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Science
Chromatography
Chromatographyisanimportantanalyticaltechniquebecauseitallowschemiststoseparatesubstancesincomplexmixtures.Thereareavarietyoftypesof
chromatography,whichcanbeusedindifferentcontexts.
Analyticalprocedures
Qualitativeanalysismeansanymethodofidentifyingthechemicalsinasample.
Quantitativeanalysismeansworkingouthowmuchofaparticularchemicalthereisinasample.
Anyanalysismustbecarriedoutonasampleofasubstancethatisrepresentativeofthebulkofthesubstance.Thismeansthattheresultsfromtheanalysiscan
beusedtodrawconclusionsabouttherestofthesubstance.
Itisimportanttomixsolutionsthoroughlybeforetesting,andtotakemanysamplesatrandomfromthesolutionwhentakingrepeatreadings.
Itisimportanttofollowstandardprocedureswhenanalysingchemicals.Thisensuresthattheresultsarereliableandvalid.
Standardprocedurescoverthefollowing:
collectingsamples
storingsamples
preparingsamplesforanalysis
Paperchromatography
Inchromatography,substancesareseparatedastheytravelinamobilephasewhichpassesthroughastationaryphase.Differentsubstancestravelat
differentspeeds,sosomemovefurtherthanothersinagiventime.
Inpaperchromatography,thestationaryphaseispaper.Themobilephasemayeitherbeanaqueous(waterbased)liquidoranonaqueousorganic(carbon
based)solvent[solvent:Asolventistheliquidinwhichthesolutedissolvestoformasolution.].Anexampleofanorganicsolventispropanonewhichisthe
mainchemicalinnailvarnishremover.
Foreachchemicalinthesample,thereisadynamicequilibrium[dynamicequilibrium:Whentherateofaforwardreactionisthesameastherateofthe
reversereaction,sotheconcentrationofproductremainsconstantovertime.]betweenthestationaryphaseandthemobilephase.Theoverallseparation
dependsuponhowstronglyattractedthechemicalsaretothemobileandthestationaryphases.
Watchthisvideocliptoseehowpaperchromatographycanbeusedtosolveacrime.
InordertoseethiscontentyouneedtohavebothJavascriptenabledandFlashinstalled.
Thinlayerchromatography(TLC)
Thinlayerchromatography(TLC)issimilartopaperchromatographybutinsteadofpaper,thestationaryphaseisathinlayerofaninert[inert:Unreactive.]
substance(egsilica)supportedonaflat,unreactivesurface(egaglassplate).
TLChassomeadvantagesoverpaperchromatography.Forexample:
themobilephasemovesmorequicklythroughthestationaryphase
themobilephasemovesmoreevenlythroughthestationaryphase
thereisarangeofabsorbenciesforthestationaryphase
TLCtendstoproducemoreusefulchromatograms[chromatogram:Toseparatedifferentsubstancesdissolvedinaliquid.]thanpaperchromatography,which
showgreaterseparationofthecomponentsinthemixtureandarethereforeeasiertoanalyse.
Analysingchromatograms
Achromatogram[chromatogram:Toseparatedifferentsubstancesdissolvedinaliquid.]canbecomparedwithachromatogramofastandardreference
materialtoidentifywhichchemicalsthemixturecontained.

Themixtureontheleftseparatedintothreesubstanceswhichmatchthe
purestandardreferencematerialsusedontheright
Locatingagents
Sometimesthesubstancesbeingseparatedarecolourless.Inthiscase,locatingagentscanbeusedtoshowwherethespotsare.
Locatingagentsbindtothechemicalsinthespots.Sometimes,anotherchemicalisthenadded,whichreactswiththelocatingagenttoproduceacolouredspot,
orthechromatogramisputunderultraviolet[ultraviolet(UV):Electromagneticwaveswithfrequencieshigherthanthoseofvisiblelight,beyondthevioletend
ofthevisiblespectrum.UVlightcausesdamagetoDNAleadingtosunburnandpossiblefutureskincancer.]lightandthelocatingagentglowstoshowwherethe
spotsare.
Rfvalue
Themovementofasubstanceduringchromatography,relativetothemovementofthesolvent[solvent:Asolventistheliquidinwhichthesolutedissolvesto
formasolution.],ismeasuredbycalculatingitsretardationfactor(Rf ).

Chromatographymeasurement
TheRf valueisworkedoutusingthisformula:
Rf value=distancetravelledbysubstance/distancetravelledbysolvent
CalculatingtheRf valueallowschemiststoidentifyunknownsubstancesbecauseitcanbecomparedwithRf valuesofknownsubstancesunderthesame
conditions.
Gaschromatography
Ingaschromatography(GC),themobilephaseisaninert[inert:Unreactive.]gas(eghelium).
Thestationaryphaseisaverythinlayerofaninertliquidonaninertsolidsupportsuchasbeadsofsilicapackedintoalongthintube(thisflexibletubeiscoiled
manytimesinsideathermostaticallycontrolledoventokeepitataconstanttemperature).
GCisusedtoseparatecomplexmixtures.Itismuchbetteratthisthanthinlayerorpaperchromatography.Thisisbecauseitismoresensitiveallowingthe
determinationnotonlyofwhatchemicalsareinthemixture,butalsohowmuchofeachchemicalthereis.

Gaschromatography
Themixturetobeanalysedisinjectedintothestreamofcarriergas[carriergas:Theinertgas(oftenhelium)usedingaschromatographytocarrythemixture
ofsubstancesthroughthecolumn.].Asitpassesalongthecolumn(longthintube)itseparatesintothedifferentsubstances.
Substanceswithagreateraffinity(attraction)forthemobilephasereachthedetectorattheendofthecolumnmorequickly.Substanceswithagreateraffinity
forthestationaryphasemovemoreslowlythroughthecolumn.
Gaschromatographyisusedtodetectbannedsubstancesinurinesamplesfromathletes.
Analysinggaschromatograms
Agaschromatogram[chromatogram:Toseparatedifferentsubstancesdissolvedinaliquid.]mightshowthetimealongthexaxisandthestrengthofresponse
alongtheyaxis.
Theamountoftimethatasubstancetakestopassthroughthecolumniscalleditretentiontime.Theretentiontimeofanunknownsubstancecanbecompared
withstandardreferencedatatohelptoidentifyit.
Threemainpiecesofinformationcanbegatheredfromagaschromatogram:
thenumberofcompounds[compound:Asubstanceformedbythechemicalunion(involvingbondformation)oftwoormoreelements.]inthemixture
representedbythenumberofpeaks
howmuchofeachcompoundispresentrepresentedbytheheightofthepeak(higher=more)
theretentiontimeindicatedbythepositionofthepeak

Gaschromatographygraph

Thisgaschromatogramshowsthat:
substanceAwaspresentinthesmallestquantity(ithasthesmallestpeak)
substanceAhadtheshortestretentiontime
substancesBandCwerepresentinequalamounts
substanceFhadthelongestretentiontime
substanceFwaspresentinthegreatestquantity(ithasthelargestpeak)
substanceFhadthegreatestaffinityforthestationaryphase
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