Electrophoresis Instrumentation and Applications

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Questions and Answers

What is the advantage of using capillary electrophoresis over gel electrophoresis?

Increased band broadening

Longer separation time

Larger tube diameter

Reduced Joule heating effect (correct)

What is the main source of band-broadening in capillary electrophoresis?

Electro-osmosis

Electrophoretic mobility

Joule heating effect

Longitudinal diffusion (correct)

What is the purpose of the Schlieren optical system in instrumentation?

To separate complex biological mixtures

To measure electrophoretic mobility

To detect the presence of analytes

To measure refractive index throughout the solution (correct)

What is isotachophoresis used for?

Separation of smaller ionic substances Signup and view all the answers

What is measured to relate to the identity of an analyte in capillary electrophoresis?

Migration time Signup and view all the answers

What is the result of higher electric fields in capillary electrophoresis?

Higher efficiency and narrower peaks Signup and view all the answers

What is the purpose of the U-shaped glass cell in instrumentation?

To hold the sample solution for analysis Signup and view all the answers

What type of electrophoresis does capillary electrophoresis belong to?

Moving boundary electrophoresis Signup and view all the answers

What is the advantage of using isotachophoresis over other electrophoresis techniques?

Ability to separate smaller ionic substances Signup and view all the answers

What is the purpose of the power pack in the general apparatus of electrophoresis?

To provide a constant voltage Signup and view all the answers

Which of the following is NOT a type of moving boundary electrophoresis?

Isoelectric focusing Signup and view all the answers

What is the purpose of the electrodes in the electrophoresis unit?

To provide a path for the electric current Signup and view all the answers

What is isotachophoresis?

A type of moving boundary electrophoresis Signup and view all the answers

What is the function of the transparent insulating cover in the electrophoresis unit?

To prevent electrical shock Signup and view all the answers

What is the main difference between moving boundary electrophoresis and zone electrophoresis?

The migration of the charged particles Signup and view all the answers

What is the function of the sample injector in the general apparatus of electrophoresis?

To apply the sample to the supporting medium Signup and view all the answers

In Capillary Electrophoresis, what is unique about the current flow?

It is carried entirely by the sample ions Signup and view all the answers

What is the main application of Isoelectric Focussing?

Separation of amphoteric substances Signup and view all the answers

What is the result of antigen-antibody reactions in Immuno Electrophoresis?

Formation of precipitin arcs Signup and view all the answers

What is the main application of Immuno Electrophoresis?

Detection of antibodies Signup and view all the answers

What type of molecules are separated in Isoelectric Focussing?

Amphoteric molecules Signup and view all the answers

What is the advantage of Isoelectric Focussing?

High resolution Signup and view all the answers

What is the purpose of the trough in Immuno Electrophoresis?

To hold the antibody Signup and view all the answers

What is the significance of the precipitin arc in Immuno Electrophoresis?

It represents the individual antigen Signup and view all the answers

Study Notes

Advantages and Disadvantages

Biologically active fractions can be recovered without denaturing agents
Minute concentrations of the sample can be detected
Costlier
Elaborate optical system required

Applications

Study homogeneity of macromolecular systems
Analysis of complex biological mixtures

Instrumentation

U-shaped glass cell with rectangular cross-section
Electrodes placed on each limb of the cell
Sample solution introduced at the bottom or through the side arm
Apparatus placed in a constant temperature bath at 40°C
Detection done by measuring refractive index throughout the solution

Capillary Electrophoresis

Capillary tube placed between two buffer reservoirs
Electric field applied, separation depends on electrophoretic mobility and electro-osmosis
Defined volume of analyte introduced into the capillary by replacing one buffer reservoir with sample vial
Electrophoretic separation measured by detector
Uses narrow bore tubes, removing joule heating effect, resulting in faster separations and less band broadening
Tubes 20-100µm diameter and 20-100cm length
Can be used with or without gel

Isotachophoresis

Development of potential gradient
Used for separation of smaller ionic substances
Sample ions migrate adjacent to each other, but not overlapping
Current flow carried entirely by sample ions
Application: separation of small anions and cations

Isoelectric Focusing

Technique for separating molecules by differences in their isoelectric point
Suitable for separation of amphoteric substances
Method has high resolution
Applications: human genetic lab, separating iso-enzymes, research in enzymology and immunology

Immuno-Electrophoresis

Study of antigen-antibody reactions under electrical field
Antibody electrophoretically separated and antigens diffuse towards each other
Precipitin arcs formed where antigen-antibody complexes form
Used to detect presence of antibodies
Mainly used to determine blood levels of three major immunoglobulins: IgM, IgG, and IgA

Process

Ag molecules separated according to electrical charge by electrophoresis
Antibody placed in a trough cut in the agar
Antibody diffuses towards antigen, forming precipitin arcs
Interpretation: precipitin arcs represent individual antigens

Factors Affecting Electrophoresis

Electric field: voltage, current, and resistance
Buffer: ionic strength and pH
Supporting medium: adsorption, electro-endosmosis

Types of Electrophoresis

Zone electrophoresis: paper, gel, thin layer, and cellulose acetate electrophoresis
Moving boundary/frontal electrophoresis: capillary electrophoresis, isotachophoresis, isoelectric focusing, and immuno-electrophoresis

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Description

This quiz covers the advantages, disadvantages, and applications of electrophoresis, a laboratory technique used to separate and analyze macromolecules. It also touches on the instrumentation involved.