column chromatography

column chromatography ppt

1. COLUMN CHROMATOGRAPHY Under the guidance of: Mr.Ch.Devadasu Assistant Professor Dept. of PA&QA Submitted by: J . Ramesh Regd No:12AB1R0088 IV/IV B. Pharm., VIGNAN PHARMACY COLLEGE (Approved by AICTE, PCI & affiliated to JNTU-K) VADLAMUDI, GUNTUR DISTRICT – ANDHRA PRADESH, INDIA PIN NO: 522213

2. CONTENTS History & Introduction to chromatography Classification of chromatography Definition of chromatography Types of chromatography Requirements for column chromatography Factors affecting efficiency of column Applications Conclusion 2 Vignan Pharmacy College, Vadlamudi, guntur Dist (AP) pin: 522213

3. A group of techniques for the separation of the compounds of mixture. The term chromatography was coined from two greek words kromatos-colour graphos -written In 1906 the Russian scientist M.Tswett separated different coloured constituents of leaves by passing an extract of the leaves through a column of calcium carbonate. HISTORY “Meaning Colour Writing” Vignan Pharmacy College, Vadlamudi, guntur Dist (AP) pin: 522213 3 INTRODUCTION

4. DEFINITION: M. Tswett (1906) defined chromatography as The method in which the components of a mixture are separated on an adsorbent column in a flowing system. The international union of pure& applied chemistry(IUPAC)definition of chromatography : “Chromatography is a physical method of separation in which the components to be separated are distributed between two phases, one of which is stationary, while the other(mobile phase)moves in a definite direction” . INTRODUCTION Vignan Pharmacy College, Vadlamudi, guntur Dist (AP) pin: 522213 4

5. The stationary phase may be a solid or a liquid supported on a solid (or) a gel - May be packed in a column eg : column chromatography. -Spread as a layer on a glass/ aluminium plate eg : TLC, HPTLC. -Distributed as a liquid film eg : GLC. The mobile phase may be a liquid / solvent/mixture of solvent or gas . Vignan Pharmacy College, Vadlamudi, guntur Dist (AP) pin: 522213 5

6. chromatography Liquid chromatography Gas chromatography Liquid -solid chromatography Liquid-Liquid chromatography eg : GLC GSC eg : column adsorption TLC,HPTLC HPLC eg : column partition Paper partition HPCPC Mobile phase: Gas Mobile phase: liquid Classification of Chromatography A/c to physical state of mobile phase 6 Vignan Pharmacy College, Vadlamudi, guntur Dist (AP) pin: 522213

7. Classification based up on physical nature of stationary phase Adsorption chromatography Partition chromatography eg : column adsorption Ion exchange Size exclusion TLC,HPTLC,HPLC eg : column partition Paper partition HPCPC GC(GLC) Stationary phase: solid Stationary phase: liquid 7 Vignan Pharmacy College, Vadlamudi, guntur Dist (AP) pin: 522213

8. Classification based on polarity of stationary phase & mobile phase Normal phase chromatography Reverse phase chromatography Stationary phase: polar(silica gel) Mobile phase: non-polar (n-hexane) Stationary phase: non polar ( Octadecylsilane or C 18 ) Mobile phase: polar (Acetonitrile, water) 8 Vignan Pharmacy College, Vadlamudi, guntur Dist (AP) pin: 522213

9. COLUMN CHROMATOGRAPHY Introduction: Column chromatography is a separation technique in which components of mixture is separated by using a glass column packed with stationary phase and the liquid mobile phase flowing continuously through the column. Principles of column chromatography: Column adsorption chromatography Partition chromatography Ion-exchange chromatography Size exclusion chromatography or Gel permeation chromatography: Affinity chromatography Vignan Pharmacy College, Vadlamudi, guntur Dist (AP) pin: 522213 9

10. Adsorption chromatography: The principle underlying the separation of the compounds is adsorption at the solid-liquid interphase . solid stationary phase like silica gel and liquid mobile phase is used. In column chromatography the interaction between adsorbent & component is usually reversible. The various components will move down the column until, they are arranged in order of their affinity move down the column at a faster rate than those with the greatest affinity for the adsorbent. Vignan Pharmacy College, Vadlamudi, guntur Dist (AP) pin: 522213 10

