ion exchange chromatography and affinity chromatography

Presented By ; BRITTO SAMUEL M

II M.Sc., BIOTECHNOLOGYDEPARTMENT OF BIOTECHNOLOGY,

AVS COLLEGE OF ARTS AND SCIENCE,SALEM

ChromatographyIon Exchange Chromatography and Affinity Chromatography

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”*Which 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.

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

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 Chromatography*Affinity Chromatography

Ion-exchange chromatography

Ion-exchange chromatography

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.

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 ExchangerType Matrix Trade Name

SC (Strong Cation)Dextran

PolystereneStyrene-Devinyl

Benzene

Sephade YDowex 50

AG50

WC (Weak Cation) DextranCelluloseAcrylic

CM. SephadexCM.Cellulase

Bio-Rex70SA (Strong Anion) Dextran AG-1

WA (Weak Anion)DextranCellulose

DEAE – 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 particles*Cellulose, Dextrin, Polyacrylamide exchangers used for

proteins and polysaccharide purification*Dextran and Polyacrylamide exchangers used for separation

of nucleotides, amino acids, Vitamins.

Affinity Chromatography

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.

Instrumentation

Instrument

Process behind Affinity Chromatography

*Three Process progressed behind Af-Chromatography.

1. Matrix :used for ligand attachment2. Ligand : used to bind on the space of interaction3. Attachment 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 Matrix

Material Name Commercial NameDextran Sephacryl SAgarose Sepharose / Biogel A

Polyacrylamide gel Biogel PPolystrene Biobeads

Spacer Arms (Used as Spacer Arms in af-Chromatography)

16 – Diaminohexane

6-Aminohexanoic acid

14-Bis Butane

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

Thanks

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