Easy to understand

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

2. *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.

3. *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. *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. *Ion Exchange Chromatography
*Affinity Chromatography

7. Ion-exchange chromatography

8. *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.

10. * 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.

12. 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. * 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

14. *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.

16. * 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.

19. *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”

21. *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.

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

24. 16 – Diaminohexane 6-Aminohexanoic acid 14-Bis Butane

25. *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

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