Size exclusion chromatography

size exclusion chromatography, its principle,instrumentations, advantages, disadvantages, applications

1. SIZE EXCLUSION CHROMATOGRAPHY Bindu Kshtriya [email protected]

2. INTRODUCTION Size exclusion chromatography is a mechanical sorting of molecules based on the size of molecules in solution. Small molecules are able to permeate more pores and are retained longer than larger molecules.

3. TYPES OF SIZE EXCLUSION CHROMATOGRAPHY Two basic types of SEC are:- GEL PERMEATION CHROMATOGRAPHY (GPC) Uses a hydrophobic column packing material and a non-aqueous mobile phase (organic solvent) to measure the molecular weight distribution of synthetic polymers. GEL FILTERATION CHROMATOGRAPHY(GFC) Uses a hydrophilic packing material and an aqueous mobile phase to separate, fractionate, or measure the molecular weight distribution of molecules soluble in water, such as polysaccharides and proteins.

4. OBJECTIVE SEC is a widely used technique for the purification and analysis of synthetic and biological polymers, such as proteins, polysaccharides and nucleic acids. Mild, non destructive method for determining molecular weight. It has become a mature and well accepted technique for characterizing both synthetic polymers and biopolymers. Standard technique for the analysis of monoclonal antibodies and their aggregates.  

5. Gel Gel is a familiar name. It refers to a fairly soft, elastic material containing water. In the scientific context, the term acquires a wider meaning. A gel consists of a three dimensional network. The structural material, often consisting of cross linked polymers, gives some mechanical stability . The space within the gel not occupied by structural material is filled with liquid. Liquid occupies the main part of gel.

6. GEL PREPARATION Two methods of Gel Preparation: Weighed amount of dry powder is mixed with excess of solvent and allowed it to swell. The mixture is left as such till the equilibrium condition is maintained. In this method, gel slurry is warmed at about 100 degree Celsius in boiling water bath. As a result gel swells in few days. The slurry is then cooled and packed in the column. The gel can stored in the wet state and there is no need to dry them, however for storage purpose, sephadex and acrylamide gels can be dried without any damage and can be brought back the wet state very easily.

7. key points The most convenient method to allow the gel to swell in a particular solvent is If swelling is completed by heating ,then the resultant slurry is allowed to cool before packing. If slurry is prepared in cold, it is necessary to deareated to vacuum.

8. MECHANISM OF ACTION Size exclusion column bed has three functional components : THE PORE VOLUME The pore volume refers to the pore-lumen space within the particles. THE VOID VOLUME It refers to the excluded volume i.e., the space between the particles. THE MATRIX VOLUME It refers to the solid component of the particles that fills the column bed.

9. Molecules larger than the pore size can not enter the pores and elute together as the first peak in the chromatogram. Molecules that can enter the pores will have an average residence time in the particles that depends on the molecules size and shape. Different molecules therefore have different total transit times through the column. Molecules that are smaller the pore size, and have the longest residence time on the column and elute together as the last peak in the chromatogram.

10. Instrumentation INJECTION VALVE PUMP COLUMN DETECTOR RECORDER

11. WORKING Pumps - for maintaining constant rates of flow Column-for separating the samples Detector – for quantifying the result Degasser

12. Commercially available columns The typical column diameters are 7.5–8 mm for analytical columns and 22–25 mm for (semi) preparative columns; usual column lengths are 25, 30, 50, and 60 cm. The packing are based on either porous silica or semi rigid (highly cross linked) organic gels, in most cases copolymers of styrene and divinyl benzene. 125Å pore size for analysis of small proteins and peptides 250Å pore size for most protein samples 450Å pore size for very large proteins and nucleic acids

13. Product pH stability Particle size Superdex Peptide Long term: 1–14 Short term: 1–14 13–15 μm Superdex 75 Long term: 3–12 Short term: 1–14 13–15 μm Superdex 200 Long term: 3–12 Short term: 1–14 13–15 μm Superdex 30 prep grade Long term: 3–12 Short term: 1–14 22–44 μm Superdex 75 prep grade Long term: 3–12 Short term: 1–14 22–44 μm Superdex 200 prep grade Long term: 3–12 Short term: 1–14 22–44 μm COMMERCIALLY AVAILABLE COLUMNS AND PROPERTIES: Superdex 200 or Superdex 200 prep grade - especially suitable for the separation of monoclonal antibodies from dimers and from contaminants of lower molecular weight

14. SIZE EXCLUSION COLUMN It consist of a hollow tube tightly packed with extremely small porous polymer beads designed to have pores of different size. Lager the particles, faster is the elusion.

