Capillary Electrophoresis docx

Capillary Electrokinetic SeparationsLecture Date: April 23 rd , 2008 Capillary Electrokinetic Separations – Brief review of theory – Capillary zone electrophoresis CZE – Capillary gel el

Capillary Electrokinetic Separations

Lecture Date: April 23 rd , 2008

Capillary Electrokinetic Separations

– 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)

– Chapter 30, Capillary Electrophoresis and Electrochromatography



What is Capillary Electrophoresis?

of ions by attraction or repulsion in an electric field

Proteins

Peptides

Amino acids

Nucleic acids (RNA and DNA)

- also analyzed by slab gel electrophoresis

The Basis of Electrophoretic Separations

Migration Velocity:

Where:

v = migration velocity of charged particle in the potential field (cm sec -1 )

Inside the Capillary: The Zeta Potential

 The inside wall of the

 SiO-attracts cations

to the inside wall of

 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)

Where:

v = electroosomotic mobility

 = dielectric constant of the buffer

– 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)

 Electroosmotic mobility:

E E

Electroosmotic Flow Profile

Cathode Anode

Electroosmotic flow profile

Hydrodynamic 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

 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 10-8m2/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)

E E

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

Surfactant Adsorbs to capillary wall through

hydrophobic or ionic interactions

Anionic surfactants increase EOF Cationic surfactants decrease EOF

Covalent coating Chemically bonded to capillary

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):

 At pH > 2, cations flow to cathode because of positive

contributions from both epand 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 eoonly

– 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):

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

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

Cu u B A

Where A, B, C are constants, and a lower

value of H corresponds to a higher

separation efficiency

CE Theory

– 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

u B

H  /

Number of theoretical plates N in CZE

uses a pressure difference between the two ends of the capillary

V c = Pd 4 t 128L t

Capillary Electrophoresis: Detectors

detector

– These have ~0.01 attomole sensitivity for fluorescent

molecules (e.g derivatized proteins)

instead, where a absorptive buffer (e.g chromate) is

displaced by analyte ions

– Detection limits are in the 50-500 ppb range

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

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

 For smaller capillaries heat is dissipated due to the large

surface area to volume ratio

– capillary internal surface area = 2 r 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 out of the

sample zone and a small plug of

“blocking agent” negates the

cells’ mobility and induces

aggregation

– Method detects whole bacterial

cellls

Detection of Bacterial Contamination with CE

show single-cell detection

of a variety of bacteria with

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

Offers new selectivity, an alternative to HPLC

Easy and predictable selectivity

Small sample sizes (1-10 ul)

Fast separations (1 to 45 min)

Cannot do preparative scale separations

Low concentrations and large volumes difficult

“Sticky” compounds

Species that are difficult to dissolve

Reproducibility 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

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 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:

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

Micellar Electrokinetic Capillary Chromatography

Analytes travel in here

Sodium dodecyl sulfate:

• 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

New Technology: Electrokinetic Pumping

P V

- Voltage controlled, pulseless

 No moving parts or seals

 Inherently microscale

 High pressure generation

 Rapid pressure response

 Inexpensive

V d

V k

P

P P

2 max

– J Tanyanyiwa, S Leuthardt, P C Hauser,

Conductimetric and potentiometric detection in

conventional and microchip capillary electrophoresis,

Electrophoresis 2002, 23, 3659–3666