Gel chromatography

definition
advantages
disadvantages
principle
theory of separation
types of gels used
components or instrumentation
separation procedures
applications

1. GEL CHROMATOGRAPHY Submitted by- Mehul H Jain M. Pharmacy 1 st Sem Pharmaceutical Analysis Modern Pharmaceutical Analytical Techniques

2. INTRODUCTION DEFINITION: Gel Chromatography (known as gel permeation, molecular sieving or size exclusion chromatography) is a chromatographic technique in which the separation of components based on the difference of molecular weight or size, and is one of the effective methods used to isolate and analyze the bio-macromolecular substances. The stationary phase consists of beads containing pores that span a relatively narrow size range. When the gel is packed into a column and percolated with a solvent, it permits the large molecular weight components to pass faster without penetration of the pores (totally excluded). Smaller molecules spend more time inside the beads and therefore is eluted later (after a larger volume of mobile phase has passed through the column).

3. When an organic solvent is used as a mobile phase, then it is tend to called as gel permeation chromatography. When an aqueous solution is used to transport the sample through the column, the technique is known as gel filtration chromatography. In 1954, the scientist Mould and Sunge showed that the separation of uncharged substances can be performed based on the size of molecules. However the systematic use of this principle was introduced by Porth and Foblin in 1954 who separated the molecules of biological origin using polysaccharide gel (Sephadex). Stationary phase: Polysaccharide gel (cross-linked to form pores) Mobile phase: Liquid solvent Sample: Molecules of biological origin (different sizes)

4. ADVANTAGES: Short analysis time. Well defined separation. Narrow bands and good sensitivity. There is no sample loss. Small amount of mobile phase required. The flow rate can be set. DISADVANTAGES: Limited number of peaks that can be resolved within the short time scale. Filtrations must be performed before using the instrument to prevent dust and other particulates from ruining the columns and interfering with the detectors. The molecular masses of most of the chains will be too close for the separation to show anything more than broad peaks.

5. PRINCIPLE The stationary phase in this method is an open network of polymers which are cross-linked to each other to form the pores of consistent size. The degree of cross-linking of polymer mainly governs the pore size. When the mobile phase containing mixture of solutes of various sizes is passed through the column, the molecules which are too large to enter the pores are “excluded” completely and get eluted with the mobile phase. The molecules which are smaller in size diffuse in and out of the pores, thus the path travelled by them through the column is quite longer and they are eluted later. The molecules which are of intermediate size cannot diffuse into the earlier pores as the space is already occupied by the smaller molecules hence they get separated quite later along the length of column. Thus, the components of mixture get eluted from the column in the order of their relative molecular sizes.

6. The one with largest molecular mass or size is eluted first, followed by elution of intermediate sized molecules. The smaller size molecules are eluted in the last order of elution. Large size > Intermediate size > Smallest size

7. THEORY OF SEPARATION A column is made up of swollen gel particles and the solvent used to swell the gel in a suitable tubular container. An equation is given below: V t = V + V i + V m Where, V t = the total volume of the column (which can be measured), V = the volume of liquid outside the gel matrix (known also void or dead volume), V i = the volume of liquid inside the matrix, V m = the volume of the gel matrix

8. TYPES OF GELS USED The gels used as molecular sieves are cross linked polymers. They are uncharged and inert i.e., don’t bind or react with the materials being analyzed. Three types of gels are used: Dextran (Sephadex) Agarose gel Acrylamide gels (synthetic gel) Nature of Gel: Chemically inert Mechanically stable Ideal porous structure Wide pore size give low resolution Uniform particle size

9. DEXTRAN (SEPHADEX): Dextran is a homopolysaccharide of glucose residues. It is prepared with various degrees of cross-linking to control pore size. It is bought as dry beads, the beads swell when water is added. 1-6 polymer of glucose is prepared by microbial fermentation of sucrose (glucose + fructose). The resulting glucose provides the required α 1-6 glucosan polymer called dextran. The resulting dextran is treated with epichlorohydrin to give several types of cross-linked dextran (sephadex). It is mainly used for separation of small peptides and globular proteins with small to average molecular mass. Sephadex is obtained in different degrees depending on the pore size. High percentage of epichlorohydrin give high degree of cross linking (small pore size). Lower percentage produce sephadex with large pore size.

10. Characters of Sephadex: Highly stable gels. Stable at pH 2-12. Their particles are free from ions. Insoluble in water and organic solvent. They swell in water and other hydrophilic solvent. They require bactericidal such as Hg acetate. AGAROSE GEL: Obtained from agar and composed of alternating units of 1,3 linked β-D-gal and 1,4 linked 3,6-anhydro-α, L-galactose. This was subjected to epichlorohydrin to give sepharose. Characters:  It dissolves in H 2 O at 50 º C and on cooling form gel. Insoluble below 40 º C. Freezing destroys the gel.

