Colum chromatography

Introduction to chromatography, Definition of Chromatography, Types of column chromatography, Theory of chromatography, Practical considerations in column chromatography , Factors affecting efficiency of a column, Applications.

1. COLUMN CHROMATOGRAPHY Under the guidance of Ms. K. Veditha Assistant professor Dept. of PA&QA Submitted by M. D urga P rasad Regd No:11AB1R0034 VIGNAN PHARMACY COLLEGE (Approved by AICTE, PCI & affiliated to JNTU-K) VADLAMUDI, GUNTUR DISTRICT – ANDHRA PRADESH, INDIA PIN NO: 522213

2. CONTENTS Introduction to chromatography Definition of Chromatography Types of column chromatography Theory of chromatography Practical considerations in column chromatography Factors affecting efficiency of a column Applications

3. INTRODUCTION Chromatography designates the generic name collectively assigned to host divergent separation techniques that have been duly recognized right from the early 1900s till date. Mikhail Tswett , a Russian Botanist first and foremost coined the terminology “ C hromatography” In 1906 he performed investigations of plant pigments, by using adsorption chromatography and has successful separated by using leaf pigments The term “Chromatography" emerged from Greek words : ‘Chroma’ means C olour and ‘Graphein’ means ‘to write’ .

4. DEFINITION Chromatography is a technique employed for separation of the components of mixture by continuous distribution of the components between two phases . Ettre(1993) vehemently recommended the IUPAC definition of chromatography which defines it as ‘A physical technique of separation where in the components required to be separated between the two phases , one of which being ‘stationary’ (stationary phase), while the other (mobile phase) that moves in a definite direction’ .

5. TYPES OF COLUMN CHROMATOGRAPHY S.No Types of column chromatography Mobile phase Stationary phase Sample phase 1 Adsorption chromatography Liquid Solid adsorbent Solution 2 Partition chromatography Liquid Immiscible solvent on solid matrix Solution 3 Ion exchange chromatography Liquid Ion exchange resin Solution 4 Gel chromatography Liquid Solvent held in the interstices of a polymetric solvent Solution

6. COLUMN ADSORPTION CHROMATOGRAPHY The principle involved in this technique is A dsorption . When a mixture of compounds (adsorbate) dissolved in the mobile phase (eluent) moves through a column of stationary phase (adsorbent) they travel according to the relative affinities towards stationary phase. The compound which has more affinity towards stationary phase travels slower and the compound which has lesser affinity towards stationary phase travels faster . In this way, the compounds are separated.

7. Separation of compounds based upon Affinities

8. COLUMN PARTITION CHROMATOGRAPHY The principle involved in this technique is partition. When two immiscible liquids are present, a mixture of solutes will be distributed according to their partition co-efficients . When the mixture of compounds dissolved in the mobile phase and passed through a column of liquid stationary phase, the component which is more soluble in stationary phase travels slower and the component that is more soluble in mobile phase travels faster. The stationary phase used cannot be a liquid. So that a solid support is used over which a thin film or coating of a liquid is made which acts as a stationary phase.

9. Substances with large differences in their partition coefficients may be completely separated by simple solvent extraction techniques involving few (one to three) extractions. As differences in partition coefficients of a mixture of substances decreases, the number of solvent extractions to complete separation increases.

10. THEORY OF CHROMATOGRAPHY Martin and synge in 1941 developed the concept of the ‘theoretical plate’ in order to establish a satisfactory theory for partition chromatography. The column is considered as being made up of large number of parallel layers of ‘ theoretical plates’. When the mobile phase passes down the column distribute themselves between the stationary and mobile phases in accordance with their partition coefficients.

11. The rate of movement of the mobile phase is assumed to be such that the equilibrium is established within each plate. The equilibrium is dynamic and the components move down the column at definite rate depending on the rate of movement of the mobile phase. N= L/H Where N= Number of theoretical plates, L = Length of the column H = Height equivalent of theoretical plate

12. PRACTICAL REQUIREMENTS Stationary phase Mobile phase Column characteristics Preparation of the column Introduction of sample Development techniques Detection of components Recovery of components

13. STATIONARY PHASE (ADSORBENT) A good number of solid compounds belonging to either ‘organic’ or ‘inorganic’ domain are being extensively employed as adsorbents in column chromatography. Examples: 1. Organic substances : Carbon, Starch , Cellulose 2. Inorganic substances : Alumina, Silica gel, Fuller’s earth, Kiesulgur

14. General requirements: Particle size and geometry : uniform size& spherical shape. (60-200 μ ) High mechanical stability Inert and should not react with the solute or other compounds Insoluble in the solvents or mobile phases used It should allow free flow of mobile phase Useful for separating a wide variety of compounds Freely available and inexpensive .

15. ADSORPTION PROCESS Adsorption is defined as the phenomenon of concentration of molecules of a gas or liquid at a solid surface. When a solid surface is exposed to gas or a liquid, molecules from the gas or the solution phase accumulate or concentrate at the surface. The substance that concentrates at the surface is called Adsorbate and the solid on whose surface the concentration occurs is called the Adsorbent.

