Vishal khamgaonkar


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Vishal khamgaonkar

  1. 1. JSPM’sCharak 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 1
  2. 2. Introduction:
  3. 3. Classification of Chromatography 3
  4. 4.  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 4
  5. 5.  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 5
  6. 6.  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. 6
  7. 7.  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. 7
  8. 8. Affinity chromatography 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. 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. 8
  9. 9.  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. 9
  10. 10.  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. 10
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