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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.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)

OH

a]

b]

OH
HO

Cl

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]

2791
899
=F

[30.0]
[15.0]

F=1.55

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