GEL CHROMATOGRAPHY

GEL CHROMATOGRAPHY also known as size exclusion chromatography or gel permeation chromatography

1. GEL CHROMATOGRAPHY (GC) PRESENTED BY: Koriya K rupali Department of Pharmaceutical Quality Assurance M.Pharm (Sem : 1) KBIPER

2. CONTENTS Introduction Gel Chromatography ( GC) GC Separation Mechanism Theory Instrumentation Applications Ref e rences

3. GEL CHROMATOGRAPHY ( GC ) Its also known as gel permeation chromatography(GPC) or Size exclusion chromatography (SEC) GC/ GPC/SEC uses columns packed with very small, round, porous particles to separate molecules contained in the solvent that is passed through them . GC/GPC/SEC Separates molecules on the basis of their size , hence ‘ Size Exclusion ’. The first GC/ GPC/SEC columns were packed with materials referred to as gels, hence ‘gel permeation’ . GC/ GPC/SEC is used to determine the molecular weight distributions of polymers .

4. GC SEPERATION MECHANISM

5. Pass through the column unhindered, without penetrating the gel matrix. Thus excluded, they travel mostly around the exterior of the packing. These will be retarded according to their penetration of the gel. Thus very small molecules diffuse into all or many of the pores accessible to them. Small molecule exit the column last Intermediate size molecule exit at intermediate times. LARGE MOLEC U L E S SMALL MOLEC U L E S IN T ERME D I A T E MOLECULES

6. Total volume of column packed with a solid matrix that has been swelled by water or other solvent is given by V t = V g + V M + V S V t = total bed volume V g = volume occupied by solid matrix. V M = void volume of mobile phase i.e. unbound solvent in interstices between the solvent loaded porous particles. V S = volume of solvent held in pores THEORY

7. Mehak 14320022 Nov 2'2015 12 MAIN COMPONENTS OF GC

8. Solvent and Solvent containers The solvent must be able to dissolve the sample, sometimes a polymer insoluble at room temperature will dissolve at higher temperature. The solvent must not induce any other interactions between the sample and the stationary phase, so that the separation is solely on the basis of sample size. The solvent container should be made of clear glass, or amber glass for solvents affected by sunlight, with a stopper to exclude dust and limit evaporation. Solvent Container

9. PUMPS The pump takes the solvent and delivers it to the rest of the system at a constant, accurate and reproducible flow rate. The pump has to be able to run the same flow rate regardless of viscosity, so that results can be compared from one analysis to another The pressure delivered by the pump also needs to be smooth so that there are no pulses in the flow.

10. OVENS GC is usually carried out at room temperature, but some instruments have heated and thermostatically controlled ovens in which the columns and detectors are placed. Higher temperatures, up to 220 °C, are necessary for some solvents that have much higher viscosities, such as trichlorobenzene or chloronaphthalene . Operating the instrument at high temperatures reduces viscosity and hence column back pressure, with a corresponding increase in efficiency.

11. SAMPLES To prepare a sample for analysis it is first dissolved in an appropriate solvent, such as tetrahydrofuran (THF) for organic GC . Since the separation obtained depends on the size of the sample molecules, it is important that they are allowed to swell and then fully dissolve in the solvent before being put through the chromatograph . Where possible, the eluent used to prepare the samples should be the same as the solvent running through the system

12. INJECTION AND INJECTORS Injectors introduce the polymer sample into the flowing solvent stream. It is important that the injector does not disturb the flow of the mobile phase

13. Different Types Of Column Packing Column packing Semi rigid Cross linked macromolecular polymers . Rigid Controlled pore- size glasses or silica Separation of the sample takes place inside the column, a hollow tube tightly packed with extremely small porous beads, typically polymer or silica. The columns vary in length from 50 mm to 300 mm , and internal diameters of 4.6 to 25 mm , depending on their intended use.

14. Semi rigid polymers These materials swell slightly Limited to a max. pressure of 300 psi. Bead diameters are usually 5 micrometer Styrene divinylbenzene polymers are used for compounds of molecular weight ranging from 100-500 million Sulphonated polystyrene beads are compatible with aqueous systems , non sulphonated with non aqueous systems.

15. Rigid porous glasses or silica Cover wide range of pore diameter Chemically resistant Used with aqueous and polar organic solvents.

16. DETECTORS So, it has to be very sensitive since the changes they measure in the mobile phase are very small. Detectors may respond to a change in the mobile phase due to the presence of the sample So, it has much greater sensitivity but often only work with specific samples Detectors may respond to a property of the sample alone

17. DETEC T ORS Measure concentration alone Differential refractive index (DRI) detector * UV detector Evaporative light scattering (ELS) detector. whose response is proportional to concentration and other properties of the polymer molecules . Static light scattering detectors or viscometers.

18. These detectors work by assessing the difference in refractive index between the mobile phase and the pure solvent Since the refractive index of polymers is usually constant above molecular weights of about 1,000 g/mol, the detector response is directly proportional to the sample concentration. WIDELY USED DETECTORS Use the fact that a beam of light will be scattered when it strikes a polymer molecule Low Angle Laser Light Scattering (LALLS), Multi-angle Laser Light Scattering (MALLS) Right Angle Laser Light Scattering (RALLS). The advantage of these detectors is that they give a response directly proportional to the molecular weight of the polymer molecules, and can provide size information Static Light Scattering Detectors: Differential Refractometer (Universal detector) :

19. APPLICATIONS Proteins fractionation Purification Molecular weight determination Separation of sugar, proteins, rubbers and others on the basis of their size. This technique can be use to determine the quaternary structure of purified proteins.

20. REFERENCES Instrumental methods of chemical analysis by: G.R.Chatwal, Sham K. Anand Instrumental Methods of Analysis, Willard, Merritt, Dean and Settle, CBS Publisher and Distributors.,1986. Principles of Instrumental Analysis, Skoog, Holder, Nieman, Fifth edition Thomson Books ,1998.

21. Thank you