Gel-filtration Chromatography:
Gel-filtration chromatography is a separation based on size. It is also called molecular exclusion or gel permeation chromatography. In gel filtration chromatography, the stationary phase consists of porous beads with a well-defined range of pore sizes. The stationary phase for gel filtration is said to have a fractionation range, meaning that molecules within that molecular weight range can be separated.
Proteins that are small enough can fit inside all the pores in the beads and are said to be included. These small proteins have access to the mobile phase inside the beads as well as the mobile phase between beads and elute last in a gel-filtration separation. Proteins that are too large to fit inside any of the pores are said to be excluded. They have access only to the mobile phase between the beads and, therefore, elute first. Proteins of intermediate size are partially included-meaning they can fit inside some but not all of the pores in the beads. These proteins will then elute between the large ("excluded") and small ("totally included") proteins.
Consider the separation of a mixture of glutamate dehydrogenase (molecular weight 290,000), lactate dehydrogenase (molecular weight 140,000), serum albumin (MW 67,000), ovalbumin (MW 43,000), and cytochrome c (MW 12,400) on a gel-filtration column packed with Bio-Gel P-150 (fractionation range 15,000 to 150,000). When the protein mixture is applied to the column, glutamate dehydrogenase would elute first because it is above the upper fractionation limit. Therefore, it is totally excluded from the inside of the porous stationary phase and would elute with the void volume (VO). Cytochrome c is below the lower fractionation limit and would be completely included, eluting last. The other proteins would be partially included and elute in order of decreasing molecular weight.
These separations can be described with this equation
Vr, = Vo + KVi
where Vr is the retention volume of the protein, Vo is the volume of mobile phase between the beads (outside the beads) of the stationary phase inside the column (sometimes called the void volume), Vi is the volume of mobile phase inside the porous beads (also called the included volume), and K is the partition coefficient (the extent to which the protein can penetrate the pores in the stationary phase, with values ranging between 0 and 1). In the mixture of proteins listed above, the partition coefficient (K) for glutamate dehydrogenase would be 0 (totally excluded), K = 1 for cytochrome c (totally included), and K would be between 0 and 1 for the other proteins, which are within the fractionation range for the column.
In practice, gel-filtration can be used to separate proteins by molecular weight at any point in purification of a protein. It can also be used for buffer exchange; a protein dissolved in a sodium acetate buffer, pH 4.8, can be applied to a gel-filtration column that has been equilibrated with Tris buffer pH 8.0. Using the Tris buffer as the mobile phase, the protein moves into the Tris mobile phase as it travels down the column, while the much smaller sodium acetate buffer molecules are totally included in the porous beads and travel much more slowly than the protein. Similarly, it can be used for the separation of salts and other small molecules from a protein sample.
Osmotic Pressure:
Molecules always move from the region of their higher concentration to the region of their lower concentration. It is applicable to both solvent and solute molecules in the case of a solution. When a solution is separated from a pure solvent by a membrane that is permeable to the solvent alone, the molecules of the solvent will move into the solution. The flow of the solvent molecules into the solution can be prevented by applying some amount of pressure that is equal to the pressure exerted by the solvent molecules to enter into the solution compartment through the membrane partition. The pressure that is needed to prevent the entry of the solvent molecule into the solution is called osmotic pressure.
The osmotic pressure of a solution depends on the concentration of a solute and the temperature of the solution. It can be used for the calculation of the molecular weight of the solute.
#V=nRT
where #is the osmotic pressure, V is the volume of the solution, n is the number of moles of solute, R is gas constant, and T is the absolute temperature.
#= n/V x RT (n/V= M, molarity of the solution)
Therefore, # = MRT
But in this equation, n-the number of moles = weight of the solute in grams/molecular weight.
i.e.#V = Wt 9 /MW x RT Therefore,
MW = Wt 9 /#V x RT. (Wt 9 /Volume = Concentration, C)
i.e. MW = CRT/#.
Thus, if the osmotic pressure and the concentration of the solution are available the molecular weight of the solute can be determined.
Tags: Bio Technology, Bio Genetics , Biochemical Techniques
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