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Capillary Electrophoresis

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Presentation on theme: "Capillary Electrophoresis"— Presentation transcript:

1 Capillary Electrophoresis

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 electrophoresis the moving boundary method was enhanced further with the development of techniques such as the paper electrophoresis (obsolete) and gel electrophoresis (joule heating).

3 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) .

4 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.

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 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

8 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

9 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

10 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

11 Total Mobility vtot = vep + veof Migration times t = lL/((μep + μeof)V
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

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.

14 Capillary Electrophoresis Instrument

15 electropherogram

16 Instrumentation Power supply Anode compartment Cathod compartment
narrow-bore fused-silica capillary tube; injection system; detector; Recorder Both with buffer reservoir

17 Capillary tube Varied length but normally 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

18 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)


20 • Pressure • Vacuum • Siphoning • Electrokinetic
Injection: • Pressure • Vacuum • Siphoning • Electrokinetic


22 Detector UV/Visible absorption Fluorescence
Radiometric (for radioactive substances) Mass Spec.



25 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) Micellar electrokinetic capillary chromatography (MEKC) Chiral Capillary Electrophoresis (CCE) Capillary electrochromatography (CEC). Free solution CE In 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.

26 Capillary isotachophoresis

27 Capillary isoelectric focusing
Separation due to differences in isoelectric point (pI). Coated column to avoide electroosmosis

28 Capillary gel electrophoresis
Separation mainly due to differences in shape and size.

29 Capillary zone electrophoresis
Separation due to differences in charge, shape and size.

30 Micellar electrokinetic chromatography
Separation due to difference in hydrophobicity.


32 Separation parameters
To achieve a good separation: Narrow bands narrow peaks efficiency:

33 Resolution:

34 Capillary Temperature
Electrode Polarity Applied Voltage Capillary Temperature Capillary Dimensions Buffers Separation Optimization Parameters Length Internal Diameter

35 The effect of separation factor ion each other
The effect of separation factors The effect of separation factor ion each other

36 Characteristics -1 Electrophoresis in narrow-bore( 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> ) 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 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)

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
Anode Cathode Detector μCD(-) μEOF k2 K 1 Inclusion R S

43 chiral selector Number of papers published using the different chiral selectors described in EKC.

44 Types of CDs Natural Cyclodextrins Charged Cyclodextrins
Anionic Cyclodextrins Ex. Highly sulfated CDs Ex.Carboxymethylated CDs Cationic Cyclodextrins Dual Cyclodextrin System

45 Part II Enantioselective 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 Dimensions Effect of BGE Concentration Effect of pH HS-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 min Application in human plasma & pharmaceutical preparation.

47 Electrophoretic Condition
Reverse polarity 7 kV voltage 25 mM triethylammonium phosphate (pH 2.5 ) 5% HS-γ-CD.

48 Results and Discussion

49 Electropherograms of spiked human plasma with 100 ng/ml of
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).

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 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.

54 500 ng/ml of (-)-tertatolol (1), (+)-tertatolol (2)
% recovery = 98.32% RSD = 0.85 % (+)-Tertatolol % recovery = % 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.

55 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.

56 mAU Minute Minute

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.

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