Ion exchange chromatography and affinity chromatography
Presented By ;
BRITTO SAMUEL M
II M.Sc., BIOTECHNOLOGY
DEPARTMENT OF BIOTECHNOLOGY,
AVS COLLEGE OF ARTS AND SCIENCE,
*Chromatography is a useful method of separating many different
kinds of chemical mixtures.
*Chrom- Color separation of chemical components based on colors
*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.
*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
*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
*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
* Column containing anion exchanger .
*The sample is poured into the column.
*Anion presented in the Column is bind with the sample which having
*During this process, unbounded samples inside the column get eluted.
*Changing pH, adding some buffers helps to elute the sample outside.
column containing anion exchanger this binds with the sample and make
the unbounded samples to be eluted.
* Two type of 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
*Resin exchanger used for separating the small particles
*Cellulose, Dextrin, Polyacrylamide exchangers used for proteins and
*Dextran and Polyacrylamide exchangers used for separation of
nucleotides, amino acids, Vitamins.
* 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.
• Works on the principle of attraction or charm between the sample and ligand Affinity
• 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.
*Three Process progressed behind Af-Chromatography.
1. Matrix :used for ligand attachment
2. Ligand : used to bind on the space of interaction
3. Attachment of Matrix with Ligand
A special tool used to bind the ligand to the matrix is “Spacer Arm”
*Addition of the sample inside the column.
*Column already containing ligand.
*Addition of sample to the column leads to binding of ligand to the
* 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.
Material Name Commercial Name
Dextran Sephacryl S
Agarose Sepharose / Biogel A
Polyacrylamide gel Biogel P
*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