24 Questions
What is the advantage of using capillary electrophoresis over gel electrophoresis?
Reduced Joule heating effect
What is the main source of band-broadening in capillary electrophoresis?
Longitudinal diffusion
What is the purpose of the Schlieren optical system in instrumentation?
To measure refractive index throughout the solution
What is isotachophoresis used for?
Separation of smaller ionic substances
What is measured to relate to the identity of an analyte in capillary electrophoresis?
Migration time
What is the result of higher electric fields in capillary electrophoresis?
Higher efficiency and narrower peaks
What is the purpose of the U-shaped glass cell in instrumentation?
To hold the sample solution for analysis
What type of electrophoresis does capillary electrophoresis belong to?
Moving boundary electrophoresis
What is the advantage of using isotachophoresis over other electrophoresis techniques?
Ability to separate smaller ionic substances
What is the purpose of the power pack in the general apparatus of electrophoresis?
To provide a constant voltage
Which of the following is NOT a type of moving boundary electrophoresis?
Isoelectric focusing
What is the purpose of the electrodes in the electrophoresis unit?
To provide a path for the electric current
What is isotachophoresis?
A type of moving boundary electrophoresis
What is the function of the transparent insulating cover in the electrophoresis unit?
To prevent electrical shock
What is the main difference between moving boundary electrophoresis and zone electrophoresis?
The migration of the charged particles
What is the function of the sample injector in the general apparatus of electrophoresis?
To apply the sample to the supporting medium
In Capillary Electrophoresis, what is unique about the current flow?
It is carried entirely by the sample ions
What is the main application of Isoelectric Focussing?
Separation of amphoteric substances
What is the result of antigen-antibody reactions in Immuno Electrophoresis?
Formation of precipitin arcs
What is the main application of Immuno Electrophoresis?
Detection of antibodies
What type of molecules are separated in Isoelectric Focussing?
Amphoteric molecules
What is the advantage of Isoelectric Focussing?
High resolution
What is the purpose of the trough in Immuno Electrophoresis?
To hold the antibody
What is the significance of the precipitin arc in Immuno Electrophoresis?
It represents the individual antigen
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
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.
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