11. The separation is based on differences in affinities towards the stationary phase. Those compounds which have greater affinity towards stationary phase will move slower rate when compared to compound with less affinity . In straight-phase chromatography, four commonly used solvents are: hexane; dichloromethane; isopropanol; methanol (Increasing strength) In reverse-phase chromatography, four commonly used solvents are: water; methanol; acetonitrile; tetrahydrofuran (Increasing strength) Vignan Pharmacy College, Vadlamudi, guntur Dist (AP) pin: 522213 11

12. Mechanism of adsorption: Vignan Pharmacy College, Vadlamudi, guntur Dist (AP) pin: 522213 12 Normal phase chromatography Reverse phase chromatography

13. Partition chromatography: Distribution of solutes between two immiscible liquids.The two immisible liquids are liquid stationary phase, liquid mobile phase (Or) gaseous mobile phase. In partition chromatography separation is based up on the differences in partition coefficient of the individual components in a mixture. According to Nernst distribution law, The ratio of activities of the solute into two immiscible liquid phases is known as a partition coefficient. a s1 k= a s2 where 1=organic phase 2=aqueous phase Vignan Pharmacy College, Vadlamudi, guntur Dist (AP) pin: 522213 13 Fig . 1:partition chromatography

14. In dilute solutions the activities can be replaced by solubilities then k is written as k D . k d = k so / k aq where s o =solubility of solute in organic phase. s aq =solubility of solute in aqueous phase. When solubilities are replaced by concentration of solute now the k D becomes D. D=Distribution ratio. D= C /C W where C o =concentration of solute in organic phase C w =concentration of solute in aqueous phase ‘D’ depends upon the nature of the both phases, nature of solute & temperature. Vignan Pharmacy College, Vadlamudi, guntur Dist (AP) pin: 522213 14

15. In case of gas chromatography the stationary phase is high boiling organic liquid ( polydimethyl siloxanes ) where as the mobile phase is inert gases ( Helium,nitrogen,Argon ) Examples: column partition Gas liquid chromatography High performance centrifugal partition chromatography Paper partition chromatography counter current extraction chromatography (counter current distribution) Vignan Pharmacy College, Vadlamudi, guntur Dist (AP) pin: 522213 15 k= concentration of solute in stationary phase concentration of solute in mobile phase

16. Ion-exchange chromatography: Ion exchange chromatography is the process by a mixture of charged ions can be separating using an ion exchange resin. The principle of separation is by reversible exchange of ions between the ions present in the solution. Example: solid matrix of solution cation retained solution cation exchange resin containing by the solid matrix cations of ion exchange resin Vignan Pharmacy College, Vadlamudi, guntur Dist (AP) pin: 522213 16 Ion exchange resin-H + M + Ion exchange resin-M + + H +

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19. Gel permeation chromatography: It is also called as gel filtration or size exclusion chromatography. In size exclusion chromatography , the stationary phase is a porous matrix made up of compounds like cross linked polystyrene, cross like dextrans , polyacrylamide gels, agarose gels etc. The separation is based on their ( analytes ) molecular sizes since the gel behaves like a moleculae sieve. This technique is used for the separation of proteins,polysaccharidees,enzymes and synthetic polymers. Vignan Pharmacy College, Vadlamudi, guntur Dist (AP) pin: 522213 19 Fig . 2:gel permeation chromatography

20. Affinity chromatography : It is based on the Lock & key mechanism prevalent in biological system. Vignan Pharmacy College, Vadlamudi, guntur Dist (AP) pin: 522213 20 Fig . 3:Affinity chromatography

21. Requirements for column chromatography : 1. C olumn characteristics & selection 2. Stationary phases 3. Mobile phases 4. P reparation of column 5. Introduction of sample 6. Development of column 7. Detection of components 8. Recovery of components Vignan Pharmacy College, Vadlamudi, guntur Dist (AP) pin: 522213 21

22. Materials of construction Good quality neutral glass,plastic or nylon Adsorbent(stationary phase)/ adsorbate ( mixture) weight ratio 30:1 Column length to diameter ratio(cm) 10-15:1 or 30-100:1 Multi component system is present Long column is used Components with similar affinities for adsorbent are present Long column is used Components with different affinities for adsorbent are present Short column is used Column characteristics & selection: Vignan Pharmacy College, Vadlamudi, guntur Dist (AP) pin: 522213 22 Table:1: selecting a suitable column dimension

