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Gel chromatography
- 1. © Ramaiah University of Applied Sciences 1 Faculty of Pharmacy © Ramaiah University of Applied Sciences 1 Faculty of Pharmacy © Ramaiah University of Applied Sciences 1 Faculty of Pharmacy © Ramaiah University of Applied Sciences 1 Faculty of Pharmacy GEL CHROMATOGRAPHY By Burhanuddin Madriwala M.Pharm – SEM I Department of Pharmaceutical Chemistry M.S Ramaiah University of Applied Sciences
- 2. © Ramaiah University of Applied Sciences 2 Faculty of Pharmacy © Ramaiah University of Applied Sciences 2 Faculty of Pharmacy © Ramaiah University of Applied Sciences 2 Faculty of Pharmacy © Ramaiah University of Applied Sciences 2 Faculty of Pharmacy GEL CHROMATOGRAPHY
- 3. © Ramaiah University of Applied Sciences 3 Faculty of Pharmacy © Ramaiah University of Applied Sciences 3 Faculty of Pharmacy © Ramaiah University of Applied Sciences 3 Faculty of Pharmacy © Ramaiah University of Applied Sciences 3 Faculty of Pharmacy CONTENTS • INTRODUCTION • HISTORICAL DEVELOPMENT • PRINCIPLE INVOLVED • PHASES OF GEL CHROMATOGRAPHY • TYPES OF GEL CHROMATOGRAPHY • INSTRUMENTATION • CHROMATOGRAM • MERITS & DEMERITS • APPLICATIONS • SUMMARY • REFERENCES
- 4. © Ramaiah University of Applied Sciences 4 Faculty of Pharmacy © Ramaiah University of Applied Sciences 4 Faculty of Pharmacy © Ramaiah University of Applied Sciences 4 Faculty of Pharmacy © Ramaiah University of Applied Sciences 4 Faculty of Pharmacy 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. © Ramaiah University of Applied Sciences 5 Faculty of Pharmacy © Ramaiah University of Applied Sciences 5 Faculty of Pharmacy © Ramaiah University of Applied Sciences 5 Faculty of Pharmacy © Ramaiah University of Applied Sciences 5 Faculty of Pharmacy 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. © Ramaiah University of Applied Sciences 6 Faculty of Pharmacy © Ramaiah University of Applied Sciences 6 Faculty of Pharmacy © Ramaiah University of Applied Sciences 6 Faculty of Pharmacy © Ramaiah University of Applied Sciences 6 Faculty of Pharmacy 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. © Ramaiah University of Applied Sciences 7 Faculty of Pharmacy © Ramaiah University of Applied Sciences 7 Faculty of Pharmacy © Ramaiah University of Applied Sciences 7 Faculty of Pharmacy © Ramaiah University of Applied Sciences 7 Faculty of Pharmacy conti….
- 8. © Ramaiah University of Applied Sciences 8 Faculty of Pharmacy © Ramaiah University of Applied Sciences 8 Faculty of Pharmacy © Ramaiah University of Applied Sciences 8 Faculty of Pharmacy © Ramaiah University of Applied Sciences 8 Faculty of Pharmacy 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. © Ramaiah University of Applied Sciences 9 Faculty of Pharmacy © Ramaiah University of Applied Sciences 9 Faculty of Pharmacy © Ramaiah University of Applied Sciences 9 Faculty of Pharmacy © Ramaiah University of Applied Sciences 9 Faculty of Pharmacy
- 10. © Ramaiah University of Applied Sciences 10 Faculty of Pharmacy © Ramaiah University of Applied Sciences 10 Faculty of Pharmacy © Ramaiah University of Applied Sciences 10 Faculty of Pharmacy © Ramaiah University of Applied Sciences 10 Faculty of Pharmacy 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.
- 11. © Ramaiah University of Applied Sciences 11 Faculty of Pharmacy © Ramaiah University of Applied Sciences 11 Faculty of Pharmacy © Ramaiah University of Applied Sciences 11 Faculty of Pharmacy © Ramaiah University of Applied Sciences 11 Faculty of Pharmacy • 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
- 12. © Ramaiah University of Applied Sciences 12 Faculty of Pharmacy © Ramaiah University of Applied Sciences 12 Faculty of Pharmacy © Ramaiah University of Applied Sciences 12 Faculty of Pharmacy © Ramaiah University of Applied Sciences 12 Faculty of Pharmacy 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.
- 13. © Ramaiah University of Applied Sciences 13 Faculty of Pharmacy © Ramaiah University of Applied Sciences 13 Faculty of Pharmacy © Ramaiah University of Applied Sciences 13 Faculty of Pharmacy © Ramaiah University of Applied Sciences 13 Faculty of Pharmacy 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
- 14. © Ramaiah University of Applied Sciences 14 Faculty of Pharmacy © Ramaiah University of Applied Sciences 14 Faculty of Pharmacy © Ramaiah University of Applied Sciences 14 Faculty of Pharmacy © Ramaiah University of Applied Sciences 14 Faculty of Pharmacy CHROMATOGRAM
- 15. © Ramaiah University of Applied Sciences 15 Faculty of Pharmacy © Ramaiah University of Applied Sciences 15 Faculty of Pharmacy © Ramaiah University of Applied Sciences 15 Faculty of Pharmacy © Ramaiah University of Applied Sciences 15 Faculty of Pharmacy MERITS Short analysis time Well defined separation Less mobile phase consumed Good sensitivity Less chance of sample loss Narrow bands formed
- 16. © Ramaiah University of Applied Sciences 16 Faculty of Pharmacy © Ramaiah University of Applied Sciences 16 Faculty of Pharmacy © Ramaiah University of Applied Sciences 16 Faculty of Pharmacy © Ramaiah University of Applied Sciences 16 Faculty of Pharmacy 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
- 17. © Ramaiah University of Applied Sciences 17 Faculty of Pharmacy © Ramaiah University of Applied Sciences 17 Faculty of Pharmacy © Ramaiah University of Applied Sciences 17 Faculty of Pharmacy © Ramaiah University of Applied Sciences 17 Faculty of Pharmacy 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.
- 18. © Ramaiah University of Applied Sciences 18 Faculty of Pharmacy © Ramaiah University of Applied Sciences 18 Faculty of Pharmacy © Ramaiah University of Applied Sciences 18 Faculty of Pharmacy © Ramaiah University of Applied Sciences 18 Faculty of Pharmacy 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.
- 19. © Ramaiah University of Applied Sciences 19 Faculty of Pharmacy © Ramaiah University of Applied Sciences 19 Faculty of Pharmacy © Ramaiah University of Applied Sciences 19 Faculty of Pharmacy © Ramaiah University of Applied Sciences 19 Faculty of Pharmacy REFERENCES • Rouessac, F. and Rouessac A. (2007) Chemical Analysis - Modern Instrumentation Methods and Techniques. 2nd Edition. England, John Wiley & Sons Ltd Publishing, pp.160-167. • https://www.chromatographyonline.com/view/early-development-size-exclusion- chromatography-historical-perspective.