16 Questions
What is the primary basis for the separation of molecules in gel permeation chromatography?
Difference in molecular weight or size
What is the purpose of the stationary phase in gel permeation chromatography?
To separate the molecules based on size
What is the characteristic of the pores in the stationary phase of gel permeation chromatography?
Well-defined range of pore sizes
What is the role of the pump in gel permeation chromatography?
To pump the mobile phase through the column
What is the characteristic of the gel beads used in gel permeation chromatography?
Chemically inert and mechanically stable
What is the result of larger molecules passing through the column in gel permeation chromatography?
They pass through the column unhindered, without penetrating the gel matrix
What is the primary application of gel permeation chromatography?
Separation of proteins, polysaccharides, enzymes, and synthetic polymers
Who developed the technique of size exclusion chromatography in 1955?
Lathe and Ruthven
What is the characteristic of the gel used in Gel Permeation Chromatography?
Uniform particle and pore size
What is the main purpose of the mobile phase in Gel Permeation Chromatography?
To dissolve the bio-molecules and permit high detection response
What is the diameter of the narrow-bore columns used in Gel Permeation Chromatography?
2-3 mm
What is the type of pumps used in Gel Permeation Chromatography?
Syringe pumps and reciprocating pumps
What is the purpose of the swelling step in the preparation of the column for gel filtration?
To allow the gel to absorb the buffer solution
What is the application of Gel Permeation Chromatography in determining the quaternary structure of proteins?
To determine the quaternary structure of purified proteins
What is the advantage of Gel Permeation Chromatography in terms of the amount of mobile phase required?
A small amount of mobile phase is required
What is the type of detector that can be used in Gel Permeation Chromatography?
All of the above
Study Notes
Gel Permeation Chromatography
- Also known as gel filtration or size exclusion chromatography
- Uses a porous matrix made up of compounds like cross-linked polystyrene, cross-linked dextrans, polyacrylamide gels, and agarose gels
- Separation based on analyte molecular sizes, with the gel behaving like a molecular sieve
Principle of Gel Permeation Chromatography
- Separation of components based on difference in molecular weight or size
- Stationary phase is a porous polymer matrix with pores completely filled with the solvent to be used as the mobile phase
- Molecules in the sample are pumped through specialized columns containing microporous packing material (gel)
- Larger molecules pass through the column unhindered, while smaller molecules are retarded according to their penetration of the gel
Components/Instrumentation of Gel Permeation Chromatography
- Stationary phase: semi-permeable, porous polymer gel beads with a well-defined range of pore sizes
- Mobile phase: a liquid used to dissolve bio-molecules and permit high detection response
- Columns: analytical, preparative, and narrow-bore columns with different diameters and lengths
- Pump: syringe pumps or reciprocating pumps with a high constant flow rate
- Detectors: concentration-sensitive detectors, bulk property detectors, and refractive index (RI) detectors
Steps in Gel Permeation Chromatography
- Preparation of column for gel filtration: swelling of the gel, packing the column, and washing
- Loading the sample onto the column using a syringe
- Eluting the sample and detection of components
Applications of Gel Permeation Chromatography
- Proteins fractionation
- Purification
- Molecular weight determination
- Separation of sugar, proteins, peptides, rubbers, and others on the basis of their size
- Determination of quaternary structure of purified proteins
Advantages of Gel Permeation Chromatography
- Short analysis time
- Well-defined separation
- Narrow bands and good sensitivity
- No sample loss
- Small amount of mobile phase required
Gel permeation chromatography, also known as gel filtration or size exclusion chromatography, is a technique used for separation of molecules based on their sizes. It is commonly used for proteins, polysaccharides, enzymes, and synthetic polymers.
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