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Capillary Electrokinetic Separations. Lecture Date: May 1 st , 2013. Capillary Electrokinetic Separations. Outline Brief review of theory Capillary zone electrophoresis (CZE) Capillary gel electrophoresis (CGE) Capillary electrochromatography (CEC) Capillary isoelectric focusing (CIEF)

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Capillary Electrokinetic Separations

Lecture Date: May 1st, 2013


Capillary electrokinetic separations
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)


What is capillary electrophoresis

Anode

Cathode

Anode

Cathode

Detector

Buffer

Buffer

E=V/d

What is Capillary Electrophoresis?

Electrophoresis: The differential movement or migration of ions by attraction or repulsion in an electric field

Basic Design of Instrumentation:

Capillary

The simplest electrophoretic separations are based on ion charge / size


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

Frictional retarding forces

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


Inside the capillary the zeta potential
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

  • 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

Bulk

Note: diffuse layer rich in + charges but still mobile


Electroosmosis
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 Chemistry

Silanols fully ionized above pH = 9


Electroosmotic flow eof
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:

ueo = electroosomotic mobility

o = dielectric constant of a vacuum

 = dielectric constant of the buffer

 = Zeta potential

 = viscosity

E = electric field


Electroosmotic flow profile

Anode

Cathode

High

Pressure

Low

Pressure

Electroosmotic Flow Profile

- driving force (charge along capillary wall)

- no pressure drop is encountered

- flow velocity is uniform across the capillary

Electroosmotic flow profile

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
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
Controlling Electroosmotic Flow (EOF)

  • Want to control EOF velocity:


Electrophoresis and electroosmosis
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


Electrophoresis and electroosmosis1
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


The electropherogram
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
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


Ce theory1
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


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
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
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

  • 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
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
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 ce1
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
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 on-line 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

  • MicellarElectrokinetic 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 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 surfactant micelles.

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


Method development in ce
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


V

P

+

-

New Technology: Electrokinetic Pumping

  • Voltage controlled, pulseless

  • No moving parts or seals

  • Inherently microscale

  • High pressure generation

  • Rapid pressure response

  • Inexpensive


Further reading
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


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