Transcript Capillary Electrophoresis

Capillary Electrokinetic Separations
Lecture Date: May 1st, 2013
Capillary Electrokinetic Separations
 Outline
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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)
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:
Capillary
Anode
Cathode
The simplest electrophoretic
separations are based on ion
charge / size
Detector
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
Migration Velocity:
V
  ep E  ep
L
Where:
v = migration velocity of charged particle in the potential field (cm sec -1)
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
 = viscosity
r = ion radius
ep 
q
6r
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 and are
fully deprotonated at
pH = 9
SiO- attracts cations
to the inside wall of
the capillary
Top figure: R. N. Zare (Stanford
University), bottom figure: Royal Society
of Chemistry
Bulk
 The distribution of
charge at the surface
is described by the
Stern double-layer
model and results in
the zeta potential
Note: diffuse
layer rich in +
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
electroosmosis
makes CE like batch
chromatography
 Excess cations in the
diffuse Stern doublelayer flow towards the
cathode, exceeding
the opposite flow
towards the anode
 Net flow occurs as
solvated cations drag
along the solution
Silanols fully
ionized above
pH = 9
Top figure: R. N. Zare (Stanford
University), bottom figure: Royal Society
of Chemistry
Electroosmotic Flow (EOF)
 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:
V
v   eo E 
L
 0
eo 
4
Where:
ueo = electroosomotic mobility
o = dielectric constant of a vacuum
 = dielectric constant of the buffer
 = Zeta potential
 = viscosity
E = electric field
Electroosmotic Flow Profile
- driving force (charge along
Anode
Cathode
Electroosmotic flow profile
High
Pressure
Low
Pressure
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
Hydrodynamic flow profile
 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
m2/Vs at pH 2 and 8.1 x 10-8 m2/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?
v
At pH = 2
v
v
At pH = 12
v
Controlling Electroosmotic Flow (EOF)
  0 
 E
v  eo E  
 Want to control EOF velocity:
 4 
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
Electrophoresis and Electroosmosis
 Combining the two effects for migration velocity of an ion
(also applies to neutrals, but with ep = 0):
V
  ep  eo E  ep  eo 
L
 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
Electrophoresis and Electroosmosis
 A pictorial representation of the combined effect in a
capillary, when EO is faster than EP (the common case):
V
  ep  eo E  ep  eo 
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
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 104 V
Assuming D = 3 x 10-9 m2/s, and  = 2 x 10-8 m2/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
Vc = Pd4 t
128Lt
Vc, calculated volume of injection
P, pressure difference
d, diameter of the column
t, injection time
, viscosity
Electrokinetic injection
uses a voltage difference between the two ends of the capillary
Qi = Vapp( kb/ka)tr2Ci
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: a broad-spectrum ISE
– Conductometric detection: like IC
J. Tanyanyiwa, S. Leuthardt, P. C. Hauser, Conductimetric and potentiometric detection in
conventional and microchip capillary electrophoresis, Electrophoresis 2002, 23, 3659–3666
Joule Heating
 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  r2
 For smaller capillaries heat is dissipated due to the large
surface area to volume ratio
– capillary internal surface area = 2 r L
– capillary internal volume =  r2 L
 End result:
high potentials can be applied for extremely
fast separations (30kV)
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

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 Contamination 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
– This approach minimizes the
effects of electrophoretic
differences between cells and
also sweeps away small
molecule contaminants
– Method detects whole bacterial
cells
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.
Detection of Bacterial Contamination 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: 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.
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
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
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
Common Modes of CE in Analytical Chemistry
Capillary zone electrophoresis (CZE, FSCE, or just CE)
Capillary gel electrophoresis (CGE)
Capillary electrochromatography (CEC)
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.
The separation relies principally on the
pH controlled dissociation of acidic
groups on the solute or the protonation
of basic functions on the solute.
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 SDS-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. For example, cITP can
be combined e.g. with NMR to produce a useful pre-concentration
technique.
Capillary Electrochromatography (CEC)
● 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 online analyte concentration that can be used to concentrate
a given sample prior to separation by CZE
Capillary Electrochromatography (CEC)
 CEC combines CE and micro-HPLC into one technique:
Actual instrument
R. Dadoo, C.H. Yan, R. N. Zare, D. S. Anex, D. J. Rakestraw,and G. A. Hux, LC-GC International 164-174
(1997).
An Example of 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
Separation was carried out on an ODS stationary phase at pH = 8:
An Example of CEC
Separation was carried out on an ODS stationary phase at pH = 2.3:
Conclusions from the CEC Example
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.
Electrokinetic Capillary Chromatography
Electrokinetic Chromatography (EKC): a family of electrophoresis
techniques named after electrokinetic phenomena, which include and
combine electroosmosis, electrophoresis and chromatography.
Examples:
• Cyclodextrin-mediated EKC. Here the differential interaction of
enantiomers with the cyclodextrins allows for the separation of chiral
compounds
• Micellar Electrokinetic Capillary Chromatography (next slides)
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.
Analytes travel in here
Sodium dodecyl sulfate:
polar headgroup, non-polar
tails
Micellar Electrokinetic Capillary Chromatography
• The MEKC surfactants are surface
active agents with polar and nonpolar 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
surfactant micelles.
• The voltage causes the negatively
charged micelles to flow slower than
the bulk flow (endoosmotic flow).
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
New Technology: Electrokinetic Pumping






Voltage controlled, pulseless
No moving parts or seals
Inherently microscale
High pressure generation
Rapid pressure response
Inexpensive
+
Pmax 

kP
V
-
V
P
32
V
2
dP
Further Reading
 Reading (Skoog et al.)
– Chapter 30, Capillary Electrophoresis and Electrochromatography
 Reading (Cazes et al.)
– Chapter 25, Capillary Electrophoresis
 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, 3659–3666