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JSPM’s Charak College of Pharmacy & Research, Wagholi.

Gat no-720/1&2 ,Wagholi, Pune-Nagar Road , Pune -412207

A Seminar On Fundamental Principles Of Seperation

Techniques

Presented By, Guided By, Mr. Vishal Khamgaonkar Dr. Rajesh J Oswal

Prof.Sandip Kshirsagar

Department Of Pharmaceutical Chemistry

CHROMATOGRAPHY (FROM GREEK WORD CHROMA "COLOR" AND GRAPHEIN "TO WRITE") IS THE COLLECTIVE TERM FOR A SET OF LABORATORY TECHNIQUES FOR THE SEPARATION OF MIXTURES. 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 VARIOUS CONSTITUENTS OF THE MIXTURE TRAVEL AT DIFFERENT SPEEDS, CAUSING THEM TO SEPARATE. THE SEPARATION IS BASED ON DIFFERENTIAL PARTITIONING BETWEEN THE MOBILE AND STATIONARY PHASES. SUBTLE DIFFERENCES IN A COMPOUND'S PARTITION COEFFICIENT RESULT IN DIFFERENTIAL RETENTION ON THE STATIONARY PHASE.CHROMATOGRAPHY MAY BE PREPARATIVE OR ANALYTICAL.

Introduction:

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Classification of Chromatography

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Adsorption Chromatography Adsorption chromatography is probably one of the oldest types of chromatography around. It utilizes a mobile liquid or gaseous phase that is adsorbed onto the surface of a stationary solid phase. The equilibriation between the mobile and stationary phase accounts for the separation of different solutes.

TLC Paper HPTLC

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Partition Chromatography This form of chromatography is based on a thin film formed on the surface of a solid support by a liquid stationary phase. Solute equilibriates between the mobile phase and the stationary liquid.

Paper Chromatography HPLC Gas Chromatography

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Ion exchange chromatography

Principle: Ion -exchange chromatography retains analyte molecules on the column based on ionic interactions. The stationary phase surface displays ionic functional groups (R-X) that interact with analyte ions of opposite charge. This type of chromatography is further subdivided into cation exchange chromatography and anion exchange chromatography. The ionic compound consisting of the cationic species M+ and the anionic species B- can be retained by the stationary phase.

Cation exchange chromatography retains positively charged cations because the stationary phase displays a negatively charged functional group:

Anion exchange chromatography retains anions using positively charged functional group.

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Size-exclusion chromatography It also known as gel filtration chromatography and separates molecules

according to their size. Smaller molecules are able to enter the pores of the media and, therefore, molecules are trapped and removed from the flow of the mobile phase. The average residence time in the pores depends upon the effective size of the analyte molecules. However, molecules that are larger than the average pore size of the packing are excluded and thus suffer essentially no retention; such species are the first to be eluted. It is generally a low-resolution chromatography technique . It is also useful for determining the tertiary structure and quaternary structure of purified proteins.

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Affinity chromatography

The Stationary phase is typically a gel matrix, often of agaros; The molecule of interest will have a defined property which can be exploited during the affinity purification process. The process itself can be thought of as an entrapment, with the target molecule becoming trapped on a stationary phase . The other molecules in solution will not become trapped as they do not possess this property.

Possibly the most common use of affinity chromotography is for the purification of recombinant proteins.

Affinity chromatography is a method of separating biochemical mixtures and based on a highly specific interaction such as that between antigen and antibody, enzyme and substrate , or receptor and ligand.

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Uses Affinity chromatography can be used to: Purify and concentrate a substance from a mixture into a buffering

solution Reduce the amount of a substance in a mixture. Purify and concentrate an enzyme solution.

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Reversed-phase chromatography includes any chromatographic method that uses a non-polar stationary phase. in RPC, polar compounds are eluted first while non-polar compounds are retained - hence "reversed phase". Today, reversed-phase column chromatography accounts for the vast majority of analysis performed in liquid chromatography.

It is similar to ion exchange chromatography. Lipophilic groups are attached to the stationary phase of the column. When a solution of proteins or molecules is passed through the column, hydrophilic proteins will flow through the column, while lipophilic proteins will remain in the column.

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Thank You.