CHROMATOGRAPHY

1. CHROMATOGRAPHY

2. Chromatography • Atechnique for the separation of a mixture by passing it in solution or suspension through a medium in which the components move at different rates.

3. Principle • It is based on the principle of the partition of the solute between two phases/solvents • It consists of a mobile phase and a stationary phase • The mobile phase is usually refers to the mixture of the substances to be separated dissolved in a liquid or a gas • The stationary phase is a porous solid matrix through which the sample contained in the mobile phase perculates

4. The interaction between the mobile and the stationary phases result in the separation of the compounds from the mixture • These interactions include the physico chemical principles such as adsorption, ion-exchange, molecular sieving and affinity

5. Chromatography- Classification 1. Based on interactions between sample component and stationary phase • Partition • Adsorption • Ion-exchange • Gel-filtration • Affinity • High performance liquid chromatography

6. 2. Based on nature of stationary phase or mobile phase , it is of two types • Planar- It may be paper or thin layer • Column- It may be gas or liquid • Planar chromatography is a separation technique in which the stationary phase is present as or on a plane • Column chromatography is a separation technique in which the stationary bed is within a tube

7. Chromatography classification

8. Partition Chromatography • The principle of partition chromatography is that the separation of components of given sample occurs due to partition of components between two liquid phases • In this process, the stationary phase is coated with a liquid surface. The solid surface must be immiscible in the mobile phase.

9. Distribution coefficients • The basis of all forms of chromatography is the distribution or partition coefficient (Kd), which describes the way in which a compound (the analyte) distributes between two immiscible phases. • For two such phases A and B, the value for this coefficient is a constant at a given temperature and is given by the expression

10. It is used for the separation of mixture of amino acids and peptides • The molecules of a mixture get partitioned between the stationary and the mobile phase depending on the relative affinity of each one of the phases • It is undertaken in two ways • Paper chromatography • Thin layer chromatography

11. Adsorption Chromatography • In this technique the separation is based on difference in adsorption at the surface of the solid stationary medium • The adsorbents such as silica gels, charcoal powder and calcium hydroxyapatite are packed in to a column in a glass tube. This serves as the stationary phase • The sample mixture in a solvent is loaded on this column • The individual components get differentially adsorbed on to the adsorbent

12. Principle • It involves the competition of components of sample mixture for active site on adsorbent. These active sites are formed in molecule due to • Cracks • Edges • The electrostatic forces present in the molecule, which hold together the crystal lattice, are directed outward • These forces and electrostatic forces of solute molecule cause separation

13. 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

15. Paper Chromatography • An analytical technique for the separation and identifying mixtures that are either coloured or can be made coloured • It is a liquid partition chromatography • Used for the separation of amino acids, sugars, sugar derivatives and peptides

16. The stationary phase is water held on a solid support of filter paper (cellulose) • The mobile phase is a mixture of immiscible solvents which are mixtures of water, a non polar solvent and an acid or base

17. Mobile Phase • Pure solvent, buffer solutions or mixture of solvents Hydrophilic mobile phase • Isopropanol: ammonia: water 9:1:2 • Methanol: water 4:1 • N-butanol: glacial acetic acid: water 4:1:5 Hydrophobic mobile phase • Dimethyl ether: cyclohexane • Kerosene: 70 % isopropanol

18. Procedure • A small spot of sample is applied to a strip of chromatography paper about two centimetres away from the base of the plate • The paper is then dipped in to a suitable solvent, such as ethanol or water, taking care that the spot is above the surface of the solvent, and placed in a sealed container • The solvent moves up the paper by capillary action and dissolves the sample mixture, which will then travel up the paper with the solvent solute sample

19. Different compounds in the sample mixture travel at different rates due to differences in solubility in the solvent, and due to difference in their attraction to the fibres in the paper Paper chromatography takes from several minutes to several hours

20. Ascending and Descending Paper Chromatography • Ascending Chromatography- In this method, the solvent is present at the bottom of the vessel in which the paper is supported • It rises up the paper by capillary action against the force of gravity

21. Descending Chromatography- In this method, the solvent is kept in a trough at the top of the chamber and is allowed to flow the paper • The liquid moves down by capillary action as well as by the gravitational force

22. Ascending and Descending Paper Chromatography

23. Paper chromatography- Analysis • The paper remaining after the experiment is known as the chromatogram • The components which have been separated differ in their retention factor • Ratio of distance travelled from the spot or origin by the solute component to that of the distance travelled from the spot or origin by the solvent

24. Rf Value • Rfis Distance travelled by sample/ distance travelled by solvent • If Rfvalue of a solution is zero, the solute remains in the stationary phase and thus it is immobile • If Rfvalue is 1, then the solute has no affinity for the stationary phase and travels with the solvent front • Retention factor can never be greater than one

