1. COLUMN CHROMATOGRAPHYUnder the guidance ofMs. K. VedithaAssistant professorDept. of PA&QASubmitted byM. Durga PrasadRegd No:11AB1R0034VIGNAN PHARMACY COLLEGE(Approved by AICTE, PCI & affiliated to JNTU-K)VADLAMUDI, GUNTUR DISTRICT ANDHRA PRADESH, INDIAPIN 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 hostdivergent separation techniques that have been duly recognized right fromthe early 1900s till date. Mikhail Tswett , a Russian Botanist first and foremost coined theterminology Chromatography In 1906 he performed investigations of plant pigments, by using adsorptionchromatography and has successful separated by using leaf pigments The term Chromatography" emerged from Greek words : Chromameans Colour and Graphein means to write. 4. DEFINITION Chromatography is a technique employed for separation of thecomponents of mixture by continuous distribution of thecomponents between two phases. Ettre(1993) vehemently recommended the IUPAC definition ofchromatography which defines it as A physical technique ofseparation where in the components required to be separatedbetween the two phases , one of which being stationary(stationary phase), while the other (mobile phase) that moves in adefinite direction. 5. TYPES OF COLUMN CHROMATOGRAPHYS.NoTypes of columnchromatographyMobilephaseStationary phase Sample phase1 AdsorptionchromatographyLiquid Solid adsorbent Solution2 PartitionchromatographyLiquid Immiscible solvent onsolid matrixSolution3 Ion exchangechromatographyLiquid Ion exchange resin Solution4 GelchromatographyLiquid Solvent held in theinterstices of apolymetric solventSolution 6. COLUMN ADSORPTION CHROMATOGRAPHY The principle involved in this technique is Adsorption. When a mixture of compounds (adsorbate) dissolved in the mobilephase (eluent) moves through a column of stationary phase(adsorbent) they travel according to the relative affinities towardsstationary phase. The compound which has more affinity towards stationary phasetravels slower and the compound which has lesser affinity towardsstationary 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 willbe distributed according to their partition co-efficients. When the mixture of compounds dissolved in the mobile phase andpassed through a column of liquid stationary phase, the componentwhich is more soluble in stationary phase travels slower and thecomponent that is more soluble in mobile phase travels faster. The stationary phase used cannot be a liquid. So that a solid supportis used over which a thin film or coating of a liquid is made whichacts as a stationary phase. 9. Substances with large differences in their partition coefficients may becompletely separated by simple solvent extraction techniques involvingfew (one to three) extractions. As differences in partition coefficients of a mixture of substancesdecreases, the number of solvent extractions to complete separationincreases. 10. THEORY OF CHROMATOGRAPHY Martin and synge in 1941 developed the concept of the theoretical plate inorder to establish a satisfactory theory for partition chromatography. The column is considered as being made up of large number of parallellayers of theoretical plates. When the mobile phase passes down the column distribute themselvesbetween the stationary and mobile phases in accordance with their partitioncoefficients. 11. The rate of movement of the mobile phase is assumed to be such that theequilibrium is established within each plate.The equilibrium is dynamic and the components move down the columnat definite rate depending on the rate of movement of the mobile phase.N= L/HWhere N= Number of theoretical plates,L = Length of the columnH = 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 organicor inorganic domain are being extensively employed as adsorbents incolumn chromatography.Examples:1. Organic substances: Carbon, Starch , Cellulose2. Inorganic substances: Alumina, Silica gel, Fullers earth, Kiesulgur 14. General requirements: Particle size and geometry: uniform size& spherical shape.(60-200) 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 ofmolecules of a gas or liquid at a solid surface. When a solid surface is exposed to gas or a liquid, moleculesfrom the gas or the solution phase accumulate or concentrate atthe surface. The substance that concentrates at the surface is calledAdsorbate and the solid on whose surface the concentrationoccurs is called the Adsorbent. 16. AdsorbateAdsorbentMechanism of AdsorptionAdsorbate(Methylene Blue)Adsorbent(Charcoal)Adsorbent atoms or molecules are not surrounded by atoms ormolecules of their kind and they have unbalanced attractiveforces on the surface which can hold adsorbate particles.Example: Silica gel, Alumina 17. The most commonly used adsorbents are Silica and Alumina.These are activated at 200 CStructure of Alumina Structure of Silica 18. ADSORPTION ISOTHERMSThe 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 amountof adsorbent is proportional to the concentration of solution. When a substance moves as a band through a column ofadsorbent there is no tendency for any portion of the band to beadsorbed more strongly than another. Therefore, a symmetrical peak is obtained as the eluate fromthe column is examined. 20. B. Convex adsorption isotherms : The convex adsorption isotherms are obtained when adsorptionfrom 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 isheld 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 whenadsorption from strong solutions is greater than from weaksolutions. So, the peak obtained in this isotherm is concave in shape. 