Columchromatography rv
- 1. COLUMN CHROMATOGRAPHY PRESENTED BY: RAGHAV DOGRA M.PHARM (ANALYSIS) 2016-2017 • GT Road (NH-95), Ghal Kalan, Moga(142001), Punjab, India1
- 2. CONTENTS 2 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 3 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 “ Chromatography”. 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 Colour and ‘Graphein’ means ‘to write or express’.
- 4. DEFINITION 4 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 5 S.N o 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 6 The principle involved in this technique is Adsorption. 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. SEPRATION OF COMPOUNDS BASED UPON AFFINITIES 7
- 8. COLUMN PARTITION CHROMATOGRAPHY 8 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. 9
- 10. 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. 11 HETP It was assumed that the chromatographic system composed of number of “distribution systems” or “equilibrations” called “Theoretical Plates”. Each theoretical plate is composed of stationary phase and mobile phase. The height of each plate is called “Height equivalent to Theoretical Plate” (HETP). The number of theoretical plates “N” is important for separation. Increasing “N” resulted in narrower bands and better separation. 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.
- 12. 12 N= L/H Where N= Number of theoretical plates, L = Length of the column H = Height equivalent of theoretical plate “N” can be increased by: 1- Increase the length of the column (impractical). 2- Decrease the HETP. How to decrease HETP: Decrease the particle size of the stationary phase. Proper selection of good mobile phase.
- 13. 13 PRACTICAL REQUIREMENTS Stationary phase Mobile phase Column characteristics Preparation of the column Introduction of sample Development techniques Detection of components Recovery of components
- 14. 14 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
- 15. General requirements: 15 Particle size and geometry: uniform size& spherical shape. (60-200μ) 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.
- 16. ADSORPTION PROCESS 16 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.
- 17. 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
- 18. 18 The most commonly used adsorbents are Silica and Alumina. These are activated at 200° C Structure of Alumina Structure of Silica
- 19. 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 19
- 20. 20 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
- 21. MOBILE PHASE 21 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
- 22. 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 22
- 23. 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 Increasingpolarity 23
- 24. 24 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
- 25. • 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. 25
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- 27. 27 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.
- 28. Apparatus of column chromatography 28
- 29. PACKING OF COLUMNS 29 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 Dry packing.
- 30. WET PACKING 30 A thin slurry of the adsorbent with the appropriate solvent (mobile phase) is prepared in a glass beaker and is poured slowly into the column Any air bubbles trapped in the slurry should be removed by the help of a long glass rod by agitation. Adsorbent once gets settled in the column, place a disc of whatman filter paper on its top layer and washed sand is added to top of disc. Solvent is continued to run down unless the level of liquid attains a height of nearly 1cm above the top level of the packed column.
- 32. DRY PACKING 32 Dry packing involves the pouring of fine powdered form of the adsorbent into the column. The column must be tapped while the filling process is going on so as to maintain the soft compactness of the adsorbent in the body of the column. The column is filled upto 3/4th of the actual height of the column. The empty head above the surface of the packed column is filled with the mobile phase.
- 33. PROCESS OF DRY PACKING 33
- 34. INTRODUCTION OF SAMPLE 34 A graduated pipette is filled up with the sample mixture and introduced by touching the top of the adsorbent layer having a filter paper with a layer of sand. The tip of the pipette is placed against the inside wall of the column just above surface of the adsorbent.
- 35. DEVELOPMENT TECHNIQUES 35 The development techniques are categorized into three types. (a) Elution analysis Isocratic elution technique Gradient elution technique (b) Frontal analysis (c) Displacement analysis
- 36. ELUTION ANALYSIS 36 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.
- 37. 37 Isocratic elution technique : In this technique, the same solvent composition or solvent of sample polarity is used throughout the process of separation. In this elution technique, solvents of Gradient elution technique : increasing polarity or increasing elution strength are used during the process of separation.
- 38. FRONTAL ANALYSIS 38 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
- 39. 39 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. the intermediate component is adsorbed later and next the most adsorbed component is passed out.
- 40. DISPLACEMENT ANALYSIS 40 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.
- 41. 41 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
- 42. DETECTION AND RECOVERY OF COMPONENTS 42 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.
- 43. 43 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
- 44. 44 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 De-activation of adsorbent decreases separation Concentration of solutes Substances of high concentration moves slowly
- 45. 45 Applications of Column Chromatography 1. In the separation of the mixtures into the pure individual components. 2. Removal of impurities and in the purification of compounds. 3. Determination of the homogeneity of chemical substances. 4. Identification of unknown compounds. 5. Used in the separation of geometrical isomers, diastereomers , racemates and tautomers. 6. In the separation and identification of inorganic anions and cations. 7. The concentrated of substance from dilute solutions such as those obtained when natural products are extracted with large volumes of the solvents from the leaves of plants, trees, roots or barks.
- 46. Applications of Column Chromatography 46 1. Column chromatography is best suited to separate active principle from plant materials 2. Isolation of metabolites from important components 3. Determination of flucinolone, acetonide, betamethasone in formulations 4. To separate natural compound mixtures like alkaloids, glycosides .
- 47. 47 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.
- 48. DISADVANTAGES OF COLUMN CHROMATOGRAPHY 48 • Time consuming. • More amount of mobile phase are required. • Automation makes the technique more complicated and expensive.
- 49. 49 REFERENCES : A.H. BECKETT & J.B. STENLAKE, Practical pharmaceutical chemistry, 4th edition, part two, page no: 86-105. ASHUTOSH KAR, Pharmaceutical analysis – II, page no: 161-181. Dr . S. RAVI SANKAR, Pharmaceutical analysis, 3rd 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
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