This is also known as “Molecular exclusion chromatography” (or) “Molecular sieve chromatography”, “Size exclusion chromatography” and “Permeation chromatography”
Principle:
In exclusion chromatography the separation of molecules is based up on the size and shape. The stationary phase is porous bead material and the mobile phase is the solvent system.
The large molecules cannot enter the pores of the beads so they are excluded out and come down rapidly. Small sized molecules enter the pores of the beads so that their speed is retarded and comedown slowly. The degree of retardation of a molecule is proportional to the time it spends inside the gel pores, which is a function of the molecule’s size and the pore diameter.
Mathematical relationships about solute behaviour on molecular sieve gels:
1) “The distribution of a solute particle between the inner and outer solvent (solvent within and
outside of the gel bead) is defined as “Distribution coefficient” (Kd).
Kd= 0 –> Solute molecule is large & excluded out completely
Kd= 1 –> Solute molecule is small, retards it in inner solvents
2) The volume of outer solvent, i.e., the solvent surrounding the gel beads is indicated as Vo. The technical term for this is “Void volume”.
The volume of solvent inside gel bead – Inner solvent = Vi
The distribution coefficient = Kd
The effluent volume = Ve
Thus
The volume of inner solvent, Vi, can be calculated if the dry weight of gel employed and the “Water regain value” of the particular gel are known. Thus,
- For two different substances possessing different molecules weights and therefore, different distribution coefficients (Kd1 & Kd2) the difference in their effluent volumes, Vs is given by
Vs =Ve1-Ve2 = (V0+Kd1.Vi)-(Vo+Kd2.Vi)
Types of gels:
Characteristics of gels:
1) The gel material should be chemically inert.
2) It should preferably contain ravishingly small number of ionic groups.
3) Gel material should provide a wide choice of pore and particle sizes.
4) The gel should have uniform particle & pore sizes.
5) The gel matrix should have high mechanical rigidity.
There are FIVE principle types of media:
a) Sephadex
b) Agarose
c)Polyacrylamide (Bio-gel.P)
d) Styragel e) Porous glass & silica granules
a) Sephadex:
1. It is most popular gel for proteins & most of the biomolecules separation.
2. When”Leuconosto mesenteroids” indulge in sucrose fermentation, large polymers of glucose are the results. These polymers are known as “Dextrans” are used to prepare sephadex.
3. It is cross-linked polymer.
4. Sephadex gels are insoluble in water, and are stable in bases, weak acids and mild reducing and oxidizing agents.
5. Sephadex gels are insoluble in water, and are stable in bases, weak acids and mild reducing and oxidizing agents.
6. Sphadex, which cannot be used to separate biopolymers larger than 300,000 Daltons.
7. Some identified gels serial number gels are G-25; G-50; G-75; G-100; G-200
b) Agarose:
1. Agarose gels are produced from AGAR.
2. They are linear polysaccharides alternating residues of D-Galactose and 3,6-anhydro-L-galactose units.
3. These gels are hydrophilic and are almost completely free of charged groups.
4. Agarose gels, due to their greater porosity, it may be used to separate molecules and participates up to a molecular weight of several million Daltons.
5. The gels are used in the study of viruses, nucleic acids and polysaccharides.
6. Some commonly used agarose gels are
--> Sepharose 2B
--> Sepharose 4B
--> Sepharose 6B
c) Polyacrylamide:
1. This is very popular medium is produced by polymerizing acrylamide into bead form.
2. polyacrylamide gels can be used to separate molecules of up to 300,000 daltons.
3. This gel is insoluble in water and common organic solvents may be used in the pH range of 2 to 11.
4. Some common gels are,
Bio-gel P 10, Bio-gel P60, Bio-gel P100,
Bio-gel P200, Bio-gel P300
d) Styragel:
1. For completely non-aqueous separations, a gel that will swell in an organic solvent is required. Styragel provides this option.
2. It is a rigid cross-linked polystyrene gel.
3. The gel structure is unaffected by temperatures as high as 1500 C
4. the gel can be used with such solvent as tetrahydrofuran, cresol, dimethyl sulfoxide, chloroform, carbon tetrachloride and others.
e) Controlled pore glass beads:
1. These fine glass spheres are manufactured from borosilicate glass to contain large number of pores within a very narrow size distribution.
2. The glass spheres have a molecular exclusion limit ranging from 3000 to 9 million Daltons.
Procedure:
This is mainly carried out in the columns. The column is filled with beads, these beads contain pores. The beads are made up of gels. The gels are made up of dextrans (Sephadex), Agarose (Biogel-A) and Polyacrylamide (Biogel-P).
The sample is discovered in buffer and allowed to flow through the column. Large molecules can not enter the pores of the beads, they are excluded out and reach down fastly. The small sized molecules enter into the pores of the beads; their speed is retarded and reaches down slowly. These particles are analyzed by spectroscopy.
Applications:
1) The main application of gel-filtration is the purification of molecules, viruses, nucleic acids, hormones, enzymes, proteins, and antibodies and can be separated and purified by this technique.
2) It is also used for the separation of vitamins, steroids, neuropeptides and drugs.
3) Separations are achieved very quickly by this technique.
4) The molecular weight of the molecule can also be determined by this technique.
5) Protein receptor binding can be understood by this technique.
6) This method is especially useful for the separation of 4S and 5S tRNA.
7) It is also the most satisfactory method for separating DNA (from bacteria, usually Gram positive) from the invariable contaminants, the “Teichoic acid”.
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