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Capillary Electrophoresis. History. 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
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History • 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).
History • 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.
Electrophoresis is a separation technique that is based on the differential migration of charged compounds in a semi-conductive medium under the influence of an electric field.
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 • 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.
Instrumentation • Power supply • Anode compartment • Cathod compartment • narrow-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 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.
Different Modes in Capillary Electrophoresis • Moving boundary CE (outdated) • (2) Steady-state CE • Isotachophoresis. (ITP) • Isoelectric focusing (IEF) • (3) Zone CE • Capillary gel electrophoresis (CGE) • Capillary zone electrophoresis (CZE) • Micellarelectrokinetic capillary chromatography (MEKC) • Chiral Capillary Electrophoresis (CCE) • Capillary electrochromatography (CEC). • Free solution CE
Capillary isoelectric focusing Separation due to differences in isoelectric point (pI). • Coated column to avoideelectroosmosis
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:
Separation Optimization Parameters • Length • Internal Diameter
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.
principle Chiral separation by capillary electrophoresis Not based on an electrophoretic mechanism because the electrophoreticmobilities 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 chiralpseudophase ( chiral selector), Electrokinetic Chromatography
Mechanism of chiralsepatation using cyclodextrine as chiral selector Detector μEOF k2 Anode R S Cathode Inclusion μCD(-) K 1
Part II Enantioselective capillary electrophoresis method for determination of tertatolol in plasma and dosage forms using highly sulphatedgamacyclodextrin as chiral selector: mechanistic and molecular modeling studies* *This part has been submitted to J. Chromatogr. A
Experimental work Application in human plasma & pharmaceutical preparation.
Electrophoretic Condition Reverse polarity 7 kV voltage 25 mMtriethylammonium 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).
