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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.

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Presentation on theme: "Capillary Electrophoresis.  Its origin can be traced back to the 1880s  it got major recognition in 1937, when Tiselius reported the separation of different."— Presentation transcript:

1 Capillary Electrophoresis

2  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  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.

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6 Principle of Capillary Electrophoresis

7 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 As a result components in the capillary are affected by physical forces coming from electro osmosis and electrophoresis

8 Electrophoretic Mobility v=Eq/f v=Eq/f E electric field strength E electric field strength f v ep = μ ep E μ = q/(6πηr) μ = q/(6πηr) q net ionic charge q net ionic charge η is buffer viscosity r is solute radius Properties that effect mobility 1. Voltage applied 2. Size and charge of the solute 3. 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 v eof = μ eof E μ eof = ɛ ζ / (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 v tot = v ep + v eof Migration times v tot = l/t l = distance between injection and detection t = migration time to travel distance l t = lL/((μ ep + μ eof )V L = length of capillary V = voltage

12 Migration of cations, anions, and neutral compounds in capillary zone electrophoresis in an ordinary fused silica capillary Electrophoretic Migration The overall migration in CE is determined by the combined effect of the effective and the electro osmotic mobility.

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  r 2  For smaller capillaries heat is dissipated due to the large surface area to volume ratio –capillary internal volume =  r 2 L -capillary internal surface area = 2  r L  End result: high potentials can be applied for extremely fast separations (30kV)

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20 Pressure Vacuum Siphoning Electrokinetic Injection:

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

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25 (1)Moving boundary CE (outdated) (2) Steady-state CE Isotachophoresis. (ITP)Isotachophoresis. (ITP) Isoelectric focusing (IEF)Isoelectric focusing (IEF) (3) Zone CE Capillary gel electrophoresis (CGE) Capillary gel electrophoresis (CGE) Capillary zone electrophoresis (CZE)Capillary zone electrophoresis (CZE) Micellar electrokinetic capillary chromatography (MEKC)Micellar electrokinetic capillary chromatography (MEKC) Chiral Capillary Electrophoresis (CCE)Chiral Capillary Electrophoresis (CCE) Capillary electrochromatography (CEC).Capillary electrochromatography (CEC). Free solution CEFree solution CE

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.

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

33 Resolution:

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

35 The effect of separation factors

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 Nature is chiral because it mainly uses one of the two enantiomers of a chiral compound. Why we need chiral separation?

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 Not based on an electrophoretic mechanism because the electrophoretic mobilities of the enantiomers of a chiral compound are equal and nonselective. Electrokinetic Chromatography This separation principle relies on the different partition of enantiomers between the bulk solution and the chiral pseudophase ( chiral selector), Electrokinetic Chromatography

42 Anode Cathode Detector μ CD(-) μ EOF k2 K 1 Inclusion R S

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

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46 Optimization one-variable Type of chiral selectore Capillary Dimensions Effect of BGE Concentration Effect of pH HS-γ-CD Concentration. Application in human plasma & pharmaceutical preparation.

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

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49 Electropherograms of spiked human plasma with 100 ng/ml of (-)-tertatolol (1), (-)-tertatolol (1), (+)- tertatolol (2) and (+)- tertatolol (2) and 400 ng/ml tolterodine L- tartarate (3). 400 ng/ml tolterodine L- tartarate (3). Rs =1.23 Rs =17.12

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51 Schematic representation of the two most probable inclusion models

52 Inclusion complex of (+)- & (-)-tertatolol with HS-γ-CD showed Model-A (upper panel) and Model-B (lower panel) from wide rings views. (+)-Model-A (wide ring) (-)-Model-A (wide ring) (-)-Model-B (wide ring) (+)-Model-B (wide ring)

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 Electropherograms of 500 ng/ml of (-)-tertatolol (1), 500 ng/ml of (-)-tertatolol (1), (+)-tertatolol (2) (+)-tertatolol (2) and 500 ng/ml tolterodine L- tartarate (3) recovered from tertatolol tablets. and 500 ng/ml tolterodine L- tartarate (3) recovered from tertatolol tablets. (+)-Tertatolol % recovery = % RSD = 0.99 % (-)-Tertatolol % recovery = 98.32% RSD = 0.85 %

55 The two drugs were subjected to thermal, photolytic, hydrolytic, and oxidative stress conditions and the stressed samples were analyzed by the proposed method. Stability study

56 mAU 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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