Paper and column and tlc

differentation paper,column,tlc.

1. PRESENTED BY V.SREE DEVI (14AB1S0407) CHROMATOHRAPHY ( PAPER, COLUMN, THIN LAYER ) UNDER GUIDENCE OF A.VISWANATH DEPARTMENT OF PHARMACEUTICAL ANALYSIS vignan pharmacy college,vadlamudi 1

2. What is Chromatography? Chromatography is a technique for separating mixtures into their components in order to analyze, identify, purify, and/or quantify the mixture or components . Separate Analyze Identify Purify Quantify vignan pharmacy college,vadlamudi 2

3. Uses for Chromatography Chromatography is used to: Analyze – examine a mixture, its components, and their relations to one another Identify – determine the identity of a mixture or components based on known components Purify – separate components in order to isolate one of interest for further study Quantify – determine the amount of the a mixture and/or the components present in the sample vignan pharmacy college,vadlamudi 3

4. Paper chromatography It is defined as the technique in which the analysis of unknown substance is carried out mainly by the flow of a solvent on specially designed filter paper . vignan pharmacy college,vadlamudi 4

5. Column chromatography It is defined as a separation process involving the uniform percolation of a liquid solute through a column packed with finely divided material. vignan pharmacy college,vadlamudi 5

6. Thin layer chromatography In TLC, partition, however occurs on a layer of finely divided adsorbent which is supported on glass plate. This chromatography using thin layer of an adsorbent held on a plate or other supporting medium is known as thin layer chromatography. vignan pharmacy college,vadlamudi 6

7. PRINCIPLES Paper chromatography Column chromatography Thin layer chromatography Partition Adsorption Adsorption Cellulose layers in filter paper contain moisture acts as stationary phase. Solid material is used as stationary phase. Solid material is used as stationary phase. Organic solvents or buffers act as mobile phase. Liquid is used as mobile phase. Liquid is used as mobile phase. The compound which is more soluble in stationar y phase will travel slower. The components move according to relative affinities. The components move according to relative affinities. Those compounds are eluted later. The compound which has more affinity towards stationary phase travels slower. The compound which has more affinity towards stationary phase travels slower. The compound which is more soluble in mobile phase travels faster. The compound which has less affinity towards stationary phase travels faster. The compound which has less affinity towards stationary phase travels faster. vignan pharmacy college,vadlamudi 7

8. vignan pharmacy college,vadlamudi 8 STATIONARY PHASE MOBILE PHASE water Isopropanol : ammonia : water (9:1:2) water N- butanol : glacial acetic acid :water(4:1:5) water Phenol saturated with water formamide Chloroform Formamide benzene Formamide Benzene : cyclohexane (9 :1) DMF Cyclohexane phenoxyethano -l Heptane kerosene 70%isopropanol Stationary phase and mobile phase use in paper chromatography

9. vignan pharmacy college,vadlamudi 9 Common Adsorbents for column chromatography Sucrose Cellulose Starch Calcium carbonate Calcium sulphate Calcium phosphate Magnesium carbonate Calcium oxide Silica gel Charcoal Magnesium oxide Alumina [ The adsorbents are given in the order of increasing adsorption power ]

10. vignan pharmacy college,vadlamudi 10 Grouping of solvents in order of chromatographic strength is known as elutropic series . (Increasing eluting power) Petroleum ether Cyclohexane Benzene Chloroform Ethyl acetate Acetone Ethanol Methanol Water Pyridine Organic acids Inorganic acids mobile phase used in column chromatography

11. Stationary phase and mobile phase use in thin layer chromatography Stationary phase : common adsorbent for thin layer chromatography are silica gel, alumina, kieselghur . Mobile phase : pure solvents or mixture of solvents are used. Petroleum ether>carbon tetra chloride> cyclohexane >carbon disulfide>ether>acetone>benzene>toluene>ethyl acetate>chloroform>alcohols> wtater >pyridine>organic acids. Name composition Silica gel H Silica gel without water Silica gel G Silica gel + caso 4 Silica gel GF Silica gel + binder + fluorescent indicator vignan pharmacy college,vadlamudi 11

12. METHODOLOGY vignan pharmacy college,vadlamudi 12

13. General procedure for paper chromatography Specially treated chromatographic paper is the inert support water absorbed by the inert support is stationary phase. While various solvents which are immiscible with water are mobile phase. A drop of mixture to be analyzed is applied to edge of a chromatographic paper and is dried. Then immersed into a cylinder with a suitable solvent level below the applied drop. vignan pharmacy college,vadlamudi 13

