ChromatographyProblemSet FromExam22002 9]DescribehowtheAtermofthevanDeemterequationcontributestobandbroadening. 10]DescribehowtheB/utermofthevanDeemterequationcontributestobandbroadening.Whyisit inverselyproportionaltomobilephaseflowrate? 11]DescribehowtheCutermofthevanDeemterequationcontributestobandbroadening.Whyisit directlyproportionaltomobilephaseflowrate? 12]TheseparationefficiencyofcapillarycolumnsoverpackedcolumnsinGCisattributableto 13]ThemostcommonmobilephasesinGCare________________________________ 14]SplitinjectionsarerequiredinGCcapillarycolumnsbecause___________________________ 17]Theresolutionterminchromatographicseparationsisproportionalto_____________________ 18]Theplateheightinchromatographyisbestdescribedas___________________________ 19]AssumethatweusingaverynonpolarstationaryphaseinGCwecanguessthatelutiontimesforthe fourfollowinganalytespeciescanberankedintermsofshortesttolongestas: I.

gestas: I.Benzene II.Isopropanol III.Ethanol 20]Thethermalconductivitydetector,flameionizationdetector,andelectroncapturedetectorare respectivelysensitiveto

Answers 9]Multiplepathsreadnotes 10]Longitudinaldiffussionpleasereadnotes 11]MTreadnotes 12]B/ueffects 13]H2,He,andN2 14]theyarelimitedintermsofsampleloading 17]L1/2 18]varianceperunitlength 19]III,II,I 20]allspecies,organics,andelectronwithdrawingorganics FromExam32005 8]PredicttheelutionorderofthefollowingsolutesinreversedphaseHPLC.(5points)

a]
Cl

OH

b] HO

OH

c]

d]

10]AGCseparationwasconductedonasamplecontainingapesticideanalyte,compoundX.This samplewastreatedwithaninternalstandardofQ,givingaconcentrationof15.0ppm.A1.0L injectionontotheGCgaveanFIDresponseof1012forQand3411forX. A1.0Lstandardsolutionof30.0ppmQwith15.0ppmofXwasinjectedgivingaresponseof899and 2791respectively. WhatistheconcentrationofQinthesample?

 AnswersToExam32005 8}B>A>C>D 10}

Aq Ax =F [X ] [Q]
F=1.55

2791 899 =F [30.0] [15.0]

3411 1012 = 1.55 [X ] [15.0]

[X]=32.6ppm FromExam32006 1]WhatwouldbetherelativeadvantagesanddisadvantagesofusingFTIRasaHPLCdetector?Discuss atleast2advantagesand2disadvantages.Commentontheuniversality(orlackof)ofthedetector(20 points) 3]Howdoesthecapillarycolumnconfigurationachieveitsadvantagesoverthepackedcolumnsetupin gaschromatography?(10points) 4]WhatispulsedflowinHPLC,whydoesitoccur,andwhyisthisnotadesirablefeature?(10points) 5]WhatisanFIDandhowdoesitwork?WhattypesofanalytesdoestheFIDrespondto?(10points) 6]ExplainelectroosmoticeffectnecessaryforflowandseparationinCE.Whatarethemigrationtime trendsforcations,anions,andneutralspecies?(10points)

7]AnHPLCanalysiswasconductedforcaffeineonSuperExtraEnergyFormula2.2withHyperDrive Now!sportsdrink.A10.1ppmmethanolISstandardwasintroducedbothintothesampleanda standardizedsolutionof304ppmofcaffeine.Themeasuredbyadiodearraydetectorateachmaxfor theabsorptionsformethanolandforcaffeinearesummarizedinthetablebelow.Whatisthe concentrationofcaffeineinthatsportsdrink?(20points) IS Caffeine Sample 23141 52777 304ppmCaffeinestandard 28441 77313 Answers 1] Advantages: CollectionoftheentireIRspectrumofanalytesispossible.FTIRdataacquisitionisrapidsoit workswellwithaflowingsystemsuchasHPLC. Itisnearlyuniversalinitsresponsetoanalytes.AfewhavenoIRactivemodes,butmost especiallylargermoleculeshavesomesortofIRactivevibration. Disadvantages: FTIRdetectionisdifficultinmobilephasesthatarehighlypolarsuchaswaterandalcoholsthat haveintenseabsorptions. ThelimitofdetectionishighrelativetootherHPLCdetectors.MolarabsorpitivityforIR transitionsislowcomparedwithelectronicones. Thereareotherpossibilitiesforbothadvantagesanddisadvantages.Illhavetoreadconsider youranswers. 3] StartingwiththevanDeemterequation:H=A+B/u+Cuweshouldconsiderallthreeterms. Athedispersionofpeakareaduetomultiplepathsinthecolumnisnotaconsiderationinthe capillarycolumnasonlyonepathpredominates.

