GEL CHROMATOGRAPHY

In this slide contains principle, instrumentation, methodology, and application of gel chromatography.
Presented by: SATHEES CHANDRA (Department of pharmaceutical analysis).
RIPER, anantapur

1. 1 Gel Chromatography A Seminar as a part of curricular requirement for I year M. Pharm I semester Presented by G. Sateesh Chandra (Reg. No. 20L81S0711 ) Under the guidance/Mentorship of Dr. P. Ramalingam , M. Pharm, Ph. D., Director-R&D Division, Professor of Pharmaceutical Analysis And Medicinal Chemistry.

2. 2 Introduction Principle Distribution theory Instrumentation Stationary phase Mobile phase Columns Pumps Detectors Methodology Applications References Content

3. 3 Gel chromatography is also known as gel permeation chromatography(GPC), Size exclusion chromatography, Gel filtration, Molecular-sieve chromatography. This is a chromatographic technique that separates, dissolved molecules on the basis of their size by pumping them through specialized columns containing a microporous packing material(gel). It is one of the effective methods used to isolate and analyze the bio-macromolecular substances. Introduction

4. 4 Principle It’s a technique that separates dissolved molecules on the basis of their size by pumping these molecules through specialized columns containing a microporous packing material(gel ). Stationary phase is a porous polymer matrix whose pores are completely filled with the solvent to be used as the mobile phase. The flow of mobile phase will cause larger molecules to pass through the column unhindered, without penetrating the gel matrix. whereas smaller molecules will be retarded according to their penetration of the gel.

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6. 6 Distribution theory Total volume of column packed with a solid matrix that has been swelled by water or other solvent is given by V t =V o +V i +V m Where V t = T otal volume of the column V o = Volume of liquid outside gel matrix V i = Volume of liquid inside matrix V m = Volume of the gel matrix Fractionation range

7. 7 If conditions are assumed such that: Time taken for solute molecules to diffuse into pore is less as compared to time spent by molecule near pore. Separation process independent of diffusion process. • Under these conditions V e = V o + K d . V i V e =vol. of effluent flowing through column between point of sample injection & sample emergence from column. K d = distribution coefficient. • For large molecules k d = 0, V e = V o • For molecules that can penetrate all the pores k d = 1, V e = V o + V i

8. Stationary phase Nature of Gel Chemically inert. Mechanically stable. Uniform particle size. Gel should have carefully formed reproducible structure . Instrumentation 8 Fractionation range of gel

9. 9 There are two main types of gel materials: 1. Xerogels: Which consists consist cross-linking polymers which swell when come in contact with the solvent to form a relatively soft porous medium. If liquid removes the structure collapse and cannot be restored. 2 . Aerogels : Rigid material which is not a gel at all. Porous material, which does not collapse when solvent is removed. eg: porous glass, porous silica The gel which commonly used included cross-linked dextrants, agarose, polyacrylamide, polystyrene, polyacryloylmorphine.

10. 10 A homopolysaccharide of glucose residues. It’s prepared with various degrees of cross-linking to control pore size. It’s bought as dry beads, the beads swell when water is added. The trade name is Sephadex. It’s mainly used for separation of small peptides and globular proteins with small to average molecular mass. Dextrants

11. 11 These gels are prepared by cross linking acrylamide with N,N- 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. Polyacrylamide

12. 12 Linear polymers of D-galactose and 3,6 anhydro-1-galactose . It forms a gel that’s held together with H bonds. It’s dissolved in boiling water and forms a gel when it’s cold. The concentration of the material in the gel determines the pore size. The pores of agarose gel are much larger than those of Sephadex or bio-gel p . It’s useful for analysis or separation of large globular proteins or long linear molecules such as DNA. Agarose

13. 13 Composed of a liquid used to dissolve the biomolecules to make the mobile phase permitting high detection response and wet the packing surface. ii. Mobile phase 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/Buffer

14. 14 Gel chromatography instrumentation.

15. 15 iii. Columns Commercially Available Columns: analytical column- 7.5–8mm diameters. Preparative columns -22–25mm. Usual column lengths-25, 30, 50, and 60 cm. Recently, narrow bore columns- 2–3mm diameter have been introduced, which save time and solve. iv. Pumps Types of pumps Syringe pump Reciprocating pumps

16. 16 V . Detectors Different detectors used in identification of components 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

17. Column preparation Gel preparation Dry powder is mixed with solvent and allowed to swell, till equilibrium is attained then go for column packing. In this method slurry is heated at 100ᵒc. It swells and go for column packing. B. Column packing: Gel material is mixed with appropriate solvent i.e, mobile phase, to get the gel suspension. Make the gel suspension free of air by bubbling it through the addition of mobile phase. Well mixed slurry is poured into column and equilibrium is attained attained by passing 2-3 volumes of mobile phase after settle. METHODOLOGY 17

18. 18 Sample preparation: Solid particles and any substances which may be strongly adsorbed on the gel should be removed from the sample by filtration. Mass and concentration of the solute is essential, as it affect the viscosity of solute. The largest volume which can be handled for complete separation in one run through column is V e1 - V e2. For most closely related samples, volume of sample is 10-30% of V t. For closely related samples, volume of samples is 1-3% of V t.

19. 19 Loading of sample into column 4. Eluting the sample and detection of components

20. 20 Group separation (Purification) The simpliest application of GC is separation of solutes of widely different sizes. E.g: Low mol.wt. materials such as urea, sugars and peptides liberated during chemical modification of proteins can be separated. Molecular weight determination The substances were eluted in the order of decreasing molecular weight initiated studies exploring relationship between chromatographic behavior of molecules and mol.wt. E.g: Separation and examination of synthetic polymers. Applications

21. 21 Solution concentration Solutions of high mol. Wt. substances can be concentrated by addition of Sephadex G-25 (coarse) Desalting Large molecules of biological origin are separated from inorganic or ionisable species is known as desalting. E. g: Separation haemoglobin and sodium chloride. Protein building studies The binding ratio can be estimated from peak area measurements obtained from the chromatogram. Group separation of hemoglobin and sodium chloride.

22. 22 Gurudeep R. Chatwal, Sham K. Anand. “Instrumentation Method of Chemical Analysis”. A. Braithwaite, F.J. Smith . “Textbook of Chromatographic Methods” 5 th edition. Ravi Shankar. “ Text book of pharmaceutical analysis” 5 th edition 2018 p.18.3-18.4 Willard, Merritt and Settle. “Instrumental Methods of Analysis” 1986. References

23. 23 Thank you