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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. 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.