Chromatography sem 2
- 1. CHCHROMROMATOATOGRAGRAPHYPHY NAME:SONIKANAME:SONIKA CLASS: MSC.SEM 2CLASS: MSC.SEM 2 ROLL NO: 06ROLL NO: 06
- 2. HistoryHistory Mikhail Tswett, Russian, 1872-Mikhail Tswett, Russian, 1872- 19191919 BotanistBotanist In 1906 Tswett used to chromatography toIn 1906 Tswett used to chromatography to separate plant pigmentsseparate plant pigments He called the new techniqueHe called the new technique chromatography because the result of thechromatography because the result of the analysis was 'written in color' along theanalysis was 'written in color' along the length of the adsorbent columnlength of the adsorbent column Chroma meansChroma means ““colorcolor”” and graphein means toand graphein means to ““writewrite””
- 3. ImportanceImportance Chromatography has application in every branch of theChromatography has application in every branch of the physical and biological sciencesphysical and biological sciences 1212Nobel prizes were awarded between 1937 and 1972 aloneNobel prizes were awarded between 1937 and 1972 alone for work in which chromatography played a vital rolefor work in which chromatography played a vital role
- 4. ChChromromatoatogragraphyphy Chromatography is a physical method ofChromatography is a physical method of separation in which the components to beseparation in which the components to be separated are distributed between twoseparated are distributed between two phasesphases one of which is stationary (one of which is stationary (stationary phasestationary phase)) while the other (thewhile the other (the mobile phasemobile phase) moves) moves through it in a definite directionthrough it in a definite direction.. The chromatographic process occurs due toThe chromatographic process occurs due to differences in thedifferences in the distribution constantdistribution constant ofof the individual sample componentsthe individual sample components..
- 5. ChChromromatoatogragraphyphy Is a technique used to separate and identify the components of a mixture. Works by allowing the molecules present in the mixture to distribute themselves between a stationary and a mobile medium. Molecules that spend most of their time in the mobile phase are carried along faster.
- 6. Classification of chromatographyClassification of chromatography according to mobile phaseaccording to mobile phase:: 11--Liquid chromatography: mobileLiquid chromatography: mobile phase is a liquid. (LLCphase is a liquid. (LLC(.(. 22--Gas chromatography : mobile phase isGas chromatography : mobile phase is a gas. (GLCa gas. (GLC(.(.
- 7. Classification according to the packing of theClassification according to the packing of the stationary phasestationary phase:: 11--Thin layer chromatography (TLC): the stationaryThin layer chromatography (TLC): the stationary phase is a thin layer supported on glass, plastic orphase is a thin layer supported on glass, plastic or aluminium platesaluminium plates.. 22--Paper chromatography (PC): the stationary phasePaper chromatography (PC): the stationary phase is a thin film of liquid supported on an inertis a thin film of liquid supported on an inert supportsupport.. 33--Column chromatography (CC): stationary phaseColumn chromatography (CC): stationary phase is packed in a glass columnis packed in a glass column..
- 8. Classification according to the force ofClassification according to the force of separationseparation:: 11--Adsorption chromatographyAdsorption chromatography.. 22--Partition chromatographyPartition chromatography.. 33--Ion exchange chromatographyIon exchange chromatography.. 44--Gel filtration chromatographyGel filtration chromatography.. 55--Affinity chromatographyAffinity chromatography..
- 10. Paper ChromatographyPaper Chromatography A method of partition chromatography using filterA method of partition chromatography using filter paper strips as carrier or inert supportpaper strips as carrier or inert support.. The factor governing separation of mixtures of solutesThe factor governing separation of mixtures of solutes on filter paper is theon filter paper is the partition between twopartition between two immiscible phasesimmiscible phases.. One is usually water adsorbed on cellulose fibres inOne is usually water adsorbed on cellulose fibres in the paper (stationary phasethe paper (stationary phase(.(. The second is the organic solvent flows past theThe second is the organic solvent flows past the sample on the paper (stationary phasesample on the paper (stationary phase(.(.