23. Stationary phases in column chromatography : Stationary phases used in column adsorption chromatography are also known as adsorbents. Requirements of an ideal stationary phase : 1.They should be insoluble in solvents or mobile phases. 2.chemically inert. 3.colourless to facilitate observation of zones. 4.should have reproducible properties from batch to batch. 5.The particle should have uniform size distribution and have spherical 6. particle size : 60-200µ Vignan Pharmacy College, Vadlamudi, guntur Dist (AP) pin: 522213 23

24. The most commonly used chromatographic adsorbent is silica or silisic acid or silica gel (80-100 mesh or 100-200 mesh size, which has a particle size of 63-200µm ) . They adsorbs polar and unsaturated substances by the formation of hydrogen bonds with the hydroxyl groups on the silicon atom. Type of mesh size in microns 60/120 mesh 120-250 micron 100/200mesh 75-150 micron 70/230 mesh 63-200 micron 230/400 mesh 37-63 micron 70/ 325 mesh 45-200 micron Vignan Pharmacy College, Vadlamudi, guntur Dist (AP) pin: 522213 24 Fig . 6: Silica gel and their mesh

25. Table 2: Adsorbents used in column chromatography Weak activity Medium activity Strong activity Sucrose Calcium carbonate Activate magnesium silicate Starch Calcium phosphate Activated alumina Inulin Magnesium carbonate Activated charcoal Talc Magnesium oxide Activated magnesia Sodium carbonate Calcium oxide Activated silica Vignan Pharmacy College, Vadlamudi, guntur Dist (AP) pin: 522213 25

26. Adsorption isotherms : The amount of a solute adsorbed from the solution by an absorbent can be determined by shaking a known weight of adsorbent with a known volume of solution at fixed temperature until equilibrium is attained. The adsorbent is filtered off & the concentration of the substance in the filtrate is determined by any suitable means. A plot of equilibrium concentration of species in a solution and that of amount adsorbed is called adsorption isotherms . Vignan Pharmacy College, Vadlamudi, guntur Dist (AP) pin: 522213 26

27. Linear adsorption isotherms: These are obtained when the amount of substance absorbed per gram of adsorbent is proportional to the concentration o f solution. The adsorption coefficient(k=m/Cs) is a constant value, in this linear adsorption isotherm. Convex adsorption isotherms: These are obtained when adsorption from weak solution is greater than from strong solutions. Concave adsorption isotherms: These are obtained when adsorption from strong solution is greater than from weak solutions. Vignan Pharmacy College, Vadlamudi, guntur Dist (AP) pin: 522213 27

28. Convex and concave adsorption isotherms: Vignan Pharmacy College, Vadlamudi, guntur Dist (AP) pin: 522213 28 Tailing is particularly likely to occur where adsorption is involved in the separation process. Both effects become more pronounced at high concentrations and are therefore symptomatic of overloading the column sample.

29. 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. Vignan Pharmacy College, Vadlamudi, guntur Dist (AP) pin: 522213 29

30. ELUENTS OR SOLVENTS OR MOBILE PHASES USED : Vignan Pharmacy College, Vadlamudi, guntur Dist (AP) pin: 522213 30 Mobile phase act as a solvent to introduce the mixture into the column as developer to develop the zones for separation and as an eluent to remove the pure component out to the column. Strain(1942) has arranged the solvents in order of eluting power. A grouping of solvents in order of chromatographic strength(polarity index) is known as elutrophic series.

31. Increasing eluting power Light petroleum ether (petroleum ether,hexane,heptane Carbon tetra chloride Cyclohexane Carbon disulphide Benzene Toluene Chloroform Ether Ethyl acetate Acetone Alcohols Water Pyridine Organic acids Inorganic acids&bases Vignan Pharmacy College, Vadlamudi, guntur Dist (AP) pin: 522213 31 Fig . 7:mobile phase

32. Preparation (packing) of the column: a) Slurry packing (wet packing method): The adsorbent is suspended in the mobile phase and stirred very well to remove all air bubbles. The resulted slurry is then poured in to the column. After slurry application, the column must be allowed to settle overnight. This is the ideal method of column packing. At the bottom portion of the column a piece of glass wool or cotton/ whattman filter paper disc must be added before the slurry application. Vignan Pharmacy College, Vadlamudi, guntur Dist (AP) pin: 522213 32

33. Vignan Pharmacy College, Vadlamudi, guntur Dist (AP) pin: 522213 33 PROCESS OF WET PACKING Fig . 8

34. b) Dry packing: In this method the dry adsorbent is poured to the column directly Vibration is applied to get rid of air bubbles then the mobile phase is passed through the adsorbent. The demerit with this method is that air bubbles are entrapped between the solvent and the stationary phase. This method cannot be applied in gel chromatography. Vignan Pharmacy College, Vadlamudi, guntur Dist (AP) pin: 522213 34