15. Increasing the column length will enhance the resolution, and increasing the column diameter increases the capacity of the column. Proper column packing is important to maximize resolution: An over packed column can collapse the pores in the beads, resulting in a loss of resolution. An under packed column can reduce the relative surface area of the stationary phase accessible to smaller species, resulting in those species spending less time trapped in pores. POINTS TO REMEMBER

16. DETECTORS TYPES Concentration sensitive detectors U.V absorption ,refractive index (R.I ) detectors, (I.R) absorption and density detectors Molecular weight sensitive detectors Low angle light scattering detectors (LALLS) ,Multi angle light scattering (MALLS) detectors . .

17. CHROMATOGRAM Extent of retention depends on the size of the included molecules relative to the pores. Small molecules will enter all pores. Intermediate molecule, due to velocity of mobile phase, will not be able to diffuse into pores that may fit, thus will bee retained less effectively. Initial peak contains totally excluded solute. Final peak contains totally included solutes.

18. Description of separation in SEC

19. ADVANTAGES It can be carried out at room temperature and the sample are not decomposed because no exposure to high temperature. Rapid, routine analysis. Identify high mass components even in low concentration. Can analyze poly disperse samples. Branching studies can be done Absolute molecular weight can be obtained.

20. Short and well defined separation times. Narrow bands, which leads to good sensitivity. Freedom from sample loss because solute do not interact with stationary phase. Absence of column deactivation brought about by interaction of solute with the packing.

21. DISADVANTAGE Filtrations must be performed before using the instrument to prevent dust and other particulates from ruining the columns and interfering with detectors. Bad response for very small molecular weights. Sensitive for flow rate variation, internal standard should be used whenever possible. High investment cost.

22. Only a limited number of bands can be accommodated because the time scale of chromatogram is short Inapplicability to samples of similar size, such as isomers. 10% difference in molecular mass is required for reasonable resolution

23. APPLICATIONS Purification Desalting Concentration of dilute solutions Molecular weight determination Protein binding study

24. PURIFICATION Purification of biological macromolecules like viruses, protein, enzymes hormones ,nucleic acids, antibodies and polypeptides. Separation of low molecular weight component from mixture. examples separation of low molecular weight Dextran from corn syrup oil.

25. DESALTING Large molecules of biological origin are separated from inorganic or ionisable species is known as desalting. By the use of a column of sephadex G-25,solution of high molecular weight compounds can be desalted. In this the high molecular weight substances move with the void volume while the low molecular weight components are distributed between the mobile and stationary phase and hence move slowly.

26. APPLICATION OF DESALTING Desalting is faster and more efficient technique than dialysis. It helps in removal of phenol from nucleic acid preparations ,ammonium sulphate from protein preparations as well as monosaccharides from polysaccharides and amino acids from proteins.

27. Solution of high molecular weight substances can be concentrated by the addition of dry G-sephadex (coarse). Water and low molecular weight substances remain in solution. After ten minutes the gel is removed by centrifugation, leaving the high molecular weight material in a solution whose concentration has increased but whose ph and ionic strength are unaltered. CONCENTRATION OF DILUTE SOLUTION

28. PROTEIN BINDING STUDIES Reversible binding of a ligand to a macromolecule such as protein including receptor proteins. A sample of proteins/ ligands mixture is applied to column of gel which has previously equilibrated with solution of ligand of same concentration in mixture. Sample is eluted with buffer and concentration of ligand and protein in the effulent are determined. Early fractions will contain unbound ligand,but the subsequent appearance of the protein will result in an increase in the total amount of ligand (bound plus unbound)

29. MOLECULAR WEIGHT DETERMINATION Sephadex G-25 and G-50 have been used in the removal of low molecular weight molecules from high molecular weight natural product molecules. The effulent volume of globular protein are largely determined by their molecular weight. The effluent volume is approximately a linear function of the logarithm of molecular weight.

30. Absolute size-exclusion chromatography Absolute size-exclusion chromatography (ASEC) is a technique that couples a  dynamic light scattering  (DLS) instrument to a size exclusion chromatography system for absolute size measurements of proteins and macromolecules as they elute from the chromatography system.

31. ADVANTAGE OF DYNAMIC LIGHT SCATTERING(DLS) A big advantage of DLS coupled with SEC is the ability to obtain enhanced DLS resolution. Batch DLS is quick and simple and provides a direct measure of the average size but the baseline resolution of DLS is 3 to 1 in diameter

32. Recent advances in SEC Size-exclusion chromatography (SEC) process is now available to purify DNA-wrapped carbon nanotubes (DNA-CNT) and to sort them into fractions of uniform length. A type of silica-based column resin was identified that shows minimum adsorption of DNA-CNT.

33. Thank you Bindu kshtriya