- 12. Techniques of development with various flowTechniques of development with various flow directionsdirections Ascending development Descending development
- 13. B- two-dimensional chromatographyB- two-dimensional chromatography:: When large numbers of substances are to be separatedWhen large numbers of substances are to be separated on a single chromatogramon a single chromatogram.. Development in a direction perpendicular to the first,Development in a direction perpendicular to the first, and with a solvent system different from that usedand with a solvent system different from that used initially is often necessaryinitially is often necessary.. The sample is applied on one corner of a square piece ofThe sample is applied on one corner of a square piece of paper and after development with the first solvent,paper and after development with the first solvent, the paper is dried , rotated 90the paper is dried , rotated 90oo and developed in theand developed in the second directionsecond direction.. Usually, different types of solvents systems are used inUsually, different types of solvents systems are used in each direction. It is essential that the first solvent beeach direction. It is essential that the first solvent be completely volatilecompletely volatile..
- 16. Thin layer chromatography (TLCThin layer chromatography (TLC(( is a method foris a method for identifyingidentifying substances andsubstances and testing the puritytesting the purity of compoundsof compounds.. TLC is a useful technique because it isTLC is a useful technique because it is relativelyrelatively quickquick and requiresand requires smallsmall quantitiesquantities of materialof material..
- 17. Separations in TLC involve distributing a mixture of twoSeparations in TLC involve distributing a mixture of two or more substances between aor more substances between a stationary phasestationary phase and aand a mobile phasemobile phase.. The stationary phaseThe stationary phase:: is a thin layer of adsorbent (usually silica gel or alumina)is a thin layer of adsorbent (usually silica gel or alumina) coated on a platecoated on a plate.. The mobile phaseThe mobile phase:: is a developing liquid which travels up the stationaryis a developing liquid which travels up the stationary phase, carrying the samples with itphase, carrying the samples with it.. Components of the samples will separate on theComponents of the samples will separate on the stationary phase according tostationary phase according to how much they adsorb on the stationary phase versushow much they adsorb on the stationary phase versus how much they dissolve in the mobile phasehow much they dissolve in the mobile phase..
- 18. Thin Layer Chromatography (TLCThin Layer Chromatography (TLC((
- 20. TLCTLC
- 21. Visualizing AgentsVisualizing Agents Alkaloids: DragendorffAlkaloids: Dragendorff’’s reagents reagent Cardiac glycosides: Antimony trichlorideCardiac glycosides: Antimony trichloride Sugar: Aniline phthalateSugar: Aniline phthalate Amino acids: NinhydrinAmino acids: Ninhydrin
- 22. Interpreting the DataInterpreting the Data The RThe Rff (retention factor) value for each spot(retention factor) value for each spot should be calculatedshould be calculated.. It is characteristic for any given compoundIt is characteristic for any given compound on the same stationary phase using the sameon the same stationary phase using the same mobile phase for development of the platesmobile phase for development of the plates.. Hence, known RHence, known Rff values can be compared tovalues can be compared to those of unknown substances to aid in theirthose of unknown substances to aid in their identificationsidentifications..
- 25. ))Note: RNote: Rff values often depend on the temperature andvalues often depend on the temperature and the solvent used in the TLC experimentthe solvent used in the TLC experiment.. the most effective way to identify a compound is to spotthe most effective way to identify a compound is to spot known substancesknown substances –– authentic - next to unknownauthentic - next to unknown substances on the same platesubstances on the same plate.(.( In addition, the purity of a sample may be estimatedIn addition, the purity of a sample may be estimated from the chromatogramfrom the chromatogram.. An impure sample will often develop as two or moreAn impure sample will often develop as two or more spots, while aspots, while a pure sample will show only one spotpure sample will show only one spot
- 26. SummarySummary A TLC plate is a sheet of glass, metal, or plastic which is coatedA TLC plate is a sheet of glass, metal, or plastic which is coated with a thin layer of a solid adsorbent (usually silica orwith a thin layer of a solid adsorbent (usually silica or aluminaalumina(.(. A small amount of the mixture to be analyzed is spotted near theA small amount of the mixture to be analyzed is spotted near the bottom of this platebottom of this plate.. The TLC plate is then placed in a shallow pool of a solvent in aThe TLC plate is then placed in a shallow pool of a solvent in a developing chamber so that only the very bottom of the plate isdeveloping chamber so that only the very bottom of the plate is in the liquidin the liquid.. This liquid, or the eluent, is the mobile phase, and it slowly risesThis liquid, or the eluent, is the mobile phase, and it slowly rises up the TLC plate by capillary actionup the TLC plate by capillary action.. As the solvent moves past the spot that was applied, anAs the solvent moves past the spot that was applied, an equilibrium is established for each component of the mixtureequilibrium is established for each component of the mixture between the molecules of that component which are adsorbedbetween the molecules of that component which are adsorbed on the solid and the molecules which are in solutionon the solid and the molecules which are in solution..
