1. Capillary Electrokinetic Separations
Lecture Date: April 23rd, 2008

2. 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

3. 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
Anode
Cathode
Detector
The cathode is defined as the electrode of an electrochemical cell at which reduction occurs (i.e. electrons are added to cations to create neutral atoms). The cathode supplies electrons to the positively charged cations in the cell.
The simplest electrophoretic separations are based on ion charge / size

4. 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

5. 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:
q = charge on ion
 = viscosity
r = ion radius
Frictional retarding forces

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
The Zeta Potential () is the potential at any given point in double layer (decreases with increasing distance from capillary wall).
The Stern model (see any good Surface Chemistry textbook for details, or look at pg. 566 in Skoog et al.) is also referred to as the electrical double-layer model.
Note: diffuse layer rich in + charges but still mobile

7. Silanols fully ionized above pH = 9
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
Silanols fully ionized above pH = 9
Top figure: R. N. Zare (Stanford University), bottom figure: Royal Society of Chemistry
Note – electroosmosis is the mobile phase “pump” of capillary zone electrophoresis (CZE)
It is the uneven distribution of cations in the diffuse layer that force the net flow towards the cathode. The net flow becomes quite large for high pH situations – a 50 mM pH 8 buffer flows through a 50-cm capillary at 5 cm/min with 25 kV applied potential (see page781 of Skoog et al.)

8. 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:
Where:
v = electroosomotic mobility
o = dielectric constant of a vacuum
 = dielectric constant of the buffer
 = Zeta potential
 = viscosity
E = electric field

9. Electroosmotic Flow Profile
- driving force (charge along capillary wall)
- no pressure drop is encountered
- flow velocity is uniform across the capillary
Cathode
Anode
Electroosmotic flow profile
Frictional forces at the column walls - cause a pressure drop across the column
High
Pressure
Low
Hydrodynamic flow profile
Result: electroosmotic flow does not contribute significantly to band broadening like pressure-driven flow in LC and related techniques

10. 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 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

11. Controlling Electroosmotic Flow (EOF)
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

12. Electrophoresis and Electroosmosis
Combining the two effects for migration velocity of an ion (also applies to neutrals, but with ep = 0):
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

13. Electrophoresis and Electroosmosis
A pictorial representation of the combined effect in a capillary, when EO is faster than EP (the common case):
Figure from R. N. Zare, Stanford

14. Figure from Royal Society of Chemistry
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

15. 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
Where A, B, C are constants, and a lower value of H corresponds to a higher separation efficiency

16. Figure from R. N. Zare, Stanford
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

17. 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 = 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.

18. Sample Injection in CE Hydrodynamic injection Electrokinetic 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
Pushing a liquid through a tube may be achieved by simply placing the end of the tube in liquid and applying a pressure at the inlet end, and you get a flow. The Hagen-Poiseuille law (top equation) says that the flow rate is directly proportional to the pressure and to the fourth power of the internal diameter of the tube, and inversely proportional to the length of the tube and the viscosity of the liquid.

19. 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 ppb range
Alternative methods involving potentiometric and conductometric detection are also used
Potentiometric detection: a broad-spectrum ISE
Conductometric detection: like IC
An attomole is moles.
See page 872, Table 30-1 of Skoog et al. 6th ed for a comparison of CE detectors.
J. Tanyanyiwa, S. Leuthardt, P. C. Hauser, Conductimetric and potentiometric detection in
conventional and microchip capillary electrophoresis, Electrophoresis 2002, 23, 3659–3666

20. 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)

21. 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

22. 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,

23. 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,

24. 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

25. Advantages and Disadvantages of CE
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

26. Common Modes of CE in Analytical Chemistry
Capillary Zone electrophoresis (CZE)
Capillary gel electrophoresis (CGE)
Capillary electrochromatography (CEC)
Capillary isoelectric focusing (CIEF)
Capillary isotachophoresis (CITP)
Micellar electrokinetic capillary chromatography (MEKC)

27. 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

28. 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.

29. 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.
The isoelectric point (pI) is the pH at which no migration occurs.

30. 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.
Currently, cITP is being combined with NMR to produce a new hyphenated techinque (Cynthia Larive)

31. 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 on-line analyte concentration that can be used to concentrate a given sample prior to separation by CZE

32. 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 (1997).

33. 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

34. An Example of CEC
Separation was carried out on an ODS stationary phase at pH = 8:

35. An Example of CEC
Separation was carried out on an ODS stationary phase at pH = 2.3:

36. 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.

37. Electrokinetic Capillary Chromatography
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.

38. 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

39. 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.
The voltage causes the negatively charged micelles to flow slower than the bulk flow (endoosmotic flow).

40. 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

41. New Technology: Electrokinetic Pumping
Voltage controlled, pulseless
No moving parts or seals
Inherently microscale
High pressure generation
Rapid pressure response
Inexpensive P V + -

42. 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