GEL PERMEATİON CHROMATOGRAPHY (GPC).pptx

GEL PERMEATİON CHROMATOGRAPHY

1. Gel Permeation Chromatography (G PC ) Prepared by: Sami FARAJALLAH Karadeniz Teknik Üniversitesi Supervisor: Prof. Dr. Murat KÜÇÜK

2. Chromatography is an important biophysical technique that enables the separation, identification, and purification of the components of a mixture for qualitative and quantitative analysis. Chromatography T ypes Gas Chromatography. High Performance Liquid Chromatography. Gel Permeation Chromatography. 2

3. GPC Size- exclusion chromatography  (SEC) M olecular sieve chromatography G el- filtration chromatography G el chromatography molecular sieve chromatography 3

4. Discovery of GPC İ nvented in 1955 by Grant Henry Lathe and Colin R Ruthven, working at Queen Charlotte's Hospital, London. Lathe and Ruthven used starch gels as the matrix . Jerker Porath  and  Per Flodin  later introduced dextran gels , agarose and polyacrylamide. J. C. Moore of the  Dow Chemical Company   - preparation o f (GPC) columns based on cross-linked  polystyrene   4

5. Components of Gel Permeation Chromatography Stationary Phase The Mobile Phase The Columns The Pump Detectors  5

6. Applications of GPC F or the analysis of large molecules such as proteins or polymers T o analyze the molecular weight distribution of organic-soluble polymers.   O ther technique should not be confused with  gel electrophoresis , where an electric field is used to "pull" or "push" molecules through the gel depending on their electrical charges . T he amount of time a solute remains within a pore is dependent on the size of the pore. Larger solutes will have access to a smaller volume and vice versa . A smaller solute will remain within the pore for a longer period of time compared to a larger solute. 6

7. Applications of GPC T o examine the stability and characteristics of natural organic matter in water . W idely utilized to study natural organic material. 7

8. GPC T heory M olecules in solution are separated by their size or  molecular weight. A pplied to large molecules or macromolecular complexes such as proteins and industrial polymers. O rganic solvent is used as a mobile phase to transport the sample through the column . C olumn is packed with fine, porous beads , composed of dextran, agarose, or polyacrylamide polymers. The pore sizes of these beads are used to estimate the dimensions of macromolecules. 8

9. GPC T heory W orks by trapping smaller molecules in the pores of the  adsorbent ("stationary phase") in the column which typically consists of a hollow tube tightly packed with micron-scale polymer beads containing pores of different size .  The larger the particles, the faster the elution. The larger molecules simply pass by the pores because those molecules are too large to enter the pores . W idely used polymer characterization method because of its ability to provide good molar mass distribution (Mw) results for polymers. 9

10. GPC

11. GPC 11

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19. Chromatogram 19

20. The resulting chromatogram is therefore a weight distribution of the polymer as a function of retention volume. 20

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22. Stationary phase   It is composed of semi-permeable, porous polymer gel beads with a well-defined range of pore sizes with the following properties: Chemically inert Mechanically stable With ideal and homogeneous porous structure (wide pore size give low resolution). A uniform particle and pore size EXAMPLE : 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. 22

23. Mobile phase ( Eluent ) C omposed of a liquid used to dissolve the bio-molecules to make the mobile phase permit high detection response and wet the packing surface. G ood solvent for the polymer , should permit high detector response from the polymer . C ommon eluents for polymers that dissolve at room temperature . Tetrahydrofuran  (THF),  o - dichlorobenzene   and   trichlorobenzene  at 130–150 °C for crystalline   polyalkynes   and   m - cresol .   o - chlorophenol  at 90 °C for crystalline condensation polymers such as  polyamides   and   polyesters . 23

24. Pump There are two types of pumps available for uniform delivery of relatively small liquid volumes for GPC: piston pumps peristaltic pumps 24

25. PİSTON PUMP 25

26. Peristaltic Pump 26

27. Detector D etector types can be divided into two main categories. C oncentration sensitive detectors which includes UV absorption,  differential refractometer  (DRI) or refractive index (RI) detectors, infrared (IR) absorption and density detectors . M olecular weight sensitive detectors, which include  low angle light scattering  detectors (LALLS) and multi angle light scattering (MALLS). 27

28. Detector The most sensitive detector is the differential UV photomete r and the most common detector is the differential refractometer (DRI). When characterizing copolymer, it is necessary to have two detectors in series. For accurate determinations of copolymer composition at least two of those detectors should be concentration detectors. The determination of most copolymer compositions is done using UV and RI detectors, although other combinations can be used. 28

29. A size exclusion column 29

30. Column Columns mostly made from hydrophilic gels of  dextran ,  agarose   or  polyacrylamide The column used for GPC is filled with a microporous packing material called gel. 30

31. Advantages of GPC Short analysis time. Well defined separation. Narrow bands and good sensitivity. There is no sample loss. The small amount of mobile phase required. The flow rate can be set. good separation of large molecules from the small molecules with a minimal volume of eluate determining the molecular weight of polymer macromolecules. The separation is based on the analyte molecular sizes since the gel behaves like a molecular sieve. 31

32. Disadvantages of Gel Permeation chromatography The number of peaks that can be resolved within the short time frame of a GPC run is small . For a satisfactory resolution of peaks , GPC as a technique needs at least a 10% difference in molecular weight . T he molecular masses of most of the polymer chains are too close together for the GPC separation to produce anything other than large peaks . For polymers it needs filtration prior to use to prevent dust and other particulates from destroying the columns and interfering with the detectors . limited number of bands can be accommodated because the time scale of the chromatogram is short , as 10% difference in molecular mass to have a good resolution. This relative data can be used to calculate molecular weights within 5% precision if comparable criteria are used . To calibrate the GPC, polystyrene standards with disparities of less than 1.2 are commonly used . 32

33. GP G C(GAZA POWER GENERATİNG COMPANY) 33