Transcript capillary electropho..

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.
is a separation technique that is based on
the differential migration of charged compounds in a semiconductive medium under the influence of an electric field.
Principle of Capillary Electrophoresis
As a result components in the capillary are affected by physical forces
coming from electro osmosis and electrophoresis
Electrophoretic Mobility
 The movement of ions solely due to the electric field,
potential difference
 Cations migrate toward cathode
 Anions migrate toward anode
 Neutral molecules do not favor either
Electrophoretic Mobility

v=Eq/f
E electric field strength
f


vep = μepE


μ = q/(6πηr)




q net ionic charge
η is buffer viscosity
r is solute radius
Properties that effect mobility
1.
2.
3.
Voltage applied
Size and charge of the solute
Viscosity of the buffer
Electroosmotic Flow
 As the buffer sweeps toward the anode due to the
electric field, osmotic flow dictates the direction and
magnitude of solute ion flow within the buffer
 All ions are then swept toward the anode.
 Negative ions will lead the neutral ions toward the
anode
 Positive ions will trail the neutral ions as the cathode
pulls them
Electroosmotic Mobility
 veof = 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 Mobility
 vtot = vep + veof
 Migration times
 vtot = 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
Electrophoretic Migration
The overall migration in CE is determined by the combined effect of
the effective and the electro osmotic mobility.
Migration of cations, anions, and neutral compounds in capillary zone
electrophoresis in an ordinary fused silica capillary
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.
Capillary Electrophoresis Instrument
electropherogram
Instrumentation
Power supply
Anode compartment
Cathod compartment
Both with buffer reservoir
narrow-bore fused-silica capillary tube;
injection system;
detector;
Recorder
 Capillary tube


Varied length but normally 25100 cm
Small bore and thickness of the
silica play a role

Using a smaller internal
diameter and thicker walls
help prevent Joule
Heating, heating due to
voltage
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 volume =  r2 L
-capillary internal surface area = 2 r L
 End result:
high potentials can be applied for extremely
fast separations (30kV)
Injection:
• Pressure
• Vacuum
• Siphoning
• Electrokinetic
 Detector
 UV/Visible absorption
 Fluorescence
 Radiometric (for radioactive substances)
 Mass Spec.
(1) Moving boundary CE (outdated)
(2) Steady-state CE
•Isotachophoresis. (ITP)
•Isoelectric focusing (IEF)
(3) Zone CE
•Capillary gel electrophoresis (CGE)
•Capillary zone electrophoresis (CZE)
•Micellar electrokinetic capillary chromatography
(MEKC)
•Chiral Capillary Electrophoresis (CCE)
•Capillary electrochromatography (CEC).
•Free solution CE
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:
Electrode
Polarity
Applied
Voltage
Capillary
Temperature
Capillary
Dimensions
Buffers
Length
Internal Diameter
The effect of separation factors
Characteristics -1
Electrophoresis in narrow-bore(25-150 μm id),
fused silica capillaries
High voltages (10-30 kV) and high electric fields
applied across the capillary
High resistance of the capillary limits current
generation and internal heating
High efficiency (N>105-106)
Short analysis time(5-20 min)
Detection performed on-capillary (no external
detection cell)
Characteristics -2
Small sample volume required (1-50 nlinjected)
Limited quantities of chemicals and reagents
required (financial and environmental benifits)
Operates in aqueous media
Simple instrumentation and method development
Automated instrumentation
Numerous modes to vary selectivity and wide
application range
Applicable to wider selection of analytes compared
to other techniques (LC, TLC, SFC, cGC)
Applicable to macro-and micromolecules
Applicable to charged and neutral solutes
Modern detector technology used (DAD, MS)
Why we need chiral separation?
Nature is chiral because it
mainly uses one of the two
enantiomers of a chiral
compound.
most biological processes have a high degree of
enantioselectivity: each enantiomer may have a different
biological activity .
drug is administered as a racemic mixture, one enantiomer
may have pharmacological effects while the other could have
antagonist effect or it could show some undesired side
effects.
All of this shows that there are many reasons to
discriminate between the enantiomers of a chiral compound
and to study them separately.
CE has been applied extensively for the separation of chiral
compounds in chemical and pharmaceutical analysis.
Not based on an electrophoretic mechanism because the
electrophoretic mobilities of the enantiomers of a chiral
compound are equal and nonselective.
This separation principle relies on the different partition of
enantiomers between the bulk solution and the chiral
pseudophase ( chiral selector), Electrokinetic
Chromatography
Detector
Cathode
μEOF
S
Inclusion
K1
R
k2
μCD(-)
Anode
Types of CDs
Natural
Cyclodextrins
Charged
Cyclodextrins
Anionic
Cyclodextrins
Dual Cyclodextrin
System
Cationic
Cyclodextrins
Ex. Highly sulfated CDs
Ex.Carboxymethylated CDs
*This part has been submitted to J. Chromatogr. A
Effect of BGE
Concentration
Effect of
pH
Capillary
Dimensions
Type of chiral
selectore
Optimization
one-variable
Application in human plasma
& pharmaceutical
preparation.
HS-γ-CD
Concentration.
Electrophoretic Condition
Reverse polarity
7 kV voltage
25 mM
triethylammonium
phosphate (pH 2.5 )
5%
HS-γ-CD.
Rs =1.23
Rs =17.12
Electropherograms of spiked human plasma with 100 ng/ml of
(-)-tertatolol (1),
(+)- tertatolol (2) and
400 ng/ml tolterodine L- tartarate (3).
X
Z
Model-B
Y
Model-A
Schematic representation of the two
most probable inclusion models
(+)-Model-A (wide ring)
(+)-Model-B (wide ring)
(-)-Model-A (wide ring)
(-)-Model-B (wide ring)
Inclusion complex of (+)- & (-)-tertatolol with HS-γ-CD
showed Model-A (upper panel) and Model-B (lower panel)
from wide rings views.
The method was linear in the range of
100-2000 ng/ ml
(r = 0.999) for each enantiomer
LOD = 50 ng/ml. LOQ = 100 ng/ml
The mean RSD of the results within-day
and intra- day precision was ≤ 5%
Accuracy of the drug were E% ≤ 2.5 % .
The method was highly specific, where the
co formulated compounds did not interfere.
(-)-Tertatolol
% recovery = 98.32%
RSD = 0.85 %
(+)-Tertatolol
% recovery = 100.96%
RSD = 0.99 %
Electropherograms of
500 ng/ml of (-)-tertatolol (1),
(+)-tertatolol (2)
and 500 ng/ml tolterodine L- tartarate (3)
recovered from tertatolol tablets.
Stability study
The two drugs were subjected to thermal, photolytic,
hydrolytic, and oxidative stress conditions and the stressed
samples were analyzed by the proposed method.
mAU
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.