Capillary Electrokinetic Separations

Lecture Date: May 1st, 2013

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?

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

Capillary

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 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

Note: diffuse layer rich in + charges but still mobile

Bulk

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:ueo = 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:

L

VEv eo

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

v

v

v

v

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

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 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

– 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

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, FSCE, or just CE)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. For example, cITP can be combined e.g. with NMR to produce a useful pre-concentration technique.

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 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)

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 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).

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

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