Gel chromatography

it is a chromatographic technique widely used in separation of chemicals.

1. GEL CHROMATOGRAPHY By Burhanuddin Madriwala M.Pharm – SEM I Department of Pharmaceutical Chemistry M.S Ramaiah University of Applied Sciences

2. GEL CHROMATOGRAPHY

3. CONTENTS INTRODUCTION HISTORICAL DEVELOPMENT PRINCIPLE INVOLVED PHASES OF GEL CHROMATOGRAPHY TYPES OF GEL CHROMATOGRAPHY INSTRUMENTATION CHROMATOGRAM MERITS & DEMERITS APPLICATIONS SUMMARY REFERENCES

4. INTRODUCTION Gel chromatography is a technique where components of a mixture are separated based on their different molecular sizes on a porous gel material used as stationary phase. Also knowns as molecular sieve or size exclusion chromatography. Mainly used for separation of macromolecules like proteins or synthetic polymers.

5. HISTORY Discovered after 50 years from Tswett’s discovery of chromatography in 1906. In 1955–1956, two British biochemists, Lathe and Ruthven – separation of polysaccharides and proteins on swollen starch granules. The next milestone occurred in 1959, when Per Flodin with Jerker Porath - demonstrated size separation of peptides and oligosaccharides through a series of cross-linked dextran packings. In early 1960s, scientists focused on the application of hydrophobic packings for the Size Exclusion Chromatography of synthetic polymers. Concept of separation of homologues series of hydrocarbons based on molecular size on swollen rubber granules was first demonstrated by Brewer. In 1962, John Moore produced a series of cross-linked polystyrene resins for the separation of synthetic polymers. In 1963 – first commercial GPC equipment developed by Jim Waters.

6. PRINCIPLE Two principles are involved: 1) Size separation2) Degree of penetration. The internal pores separates small molecules from large size molecules. The small molecules having size range less than internal pore size of gel beads get stuck in the them while large molecules i.e. size greater than internal pores passes through void volumes. Large molecules are separated based on the movement through void space. Small molecules are separated based on degree of penetration inside the pores.

7. conti….

8. PHASES OF GEL CHROMATOGRAPHY STATIONARY PHASE on which separation takes place. Microporous gel material having bead like structure with internal pores. Rigid, semi rigid and soft gels used. Size of gel beads range from 3-20 µm with internal pore size of 4-200 nm. Examples include dextran(sephadex ) , agarose(sepharose) , polyacrylamide gel ,porous silica gels etc. Type of gel to be used depends on size range of molecules to be separated.

9. Conti….. Properties of gel Chemically inert. Mechanically stable. Should withstand pressure of mobile phase. Should be stable at temperature of up to 100°C. Should have uniform porosity. Should have larger size of beads to allow proper separation of higher molecules. Mobile phase Aqueous &non-polar solvents both are used. Examples of non-polar solvents include tetrahydrofuran, trichloromethane , hot trichlorobenzene etc.

10. Examples of aqueous solvents include water or buffer solutions. Buffer solutions used are: - 1. Tris buffer (tris(hydroxymethyl)aminomethane) – pH:7.5-9.0 2. Sodium phosphate buffer mostly used – pH: 5.8-7.4 3. MOPS buffer (3-(N-morpholino)propane sulfonic acid) – pH: 6.5-7.9 4. MES buffer (2-(N-morpholino)ethane sulfonic acid) - pH: 5.5-6.7 5. BES buffer (N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid) - pH: 6.4-7.8

11. TYPES OF GEL CHROMATOGRAPHY Gel filtration chromatography Stationary phase is hydrophilic. Aqueous mobile phase used. Polyvinyl alcohols used as gel. Copolymers of polyglycerometh-acrylates or vinyl polyacetates are also used as gels. Water or buffer solutions are used as mobile phase. Soft gels are used. Biomolecules are separated. Gel permeation chromatography Stationary phase is hydrophobic. Organic solvents used as mobile phase. Styrene-divinylbenzene copolymer gel is mostly used. Tetrahydrofuran used as mobile phase. Semi-rigid & rigid gels are used. Synthetic polymers are separated.

12. INSTRUMENTATION Solvent delivery system Pumps Autosampler Sample injector Column(30 cm with internal diameter of 7.5 mm) Detector( refractive index , light scattering ,uv absorption detectors etc.) Recorder

13. CHROMATOGRAM

14. MERITS Short analysis time Well defined separation Less mobile phase consumed Good sensitivity Less chance of sample loss Narrow bands formed

15. DEMERITS Low resolution Proteolysis problem in protein separation Pre-filtration of sample required Less efficient than other techniques Limited number of peaks Difference(>10%) in size of different molecules required

16. APPLICATIONS Determination of molecular weight of different compounds using hyphenated techniques like LC-MS or molar mass sensitive detectors. Used for separation of biomolecules like proteins, polysaccharides, nucleic acids, hormones, enzymes etc. Estimating molecular weights of synthetic polymers, elastomers, polyisoprene, rubbers etc. Characterisation of properties of different macromolecules. Study of quaternary structure of proteins.

17. SUMMARY Gel chromatography separates molecules based on their different sizes using gel as a stationary phase. No distribution or chemical interaction involved in the technique. It is divided into gel permeation & gel filtration chromatography based on type of stationary & mobile phase used. Stationary & mobile phase can be hydrophilic & hydrophobic in nature. Mostly used for separation of macromolecules. Used for separation of wide range of polymers, biomolecules etc.

18. REFERENCES Rouessac, F. and Rouessac A. (2007) Chemical Analysis - Modern Instrumentation Methods and Techniques. 2 nd Edition. England, John Wiley & Sons Ltd Publishing, pp.160-167. https://www.chromatographyonline.com/view/early-development-size-exclusion-chromatography-historical-perspective.