25. Detecting/Visualising Agents • If the substance are coloured they are visually detected easily • For colourless substances, physical and chemical methods are used to detect the spot Physical methods (Non-specific methods) • Iodine chamber method • UV chamber for fluorescent compounds at 254 or at 365 nm

26. Chemical method (Spraying or specific method) • Ferric chloride - Phenolic comp. & tannins • Ninhydrin in acet. - Amino acids • Dragendroff’sReag.- Alkaloids • 3,5 dinitro benzoic acid - Cardiac glycosides

27. Two Dimensional Chromatography • It is used to separate a complex mixture of substances which was not resolved by a single runs with one solvent system • In two-dimensional chromatography, a second run is carried out by a different solvent system, in a direction perpendicular to the first run which enhances the separation of a mixture in the individual components

29. Significance of Paper Chromatography • Paper chromatography is a very easy, simple, rapid and highly efficient method of separation • Can be applied even in microgram quantities of the sample • Can also be used for the separation of a wide variety of materials such as amino acids, oligopeptides, sugars, oligosaccharides, glycosides, purines and pyrimidines, steroids, vitamins and some alkaloids such as penicillin, tetracyclin and streptomycin

30. Not preferred for separating proteins because they are not soluble in many of the solvent systems and are denatured by them • Paper chromatography is inferior to thin layer chromatography in resolving power

31. Thin Layer Chromatography • It is the one of the simplest, fastest, easiest and least expensive of several chromatographic techniques used in qualitative and quantitative analysis to separate organic compounds and to test the purity of compounds

32. Principle of TLC • It is based on the principle of adsorption chromatography or partition chromatography or combination of both, depending on adsorbent, its treatment and nature of solvents employed • The components with more affinity towards stationary phase travels slower • Components with less affinity towards stationary phase travels faster • Qualitative and quantitative analysis of the eluted substances

33. TLC requires • A mobile phase (developing solvent) • A stationary phase (a plate or strip coated with a form of silica gel) • Analysis is performed on a flat surface under atmospheric pressure and room temperature

34. In TLC, a solid phase, the adsorbent, is coated onto a solid support (thin sheet of glass, plastic or aluminium) as a thin layer (about 0.25 mm thick) • Mostly, a small amount of a binder such as plaster of Paris is mixed with the absorbent to facilitate the coating

35. Selection of Stationary Phase • The choice of the stationary phase for a given separation problem is the most difficult decision in TLC • To obtain satisfactory separation efficiency, the mean particle size, the particle size distribution and the morphology of the particle must be considered

36. Stationary phases used for thin-layer chromatography • Silica gel, modified silica gels, alumina, cellulose powder – adsorption chromatography • Cellulose powder, Kieselguhr – partition chromatography

37. Selection of adsorbents • Solubility of compound (hydrophilic or lipophilic) • Nature of substance to be separated (acidic, basic or amphoteric) • Adsorbent particle size • Adsorbent should not adhere to glass plate • Reactive of compound with the solvent or adsorbent • Chemical reactivity of compounds with binders

38. Plate preparation • TLC plates are prepared by mixing the adsorbent, such as silica gel, with a small amount of inert binder like calcium sulfate (gypsum) and water • This mixture is spread as a thick slurry on an unreactive carrier sheet, usually glass, thick aluminumfoil • The resultant plate is dried and activated by heating in an oven for thirty minutes at 110 °C • The thickness of the absorbent layer is typically around 0.1 – 0.25 mm for analytical purposes and around 0.5 – 2.0 mm for preparative TLC

39. Mobile phase • Nature of the substance to be separated (polar or non-polar) • Nature of stationary phase used • Mode of chromatography • Nature of separation (analytical or preparative) • Solvents used should be of high purity • Solvents should be miscible

41. Development of a TLC plate

42. TLC plate visualization

43. Factors affecting Rf value • It depends upon the following factors • Nature of adsorbent • Mobile phase • Activity • Thickness of layer • Temperature • Equilibrium • Loading • Dipping zone • Chromatographic Techniques

44. Advantages of TLC • Short analysis time • All spots can be visualized • Adoptable to most pharmaceuticals • Low cost • Uses small quantities of solvent • Requires minimal training • Reliable and quick • Minimum amount of equipment is needed

45. Column Chromatography

49. Ion-exchange chromatography • Ion chromatography (or ion-exchange chromatography) is a chromatography process that separates ions and polar molecules based on their affinity to the ion exchanger. • The stationary phase is an ion exchange resin that carries charged functional groups which interacts with oppositely charged groups of the compound to retained • It works on almost any kind of charged molecule—including large proteins, small nucleotides, and amino acids.

50. In this type of chromatography, a resin (the stationary solid phase) is used to covalently attach anions or cations onto it • Solute ions of the opposite charge in the mobile liquid phase are attracted to the resin by electrostatic forces • Ion exchange mechanism separates analytes based on their respective charges