Chromatography; history and its types.

Chromatography; history and its types.

1. By Group 2

2. Contents Introduction History Principle Types of chromatography Conclusion References

3. Introduction By Zeeshan Khalid Reg # 634/BSBT/F14

4. Introduction Chromatography Chroma Color Graphein To write

5. Chromatography Chromatography is used to separate mixtures of substances into their components.

6. History It was first employed in Russia by the Italian- scientist Mikhail Tsvet in 1900. The separation of plant pigments such as Chlorophyll. New types of chromatography developed.

7. Principle All forms of chromatography work on the same principle. Stationary phase (a solid, or a liquid supported on a solid) Mobile phase (a liquid or a gas)

8. High performance liquid chromatography Asfand Yar Khan Reg # 635/BSBT/F14

9. Introduction HPLC is a form of liquid chromatography used to separate compounds that are dissolved in solution . HPLC instruments consist of a reservoir of mobile phase, a pump, an injector, a separation column, and a detector. Compounds are separated by injecting a sample mixture onto the column. The different component in the mixture pass through the column at differentiates due to differences in their partition behavior between the mobile phase and the stationary phase. The mobile phase must be degassed to eliminate the formation of air bubbles.

10. What is Liquid Chromatography? Liquid chromatography is a separation technique that involves: • the placement (injection) of a small volume of liquid sample • into a tube packed with porous particles (stationary phase) • where individual components of the sample are transported along the packed tube (column) by a liquid moved by gravity. The components of the sample are separated from one another by the column packing that involves various chemical and/or physical interactions between their molecules and the packing particles. The separated components are collected at the exit of this column and identified by an external measurement technique , such as a spectrophotometer that measures the intensity of the color , or by another device that can measure their amount.

11. Principles of Liquid Chromatography

12. Principles of HPLC Principle; Tabel shows the relationship b/w various parameters of HPLC Trendline : Stationary phase have small particulate size and high surface areas. Columns: 20 cm or less Mobile phase pumped at high pressures of 200Bar, 3000 psi. Flow rates: 1-3 cm 3 per min Column length No. of theoretical plates per unit area Resolving power Column length Particle size Surface area

13. What is HPLC? HPLC is a separation technique that involves: •the injection of a small volume of liquid sample •into a tube packed with tiny particles (3 to 5 micron ( μm ) in diameter called the stationary phase) •where individual components of the sample are moved down the packed tube ( column) with a liquid (mobile phase) forced through the column by high pressure delivered by a pump. These components are separated from one another by the column packing that involves various chemical and/or physical interactions between their molecules and the packing particles. These separated components are detected at the exit of this tube ( column) by a flow-through device (detector) that measures their amount. An output from this detector is called a “liquid chromatogram”.

14. Gas chromatography Muhammad Jawad Reg # 636/BSBT/F14

15. Ion exchange chromatography Zeeshan Khan Reg # 637/BSBT/F14

16. inTro This technique was developed in 19 th century and was used to purify the drinking water. Reversible exchange of ions in the solution with ions electrostatically bound to some sort of insoluble matrix or stationary phase . It is extremely useful in the separation of charged compounds like proteins differing from only one charged amino acid. Ion exchanged is performed in the column.

17. Principle The principle of separation is by reversible exchange of ions between ions present in the solution and those present in the ion exchange resin.

18. Ion exchangers There are two types of ion exchangers. Cations exchanger. Anions exchanger.

19. Cation exchange chromatography Positively charged molecules are attracted to a negatively charged solid support. Commonly used Cation exchange resins are S-resin, sulfate derivatives; and CM resins, carboxylate derived ions

20. Anion exchange chromatography Negatively charged molecules is attracted to a positively charged solid support. Commonly used anion exchange resins are Q-resin, a Quaternary amine; and DEAE resin, Di- EthylAminoEthane .

21. CATION AND ANION EXCHANGER

22. Uses Used in the analyses of aminoacid To determine the base composition of the nuclic acid. Proteins are also successfully seprated by this technique Used for sepration of vitamens This is most effective method for water purification. Complete deionization of water (or) a non-electrolyte solution is performed by exchanging solute cations for hydrogen ions and solute anions for hydroxyl ions. This is usually achieved by method is used for softening of drinking water.

23. Thin layer chromatography Hammad Ali Reg # 638/BSBT/F14

24. Paper chromatography Muzaffar Latif Reg # 639/BSBT/F14

25. Definition Paper chromatography is a type of chromatography. It is an analytical method used to separate colored chemicals or substances. A method of separating components of a mixture by differential movement through a two-phase system: the mobile phase and the secondary phase. The separation of an unknown substance is mainly carried out by the flow of solvents on the specially designed chromatographic paper

26. Principle Paper chromatography works on the principle of partition, i.e., it is based upon continuous differential distribution of the various components of the mixture between the stationary and the mobile phase. Cellulose layers in filter paper contains moisture which acts as stationary phase & organic solvents/buffers are used as mobile phase. In paper chromatography the results are represented by Rf value which represents the movement of component of the mixtures relative to the solvent front. Rf value = Distance traveled by the solute Distance traveled by the solvent

27. Procedure Solvent front Filter paper Ink spots Solvent Beaker Separated dyes

28. Observing the Chromatograms Concentration of Isopropanol 0% 20% 50% 70% 100%

29. Uses of Paper chromatography To Analyze – examine a mixture, its components, and their relations to one another To Identify – determine the identity of a mixture or components based on known components To Purify – separate components in order to isolate one of interest for further study To Quantify – determine the amount of the a mixture and/or the components present in the sample

30. Column chromatography Muhammad Sharif Reg # 640/BSBT/F14

31. Column chromatography An impure sample is loaded onto a column of adsorbant , such as silica gel . An organic solvent or a mixture of solvents (the eluent ) flows down through the column. Components of the sample separate from each other by partitioning between the stationary packing material.

32. Packing a (silica gel) column: 1- Add cotton to bottom of column. 2-Add sands to the column 3- Prepare a slurry of silica in the intial eluent by pouring dry silica into a beaker of eluent . 4-Quickly but carefully pour the slurry into the column. 5-Drain eluent from the column until no solvent remains . 6- Loading a sample onto the column

33. Column

34. Precautions

35. Affinity chromatography Umer Awais Reg # 641/BSBT/f14

36. Affinity Chromatography Affinity chromatography is one of the most diverse and powerful chromatographic methods for purification of a specific molecule or a group of molecules from complex mixtures.

37. Principle Affinity chromatography is based on highly specific biological interactions between two molecules, such as interactions between enzyme and substrate, receptor and ligand , or antibody and antigen.  These biological interactons are typicaly reversible these reversible interactions are used for purification .

38. Procedure Purification is done by placing one of the interacting molecules, referred to as affinity ligand , onto a solid matrix to create a stationary phase while the target molecule is in the mobile phase. Successful affinity purification requires a certain degree of knowledge and understanding of the nature of interactions between the target molecule and the ligand to help determine the selection of an appropriate affinity ligand and purification procedure.

39. Conclusion Separation technique Many types of chromatography Working depend on stationary and mobile phase Many applications in the field of biochemistry, organic chemistry, etc

40. Thanks