16. Adsorbate Adsorbent Adsorbent atoms or molecules are not surrounded by atoms or molecules of their kind and they have unbalanced attractive forces on the surface which can hold adsorbate particles. Example : Silica gel, Alumina Adsorbate (Methylene Blue) Adsorbent (Charcoal) Mechanism of Adsorption

17. The most commonly used adsorbents are Silica and Alumina. These are activated at 200 C Structure of Alumina Structure of Silica

18. ADSORPTION ISOTHERMS The adsorption isotherms are of three types. They are (a) Linear adsorption isotherms. (b) Convex adsorption isotherms. (c) Concave adsorption isotherms .

19. A. Linear adsorption isotherms : The linear adsorption isotherms are obtained when the amount of adsorbent is proportional to the concentration of solution . When a substance moves as a band through a column of adsorbent there is no tendency for any portion of the band to be adsorbed more strongly than another . Therefore, a symmetrical peak is obtained as the eluate from the column is examined .

20. B. Convex adsorption isotherms : The convex adsorption isotherms are obtained when adsorption from weak solutions is greater than from strong solutions. The pattern of the substance is symmetrical initially. But the substance in low concentration, the front of the band is held strongly by adsorption than in the center of the band . Therefore, a sharp leading edge to the band is obtained.

21. C. Concave adsorption isotherms : The concave adsorption isotherms are obtained when adsorption from strong solutions is greater than from weak solutions. So, the peak obtained in this isotherm is concave in shape.

22. Adsorption isotherms and related elution patterns of substances from a column of adsorbent. m = weight of substance adsorbed per g of adsorbent. Cs = Concentration of solution. Cf = Concentration of each fraction. f = number of each fraction.

23. S.No Adsorbent Example 1 Strong adsorbent Alumina, Fuller’s Earth, Activated charcoal 2 Intermediate adsorbent Calcium carbonate, Calcium phosphate, Magnesia, Slaked lime, Silica gel 3 Weak adsorbent Cellulose, Starch, Talc, Sucrose powder Types of Adsorbents

24. S.No Adsorbent Separable chemical constituents 1. Alumina, Magnesia Alkaloids, Sterols, Vitamins 2. Aluminium chloride Sterols 3. Calcium carbonate Carotenoids, Xanthophylls 4. Carbon Amino acids, Carbohydrates, Peptides 5. Magnesium carbonate Porphyrins 6. Magnesium silicate Alkaloids, Glycerides, Sterols 7. Silica gel Amino acids, Sterols 8. Starch Enzymes Commonly used adsorbents for separation of chemical constituents in Column chromatography

25. MOBILE PHASE Mobile phase is very important and they serve several functions. They act as solvent, developer and as a eluent. The functions of the mobile phase are: As developing agent To introduce the mixture into the column – as solvent To remove pure components out of the column – as eluent

26. Choice of the solvent: Depend on the solubility characteristics of the mixture. Should also have sufficiently low boiling points which permit ready recovery of eluted material. Polarity

27. SOLVENTS POLARITY Propanol 3.9 Tetrahydro furan 4.0 Ethanol 4.3 Acetone 5.1 Acetonitrile 5.8 Ethylene glycol 6.9 Dimethyl sulfoxide 7.2 Water 10.2 Increasing polarity

28. COLUMN CHARACTERISTICS & SELECTION Chromatographic columns were made up of good quality of glass that should be neutral because to avoid the affects of solvents, acids or alkalies. Column selection Multi-component system Long column Components with similar affinities Long column Components with different affinities Short column More no. of compounds Long column Weak adsorbent few compounds Short column

29. The column dimensions are very important for effective separation. The length : diameter ratio from 10:1 to 30:1. For more efficiency 100:1 can be used. The length of the column depends upon : Number of compounds to be separated. Type of adsorbent used. Quantity of sample Affinity of the compounds towards adsorbent used. Better separation will be obtained with a long narrow column than short thick column because number of plates will be more.

30. PREPARATION OF THE COLUMN It consists of a glass tube with the bottom portion of the column packed with glass wool / cotton wool or may contain asbestos pad. Above this the adsorbent is packed. After packing a paper disc is kept on the top, so that the adsorbent layer is not disturbed during the introduction of sample. Slurry is introduced into the column using funnels. The level of solvent must never be allowed to fall below the level of adsorbent to prevent cracks.

31. Apparatus of column chromatography

32. PACKING OF COLUMNS The packing of column is an exceptional art that essentially needs a lot of skill, wisdom and talent. A careful attention should always be given to the perfect uniform packing of the selected adsorbent into the chromatographic column so as to achieve the maximum efficiency. The packing of column is carried out in two different manners. Wet packing and Dry packing.

33. WET PACKING

34. Wet packing process

35. DRY PACKING

36. PROCESS OF DRY PACKING

37. INTRODUCTION OF SAMPLE

38. DEVELOPMENT TECHNIQUES The development techniques are categorized into three types. (a) Elution analysis Isocratic elution technique Gradient elution technique (b) Frontal analysis (c) Displacement analysis

39. ELUTION ANALYSIS Elution analysis refers to the specific removal of chemical entities from a chromatographic support by the aid of solvent. This method makes use of a small volume of mixture that need to be separated and the respective ‘mobile phase’ is permitted to flow through the column downward due to gravity. With the passage of time the ‘mobile phase’ moves down the column and the mixture of ‘analytes’ undergo resolution into various ‘distinct zones’ by the fact that the analytes in the mixture get adsorbed to various degree.