35. Fig 9:PROCESS OF DRY PACKING Vignan Pharmacy College, Vadlamudi, guntur Dist (AP) pin: 522213 35

36. c) Sample introduction in column chromatography: 1) Wet application : D issolve the sample in the initial mobile phase and apply by pipette to the top of the column. This is very good method but in most of cases the samples are not soluble in the initial mobile phase. Vignan Pharmacy College, Vadlamudi, guntur Dist (AP) pin: 522213 36

37. Vignan Pharmacy College, Vadlamudi, guntur Dist (AP) pin: 522213 37 2 ) Dry loading: Dissolve sample in any volatile solvent. The sample solution is then adsorbed an small weight of adsorbent and the solvent is allowed to evaporate. The dry adsorbent loaded with the sample is then applied to the column. Fig . 10:sample adsorbed in silica gel Fig.11: column loaded with dry sample

38. Development of column : Removal of individual components from a column is called development of column. Frontal analysis : This technique was developed by Tiselius in 1940 In this method, the solution of sample mixture is added continuously on the column. No mobile phase (solvent) is used for development of column. A mixture containing A,B,C is added on the column. If component A is least adsorbed, component B is adsorbed to intermediate extent and component C most strongly to the column adsorbent material. Vignan Pharmacy College, Vadlamudi, guntur Dist (AP) pin: 522213 38

39. The mixture flows through the column, the least adsorbed A runs down the column fast, component B to intermediate extent while ‘c’ is retained at the top of the column. A plot of amount of substance against volume of eluate is gives a chromatogram. Vignan Pharmacy College, Vadlamudi, guntur Dist (AP) pin: 522213 39

40. Displacemental Analysis: In this method, a small volume of mixture is added to the column and elution is carried out by a solvent containing a solute which has adsorptivity for column material. The adsorbed constituents of mixture are displayed by the solute from mobile phase. Each solute in the mixture in turn displaces another substance solute which is less firmly adsorbed. The least adsorbed constituent is pushed out of the column. The substance used in mobile phase is called as displacer. Vignan Pharmacy College, Vadlamudi, guntur Dist (AP) pin: 522213 40

41. The plot of amount of substance (conc. In eluate ) against volume of eluate is given by: Displacement analysis technique is mainly used in preparative work and is not suitable for analysis since some overlapping may occur Vignan Pharmacy College, Vadlamudi, guntur Dist (AP) pin: 522213 41 Fig.12:Displacement analysis

42. Elution analysis: It is a common method used in column chromatography. In this method a small volume of mixture to be separated is added on the top of column& mobile phase is allowed to flow through the column. T he mixture introduced on the column gets separated into individual as the components of mixture are adsorbed to the column material to different extent. on other phase of mobile phase, each component of mixture is eluted out as separated components (called eluate ). Vignan Pharmacy College, Vadlamudi, guntur Dist (AP) pin: 522213 42

43. A plot of amount of substance per ml of eluate against volume of eluate will gives the following chromatogram. Vignan Pharmacy College, Vadlamudi, guntur Dist (AP) pin: 522213 43 Fig13:Elution analysis

44. a) Isocratic elution: ( Iso means same or similar ) In this elution technique, the same solvent composition or solvent of sample polarity is used throughout the process of separation.e.g .(chloroform only as a solvent or CHCL3:MeOH=1:1 ) b) Gradient elution:( gradient means gradual) In this elution tecenique,solvents of gradually increasing polarity or increasing elution length are used during the process of separation. Vignan Pharmacy College, Vadlamudi, guntur Dist (AP) pin: 522213 44

45. Vignan Pharmacy College, Vadlamudi , guntur Dist (AP) pin: 522213 45 DETECTION & RECOVERY OF COMPONENTS : Fractions are collected by elution analysis Each fraction is examined by TLC using suitable experimental conditions Those fractions which give same R f values in TLC are added as a common fraction From the column fraction, solvent is evaporated, dried & the materialis collected in eppendorf container After spectral analysis (NMR, MS, X-RD etc.)the compound is identified

46. Vignan Pharmacy College, Vadlamudi , guntur Dist (AP) pin: 522213 46

47. Table 3: Factors affecting column efficiency 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 Vignan Pharmacy College, Vadlamudi, guntur Dist (AP) pin: 522213 47

