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Gel filtration chromatography can be used to separate compounds such as small molecules, proteins, protein complexes, polysaccharides, and nucleic acids when in aqueous solution.
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CHROMATOGRAPHY Chromatography literally means “colour writing” Chromatography was invented by Russian botanist Mikhail Tsvet in 1900. He used it to separate chlorophyll containing extracts of plants. Chromatography is process in which separation of mixture compounds using the stationary phase and mobile phase.
Techniques of chromatography 1.Plane chromatography. 2. Column chromatography. Column chromatography are of different types: Affinity chromatography. Ion Exchange chromatography. Size Exclusive chromatography. Adsorption chromatography.
Gel filtration chromatography Size-exclusion chromatography(SEC) also called Gel filtration or Gel permeation chromatography is a chromatographic method in which molecules in solution are separated by their size, and in some cases molecular weight. It is usually applied to larger molecules or macromolecular complexes such as proteins and industrial polymers Gel filtration medium is packed into a column to form a packed bed.
The medium is a porous matrix in the form of spherical partials that have been chosen for their chemical and physical stability and inertness. A mixture of molecules dissolved in liquid (the mobile phase) is applied to a chromatography column which contains a solid support in the form of microscopic spheres, or “beads” (the stationary phase). The mass of beads within the column is often referred to as the column bed. The beads act as “traps” or “sieves” and function to filter small molecules which become temporarily trapped within the pores.
Column parameter Vo = void volume Vt = total volume Vs= volume of solvent held in the pores. This is normally approximated to Vt-Vo = volume of beads
Concept of Plates: Column Efficiency Column can be divided into a number of identical segments such that one equilibrium distribution takes place on each of these segments. Each of these segments is called theoretical plate. Thus, the number of equilibrations taking place in a column is equal to the number of plates that the column possesses. The column which has a larger number of plates will be better suited for resolving these compounds whereas a column which has small number of plates will not be as efficient. Therefore column efficiency is directly related to the number of plates it possesses
Types of gels used Sephadex Gel : It’s mainly used for separation of small peptides and globular protein with small to average molecular mass.its separate molecular weight limit up to 5000-200000 Daltons. Polyacrylamide gel : This type of gel can be used to separate molecules of up to 300000 Daltons, Agarose gel : Agarose gel produced from agar. It's useful for analyses or separation of large globular proteins or long linear molecules such as DNA. Agarose gel can be used to separate molecules and particles up to a molecular weight more than 300000 Daltons.
Separation procedures 1. Preparation of column for gel filtration Swelling of the gel Packing of the column Washing with the buffer 2. Loading the sample onto the column 3. Eluting the sample and detecting of compounds
If the mixture of molecules of different size is placed on the top of such an equilibrated column. Molecules larger than the pore size can not enter the pores and elute together as the first peak in the chromatogram. Different molecules therefore have different total transit times through the column. Molecules that are smaller than the pore size can enter all pores, and have the longest residence time on the column and elute together as the last peak in the chromatogram. The collected fraction are often examined by spectroscopic technique to determine the concentration of partial eluted.
Aaa Theoretical chromatogram of a high resolution fractionation (UV absorbance)
Applications of Gel Filtration Chromatography It’s the best method for separation of molecules differing in molecular weight because: It doesn’t depend on temperature, pH, ionic strength and buffer composition. So separation can be carried out under any condition. The elution volume is related to the molecular weight. Purification of biomolecules. Estimation of molecular weight mainly for proteins.
Molecular weight determination by gel filtration In gel filtration experiment if one determines the effluent volume for a macromolecules, one can calculate its distribution coefficient. If distribution coefficient of standard proteins of known molecule weight are plotted against the log of their molecular weights. The position of the distribution coefficient of the unknown protein on the plot will lead to the determination of its molecular weight.
Molecular weight and elution volume for proteins from a gel permeation chromatography
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