11. ACRYLAMIDE GELS (SYNTHETIC GEL): It is not dextran polymer. It is polymerized acrylamide or methylene-bis-acrylamide. The pore size is determined by the degree of cross-linking. The separation properties of polyacrylamide gels are mainly The same as those of dextrans. They are sold as bio-gel P. They are available in wide range of pore sizes. According to the swelling process, the gels are two types: Soft gels (Xerogel i.e., gel only on swelling): Example: Polyacrylamide gels, dextran or agarose (used for separation of proteins in aqueous mobile phase). Semirigid or rigid gels (aerogel i.e., gel in air): Polystyrene gels (separation of non-polar polymers in non-polar solvents). Porous glass gels (separation of polar systems).

12. COMPONENTS Stationary Phase The Mobile Phase The Columns The Pump Detectors STATIONARY PHASE: Composed of semi-permeable, porous polymer gel beads with well defined range of pore sizes. Properties of gel beads: Chemically inert. Mechanically stable. Has ideal and homogeneous porous structure (wide pore size give low resolution). Uniform particle and pore size. The pore size of the gel must be carefully controlled.

13. Examples of gel: Dextran(Sephadex) gel: An α 1-6-polymer of glucose natural gel. Agarose gel: A 1,3 linked β-D-galactose and 1,4 linked 3,6-anhydro-α, L-galactose natural gel. Acrylamide gel: A polymerized acrylamide, a synthetic gel. THE MOBILE PHASE: Composed of a liquid used to dissolve the bio-molecules to make the mobile phase permitting high detection response and wet the packing surface. MATERIAL SOLVENT Synthetic elastomers (polybutadiene, polyisoprene) Toluene PS, PVC, Styrene-Butadiene Rubber, Epoxy resins Tetrahydrofuran (THF) Polyolefins Tri-chloro-benzene Polyurethane Di-methylformamide (DMF) Proteins, polysaccharides Water / Buffers

14. COLUMNS: Commercially Available Columns include Analytical column- 7.5–8mm diameters. Preparative columns-22–25mm. Usual column lengths-25, 30, 50, and 60 cm. Narrow bore columns- 2–3mm diameter have been introduced. THE PUMP: These are either syringe pumps or reciprocating pumps with a highly constant flow rate.

15. DETECTORS: CONCENTRATION SENSITIVE DETECTORS: Bulk Property Detectors- Refractive Index (RI) Detector. Solute Property Detectors- Ultraviolet (UV) Absorption Detector. Evaporative Detectors- Evaporative Light Scattering Detector (ELSD). MOLAR MASS SENSITIVE DETECTORS: Light Scattering Detectors: Low Angle Light Scattering (LALS) Detectors. Multi Angle Light Scattering (MALS) detectors. Viscosity Detectors- Differential Viscometers.

16. SEPARATION PROCEDURE PREPARATION OF COLUMN FOR GEL FILTRATION: Swelling of the gel: Some resin come in a powder form. These must be sonicated first in the eluent or the desired buffer to swell. Packing the column: Make a slurry of gel + buffer and pour it into column which is one third filled with the buffer. Washing the resin: After packing, pass several column volumes of the buffer through the column to remove any air bubbles and to test the column homogeneity. LOADING THE SAMPLE ONTO THE COLUMN: The sample must enter the resin in the form of solution using a syringe. ELUTING THE SAMPLE AND DETECTION OF COMPONENTS: Fractions are collected as the sample elutes from the column.

17. APPLICATIONS Gel chromatography is mainly used for the separation of sugars, polysaccharides, proteins, lipids, polymers and other materials. PURIFICATION: This technique is used for purification of biological molecules. Different proteins, enzymes, hormones, antibodies, polysaccharides have been separated and purified by using appropriate gels. Low molecular weight dextrans can be separated from corn syrup oil. DESALTING: This method of desalting is faster and more efficient than dialysis. Examples of desalting process include separation of monosaccharides from polysaccharides and separation of amino acids from proteins.

18. FRACTIONATION: In this method of separation, the similar substance are eluted closer to each other. Thus, the separation of substances which has nearly equal molecular size can be separated. PROTEIN-BINDING STUDIES DETERMINATION OF MOLECULAR WEIGHT: It is assumed that the size of molecule is proportional to the molecular weight. Their relation is expressed by an equation V E = a + b logM Where, V E = elution time M = molecular weight a, b = constants that depend on stationary phase and mobile phase.

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