Thin Layer Chromatography and HighPerformance Thin Layer chromatography

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Transcript of Thin Layer Chromatography and HighPerformance Thin Layer chromatography

  • 1. Thin Layer Chromatography & High Performance Thin Layer Chromatography (TLC & HPTLC) K V GOPINATH M Pharm PhD,CPhT Tirumala Tirupati Devasthanams TIRUPATI e-mail:gopinath.karnam@gmail.com

2. Introduction Chromatographyisaphysicalmethodofseparationinwhichthe componentstobeseparatedaredistributedbetweentwophases,one ofwhichisstationary(stationaryphase)whiletheother(themobile phase)movesinadefinitedirection. Types of Chromatographic Techniques: Technique Stationary Mobile Phase Column/AdsorptionChromatography solid Liquid PartitionChromatography Liquid Liquid PaperChromatography Liquid Liquid ThinLayerChromatography(TLC) Liquid/Solid Liquid GasLiquidchromatography(GLC) Liquid gas GasSolidChromatography(GSC) Solid gas IonExchangeChromatography Solid Liquid 3. Definitions Thin Layer Chromatographycanbedefinedasamethodof separationoridentificationofamixtureofcomponentsintoindividual componentsbyusingfinelydividedadsorbentsolid/(liquid)spread overaglassplateandliquidasamobilephase. Synonyms:Drop,strip,spreadlayer,surfacechromatographyandopen columnchromatography Principle: - Adsorptionorretentionorpartition orbothoranyother principleofasubstance(s)onthestationaryphase -Separationoftheadsorbedsubstancesbythemobilephase -Recoveryoftheseparatedsubstancesbyacontinuousflowof themobilephase(elution) -Qualitativeandquantitativeanalysisoftheelutedsubstances 4. MechanismofThinLayerChromatography AdsorptionThinLayerChromatography IonExchangeThinLayerChromatography PartitionThinLayerChromatography ReversedPhasedPartitionThinLayerChromatography HighPerformanceThinLayerChromatography 5. ClassesofchemicalsinChromatography MostStrongly Adsorbent Alumina Al2O3 Charcoal C Florisil MgO/SiO2 (anhydrous) LeastStrongly Adsorbent Silicagel SiO2 Therearethreeclassesof chemicalsinchromatography: -The stationary phase (silica), -The mobile phase (eluents/organic solvents), and -the analyte mixture. Theseparationofthedifferent analytecompoundsresultsfrom thecompetingintermolecular forcesofthestationaryand mobilephasesfortheanalyte compounds. 6. SilicaAsaStationaryPhase OHOHOH SiOSiO-Si- OOO- Silica(SiO2)isasolidwithanextendedstructure oftetrahedralsilicaatomsbridgedtogetherbybent oxygenatoms. Onthesurfaceofthesilicaparticles,thesolid terminatesinverypolarsilanol(Si-O-H)groups. Thesilicaisthestationaryphasebecauseit remainsadheredtotheglassplateanddoesnot moveduringthechromatographyprocess. 7. MobilePhase Least Eluting Power (alumina as adsorbent) -Petroleumether(hexane; pentane) -Cyclohexane -Carbontetrachloride -Benzene -Dichloromethane -Chloroform;-Ether -Ethylacetate(anhydrous) -Acetone(anhydrous) -Ethanol; -Methanol -Water; -Pyridine Greatest Eluting Power (alumina as adsorbent) -Organicacids Theelutingsolventshouldalsoshowa maximumofselectivityinitsabilitytodissolve ordesorbsthesubstancesbeingseparated. Amoreimportantpropertyofthesolventisits abilitytobeitselfadsorbedontheadsorbent. Anumberofcommonsolventsinapproximate orderofincreasingadsorbability,andhencein orderofincreasingelutingpower. Mixturesofsolventscanbeusedand,since increasingelutingpowerresults(0.5to2%by volume) solventstobeusedinchromatographyshould bequitedry 8. Adsorbabilityoforganiccompounds Absorbabilityoforganiccompoundsbyfunctionalgroup Least Strongly Adsorbed : -Saturatedhydrocarbons;alkylhalides -Unsaturatedhydrocarbons;aIkenylhalides -Aromatichydrocarbons;arylhalides -Polyhalogenatedhydrocarbons -Ethers -Esters -Aldehydesandketones -Alcohols Most Strongly Adsorbed : -Acidsandbases(amines) 9. TheoryofThinLayerChromatography InTLC,asolidphase,theadsorbent, iscoatedontoasolidsupport (thinsheetofglass,plastic,andaluminum)asathinlayer(about0.25 mmthick).Inmanycases,asmallamountofa bindersuchasplaster ofParisismixedwiththeabsorbenttofacilitatethecoating. Themixture(A+B)tobeseparatedisdissolvedinasolventandthe resultingsolutionisspottedontothethinlayerplatenearthebottom.A solvent,ormixtureofsolvents,calledtheeluatant, is allowed to flow up the plate by capillary action. Atalltimes,thesolidwilladsorba certainfractionofeachcomponentofthemixtureandtheremainder willbeinsolution.Anyonemoleculewillspendpartofthetimesitting stillontheadsorbentwiththeremaindermovinguptheplatewiththe solvent.Asubstancethatisstronglyadsorbed(say,A)willhavea greaterfractionofitsmoleculesadsorbedatanyonetime,andthusany onemoleculeofAwillspendmoretimesittingstillandlesstime movingandviceversa. 10. HowtoRunThinLayerChromatography Step1:Preparethedevelopingcontainer Step2:PreparetheTLCplate Step3:SpottheTLCplate Step4:Developtheplate Step5:Visualizethespots 11. Preparationofthedevelopingcontainer Itcanbeaspeciallydesignedchamber,ajar withalid,orabeakerwithawatchglassonthe top Poursolventintothechambertoadepthofjust lessthan0.5cm. ToaidinthesaturationoftheTLCchamber withsolventvapors,youcanlinepartofthe insideofthebeakerwithfilterpaper. Coverthebeakerwithawatchglass,swirlit gently. AllowittostandwhileyouprepareyourTLC plate. 