Ion exchange chromatography PPT.

Ion exchange chromatography PPT.

1. Ion exchange chromatography Submitted by: Humna Mehmood BS Chemistry (2017-2021) 1

2. Contents Introduction Principle Classification of ion exchange resin Requirements of ion exchange resin Selection of resin Practical requirements of IEC Steps in ion exchange chromatography Methods of ion exchange chromatography Chromatographic parameters Factors affecting ion exchange chromatography Ion exchange constant Applications of ion exchange chromatography Advantages of ion exchange chromatography 2

3. Ion exchange chromatography Introduction: Ion exchange chromatography is a process by which a mixture of similar charged ions can be separated by using an ion-exchange resin which exchanges ions according to their relative affinities. It is a process that allows the separation of ions and polar molecules based on their charge. A form of liquid chromatography in which the stationary phase is an ion exchange resin which may be cation exchange resin or anion exchange resin. It can be used for almost any kind of charged molecule including large proteins, small nucleotides and amino acids. 3

4. Principle Reversible exchange of ions between ions present in solution and ion exchange resin. Cation exchange chromatography: Positively charged proteins are reversibly adsorbed to immobilized negatively charged beads/polymers. Anion exchange chromatography: Negatively charged proteins are reversibly adsorbed to immobilized positively charged beads/polymers. 4

5. Animated view Cation exchange: Anion exchange: 5

6. Types of ion exchange resin on basis of chemical nature Types Functional group Examples PH value Strong acid cation exchanger sulfonic acid Methylsulphonate –CH 2 CH 2 SO 3 – 1 to 14 Weak acid cation exchanger Carboxylic acid Carboxymethyl –CH 2 COO– 5 to 14 strong base anion exchanger Quaternary amine –CH 2 CH 2 N(CH 2 CH 3 ) 3 + 0 to 12 Weak base anion exchanger Amine Diethylaminoethyl (DEAE) –CH 2 CH 2 NH(CH 2 CH 3 ) 2 + to 9 Cation exchange resin: It exchanges cations . Anion exchange resin :   It exchanges negatively charged ions such as chloride etc.   6

7. Anion exchange Cation exchange - in softening of water 7

8. Types of ion exchange resin on basis of source Natural resin: Cation exchange resin: Zeolites, clay Anion exchange resin: Dolomite Synthetic resin: Organic and inorganic resin Epoxy resins Acetal resin solvent impregnated resins 8

9. Types of ion exchange resin on basis of Structure A. Peculiar types with ion exchange film Peculiar size of 30-40 micrometer film thickness Ion exchange efficiency is 0.01-0.1 meq /g of ion exchange resin. B. Porous resin coated with exchange beads Partical size of 5-10 micrometer Ion exchange efficiency is 0.5-2 meq /g of ion exchange resin. 9

10. Types of ion exchange resin on basis of Structure C. Macroreticular resin bead: Not highly efficient Very low exchange capacity D. Surface sulphonated and bounded e lectrosctatically with anion exchange: Less efficient and low exchange capacity Ion exchange capacity is 0.02meq/g 10

11. Ion exchange resin should have following requirements: It must be chemically stable Insoluble in common solvents It should have a sufficient degree of cross linking The swollen resin must be denser than water It must contain sufficient no. of ion exchange groups A resin of known weight should be used Analytical grade resins are prefered as they are carefully sized and washed. Selection of a resin If a protein is most stable below its pH, a cation exchanger should be used If a protein is most stable above its pH, an anion exchanger should be used If stability of the protein is known to be good over a wider pH range then either type of ion exchanger can be used 11

12. Practical requirements of ion exchange chromatography 1. Column material and Dimentions Material: Glass (for laboratories), high quality stainless steel or polymers(Industries) Dimentions : Length diameter ratio of 20:1 to 100:1 2. Types of ion exchange resin and physical resin Types: Cation and anion Nature of ions to be seperated : Weak or strong Efficiency of resin: Measured by ion exchangge capacity Particle size of resin: 50 to 100 mesh 0r 100 to 200 mesh Structural types of resin: Porous, peculiar atc Amount of cross linking agent present which decides swelling of resin 12

13. Practical requirements of ion exchange chromatography 3. Stationary Phase It is composed of two structural elements; Charged groups involved in ion exchange and matrix on which charged groups are fixed. Some matrix are; Cellulose, sillia , polyacrylamide, acrylate co-polymer, coated sillica 4. Mobile phase Generally, eluent which consists of aqueous solution of suitable salt or mixture of salt with small percentage of organic compounds is used in which all ionic compounds are dissolved. Some eluents used in ion exchange chromatography are EDTA, polyols , glycerol, glucose, detergents, lipids, organic solvents, urea 13