B/uthisisthelongitudinaldiffusiontermwhichtellsusthatthelongerthetimetheanalyte bandspendsinthecolumnthemoredispersion.Thisisthemostsignificantofthethreetermsin thebandbroadeningcharacteristicsofthevanDeemterequation.Flowofthemobilephase throughacapillarycolumnisrelativelyunimpededwhenincomparisonwithapackedcolumn. Thisallowsforafastermobilephaseflowratethroughthecolumn. CuThemasstransferbetweenthem.p.thes.p.termisoflessimportanceintheconsiderations oftheGCcapillarycolumnhowever,thes.p.ismadeasthinaspossibletoaccommodatefacile kineticsbetweenthes.p.andthem.p. 4] Pulsed flow is the rhythmic flow pattern that occurs due to the cycles within a reciprocating pump. This is not a desirable feature as it causes remixing of the solutes in HPLC. Note it is partiallyaddressedbypulsedampers. 5] Theflameionizationdetector(FID)forGCisbasedontheformationoforganicradicals,CHand CHO+withinaflame.Theseradicalsarereducedatacathodeandthecurrentflowis proportionalroughlytothenumberoforganiccarbonsintheanalyte.Thefloworeffluentfrom theseparationcolumnisfedtoaflowairandfuel(H2)wheretheanalytesarecombusted.A cathodeisfurtherupstreamfromtheflame.TheFIDisresponsiveonlytoorganiccarbons. 6] TheelectroosmoticflowisbasedontheflowofadsorbedofcationstotheOsitesonthe surfaceofthesilicacapillary.Thesecationswillmigratetowardsthecathodedraggingalong withitasolvationsphereofwatermolecules(andthusneutrals)alongwithsolvatedanions.All threemajortypesarepulledtothecathodewithmigrationtimetrendsof t(cations)<t(neutrals)<t(anions) 7] FirstnormalizecaffeinepeakswiththeresponsebytheIS. IS Caffeine Caffeine/IS Sample 23141 52777 2.2807 304ppmCaffeinestandard 28441 77313 2.7184 Assumethat y=mx+b withythenormalizeddetectorresponseandxtheconcentration.This onepointanalysisassumesthatb=0.

 2.7184=m(304) m=8.927e3 2.2807=8.927e3(x) x=255ppm

Nowcalculatetheanalyteconcentration.

seehttp://wilstar.com/caffeine.htmfortypicalcaffeineconcentrations.

AlsousingFormula519fromHarris: Ax/Cx=F(As/Cs) 77313/304ppm=F(28441/10.1ppm) F=9.031e2 Forthesample: 52777/Cx=9.031e2(23141/10.1) Cx=255ppm FromExam32008 2]Whatistheelectroosmoticeffectincapillaryelectrophoresis?Whydoallspeciesmigratetoone electrode?Dotheymigratetothecathodeortheanode?Whatisthegeneralorderofmigrationtime forcations,anions,andneutrals?Whyareneutralspoorlyresolved? 3]WhenconsideringthevanDeemterequation,whydoesHPLCrequiresmallcolumnpackingparticles? 4]Whatisgradientelutionandhowdoesthisdifferfromanisocraticone?Whatadvantagedoes gradientelutionhaveoverisocraticseparations?WhenusingaC18stationaryphaseisitmore beneficialtoincreaseordecreasem.p.compositionpolarityduringelution? 5]Whatismeantbyabulkpropertydetector?GiveanexampleofanHPLCdetectorthatisbasedon bulkpropertiesandonethatisnot. 6]WhydocapillarycolumnspredominateinanalyticalGC? 7]WhatistemperatureprogramminginGC?HowdoesitgainanadvantageoversingleTseparations?