48. Inorganic ions Separeation of copper,cobalt,nickel etc Organic Purification of dyes e.g.sudan red,methylene blue,malachite green Separation of diastereomers Separation of tautomers and racemates Separation of cis and trans- pyrazolines Plant constituents Separation of geometrical isomers of cis and trans isomers of bixin and crocetin dimethyl ether using alumina Purification of steroids carotenoids,chlorophils and xanthophylls Separation of alkaloids and glycosides. Drugs and pharmaceuticals Purification of vitamin-B12 Oxytetracyclin and oleandomycin Separation of the urinary 17-ketosteroids Separation of cartisol from plasma Separation of amino acids,proteins,phospholipids and triglycerides. APPLICATIONS : Vignan Pharmacy College, Vadlamudi, guntur Dist (AP) pin: 522213 48

49. 2.column chromatography can be used for the estimation of drugs in formulations or crude extracts:e.g . 1) Determination of % w/w of strychnine in syrup of ferrous phosphate with quinine and strychinine . 2) Determination of primary and secondary glycosides in digitalis leaf. 3) Determination of phytomeandione in injection and tablets. 4) Determination of flucinnolone acetonide or betamethasone 17-valerate in formulated products. Vignan Pharmacy College, Vadlamudi, guntur Dist (AP) pin: 522213 49

50. Cholestenone Cholesterol Examples : 1. Separation of cholestenone from cholesterol: Stationary phase :copper (n) oxide Mobile phase : Light petroleum Temp. conditions :300-314°c Vignan Pharmacy College, Vadlamudi, guntur Dist (AP) pin: 522213 50

51. Stationary phase :Activated silica Mobile phase :Light petroleum Temp. conditions :40-60°c 2.Separation of (E)-isomer from the (Z)- azobenzene : Vignan Pharmacy College, Vadlamudi, guntur Dist (AP) pin: 522213 51

52. Vignan Pharmacy College, Vadlamudi , guntur Dist (AP) pin: 522213 52 CONCLUSION Column chromatography is a conventional tool for separation of phytohemicals, removal of impurities and purification of drugs. Effective separation of constituents from different sources in preparative scale (milligram to gram) can be achieved by column chromatography. Availability of wide range of stationary phases makes the technique to be used for different kinds of mechanisms. Understanding the basic principles of column chromatography enables us to find solutions for current research problems.

53. Vignan Pharmacy College, Vadlamudi , guntur Dist (AP) pin: 522213 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. DAVID G.WATSON, Pharmaceutical analysis, page no: 270 - 271. 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. VOGEL’S, Text book of quantitative analysis, page no:289- 314. G.DEVALA RAO, A text book of advanced pharmaceutical analysis, page no:332- 335. Vignan Pharmacy College, Vadlamudi , guntur Dist (AP) pin: 522213 54

54. Vignan Pharmacy College, Vadlamudi , guntur Dist (AP) pin: 522213 54 Mahfouz m abdel-gawad , maher a el- hashash , mortada m el- sayed . ., Chromatographic isolation of Allium cepa (ssp. Red onion) and its cytotoxic activity against human liver carcinoma cell lines (HEPG2); International Journal Of Pharmcy and Pharmaceutical Sciences, vol 6, Page no: 109-110. Rachana gohel , bhavna solanki , nilesh gurav .., Isolation and characterization of shatavarin iv from root of asparagus racemosus willd ; International Journal Of Pharmcy and Pharmaceutical Sciences, vol 7, page no: 363-365. ARTICLES :

55. Thank you for paying attention. I sincerely thank my guide CH.DEVADASU sir for giving me the valuable guidance. My honored thanks to principal DR.P.SRINIVASA BABU sir for giving me this opportunity. A special thanks to seminar committee. Vignan Pharmacy College, Vadlamudi, guntur Dist (AP) pin: 522213 55 ACKNOWLEDGEMENT

56. Vignan Pharmacy College, Vadlamudi , guntur Dist (AP) pin: 522213 56 QUESTIONS 1) what are the temperature conditions used In separation of (E)-isomer from the (z)- azobenzene …………….?   2) silica gel is also called as…………….?   3) In reverse phase chromatography what is the polarity of stationary phase…………..?   4) write the formula for Nernst distribution law…………..?   5) what is the stationary phase used in gel permeation chromatography……..?

57. Vignan Pharmacy College, Vadlamudi, guntur Dist (AP) pin: 522213 57