Capillary Electrophoresis

Its origin can be traced back to the 1880s

it got major recognition in 1937, when Tiselius

reported the separation of different serum proteins

by a method called moving boundary

electrophoresis

the moving boundary method was enhanced

further with the development of techniques such

as the paper electrophoresis (obsolete) and gel

electrophoresis (joule heating).

in 1967, Hjerten used glass tubes with an internal diameter

(I.D.) around 3 mm (tube improves the dissipation of heat).

In 1979, Mikkers provided a theoretical basis for migration

dispersion in free zone electrophoresis

in 1981, Jorgenson and Lukacs was introduced the term

"capillary electrophoresis (CE)“ fused silica-100um -30kV

major challenge toward practical applications of CE Coupling

with mass spectrometry (MS) .

Electrophoresis

Electro = flow of electricity, phoresis, from the Greek = to carry across

A separation technique based on a solute’s ability to move through a conductive medium under the influence of an electric field.

The medium is usually a buffered aqueous solution

In the absence of other effects, cations migrate toward the cathode, and anions migrate toward the anode.

Principle of Capillary Electrophoresis

Electrophoretic MobilityElectrophoretic MobilityThe movement of ions solely due to the electric

field, potential differenceCations migrate toward cathodeAnions migrate toward anodeNeutral molecules do not favor either

As a result components in the capillary are affected by physical forces coming from electro osmosis and electrophoresis

Electrophoretic MobilityElectrophoretic Mobility v=Eq/fv=Eq/f

E electric field strength E electric field strength f f vep = μepE

μ = q/(6πηr)μ = q/(6πηr) q net ionic charge q net ionic charge

η is buffer viscosity r is solute radius

Properties that effect mobility1. Voltage applied2. Size and charge of the solute3. Viscosity of the buffer

Electroosmotic FlowElectroosmotic FlowAs the buffer sweeps toward the anode due to

the electric field, osmotic flow dictates the direction and magnitude of solute ion flow within the bufferAll ions are then swept toward the anode.Negative ions will lead the neutral ions toward

the anodePositive ions will trail the neutral ions as the

cathode pulls them

Electroosmotic MobilityElectroosmotic Mobilityveof = μeofE

μeof = ɛζ / (4πη)

ɛ = buffer dielectric constant

ζ = zeta potential

Zeta Potential The change in

potential across a double layer

Proportional to the charge on the capillary walls and to the thickness of the double layer. Both pH and ion

strength affect the mobility

Total MobilityTotal Mobilityvtot = vep + veof

Migration timesvtot = l/t

l = distance between injection and detection t = migration time to travel distance l

t = lL/((μep + μeof)V L = length of capillary V = voltage

Migration of cations, anions, and neutral compounds in capillary zone electrophoresis in an ordinary fused silica capillary

Electrophoretic MigrationThe overall migration in CE is determined by the combined effect of the effective and the electro osmotic mobility.

As a result, the EOF has a flat plug-like flow profile, compared to the parabolic profile of hydrodynamic flows (Fig. 4). Flat profiles in capillaries are expected when the radius of the capillary is greater than seven times the double layer thickness (Schwer and Kenndler, 1990) and are favorable to avoid peak dispersion. Therefore, the flat profile of

the EOF has a major contribution to the high separation efficiency of CE .

electropherogram

Instrumentation Power supplyAnode compartmentCathod compartmentnarrow-bore fused-silica capillary tube; injection system; detector; Recorder

Both with buffer reservoir

Capillary tube Varied length but normally

25-100 cm Small bore and thickness of

the silica play a roleUsing a smaller

internal diameter and thicker walls help prevent Joule Heating, heating due to voltage

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 volume = r2 L

-capillary internal surface area = 2 r L

End result: high potentials can be applied for extremely fast separations (30kV)

• Pressure

• Vacuum

• Siphoning

• Electrokinetic

Injection:Injection:

DetectorUV/Visible absorptionFluorescenceRadiometric (for radioactive substances)Mass Spec.

Capillary isotachophoresis

Capillary isoelectric focusing

Separation due to differences in isoelectric point (pI).

Coated column to avoide electroosmosis

Capillary gel electrophoresis

Separation mainly due to differences in shape and size.

Capillary zone electrophoresis

Separation due to differences in charge, shape and size.

Micellar electrokinetic chromatography

Separation due to difference in hydrophobicity.

Separation parameters

To achieve a good separation:Narrow bands narrow peaks efficiency:

Resolution:

Length

Internal Diameter

The effect of separation factors

Characteristics -1

Electrophoresis in narrow-bore(25-150 μm id), fused silica capillariesHigh voltages (10-30 kV) and high electric fields applied across the capillaryHigh resistance of the capillary limits current generation and internal heatingHigh efficiency (N>105-106)Short analysis time(5-20 min)Detection performed on-capillary (no external detection cell)

Characteristics -2Small sample volume required (1-50 nlinjected)Limited quantities of chemicals and reagents required (financial and environmental benifits)Operates in aqueous mediaSimple instrumentation and method developmentAutomated instrumentationNumerous modes to vary selectivity and wide application rangeApplicable to wider selection of analytes compared to other techniques (LC, TLC, SFC, cGC)Applicable to macro-and micromoleculesApplicable to charged and neutral solutesModern detector technology used (DAD, MS)

Nature is chiral because it mainly uses one of the two enantiomers of a chiral compound.

Why we need chiral Why we need chiral separation?separation?

AnodeCathode

Detector

μCD(-)

μEOF k2

K 1 Inclusion

R S

Types of CDsTypes of CDs

Electrophoretic Condition

7 kV voltage

Reverse polarity

25 mM triethylammonium phosphate (pH

2.5 )

5% HS-γ-CD.

Electropherograms of spiked human plasma with 100 ng/ml Electropherograms of spiked human plasma with 100 ng/ml

of of

(-)-tertatolol (1), (-)-tertatolol (1),

(+)- tertatolol (2) and (+)- tertatolol (2) and

400 ng/ml tolterodine L- tartarate (3). 400 ng/ml tolterodine L- tartarate (3).

Rs =1.23

Rs =17.12

X

YModel-B

Z

Model-A

Schematic representation of the two most probable inclusion models

Inclusion complex of (+)- & (-)-tertatolol with HS-γ-CD showed Model-A (upper panel) and Model-B (lower

panel) from wide rings views.

-(+)-(+)Model-A (wide ring)Model-A (wide ring) -(-)-(-)Model-A (wide ring)Model-A (wide ring)

-(-)Model-B (wide ring)-(+)Model-B (wide ring)

Electropherograms of Electropherograms of 500 ng/ml of (-)-tertatolol (1),500 ng/ml of (-)-tertatolol (1), (+)-tertatolol (2)(+)-tertatolol (2) and 500 ng/ml tolterodine L- tartarate (3) and 500 ng/ml tolterodine L- tartarate (3) recovered from tertatolol tablets.recovered from tertatolol tablets.

(+)-Tertatolol% recovery = 100.96%

RSD = 0.99 %

(-)-Tertatolol% recovery = 98.32%

RSD = 0.85 %

The two drugs were subjected to thermal, photolytic,

hydrolytic, and oxidative stress conditions and the

stressed samples were analyzed by the proposed

method.

Stability study

mA

U

Minute

Minute

Degradation products (UK-55-410), (PD 0162910-00)

for AM and AT respectively produced as a result of

stress studies did not interfere with the detection of

AM and AT and the assay can thus be considered

stability indicating.