Capillary Electrophoresis

Its origin can be traced back to the 1880sit 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).

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 electrophoresisin 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 solutes 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.

Principle of Capillary Electrophoresis

Electrophoretic MobilityThe movement of ions solely due to the electric field, potential differenceCations migrate toward cathodeAnions migrate toward anodeNeutral molecules do not favor eitherAs a result components in the capillary are affected by physical forces coming from electro osmosis and electrophoresis

Electrophoretic Mobilityv=Eq/fE electric field strength f vep = epE = q/(6r)q net ionic charge is buffer viscosityr is solute radiusProperties that effect mobilityVoltage appliedSize and charge of the soluteViscosity of the buffer

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

Electroosmotic Mobilityveof = eofEeof = / (4) = buffer dielectric constant = 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

Total Mobilityvtot = vep + veofMigration timesvtot = l/tl = distance between injection and detectiont = migration time to travel distance lt = lL/((ep + eof)VL = length of capillaryV = voltage

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

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.

electropherogram

Instrumentation Power supplyAnode compartmentCathod compartmentnarrow-bore fused-silica capillary tube; injection system; detector; Recorder

Both with buffer reservoir

Capillary tubeVaried length but normally 25-100 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

Joule 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 L

End result: high potentials can be applied for extremely fast separations (30kV)

Pressure

Vacuum

Siphoning

Electrokinetic

Injection:

DetectorUV/Visible absorptionFluorescenceRadiometric (for radioactive substances)Mass Spec.

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:

Length Internal Diameter

Characteristics -1

Electrophoresis in narrow-bore(25-150 m 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>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 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)

Nature is chiral because it mainly uses one of the two enantiomers of a chiral compound.

Why we need chiral separation?

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

Schematic representation of the two most probable inclusion models

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

Electropherograms of 500 ng/ml of (-)-tertatolol (1), (+)-tertatolol (2) and 500 ng/ml tolterodine L- tartarate (3) recovered from tertatolol tablets.

* 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). *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 eof is greater than eff 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.

*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.**The effect of separation factor ion each other*Number of papers published using the different chiral selectors described in EKC.

*Neutral CDs such as -CD, -CD, -CD and their derivatives hydroxy propyl-CD (HP-CD) and dimethyl--CD (DM--CD) were examined. The charged CDs investigated were sulfated- -CD (S--CD), carboxy methyl--CD (CM--CD), highly sulfated--CD (HS--CD), highly sulfated--CD(HS--CD) and highly sulfated--CD (HS--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--CD in 25mM triethylammonium phosphate (TEAP) buffer at pH 2.5 where base line separations was achieved within 20 min

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