14. The solvent rises by capillary action of the paper and various components of the mixture are carried with it at different rates. Here the solute distributed between the stationary phase and mobile phase. According to partition coefficient for each component. Hence the compounds are separated that is they are distributed by zones. If it is colour less the chromatogram has to be developed by applying visualizing reagents forming coloured compounds with ions are to be detected. vignan pharmacy college,vadlamudi 14

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16. vignan pharmacy college,vadlamudi 16 CHOICE OF THE FILTER PAPER: The filter paper plays an important role in the success of paper chromatography . Various types of whatmann chromatography papers are available. The choice of a particular whatmann chromatography paper depends upon the type of separation. Characteristics of whatmann chromatography papers are as follows. FAST MEDIUM SLOW Thin paper Grade no:4 Grade no:7 Grade no:2 Grade no:54 Grade no:1 Grade no:20 Thick paper Grade no:31 Grade no:3 - Grade no:17 Grade no:3MM -

17. vignan pharmacy college,vadlamudi 17 Type Typical uses Carboxyl papers Cationic separation of protonated amines & amino acids Acetylated papers Rp -chromatography of lipophilic substances like steroids , insecticides, Pigments & metal cations Keiselguhr alumina,silica , zirconia papers Separation of low polarity substance such as amines , fatty acids , steroids , triglycerides , vitamins etc Ion exchange papers Ion exchange paper chromatography of various ionic species Hydrophilic papers Papers modified with methanol , formamide , glycol etc

18. R f VALUE vignan pharmacy college,vadlamudi 18 The R f value is calculated for identifying the spots. Rf value is the ratio of distance travelled by the solute to the distance travelled by the solvent front. R f = Distance travelled by solute Distance travelled by solvent front The R f value ranges from 0 to 1. but ideal values ranges from 0.3 to 0.8. R f value is constant for every compound in a particular combination of stationary and mobile phase.

19. General procedure for column chromatography In the column chromatography the stationary phase is silica gel is placed as slurry, mobile phase is liquid. The sample is dissolved in a minimum amount of solvent and applied to the column and passed into the column with liquid mobile phase. The fundamental principle of column chromatography is the selective adsorption of various compounds of the mixture on the solid stationary phase. It is based on the fact that when a solution of complex mixture is passed through a column of adsorbent the various components of the mixture are adsorbed to different extents on the adsorbent. vignan pharmacy college,vadlamudi 19

20. The mixture to be analyzed is dissolved in a suitable solvent and then the mobile phase is allowed to pass through the column containing the stationary phase. The component which has the greatest adsorbing power is adsorbed first, that is absorbs at the upper part of the column. In this way the various components of the mixture are separated in order of their decreasing adsorptive powers. The process of separation of various components of the mixture into different bands or zones of pure substance, each located at different region in the column. vignan pharmacy college,vadlamudi 20

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22. vignan pharmacy college,vadlamudi 22 FACTORS AFFECTING COLUMN EFFICIENCY Effect Factor Decrease of size improves separation (but very small particles need high pressure). Particle size of solid stationary phase (or of support) Efficiency increases as ratio length / width increases. Column dimensions Non uniform packing results in irregular movement of solutes through column & less uniform zone formation. Uniformity of packing Increase in column temperature results in speed of elution but does not improve separation . Column temperature Solvents should be of low viscosity (to give efficient resolution) & h igh volatility (to get rapid recovery of the substances). solvent Uniform & low flow rate gives better resolution. Solvent flow rate Discontinuous flow disturbs resolution Continuity of flow Deactivation of adsorbent decreases separation. Condition of adsorbent Substances of high concentration move slowly. Concentration of solutes

23. General procedure for thin layer chromatography A glass plate is coated with a loose powder or with a slurry of an adsorbent slurries will adhere to the surface of the glass plate, after drying as a thin layer. The unknown substance and reference materials are dissolved in water or mobile phase and the solution is applied in a row of spots, 1-2cm from the edge of the plate with the help of capillary tube or micro syringe. The chromatographic plate is placed in a jar containing the solvent for development and the jar with solvent vapours. vignan pharmacy college,vadlamudi 23

24. The jar is covered with an air tight lid. As the solvent ascends through the layer by capillary action. The sample is resolved into fractions. The plate is carefully with drawn after the solvent front has migrated about 75%of the length of the plate. The plate is then dried and sprayed with a reagent for detection of components or more commonly exposed to iodine vapours. Solute position is indicated by brown vapours. vignan pharmacy college,vadlamudi 24

25. Preparation and activation of TLC plates vignan pharmacy college,vadlamudi 25 Pouring technique, the slurry prepared and poured on to a glass plate and is spread uniformly on the glass plate. After setting plates are dried in an oven. Dipping technique two plates are dipped in to the slurry and separated after removing from slurry and dried. Spraying technique is like perfume spray on a cloth. the slurry is sprayed on a glass plate using sprayer. Spreading technique the slurry is poured inside the reservoir of tlc spreader. the spreader is rolled only once on the plate after setting the plates are activated by keeping in an oven. Activation of tlc plates is nothing but removing moisture from the surface of any adsorbent at high temperature .

