Chromatography (Pharmacognosy)

Chromatography by Dr. Tahir ALi
Pharmacognosy department, RIPS, Riphah International University.

1. Chromatography Dr. Tahir Ali RIPS Riphah I nternational University

2. For more detail follow the link: https :// www.youtube.com/watch?v=e8i374hrWQg

3. What is Chromatography? Chromatography, literally " color writing ", was first employed by Russian scientist Mikhail Tsvet  in 1900.

4. DEFINITION “ It is a physical separation method in which the components of a mixture are separated by differences in their distribution between two phases, one of which is stationary ( stationary phase ) while the other ( mobile phase ) moves through it in a definite direction . The substances must interact with the stationary phase to be retained and separated by it . What is Chromatography?

5. What is Chromatography? Chromatography is a technique for separating mixtures into their components in order to analyze, identify, purify, and/or quantify the mixture or components. Separate Analyze Identify Purify Quantify Components Mixture

6. Applications of Chromatography Forensics Research Pharmaceutical industry

7. Types of Chromatography… Paper HPLC Gas Thin layer Column

8. CLASSIFICATION According to mechanism of separation Ion-exchange chromatography Affinity chromatography Size-exclusion chromatography Adsorption chromatography Partition chromatography

9. ION-EXCHANGE CHROMATOGRAPHY DEFINITION : Ion - exchange chromatography  (or  ion chromatography ) is a  chromatography process that separates  ions  and polar molecules based on their affinity to the  ion exchanger . It works on almost any kind of charged molecule—including large proteins, small nucleotides, and amino acids . For more detail visit the following links: https:// www.youtube.com/watch?v=efUrl_djzQ0 https:// www.youtube.com/watch?v=q3fMqgT1do8

10. If the stationary phase is represented by R− or R+ and the sample by X+ and X−, retention in IEC can be represented as X + + R − K + X+R − + K + (cation exchange) X - + R + Cl - X-R + + Cl - (anion exchange)

11. APPLICATIONS : It can be used for almost any kind of charged molecule including large proteins, small nucleotides and amino acids. Protein purification Water analysis Quality control

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

13. APPLICATIONS : Purify and concentrate an enzyme solution Purification of recombinant proteins Purification of antibodies

14. SIZE-EXCLUSION CHROMATOGRAPHY It is also known as gel permeation or gel filtration chromatography . This type of chromatography lacks an attractive interaction between the stationary phase and solute. The liquid or gaseous phase passes through a porous gel which separates the molecules according to its size . The pores are normally small and exclude the larger solute molecules , but 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. This causes the larger molecules to pass through the column at a faster rate than the smaller ones

15. SIZE-EXCLUSION CHROMATOGRAPHY

16. APPLICATIONS : Purification and analysis of synthetic and biological polymers, such as; Proteins, Polysaccharides, Nucleic acids . It is also useful for determining the tertiary structure and quaternary structure of purified proteins .

17. ADSORPTION CHROMATOGRAPHY

18. DEFINITION “It is a type of chromatography in which a mobile liquid or gaseous phase is adsorbed onto the surface of a stationary solid phase. The equilibration between the mobile and stationary phase accounts for the separation of different solutes.”

19. PRINCIPLE Separation occurs because of the fact that an equilibrium is established between molecules adsorbed on stationary phase and those which are flowing freely in mobile phase. The more the affinity of the molecule of particular component, less will be its movement.

20. TYPES

21. ADSORBENTS “An adsorbent is a substance, usually porous in nature and with a high surface area that can adsorb substances onto its surface by intermolecular forces .”

22. AN IDEAL ADSORBENT The Ideal adsorbent must fulfill the following requirements: Insoluble in mobile phase Inert to solutes (adsorptive) Colorless especially when work with colored mixtures Suitable particle size enough to give good separation and reasonable flow rate

23. COMMON ADSORBENTS Hydrated silica gel Silica gel G Silica gel S Silica gel GF 254 Silica gel H Silica gel N Silica gel HF 254 Silica gel PF 254

24. COMMON ADSORBENTS Modified silica gel Alumina Kieselghur (Diatomaceous earth) Cellulose MN 300 Cellulose microc rystalline

25. THIN-LAYER CHROMATOGRAPHY “The technique which involves flowing of mobile phase over a thin layer of adsorbent , applied on solid support, where separation of components occur by differential migration which occurs when solvent flows along fine powder spread on glass plates, is called thin –layer chromatography.”

