The document summarizes different types of separation techniques including: 1) Adsorption chromatography which utilizes a mobile and stationary solid phase for separation. Examples include TLC, paper, and HPTLC. 2) Partition chromatography which uses a thin film stationary liquid phase for separation like paper chromatography, HPLC, and gas chromatography. 3) Ion exchange chromatography which separates molecules based on ionic interactions using cation or anion exchange. 4) Size-exclusion chromatography which separates molecules by size using a porous stationary phase. 5) Affinity chromatography which separates using highly specific interactions like antigen-antibody binding and is commonly used to purify recombinant proteins. 6

1. 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
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2. Introduction:

3. Classification of Chromatography
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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
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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
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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.
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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.
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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.
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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.
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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.
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