GEL CHROMATOGRAPHY : Principles and Applications

There are other names for gel chromatography, such as size exclusion and gel permeation. Pumping dissolved molecules through columns allows for the size-based separation of molecules in a process known as gel chromatography. This technique is highly efficient for separating and examining bio-macromolecular materials. The fundamentals, columns, pumps, detectors, stationary phase, and mobile phase instruments. The mobile phase's solvent is entirely contained within the pores of the stationary phase, which is a porous polymer matrix. Conversely, the rate at which smaller molecules permeate the gel will determine how slowly they progress.

1. GEL CHROMATOGRAPHY PRESEMTED BY : M. SOWMIYA M.PHARM(PHARMACEUTICAL ANALYSIS) I YR – II SEMESTER

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

3. 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.

4. Separation based on their size of molecules

5. 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 = Vg + V i + V 0 Where V t -Total column volume Vg – is the volume occupied by the packing V i – is the volume of solvent in the pores V 0 – is the free solvent volume Volume occupied by gel space = Vt –V0

6. INSTRUMENTATION:

7. COMPONENTS: Stationary phase Mobile phase Columns Pump Detectors

8. STATIONARY PHASE It is composed of semi permeable porous polymer gel beads with well defined range of pore sizes. Following all the properties of gel beads: Chemically inert Mechanically stable Ideal and homogeneous porous structure (wide pore size give low resolution) Uniform particle and pore size Pore size of the gel must be carefully controlled MOBILE PHASE An organic solvent is used as a mobile phase. Buffer is used to maintain the pH. The samples are eluted isocratically , i.e. there is no need to use different buffers during the separation.

9. COLUMNS Any of the following kinds may be used: Analytical column – 7.5-8mm diameters. Preparative columns – 22-25mm PUMPS They are either syringe pump or reciprocating pump with high constant flow rate. DETECTORS The various type of detectors are: Concentration sensitive detector Bulk property detector Refractive index detector and many more

10. SEPARATION PROCEDURE Preparation of column for gel filtration Swelling of the gel: Some resin come in a powder form, these must be sonicated first in the eluent or the desired buffer to swell. Packing the column: Make a slurry of gel plus buffer and pour it into column which is one third filled with the buffer. Washing the resin: After packing, pass several column volumes of the buffer through the column to remove any air bubbles and to test the column homogeneity.

11. Loading the sample onto the column: The sample must enter the resin in the form of solution using a syringe. Eluting the sample and detection of components: Fractions are collected as the sample elutes from the column .

12. APPLICATIONS: Proteins fractionation Purification Molecular weight determination. Separation of sugar, proteins, peptides, rubbers, and others on the basis of their size. Can be used to determine the quaternary structure of purified proteins.

13. ACKNOWLEDGEMENT: I would like to express my sincere Gratitude to My esteemed professor Dr.S.S.Rajendran HOD, Department of Pharmaceutical analysis , RVS College of pharmaceutical sciences. My friends Ms.N.Abinaya , K.Supriya and Ms.K.Vytheeswari for their encouragement and assistance.

14. REFERENCES: Dr.S.Ravi Shankar. “Text book of of pharmaceutical analysis” N.Santhi , Dr.S.S.Rajendran , Dr.S.Muthu kumar . “Instrumental Methods of Analysis”

15. THANK YOU