 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 J oule 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
Electrophoresis and Electroosmosis
- 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
+ = + =
- 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
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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
eo ep eo ep
+ = + =
FigurefromR. 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:
FigurefromRoyal Societyof Chemistry
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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
Cu u B A H + + = /
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:
- Cross-section of a capillary:
FigurefromR. N. Zare,Stanford
u B H / =
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Number of theoretical plates N in CZE
N = L/H
H = B/v = 2D/v
v = E = V/L
Therefore, N = L/[2D/(V/L)] = V/2D
The resolution is INDEPENDENT of the length of the
column!
Moreover, for V =3 000 V/cm x 100 cm =3 x 10
4
V
D =3 x 10
-9
m
2
/s , and =2 x 10
-8
m
2
/Vs,
we find that
N =100, 000 theoretical plates.
Sample Injection in CE
Hydrodynamic injection
uses a pressure difference between the two ends of the capillary
V
c
= Pd
4
t
128L
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
)tr
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
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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: a broad-spectrum ISE
Conductometric detection: like IC
J. Tanyanyiwa, S. Leuthardt, P. C. Hauser, Conductimetricandpotentiometricdetectionin
conventional andmicrochipcapillary electrophoresis, Electrophoresis 2002, 23, 36593666
Joule Heating
- J oule 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)
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Capillary Electrophoresis: Applicati ons
- 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
Detection of Bacterial Contaminati on with CE
- 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: Testof Microbial ContaminationUsingCapillaryElectrophoresis,Anal.Chem. 2007, ASAP Article.
Rodriguez,M. A.; Lantz, A. W.; Armstrong,D. W., CapillaryElectrophoreticMethodfor theDetectionof Bacterial Contamination,Anal. Chem. 2006, 78, 4759-4767.
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Detection of Bacterial Contaminati on with CE
- The electropherograms
show single-cell detection
of a variety of bacteria with
good S/N
- 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: Testof Microbial ContaminationUsingCapillaryElectrophoresis,Anal.Chem. 2007, ASAP Article.
Rodriguez,M. A.; Lantz, A. W.; Armstrong,D. W., CapillaryElectrophoreticMethodfor theDetectionof Bacterial Contamination,Anal. Chem. 2006, 78, 4759-4767.
Capillary Electrophoresis: Summary
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
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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 Anal yti cal Chemistry
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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 Electrophoresi s (CZE)
Figurefromdelfin.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)
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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.
(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.
Currently, cITP is being combined with NMR to produce a new
hyphenated techinque (Cynthia Larive)
Capillary Isotachophoresi s (CITP)
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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)
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).
- CEC combines CE and micro-HPLC into one technique:
Actual instrument
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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
Separation was carried out on an ODS stationary phase at pH = 8:
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An Example of CEC
Separation was 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
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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
Mi cel lar El ectroki neti c Capi l l ary
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 Electrokineti c Capillary Chromatography
Analytes travel in here
Sodiumdodecyl sulfate:
polar headgroup, non-polar
tails
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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 Electrokineti c Capillary Chromatography
Method Development in CE
- Frameworks for
CE method
development
allow for a
structured
approach.
- For example, this
is a method
development
flowchart from
the Agilent CE
system
documentation
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New Technology: Electrokineti c 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
32
= =
Homework Problems (Optional) and Further
Reading
- Homework problems (for study only):
30-1, 30-2
- For more information about CE detectors, see:
J . Tanyanyiwa, S. Leuthardt, P. C. Hauser,
Conductimetric and potentiometric detection in
conventional and microchip capillary electrophoresis,
Electrophoresis 2002, 23, 36593666