Ion exchange chromatography and affinity chromatography

Easy to understand

1. Presented By ; BRITTO SAMUEL M II M.Sc., BIOTECHNOLOGY DEPARTMENT OF BIOTECHNOLOGY, AVS COLLEGE OF ARTS AND SCIENCE, SALEM Chromatography Ion Exchange Chromatography and Affinity Chromatography

2. Introduction Chromatography is a useful method of separating many different kinds of chemical mixtures . Chrom - Color separation of chemical components based on colors (Paper Chromatography). Components ; The mixture is dissolved in a fluid called the  ”mobile phase” W hich carries it through a structure holding another material called the stationary phase The separation is based on differential partitioning between the mobile and stationary phases . Chromatography may be preparative or analytical .

3. History Chromatography was first employed in Russia by the Italian-born scientist  Mikhail Tsvet  in 1900 research in plant pigments such as chlorophyll, carotenes, and xanthophylls New types of chromatography developed during the 1930s and 1940s made the technique useful for manyseparation processes .   Archer John Porter Martin and Richard Laurence Millington Synge during the 1940s & 50s established the principles and basic techniques of partition chromatography Tsvet's chromatography could be applied in many different ways, resulting in the different varieties of chromatography Separated color of plant pigment

4. Why Chromatography Special In any chemical or bioprocessing industry, the need to separate and purify a product from a complex mixture is a necessary and important step in the production line. chromatography can purify basically any soluble or volatile substance if the right adsorbent material, carrier fluid, and operating conditions are employed. chromatography can be used to separate small products since the conditions under which it is performed are not typically severe. For these reasons, chromatography is quite well suited to a variety of uses in the field of biotechnology, such as separating mixtures of proteins.

5. Reaction based chromatography Ion Exchange Chromatography Affinity Chromatography

6. Ion-exchange chromatography

7. Ion-exchange chromatography

8. Ion-exchange chromatography Ion chromatography (or ion-exchange chromatography) is a chromatography process that separates ions and polar molecules based on their affinity to the ion exchanger . Proteins , small nucleotides, and amino acids are also purified using Ion exchange Chromatography The water-soluble and charged molecules such as proteins, amino acids, and peptides bind to  oppositely charged by forming covalent bonds to the insoluble stationary phase This method applies the idea of the interaction between molecules and the stationary phase which are charged oppositely to each other . The bound molecules then can be eluted and collected using an eluent which contains anions and cations by running higher concentration of ions through the column or changing pH of the column.

9. Principle

10. How it works ? ( Step by Step process) Column containing anion exchanger . The sample is poured into the column. Anion presented in the Column is bind with the sample which having cations. During this process, unbounded samples inside the column get eluted. Changing pH, adding some buffers helps to elute the sample outside. Information: column containing anion exchanger this binds with the sample and make the unbounded samples to be eluted.

11. Anion Exchanger

12. Type of Ion Exchanger Type Matrix Trade Name SC (Strong Cation ) Dextran Polysterene Styrene- Devinyl Benzene Sephade Y Dowex 50 AG50 WC (Weak Cation ) Dextran Cellulose Acrylic CM. Sephadex CM.Cellulase Bio-Rex70 SA (Strong Anion) Dextran AG-1 WA (Weak Anion) Dextran Cellulose DEAE – Sephadex DEAE – Cellulose (DEAE: Diethyl Amino ethyl) ©Britto Samuel

13. Buffer Choices Two type o f buffers to be used in Ion Exchange Chromatography. > Cation Buffer > Anion Buffer Cation Buffer: used for anion exchanger for product retrieval Anion Buffer: Used for cation exchanger for product retrieval

14. Applications: Resin exchanger used for separating the small particles Cellulose, Dextrin, Polyacrylamide exchangers used for proteins and polysaccharide purification Dextran and Polyacrylamide exchangers used for separation of nucleotides, amino acids, Vitamins.

15. Affinity Chromatography

16. Affinity Chromatography The process of chromatography depends upon affinity between sample and ligands. Based on attraction between the sample and ligand this process succeed. Otherwise said to be Preparative Chromatography. Process fast separation. Principle Works on the principle of attraction or charm between the sample and ligand Affinity chromatography works. Affinity – Attraction , Kinship, Relationship. Because of the process of attraction, this process termed to be Affinity Chromatography. The principle of affinity chromatography is that the stationary phase consists of a support medium (e.g. cellulose beads) on which the substrate (or sometimes a coenzyme) has been bound covalently, in such a way that the reactive groups that are essential for enzyme binding are exposed.

17. Instrumentation

18. Instrument

19. Process behind Affinity Chromatography Three Process progressed behind Af -Chromatography. Matrix :used for ligand attachment Ligand : used to bind on the space of interaction Attachment o f Matrix with Ligand A special tool used to bind the ligand to the matrix is “Spacer Arm”

20. How it works? (Step by Step process)

21. How it works? (Step by Step process) Addition of the sample inside the column. Column already containing ligand. Addition of sample to the column leads to binding of ligand to the sample. M atrix helps to bind the sample to the ligand. Spacer arm present between the matrix and ligand helps to hold the ligand matrix this leads to binding of the sample to the ligand. In this process, the purified materials like proteins get attached with the ligand. Rest of the rusts eluted out. Due to changing of pH or addition of buffers in the column helps to elute our desisted product.

22. Simplified Process

23. Commercially available Matrix Material Name Commercial Name Dextran Sephacryl S Agarose Sepharose / Biogel A Polyacrylamide gel Biogel P Polystrene Biobeads

24. Spacer Arms (Used as Spacer Arms in af -Chromatography) 16 – Diaminohexane 6-Aminohexanoic acid 14-Bis Butane

25. Applications Used for the separation of enzymes and proteins Heparin agarose ;used for the separation of collagenous, Hepatitis B Surface antigen Polynucleotide Lysine agarose ; for separation of RNA Protein A agarose ;used for the Purification of Immunoglobulin G

26. Thanks Follow me at facebook.com/ Britto.Samuel