ion exchange chromatography and affinity chromatography
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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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