8]Whatistheelectroncapturedetector?Explainitsbasisforoperation,whyisN2necessary?What typesofspeciesaredetectedwiththeECD?

9]Generally,itisthoughtbymanychromatographydilettantesthattwicethecolumnlength willgiveyoutwicetheseparationpower.Commentonwhythisisfalse. 10]AGCFIDanalysiswasconductedonasoilsamplecontainingpollutantX.Thefollowing separationswereconducted: Injection1 Injection2 21.1ppmTolueneInternalStandard 33.4ppmX 21.1ppmTolueneInternalStandard unknownconcentrationX tr(minutes) 10.11 14.82 10.05 14.77 peakarea 36,242 45,997 38,774 39,115

WhatistheconcentrationofXinthesample?
ANSWERStoExam32008 2]Whatistheelectroosmoticeffectincapillaryelectrophoresis?Whydoallspeciesmigratetoone electrode?Dotheymigratetothecathodeortheanode?Whatisthegeneralorderofmigrationtimefor cations,anions,andneutrals?Whyareneutralspoorlyresolved? http://en.wikipedia.org/wiki/Capillary_electrophoresis 3] H=A+B/u+Cu InthevDequationtheMTeffectspredominate,i.e.Cu.Increasingthesurfacearea/bulkratioof thes.p.isawaytogreatimprovetheMTbetweenthetwophases.Thisrequiressmalldiameter supportsforthes.p.Thecostisthepressurerequiredtosqueezethem.p.throughthespace betweenthesmallerdiameterparticles. 4] Gradientelutionsvarythem.p.solventcompositionandpolarityduringseparation.Thishasan advantageoverisocraticseparationswheresolventcompostionsarekeptconstant.Agradient elutionwillallowfortheseparationofalargevarietyofspecieswithabroadspectrumof polaritieswithamuchshortertimesthanisocraticones.Generallyitsbestthatpolaritydecrease duringseparationwhenusingaC18s.p.Ifanonpolarm.p.isusedatthebeginningofthe elution,therewillbenoretentionbetweenthesolutesandtheC18s.p.

5] Seeproblem287h.Measureaphysicalpropertyofthem.p.ExampleUVvisabsorbance, fluorescencetechniquesareexamplesofbulkpropertydetectors.Electrochemicaldetectorsare not,sincetheyarebasedonredoxexchangeswithsolutesneartheelectrodesurface. 6] AgainthisgetsbacktothevDeqn.TheB/u,longitudinaldiffusiontermcontributesmostto bandbroadeninginthegasphase.Capillarycolumnsallowfortheunobstructedandtherefore fasterflowofthegaseousm.p.overtheirpackedcounterparts. 7] Bygoingfromcoldertowarmertemperatures,itispossibletoaddanotherdimension separationofsolutesbeyondthechromatographicones.Thisisbasedonboilingpoint differences.GenerallytheinitialTisbelowthatofthesolutesspeciesandslowlyrampedup.See alsoproblem273. 8] Seehttp://www.instrumentalchemistry.com/gasphase/pages/ecd.htmNickel63sourceemits

energeticelectronscollideswithN2(introducedasmakeupgasorcanbeusedascarrier gas)producingmoreelectrons: Ni63=>e

e+N2=>2e+N2+

Theresultisaconstantcurrentthatisdetectedbytheelectroncollector(anode). AsananalyteflowsthroughpasttheNi63sourceelectroncaptureispossibleby electronwithdrawingspecies: A+e=>A

Currentdecreasesasaresultofecapturebyanalyte.Thisisoneofthefewinstancesin whichasignalisproducedbyadecreaseindetectablephenomenon. SensitivetoelectronwithdrawinggroupsespeciallytowardsorganicscontainingF,Cl, Br,Ialso,CN,NO2 9] RememberthatRsL1/2.Seechapter26pages776782.So2xthecolumnlength increaseresolutionby1.4.AlsorememberthatB/ueffectsincreasewithseparationtime and2xwillincreasetby2x.Also,usingalongercolumnusesmorem.p.anddecreases experimentalthroughput. 10] 39,115*(36,242/39,115)*(33.4ppm/45,997)=26.3ppm