22. Adsorption isotherms and related elution patterns of substances from acolumn 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. Types of AdsorbentsS.No Adsorbent Example1 Strong adsorbent Alumina, Fullers Earth,Activated charcoal2 Intermediate adsorbent Calcium carbonate, Calciumphosphate, Magnesia, Slakedlime, Silica gel3 Weak adsorbent Cellulose, Starch, Talc, Sucrosepowder 24. Commonly used adsorbents for separation ofchemical constituents in Column chromatographyS.No Adsorbent Separable chemical constituents1. Alumina, Magnesia Alkaloids, Sterols, Vitamins2. Aluminium chloride Sterols3. Calcium carbonate Carotenoids, Xanthophylls4. Carbon Amino acids, Carbohydrates, Peptides5. Magnesium carbonate Porphyrins6. Magnesium silicate Alkaloids, Glycerides, Sterols7. Silica gel Amino acids, Sterols8. Starch Enzymes 25. MOBILE PHASEMobile 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 readyrecovery of eluted material. Polarity 27. SOLVENTS POLARITYPropanol 3.9Tetrahydro4.0furanEthanol 4.3Acetone 5.1Acetonitrile 5.8Ethylene glycol 6.9Dimethyl7.2sulfoxideWater 10.2Increasing polarity 28. COLUMN CHARACTERISTICS & SELECTION Chromatographic columns were made up of good quality of glassthat should be neutral because to avoid the affects of solvents, acidsor alkalies.Column selectionMulti-component system Long columnComponents with similaraffinitiesLong columnComponents with differentaffinitiesShort columnMore no. of compounds Long columnWeak 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 narrowcolumn 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 columnpacked 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 adsorbentlayer 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 ofadsorbent to prevent cracks. 31. Apparatus of column chromatography 32. PACKING OF COLUMNS The packing of column is an exceptional art that essentially needs alot of skill, wisdom and talent. A careful attention should always be given to the perfect uniformpacking of the selected adsorbent into the chromatographic columnso as to achieve the maximum efficiency. The packing of column is carried out in two different manners.Wet packing andDry packing. 33. WET PACKINGA thin slurry of the adsorbent with the appropriatesolvent (mobile phase) is prepared in a glassbeaker and is poured slowly into the columnAny air bubbles trapped in the slurry should beremoved by the help of a long glass rod byagitation.Adsorbent once gets settled in the column, placea disc of whatman filter paper on its top layer andwashed sand is added to top of disc.Solvent is continued to run down unless thelevel of liquid attains a height of nearly 1cmabove the top level of the packed column. 34. Wet packing process 35. DRY PACKINGDry packing involves the pouring of fine powderedform of the adsorbent into the column.The column must be tapped while the fillingprocess is going on so as to maintain the softcompactness of the adsorbent in the body of thecolumn.The column is filled upto 3/4th of the actual heightof the column. The empty head above the surface ofthe packed column is filled with the mobile phase. 36. PROCESS OF DRY PACKING 37. INTRODUCTION OF SAMPLEA graduated pipette is filled up withthe sample mixture and introduced bytouching the top of the adsorbentlayer having a filter paper with alayer of sand.The tip of the pipette is placed againstthe inside wall of the column justabove surface of the adsorbent. 38. DEVELOPMENT TECHNIQUES The development techniques are categorized into three types.(a) Elution analysisIsocratic elution techniqueGradient elution technique(b) Frontal analysis(c) Displacement analysis 39. ELUTION ANALYSIS Elution analysis refers to the specific removal of chemical entities from achromatographic support by the aid of solvent. This method makes use of a small volume of mixture that need to beseparated and the respective mobile phase is permitted to flow through thecolumn downward due to gravity. With the passage of time the mobile phase moves down the column andthe mixture of analytes undergo resolution into various distinct zones bythe 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 samplepolarity is used throughout the process of separation.Gradient elution technique : In this elution technique, solvents of increasing polarity or increasingelution strength are used during the process of separation. 41. 6FRONTAL ANALYSIS Tiselius (1940) first and foremost developed this method. The Frontal analysis employs the solution of the respective samplemixture which is incorporated continuously onto the column. In this particular instance there is no mobile phase (i.e., elutingsolvent) is used at all for the development of the analytes on thecolumn. At first the least adsorbed component passes out of the column and 42. the intermediate component is adsorbed later and next the most adsorbedcomponent is passed out. The graphical representation provides separation profile of thecomponents. The extrapolation of the various points clearly showsthe presence of other components along with the first one and needsfurther separation. 43. DISPLACEMENT ANALYSIS The principle involved in this method is that small volume ofmixture of components is introduced into the column and the usualelution is performed by means of a solvent consisting of a solutethat possesses high degree of adsorptivity for the adsorbent packedin the column. Then the adsorbed components present in the sample mixture aredisplaced by the added solute from the eluting mobile phase. Each component present in the sample mixture helps to displaceanother 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 criticalseparation of a sample mixture comprising of three components(assuming adsorption of X