2 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 electrophoresisthe moving boundary method was enhanced further with the development of techniques such as the paper electrophoresis (obsolete) and gel electrophoresis (joule heating).
3 Historyin 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 electrophoresisin 1981, Jorgenson and Lukacs was introduced the term "capillary electrophoresis (CE)“ fused silica-100um -30kVmajor challenge toward practical applications of CE Coupling with mass spectrometry (MS) .
4 ElectrophoresisElectro = flow of electricity, phoresis, from the Greek = to carry acrossA 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 solutionIn the absence of other effects,cations migrate toward the cathode,and anions migrate toward the anode.
5 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.
6 Principle of Capillary Electrophoresis A semiconducting medium and an electric field are the basic needs electrophoresis. In the case of CE the semi conducting medium is composed of a capillary filled with an electrolyte or a gel. An electric field is generated by applying a voltage difference across the capillary. As a result components in the capillary are affected by physical forces coming from electro osmosis and electrophoresis (Fig. 1).
7 Electrophoretic Mobility As a result components in the capillary are affected by physical forces coming from electro osmosis and electrophoresisElectrophoretic MobilityThe movement of ions solely due to the electric field, potential differenceCations migrate toward cathodeAnions migrate toward anodeNeutral molecules do not favor either
8 Electrophoretic Mobility v=Eq/fE electric field strengthfvep = μepEμ = q/(6πηr)q net ionic chargeη is buffer viscosityr is solute radiusProperties that effect mobilityVoltage appliedSize and charge of the soluteViscosity of the buffer
9 Electroosmotic 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
10 Electroosmotic Mobility veof = μeofEμeof = ɛζ / (4πη)ɛ = buffer dielectric constantζ = zeta potentialZeta PotentialThe change in potential across a double layerProportional to the charge on the capillary walls and to the thickness of the double layer.Both pH and ion strength affect the mobility
11 Total Mobility vtot = vep + veof Migration times t = lL/((μep + μeof)V vtot = l/tl = distance between injection and detectiont = migration time to travel distance lt = lL/((μep + μeof)VL = length of capillaryV = voltage
12 Electrophoretic Migration The overall migration in CE is determined by the combined effect of the effective and the electro osmotic mobility.In CE using fused silica capillaries the EOF is directed toward the cathode; therefore, the apparent migration velocity of cations is positively affected, while the migration of anions is negatively affected. Neutral compounds are also transported through the capillary toward the cathode because of the EOF. When the electroendosmotic mobility is sufficiently high it is ever possible to separate both cations and anions in one single run (Fig. 5). When µeof is greater than µeff anions that originally migrate toward the anode are still carried toward the cathode: due to a positive apparent velocity. Neutral compounds migrate with the velocity of the EOF, but are unresolved under one peak in the electropherogram. Migration of cations, anions, and neutral compounds in capillary zone electrophoresis in an ordinary fused silica capillary
13 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.
16 Instrumentation Power supply Anode compartment Cathod compartment narrow-bore fused-silica capillary tube;injection system;detector;RecorderBoth with buffer reservoir
17 Capillary tubeVaried length but normally cmSmall bore and thickness of the silica play a roleUsing a smaller internal diameter and thicker walls help prevent Joule Heating, heating due to voltage
18 Joule HeatingJoule heating is a consequence of the resistance of the solution to the flow of currentif heat is not sufficiently dissipated from the system the resulting temperature and density gradients can reduce separation efficiencyHeat dissipation is key to CE operation:Power per unit capillary P/L r2For smaller capillaries heat is dissipated due to the large surface area to volume ratiocapillary internal volume = r2 L-capillary internal surface area = 2 r LEnd result: high potentials can be applied for extremely fast separations (30kV)
25 Different Modes in Capillary Electrophoresis Moving boundary CE (outdated)(2) Steady-state CE Isotachophoresis. (ITP)Isoelectric focusing (IEF)(3) Zone CECapillary gel electrophoresis (CGE)Capillary zone electrophoresis (CZE)Micellar electrokinetic capillary chromatography (MEKC)Chiral Capillary Electrophoresis (CCE)Capillary electrochromatography (CEC).Free solution CEIn a steady state process, the composition of the background electrolyte is not constant. Both the electric field and the effective mobilities may change along the migration path. The most common practical realization of this type of separation process is to form a pH gradient along the migration path.
35 The effect of separation factor ion each other The effect of separation factorsThe effect of separation factor ion each other
36 Characteristics -1Electrophoresis in narrow-bore( 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> )Short analysis time(5-20 min)Detection performed on-capillary (no external detection cell)
37 Characteristics -2 Small 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)
38 Why we need chiral separation? Nature is chiral because it mainly uses one of the two enantiomers of a chiral compound.
39 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.
40 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.
41 principle Chiral separation by capillary electrophoresis 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
42 Mechanism of chiral sepatation using cyclodextrine as chiral selector AnodeCathodeDetectorμCD(-)μEOFk2K 1InclusionRS
43 chiral selectorNumber of papers published using the different chiral selectors described in EKC.
44 Types of CDs Natural Cyclodextrins Charged Cyclodextrins Anionic CyclodextrinsEx. Highly sulfated CDsEx.Carboxymethylated CDsCationic CyclodextrinsDual Cyclodextrin System
45 Part IIEnantioselective capillary electrophoresis method for determination of tertatolol in plasma and dosage forms using highly sulphated gama cyclodextrin as chiral selector: mechanistic and molecular modeling studies**This part has been submitted to J. Chromatogr. A
46 Experimental work Optimization one-variable Type of chiral selectore Capillary DimensionsEffect of BGE ConcentrationEffect of pHHS-γ-CD Concentration.Neutral CDs such as α-CD, β-CD, γ-CD and their derivatives hydroxy propyl-CD (HP-CD) and dimethyl-β-CD (DM-β-CD) were examined. The charged CDs investigated were sulfated- β-CD (S-β-CD), carboxy methyl-β-CD (CM-β-CD), highly sulfated-α-CD (HS-α-CD), highly sulfated-β-CD(HS-β-CD) and highly sulfated-γ-CD (HS-γ-CD). Tow concentration (3 and 5 mM) from each bile salt, taurocholic sodium (STDC), taurodeoxycholate (STC), deoxycholate (SDC) and cholate (SC) were also studied under normal and reverse polarity at pH (2.5 and 8) of phosphate and acetate buffer, respectively. No separations were obtained using bile salts and CDs except for HS-γ-CD in 25mM triethylammonium phosphate (TEAP) buffer at pH 2.5 where base line separations was achieved within 20 minApplication in human plasma & pharmaceutical preparation.
49 Electropherograms of spiked human plasma with 100 ng/ml of Rs =1.23Rs =17.12Electropherograms of spiked human plasma with 100 ng/ml of(-)-tertatolol (1),(+)- tertatolol (2) and400 ng/ml tolterodine L- tartarate (3).
50 Study the molecular mechanics for both chiral drugs
51 Schematic representation of the two most probable inclusion models
52 (+)-Model-A (wide ring) (-)-Model-A (wide ring) (-)-Model-B (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.
53 The method was linear in the range of 100‑2000 ng/ ml (r = 0.999) for each enantiomerLOD = 50 ng/ml. LOQ = 100 ng/mlThe 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.
57 Degradation products (UK ), (PD ) 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.