Capillary electrophoresis

Sandhya TallaM.Pharm (Pharmacology)

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 = viscosityr = ion radius Frictional retarding forces

LVE epep

rq

ep

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

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: 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

Electrophoresis and Electroosmosis Combining the two effects for migration velocity of an ion

(also applies to neutrals, but with ep = 0):

LVE 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

Electrophoresis and Electroosmosis A pictorial representation of the combined effect in a

capillary, when EO is faster than EP (the common case):

LVE 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 /

Sample Injection in CEHydrodynamic 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– Conductometric detection

J. Tanyanyiwa, S. Leuthardt, P. C. Hauser, Conductimetric and potentiometric detection inconventional and microchip capillary electrophoresis, Electrophoresis 2002, 23, 3659–3666

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

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

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 MS

Disadvantages

Cannot do preparative scale separations“Sticky” compoundsSpecies that are difficult to dissolveReproducibility problems

Advantages and Disadvantages of CE

Capillary Zone electrophoresis (CZE)Capillary gel electrophoresis (CGE)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.

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 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.

Capillary Isotachophoresis (CITP)

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.

Micellar Electrokinetic Capillary Chromatography

References

1.Watson G.David,pharmaceutical analysis,2nd edi.2005,Churchill

Livingstone,Pno.333-353.

2.Frank A. Settle,Handbook of Instrumental Techniques for

Analytical chemistry,1st edi.,2004,Pearson education,Pno.165.

3. http://www.ceandcec.com/presentation.htm

4.http://www.hbc.ukans.edu/CBAR/Electrochrom.htm