12. PreparationoftheTLCplate Pouring,Dipping,Spraying,Spreading TLCplatesusedarepurchasedas5cmx20cmsheets. Eachlargesheetiscuthorizontallyintoplateswhichare5 cmtallbyvariouswidths; Handletheplatescarefullysothatyoudonotdisturbthe coatingofadsorbentorgetthemdirty. Measure0.5cmfromthebottomoftheplate. Usingapencil,drawalineacrosstheplateatthe0.5cm mark.Thisistheorigin:thelineonwhichyouwillspot theplate.Takecarenottopresssohardwiththepencil thatyoudisturbtheadsorbent. Undertheline,marklightlythesamplesyouwillspoton theplate,ormarknumbersfortimepoints.Leaveenough spacebetweenthesamplessothattheydonotrun together;about4samplesona5cmwideplateisadvised. 13. Spot the TLC plate Prepare 1% solution of drug dissolving in volatile solvents like hexanes, ethyl acetate, or methylene chloride. Dip the microcap or microcapillary into the solution and then gently touch the end of it onto the proper location on the TLC plate. Don't allow the spot to become too large - if necessary, you can touch it to the plate, lift it off and blow on the spot. If you repeat these steps, the wet area on the plate will stay small. This example plate has been spotted with three different quantities of the same solution and is ready to develop 14. Thin Layer Chromatography Column Development Place the prepared TLC plate in the developing beaker, cover the beaker with the watch glass, and leave it undisturbed on your bench top. The solvent will rise up the TLC plate by capillary action. Make sure the solvent does not cover the spot. Allow the plate to develop until the solvent is about half a centimeter below the top of the plate. Remove the plate from the beaker and immediately mark the solvent front with a pencil. Allow the plate to dry. 15. Visualize the spots If there are any colored spots, circle them lightly with a pencil. Most samples are not colored and need to be visualized with a UV lamp. Hold a UV lamp over the plate and circle any spots you see. Make sure you are wearing your goggles and do not look directly into the lamp. Protect your skin by wearing gloves. 16. The Retention Factor / Rf It is defined as the distance traveled by the compound divided by the distance traveled by the solvent. The Rf for a compound is a constant from one experiment to the next only if the chromatography conditions below are also constant: - solvent system ; - adsorbent - thickness of the adsorbent; - amount of material spotted - temperature Relative Retention Factor The Rf can provide corroborative evidence as to the identity of a compound. If two substances have the same Rf value, they are likely (but not necessarily) the same compound. If they have different Rf values, they are definitely different compounds 17. General Review of preparation of materials The thin layer chromatography plates are commercial pre-prepared ones with a silica gel layer on a glass, plastic, or aluminum backing. Use the wide plates for spotting several compounds on the same plate. This allows for more precise comparison of the behavior of the compounds. The samples are spotted on the thin layer plates using fine capillaries drawn from melting point capillaries. You will need to draw several spotters. Samples for spotting are prepared by dissolving approximately 0.1 g (the amount on the tip of a spatula) of the compound in less than 0.5 mL of a solvent (ethyl acetate, dichloromethane, or ether work well). 18. General Review of preparation of materials When spotting samples on the TLC plates, it is a good idea to check if enough sample has been spotted on the plate. Allow the solvent to evaporate and then place the plate under a short wavelength ultraviolet lamp. A purple spot on a background of green should be clearly visible. If the spot is faint or no spot is apparent, more sample will have to be applied to the plate. The chromatograms are developed in a 150-mL beaker or jar containing the developing solvent. The beaker is covered with a small watch glass. A wick made from a folded strip of filter paper is used to keep the atmosphere in the beaker saturated with solvent vapor. 19. General Review of preparation of materials When the plates are removed from the developing solvent, the position of the solvent front is marked, and the solvent is allowed to evaporate. The positions of the spots are determined by placing the plates under a short wavelength ultraviolet lamp. The silica gel is mixed with an inorganic phosphor which fluoresces green in the UV light. Where there are compounds on the plates, the fluorescence is quenched and a dark purple spot appears. 20. Applications of TLC It is used for separation of all classes of natural products and is established as an analytical tool in modern pharmacopoeias. - E.g. Acids, alcohols, glycols, alkaloids, amines, macromolecules like amino acids, proteins and peptides, and antibiotics - for checking the purity of samples - as