Ion exchange chromatography and affinity chromatography

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Presented By ; BRITTO SAMUEL MII M.Sc., BIOTECHNOLOGYDEPARTMENT OF BIOTECHNOLOGY,AVS COLLEGE OF ARTS AND SCIENCE,SALEMChromatographyIon Exchange Chromatography and Affinity Chromatography

1

IntroductionChromatography 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 themobile phaseWhich carries it through a structure holding another material called thestationary phaseThe separation is based on differential partitioning between the mobile and stationary phases.Chromatography may be preparative or analytical.

History

Chromatography was first employed in Russia by the Italian-born scientistMikhail Tsvetin 1900 research in plantpigmentssuch aschlorophyll,carotenes, andxanthophyllsNew types of chromatography developed during the 1930s and 1940s made the technique useful for manyseparation processes.Archer John Porter MartinandRichard Laurence Millington Synge during the 1940s & 50s established the principles and basic techniques of partition chromatographyTsvet's chromatography could be applied in many different ways, resulting in the different varieties of chromatography

Separated color of plant pigment

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.

Reaction based chromatography

Ion Exchange ChromatographyAffinity Chromatography

Ion-exchange chromatography

Ion-exchange chromatography

Ion-exchange chromatographyIon chromatography(orion-exchange chromatography) is achromatographyprocess that separatesionsandpolar moleculesbased on their affinity to theionexchanger. Proteins, smallnucleotides, andamino acids are also purified using Ion exchange Chromatography The water-soluble and charged molecules such as proteins, amino acids, and peptides bind tooppositely charged by forming covalent bonds to the insoluble stationary phaseThis 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.

Principle

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.

Anion Exchanger

Type of Ion ExchangerTypeMatrixTrade NameSC (Strong Cation)DextranPolystereneStyrene-Devinyl BenzeneSephade YDowex 50AG50WC (Weak Cation)DextranCelluloseAcrylicCM. SephadexCM.CellulaseBio-Rex70SA (Strong Anion)DextranAG-1WA (Weak Anion)DextranCelluloseDEAE SephadexDEAE Cellulose

(DEAE: Diethyl Amino ethyl)

Britto Samuel

Buffer Choices Two type of buffers to be used in Ion Exchange Chromatography. > Cation Buffer > Anion Buffer

Cation Buffer: used for anion exchanger for product retrievalAnion Buffer: Used for cation exchanger for product retrieval

Applications:Resin exchanger used for separating the small particlesCellulose, Dextrin, Polyacrylamide exchangers used for proteins and polysaccharide purificationDextran and Polyacrylamide exchangers used for separation of nucleotides, amino acids, Vitamins.

Affinity Chromatography

Affinity ChromatographyThe 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. Theprincipleofaffinity chromatographyis 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.

Instrumentation

Instrument

Process behind Affinity Chromatography

Three Process progressed behind Af-Chromatography.

Matrix :used for ligand attachmentLigand : used to bind on the space of interactionAttachment of Matrix with Ligand

A special tool used to bind the ligand to the matrix is Spacer Arm

How it works? (Step by Step process)

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. Matrix 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.

Simplified Process

Commercially available MatrixMaterial NameCommercial NameDextranSephacryl SAgaroseSepharose / Biogel APolyacrylamide gelBiogel PPolystreneBiobeads

Spacer Arms (Used as Spacer Arms in af-Chromatography)16 Diaminohexane6-Aminohexanoic acid14-Bis Butane

ApplicationsUsed for the separation of enzymes and proteinsHeparin agarose ;used for the separation of collagenous, Hepatitis B Surface antigen Polynucleotide Lysine agarose; for separation of RNAProtein A agarose ;used for the Purification of Immunoglobulin G

Thanks

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