40. Isocratic elution technique : In this technique, the same solvent composition or solvent of sample polarity is used throughout the process of separation. Gradient elution technique : In this elution technique, solvents of increasing polarity or increasing elution strength are used during the process of separation.

41. 6 FRONTAL ANALYSIS Tiselius (1940) first and foremost developed this method. The ‘Frontal analysis’ employs the solution of the ‘respective sample mixture’ which is incorporated continuously onto the column. In this particular instance there is no mobile phase (i.e., eluting solvent) is used at all for the development of the ‘analytes’ on the column. At first the least adsorbed component passes out of the column and

42. the intermediate component is adsorbed later and next the most adsorbed component is passed out. The graphical representation provides separation profile of the components. The extrapolation of the various points clearly shows the presence of other components along with the first one and needs further separation.

43. DISPLACEMENT ANALYSIS The principle involved in this method is that ‘small volume of mixture of components’ is introduced into the column and the usual ‘elution’ is performed by means of a solvent consisting of a solute that possesses high degree of adsorptivity for the adsorbent packed in the column. Then the adsorbed components present in the ‘sample mixture’ are displaced by the ‘added solute’ from the eluting mobile phase. Each component present in the sample mixture helps to displace another solute that is less adsorbed.

44. In this way, the least adsorbed component is flushed out of the column. The graphic representation of the plot is obtained by the critical separation of a sample mixture comprising of three components (assuming adsorption of X<Y<Z). In the event, when D is designated as displacer, the graph is

45. DETECTION AND RECOVERY OF COMPONENTS The coloured components are detected by visual examination. The colourless components may also be detected visually if they fluorescence. Ex : Quinine & Ergotamine. Recovery of the components after detection on the column requires ‘extrusion’ of the column of adsorbent and isolation of each zone for extraction with solvents. In case of plastic tubing the zones are isolated by cutting tubing into sections.

46. For colourless compounds the eluate is collected as a large number of fractions, each of small volume. Each fraction is examined appropriately for the presence of a compound. The examination may by Evaporation of the solvent from each fraction and weighing the residue By simple spot tests By examination of the fraction by paper or thin layer chromatography By spectrophotometry

47. FACTORS AFFECTING EFFICIENCY OF A COLUMN Factor Effect Particle size of solid stationary phase Decrease in size improves separation Column dimensions Efficiency increases as ratio length Column temperature Increase in column temperature results in speed of elution but does not improve separation Solvent It should be of low viscosity & high volatility Solvent flow rate Uniform and low flow rate gives better resolution Conduction of adsorbent Deactivation of adsorbent decreases separation Concentration of solutes Substances of high concentration moves slowly

48. APPLICATIONS OF COLUMN CHROMATOGRAPHY Column chromatography is best suited to separate active principle from plant materials. To separate impurities along with the important constituents Isolation of metabolites from important components Used for determination of phytomenadione in tablets and injections Determination of flucinolone, acetonide, betamethasone in formulations.

49. Used for separation of inorganic ions like copper ion, cobalt ion, nickel ion. Determination of the percentage w/w of strychnine in syrup of iron phosphate with quinine and stychnine. Determination of quinine in ethanolic solution. Useful in the separation of carbohydrates and their derivatives. To separate natural compound mixtures like alkaloids, glycosides.

50. Determination of phenothiazine in the presence of diphenylamine and carbazole. The chromatogram for a mixture of diphenylamine and phenothiazine.

51. ADVANTAGES OF COLUMN CHROMATOGRAPHY Any type of mixture can be separated. Wider choice of mobile phase. Automation is possible Any quantity of mixture can be separated.

52. DISADVANTAGES OF COLUMN CHROMATOGRAPHY Time consuming. More amount of mobile phase are required. Automation makes the technique more complicated and expensive.

53. REFERENCES : A.H. BECKETT & J.B. STENLAKE, Practical pharmaceutical chemistry, 4 th edition, part two, page no: 86-105. ASHUTOSH KAR, Pharmaceutical analysis – II, page no: 161-181. Dr . S. RAVI SANKAR, Pharmaceutical analysis, 3 rd edition, page no: 13-4 to 13-13. B.K. SHARMA, Instrumental methods of chemical analysis, page no : C-8 to C-15. Dr. A.V. KASTURE, Dr. K.R. MAHADIK, Dr. S.G. WADODKAR, Dr. H.N. MORE, Pharmaceutical analysis volume – II, page no: 10-17

54. ACKNOWLEDGEMENT Thank you for paying attention. I sincerely thank my principal Dr. P. Srinivasa babu sir for the honoured encouragement. I also sincerely thank my guide K. Veditha madam for the valuable guidance. A special thanks for Ch. Devadas sir and Seminar committee.

55. THANK YOU