26. Instrumentation Chromatography jar Capillary tube Thin layer chromatography plate Stationary phase Mobile phase

27. Instrumentation Chromatography jar: It is made of glass and has a lid on it. Jar maintains proper environment that is required for separation. Capillary tube: It is used to apply sample mixture on TLC plate. Stationary phase: Adsorbents

28. Instrumentation TLC plate: Borosilicate glass plates are preferred. Most commonly used sizes are; 20 X 20cm 20 X 10cm 20 X 5cm Microscopic slides are also used.

29. Instrumentation Mobile phase: Mobile phase may be a single liquid or a mixture of liquids . Commonly used mobile phases are; Methanol Ethanol Ethyl acetate Diethyl ether Acetone Chlorofor m

30. Procedure Clean and dried chromatography jar is taken. A paper impregnated in the mobile phase is applied to the walls to ensure that atmosphere of the jar is saturated with solvent vapors. Mobile phase is added to the jar at a length of 0.5-1cm from the bottom. Jar is closed. Equilibrium is allowed to be maintained. Base line is marked on adsorbent.

31. Procedure Sample is applied on TLC plate with help of capillary tube. Sample spot is air dried. TLC plate is put in the chromatography jar and lid is closed. The system is allowed to be static until the solvent move to a proper distance from baseline. TLC plate is taken out and dried.

32. Location of separated components If the sample is separated into colored components, then the location is dried in ordinary light. But in case of colorless components following are used ; Uv lamp Iodine crystals Spraying agents

33. Documentation Storage of chromatogram for TLC is difficult. It is usually undesirable since plates are employed for repeated use. Various methods for documentation include; Rf value in TLC Preservation of chromatogram by peeling off adsorbent. Graphical copying i.e. tracing on transparent paper. Photography

34. Applications It is used for separation and identification of; Amino acids Peptides and proteins Alkaloids Carbohydrates Fats and fatty acids Antibiotics Narcotic analgesics Glycosides

35. PARTITION CHROMATOGRAPHY

36. DEFINITION “ This form of chromatography is based on a thin film formed on the surface of a solid support by a liquid stationary phase. Solute equilibrates between the mobile phase and the stationary liquid .”

37. PRINCIPLE Separation of components of a sample mixture occurs because of partition. Stationary phase is coated with a liquid which is immiscible in mobile phase. Partition of component of sample between sample and liquid/ gas stationary phase retard some components of sample more as compared to others.

38. PRINCIPLE The stationary phase immobilizes the liquid surface layer, which becomes stationary phase. Mobile phase passes over the coated adsorbent and depending upon relative solubility in the coated liquid, separation occurs. The component of sample mixture appear separated because of differences in their partition coefficient.

39. TYPES

40. PAPER CHROMATOGRAPHY

41. Instrumentation Chromatography jar Capillary tube Stationary phase (liquid impregnated paper) Mobile phase

42. Instrumentation Chromatography jar: It is made of glass and has a lid on it. Jar maintains proper environment that is required for separation. Capillary tube: It is used to apply sample mixture. Stationary phase: liquid impregnated paper

43. Instrumentation Mobile phase: Mobile phase may be a single liquid or a mixture of liquids. Commonly used mobile phases are; Methanol Ethanol Ethyl acetate Diethyl ether Acetone Chloroform

44. Procedure Clean and dried chromatography jar is taken. A paper impregnated in the mobile phase is applied to the walls to ensure that atmosphere of the jar is saturated with solvent vapors. Mobile phase is added to the jar at a length of 0.5-1cm from the bottom. Jar is closed. Equilibrium is allowed to be maintained. Base line is marked on adsorbent.

45. Procedure Sample is applied on paper with help of capillary tube. Sample spot is air dried. Paper is put in the chromatography jar and lid is closed. The system is allowed to be static until the solvent move to a proper distance from baseline. Paper is taken out and dried.

46. Location of separated components If the sample is separated into colored components, then the location is dried in ordinary light. But in case of colorless components following are used; Uv lamp Iodine crystals Spraying agents

47. Documentation Storage of chromatogram. Calculating Rf values

48. Applications It is used for separation and identification of; Amino acids Carbohydrates Tannins Glycosides Alkaloids etc.