14. Practical requirements of ion exchange chromatography 5.Buffers In IEX, PH value is an important parameter and can be controlled by means of buffer. For cation exchange; Citric acid, lactic acid, acetic acid, formic acid For anion exchange; Piperazine , N-methyl piperzine , triethanolamine , Ethanolamine 6. Sample preparation For sample preparation, sample must be soluble in eluent and should ideally dissolve in mobile phase itself To protect column from possible damage, sample must be filtered before use to remove particulates. 14

15. Practical requirements of ion exchange chromatography 7. Packing of column Wet packing method is th ideal technique of column packing. Slurry is prepared be mixing silica(10-20g) and least polar solvent in a beaker and poured in the column. When the packing is complete, the eluent is allowed to pass through colum for certein time. 8. Development of chromatogram and elution After introduction of sample, development of the chromatogram is done by using different mobile phases. There are two elution techniques; isocratic and gradient elution 15

16. Practical requirements of ion exchange chromatography 9. Analysis of elute Different fractions collected with respect to volume or time is analysed for their contents by several methods. Spectrophotometric method Polarographic method Conductometric method Radiochemical method 10. Regeneration of ion exchange resin It refers to replacement of exchangeable cations or anions present in the original resin Regeneration of cation exchange resin is done by charging the column with strong acid like HCl. 16

17. Steps in ion exchange chromatography An IEX medium comprises a matrix of spherical particles substituted with ionic groups. The matrix is usually porous to give a high internal surface area. The medium is packed into a column to form a packed bed. The bed is then equilibrated There are four steps: Equilibrium Sample application and wash Elution Regeneration 17

18. 1. Equilibrium : The first step is the equilibration of the stationary phase to the desired start conditions. When equilibrium is reached, all stationary phase charged groups are bound with exchangeable counter ions, , such as chloride or sodium. The pH and ionic strength of the start buffer are selected to ensure that, when sample is loaded, proteins of interest bind to the medium and as many impurities as possible do not bind. 18

19. 2. Sample application and wash The goal in this step is to bind the target molecule(s) and wash out all unbound material. The sample buffer should have the same pH and ionic strength as the start buffer in order to bind all charged target proteins. Oppositely charged proteins bind to ionic groups of the IEX medium, becoming concentrated on the column. Uncharged proteins, or those with the same charge as the ionic group, pass through the column at the same speed as the flow of buffer, eluting during or just after sample application, depending on the total volume of sample loaded. 19

20. 3. Elution After sample application and washing, conditions are altered in order to elute the bound proteins. Most frequently, proteins are eluted by increasing the ionic strength of the buffer or, occasionally, by changing the pH. As ionic strength increases the salt ions compete with the bound components for charges on the surface of the medium and one or more of the bound species begin to elute and move down the column. The proteins with the lowest net charge at the selected pH will be the first ones eluted from the column as ionic strength increases. T he proteins with the highest charge at a certain pH will be most strongly retained and will be eluted last. The higher the net charge of the protein, the higher the ionic strength that is needed for elution. By controlling changes in ionic strength proteins are eluted differently in a purified, concentrated form. 20

21. 3. Elution 21

22. 4 . Regeneration A final wash with high ionic strength buffer regenerates the column and removes any molecules still bound. This ensures that the full capacity of the stationary phase is available for the next run. The column is then re-equilibrated in start buffer before starting the next run. 22

23. Ion exchange techniques Two techniques are generally used to bring the solution in contact with ion exchange resins. They are: 1.    Batch method 2.    Column method 1. Batch method This method involves single step equilibrium. The resin and the solution are mixed in a vessel until equilibrium is attained. The solution which is obtained filtered. In single step equilibrium of this type, only a single portion of the exchange capacity of the resin is utilized . This method is used for softening of water and the production of deionized or demineralised water  23

24. Ion exchange techniques The apparatus used in the column method consist of a glass column fitted with a glass wool plug or a sintered glass disc at the lower end.  An ordinary burette can also be used. The resin used should have small particle size and the diameter of the resin should be less than one tenth of the column. The slurry is slowly poured into the column containing some water. The resin is allowed to settle and then excess water is drained off  2. Column Method 24

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26. Chromatographic parameters 1. Rate of ion exchange process: Its depends on the rate of diffusion which is the slowest state on ion exchange process. 2. Flow rate: Due to difference in rate of exchange and the fact that they may vary significantly for different kinds of seperation , flow rates are to be controlled ( 0.5-5 ml/min) 3. Mechanical strength: Polystyrene bead would have little mechanical strength and upon adding functional group such as sulphonic acid to ploymer , solubility is enchance greatly If polymer is cross linked by incorporation of divinyl benzene, mechanical strength is imparted to resin . 26

