capillary electrophoresis. its origin can be traced back to the 1880s it got major recognition in...
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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.