- 27. In principle, the components will differ in solubility and inIn principle, the components will differ in solubility and in the strength of their adsorption to the adsorbent and somethe strength of their adsorption to the adsorbent and some components will be carried farther up the plate thancomponents will be carried farther up the plate than othersothers.. When the solvent has reached the top of the plate, the plate isWhen the solvent has reached the top of the plate, the plate is removed from the developing chamber, dried, and theremoved from the developing chamber, dried, and the separated components of the mixture are visualizedseparated components of the mixture are visualized.. If the compounds are colored, visualization isIf the compounds are colored, visualization is straightforward. Usually the compounds are not colored,straightforward. Usually the compounds are not colored, so a UV lamp is used to visualize the platesso a UV lamp is used to visualize the plates..
- 28. Columnar Chromatography (CCColumnar Chromatography (CC(( This includes chromatographic methods inThis includes chromatographic methods in whichwhich:: The stationary phase is packed into a columnThe stationary phase is packed into a column.. The mobile phase is a moving liquid or gasThe mobile phase is a moving liquid or gas.. According to the mechanism of separation ofAccording to the mechanism of separation of solutes, five major types of CC aresolutes, five major types of CC are ditinguished. Usually, one mechanismditinguished. Usually, one mechanism predominates but does not exclude the otherspredominates but does not exclude the others
- 29. Different Types of chromatography Mode or type Stationary phase Mobile phase Mechanism Adsorption Chromatography Solid that attracts the solutes Liquid or gas Solutes move at different rates according to the forces of attraction to the stationary phase. Partition Chromatography Thin film of liquid formed on the surface of a solid inert support Liquid or gas Solutes equilibrate between the 2 phases according to their partition coefficients Ion Exchange Chromatography Solid resin that carries fixed ions & mobile couterions of opposite charge attached by covalent bonds Liquid containing electrolytes Solute ions of charge opposite to the fixed ions are attracted to the resin by electrostatic forces & replace the mobile counterions. Molecular Exclusion Chromatography Porous gel with no attractive action on solute molecules Liquid Molecules separate according to their size: 1.Smaller molecules enter the pores of the gel, and need a larger volume of eluent. 2.Larger molecules pass through the column at a faster rate. Affinity Chromatography Solid on which specific molecules Liquid or gas Special kind of solute molecules interact with those immobilized on
- 30. Column ChromatographyColumn Chromatography ColumnColumn chromatographychromatography Stationary phase isStationary phase is held in a narrowheld in a narrow tube throughtube through which the mobilewhich the mobile phase is forcedphase is forced under pressure orunder pressure or under the effectunder the effect of gravityof gravity
- 31. Term Definition Solvent Mobile liquid phase with no affinity to the stationary phase (i.e. inert towards it) & no effect on solutes. Developer Any liquid with more affinity to the stationary phase than the solvent but less than solutes and just capable to move them through the column. Effluent Any liquid that passes out of the column. Eluent Any liquid that has lesser affinity to the stationary phase than solutes but is capable to move them out of the column. Eluate Fraction of eluent containing a required specific substance. Retention volume (VR ) (or retardation volume): Volume of mobile phase that passes out of the column, before elution of a specific substance.
- 32. Number of Theoretical PlatesNumber of Theoretical Plates (N(N(( H = Theoretical Plate Height L = Length of the Column. N = L / H As HETP decreases efficiency of the column increases.
- 34. THANK YOUTHANK YOUTHANK YOUTHANK YOU