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27. vignan pharmacy college,vadlamudi 27 Parameters TLC TYPE OF CHROMATOGRAPHIC PLATE HAND MADE / PRECOATED PARTICAL SIZE DISTRIBUTION WIDE PARTICAL SIZE RANGE 5 – 20 μ m SHAPE SPOT SPOT SIZE 2 – 4 mm LAYER THIKNESS 250 μ m SOLVENT CONSUMPTION 50 ml NO. OF SAMPLES MAXIMUM 12 OPTIMUM DEVELOPMENT DISTANCE 10 - 15 cm SAMPLE VOLUME 1-10 μ l NO. OF SAMPLE PER PLATE 15 – 20 PARAMETERS AFFECTING TLC

28. vignan pharmacy college,vadlamudi 28 Paper Column Thinlayer Paper chromatography used for separation of amino acids and oligosaccharides. Removal of impurities and in the purification of compounds. TLC has high speed of separation. It is also used for structural analysis for unknown compound. Isolation of active constituents. A large number of vitamins, antibiotics & food products have also been separated by using TLC. Separation of carbohydrates, vitamins, antibiotics, alkaloids, glycosides. Isolation of metabolites from biological fluids. TLC used for isolation & characterization of organic compounds Identification of foreign substance in drugs. Estimation of drugs in formulations or crude extracts. TLC is very sensitive gives sharper zones &better resolution. Identification of decomposition products. The greatest application of column chromatography has been in the separation &identification of inorganic anions & cations. Purification process. APPLICATIONS

29. vignan pharmacy college,vadlamudi 29 Chromatography Advantages Disadvantages Paper It is an inexpensive but powerful analytical tool that requires very small quantities of material. They are used in many scientific studies to identify unknown organic and inorganic compounds. Paper chromatographic techniques can not be used in separation of volatile substances such as hydrocarbons and volatile fatty acids. The lower limit for the detection of most compounds is 1-5 microgram . Column Any type of mixture can be separated by column chromatography. Any quantity of the mixture can be separated (  g to mg). Time consuming method. More amount of solvents are required which are expensive Thin layer The components are separated in very little time as the components will elute out very quickly. The solvents for the TLC plate can be changed easily. In this method the plate length is limited and hence separation takes place only up to certain length. The separation takes place in an open system there are chances that sample may be affected by the humidity and temperature.

30. Development technique in paper chromatography Ascending chromatography : Descending chromatography : Ascending- descending mode : vignan pharmacy college,vadlamudi 30 Descending chromatography

31. vignan pharmacy college,vadlamudi 31 Radial chromatography Two dimensional chromatography

32. Development technique for column chromatography vignan pharmacy college,vadlamudi Isocratic elution technique: Eg. Chloroform only, pet ether:benzene =1:1 only Gradient elution technique: Eg. Intially benzene, then chloroform, then ethyl acetate, then methanol. Other techniques like frontal analysis and displacement analysis are also used. Frontal analysis : Solution of sample mixture is added continuously on the column. no mobile phase is used for development of column. Displacement analysis : a small volume of mixture is added to the column and elution is carried out by a solvent containing solute which has high adsorptivity for column material. 32

33. Development technique for thin layer chromatography vignan pharmacy college,vadlamudi One dimensional development. Two dimensional development. Multiple development technique. Step wise development. 33

34. CONCLUSION vignan pharmacy college,vadlamudi By using Wider choice of mobile phase any type of mixture can be separated & any quantity of the mixture can be separated( µg to mg). separation of amino acids ,food dyes , pigments can be done 34

35. REFERENCES vignan pharmacy college,vadlamudi Practical pharmaceutical chemistry by Beckett & stenlake (part two). Instrumental methods of chemical analysis by B .k . Sharma. ………. Pharmaceutical Analysis by P.PARIMO. Pharmaceutical Analysis by Higuchi . 35

36. vignan pharmacy college,vadlamudi 36 Thank you