27. Chromatographic parameters 3. Swelling: Swelling is due to the tendency of particles to hydrate and electrostatic repulsion of fixed with like charges . Swelling is continued until an equilibrium is reached between the osmotic pressure in the system and opposing elastic force of flexible hydrocarbon chains. 4. Particle size: Large surface area and small particle size will increase the rate of ion exchange process 5. Porosity : High porosity offers a large surface area covered by charged groups ans so provides a high binding capacity. 27

28. Chromatographic parameters 7. Selectivity : The ion exchange in solution is a selective process. As absolute conc. Of solution decreases, polyvalent are absorbed and at higher concentration, monovalent are adsorbed 8. Total capacity: Total capacity of resin is determined by taking a weighed sample of resin, placing it in a colum and passing through a solution of KCl through the column in excess. 9. Exchange capacity: It depends on the wuality of the ions extracted from water by ine gram of air dry ion exchanger. 10. Crosslinking : As crosslinking decreases, resin swelling increases . Divinyl is the most commonly employed crosslinking agent. 28

29. Factors affecting ion exchange chromatography Three major factors are affecting ion exchange selectivity in IEC. Cross linking and swelling are important factors, when more cross linking agent is present, the resin becomes more rigid and swells less (has small pore size). This makes separations of ions of different sizes more difficult as they can not pass through the pores present and it becomes selective to ions of different (smaller) sizes. The nature of resin whether cationic or anionic exchanger, which determines strongly its selectivity. Cationic resin is selective for cations and vice versa. T he resin capacity (number of me-equivalents of replaceable ions per gram of dry resin) is important. 1. Nature and properties of ion exchange resins 29

30. 2.Nature of exchanging ions in the sample a. Valence of ions: At low concentrations, the extent of exchanges increases with increase in valence ; Ions with higher charge is more selective   b. Size of ions: For similarly charged ions; the exchange selectivity increases with decrease in the size of hydrated ions ; < < < < < < c. Polarizability : Highly polarizable ions are more selective. Exchange is preferred for greater polarizable ions, as; d. Concentration of solutions : In dilute solutions poly- valent ions are generally absorbed preferentially.   30

31. 3.Nature of mobile phase and pH Exchange constant or selectivity coefficient (K) The presence of other ions that compete with the sample for binding to the ion exchanger (using of electrolyte). The pH of the solution which influences the net charge of the sample (as in case of amino acids). For above equilibrium For above equilibrium   31

32. Exchange constant or selectivity coefficient (K ) 32

33. Applications of ion exchange chromatography IEC is utilized in numerous industrial and research settings including a test for authentic tequila and for environmental analyses such as the determination of anions (phosphate, chloride, etc ) in surface waters. It is used in the treatment of water for drinking, use (commercial, industrial, and residential), and wastewater treatment. Ion exchangers can soften the water, deionize it, and even be used in desalination. Preparation of various acids, bases, salts, and solutions is also aided by ion exchange. The recovery of valuable metals is also possible with resins. I ndustrial drying of treatment of gases is accomplished often with ion exchange. The food industry uses ion exchange in a variety of ways, ranging from wine-making to sugar manufacture. 33

34. Applications of ion exchange chromatography In the medical world, it is used for development and preparation of key drugs and antibiotics, such as streptomycin and quinine, for treatments of ulcers, TB, kidneys, and much more. Ion exchange is used to prevent coagulation in blood stores and in dextrose, as well. An ion exchange is also useful in death, as it plays a role in the treatment of formaldehyde. In soil science, cation exchange capacity is the ion exchange capacity of soil . Soils can be considered as natural weak cation exchangers. A very important case is the PUREX process (plutonium-uranium extraction process), which is used to separate the plutonium and the uranium from the spent fuel products from a nuclear reactor. In planar waveguide manufacturing, ion exchange is used to create the guiding layer of higher index of refraction. 34

35. Applications of ion exchange chromatography An important use of ion-exchange chromatography is in the routine analysis of amino acid mixtures. The 20 principal amino acids from blood serum or from the hydrolysis of proteins are separated and used in clinical diagnosis. In the analysis of products of hydrolysis of nucleic acids. In this way, information is gained about the structure of these molecules and how it relates to their biological function as carriers of hereditary information.  Chelating resins are used to collect trace metals from seawater. To analyze lunar rocks and rare trace elements on Earth . Removal of interfering ions For estimation of , ions by the oxalate or sulphate method in quantitative analysis, phosphate ions are found to interfere and can be removed by passing the solution through sulphuric acid.   35

36. Advantages of ion exchange chromatography Has highest resolving power Has highest loading capacity Widespread applicability (almost universal) Most frequent chromatographic technique for protein purification Used in approximately 75 % of all purifications It is one of the most efficient methods for the separation of charged particles. C an be used for almost any kind of charged molecule including large proteins, small nucleotides and amino acids. Ion exchange is used for both analytical and preparative purposes in the laboratory, the analytical uses being the more common. Inorganic ions also can be separated by ion-exchange chromatograph.y 36

37. Thank you 37