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Capillary Electrokinetic Separations
Lecture Date: April 23rd, 2008
Capillary Electrokinetic 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
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
DetectorThe simplest electrophoretic separations are based on ion charge / size
Proteins Peptides Amino acids Nucleic acids (RNA and DNA)
- also analyzed by slab gel electrophoresisInorganic ions Organic bases Organic acids Whole cells
Types of Molecules that can be Separated by Capillary Electrophoresis
The Basis of Electrophoretic Separations
Migration Velocity:
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 Frictional retarding forces
L
VE epep
r
qep
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
Top figure: R. N. Zare (Stanford University), bottom figure: Royal Society
of Chemistry
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 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 ChemistrySilanols fully
ionized above pH = 9
Electroosmotic Flow (EOF)
Where:v = electroosomotic mobilityo = dielectric constant of a vacuum = dielectric constant of the buffer = Zeta potential = viscosityE = electric field
4
0eo
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:
EEv eo
40
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
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?
At pH = 2
At pH = 12
Controlling Electroosmotic Flow (EOF)
EEv eo
40 Want to control EOF velocity:
Variable Result Notes
Electric Field Proportional change in EOF Joule heating may result
Buffer pHEOF decreased at low pH,
increased at high pHBest method to control EOF, but may change charge of analytes
Ionic StrengthDecreases and EOF with
increasing buffer concentrationHigh ionic strength means high
current and Joule heating
Organic ModifiersDecreases and EOF with
increasing modifierComplex effects
SurfactantAdsorbs to capillary wall through hydrophobic or ionic interactions
Anionic surfactants increase EOFCationic surfactants decrease EOF
Neutral hydrophilic poymer
Adsorbs to capillary wall via hydrophobic interactions
Decreases EOF by shielding surface charge, also increases viscosity
Covalent coatingChemically bonded to capillary
wallMany 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
VE eoepeoep
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):
L
VE eoepeoep
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
CuuBAH /
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:
Figure from R. N. Zare, Stanford
uBH /
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
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 injectionuses a pressure difference between the two ends of the capillary
Vc = Pd4 t 128Lt
Vc, calculated volume of injectionP, pressure differenced, diameter of the columnt, injection time, viscosity
Electrokinetic injectionuses a voltage difference between the two ends of the capillary
Qi = Vapp( kb/ka)tr2Ci
Q, moles of analytevapp, velocityt, injection timekb/ka ratio of conductivities (separation buffer and sample)r , capillary radiusCi 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 ICJ. 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
– 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.
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
AdvantagesOffers 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 automatedQuantitation (linear) Easily coupled to MSDifferent “modes” (to be discussed)
Disadvantages
Cannot do preparative scale separationsLow concentrations and large volumes difficult“Sticky” compoundsSpecies that are difficult to dissolveReproducibility 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 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.
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 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)
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 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)
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
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 CECSeparation was carried out on an ODS stationary phase at pH = 8:
An Example of CECSeparation 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
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
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
PV+ -
Voltage controlled, pulseless No moving parts or seals Inherently microscale High pressure generation Rapid pressure response Inexpensive
Vd
Vk
PPP2max
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, 3659–3666
Recommended