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2/14/2018

The Theory Of HPLC ‐ Reverse phase chromatography ‐ Reversed Phase HPLC of Ionizable Samples

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TheTheoryOfHPLC­Reversephasechromatography­e­Learningmodule
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ReversedPhaseHPLCofIonizableSamples
1. AimsandObjectives
2. MechanismofReversed
PhaseHPLC
SofarinthissectionthefocushasbeenontheanalysisofneutralsamplesbyreversedphaseHPLC. Ionizableanalytespresent
theirownparticularseparationchallengesandopportunities.
3. ApplicationsofReversed
Beforeweexaminehowionizablesamplesaretypicallyanalyzed,afewfundamentalprinciplesshouldbereviewed.
PhaseHPLC
pH
4. AnalyteRetentionin
ReversedPhaseHPLC
TheconceptofpHisusedtoindicatetherelativeacidityorbasicity(ofamobilephaseinHPLC),andisdefinedasthenegative
logarithmofthehydrogenionconcentrationinanaqueoussolution.Addinganacidorbasetoamobilephasewillchangethe
solutionpH(Figure1­2).
5. RetentionOrderin
ReversedPhaseHPLCi
6. RetentionOrderin
ReversedPhaseHPLCii
7. ReversedPhaseMobile
phaseSolvents
8. Mobilephasestrengthand
retention
9. ChangingtheOrganic
Modifier
10. EluotropicSeries
11. SelectingReversePhase
Columns
12. ReversePhaseHPLCof
IonisableSamples
13. ControllingExtentof
Ionisation
Figure1:ThelinkbetweenpHandhydrogenionconcentrationinsolution.
14. The2pHRule
15. pHvsRetentioninReverse
PhaseHPLC
16. pHvsRetentioninReverse
PhaseHPLCQ2
17. pHvsRetentioninReverse
PhaseHPLCQ3
18. pHvsRetentioninReverse
PhaseHPLCQ4
19. pHvsRetentioninReverse
PhaseHPLCQ5
20. BasicAnalytes&Ion
Suppression
21. BuffersforReversePhase
HPLC
22. Henderson­Hasselbalch
Equation
23. BufferPreparationUsingthe
Henderson­Hasselbalch
Equation
24. GettingStartedwith
IonizableCompounds

2/14/2018

The Theory Of HPLC ‐ Reverse phase chromatography ‐ Reversed Phase HPLC of Ionizable Samples

25. OptimizingSeparation Figure2:EffectofadditionofacidicandbasicmodifierstomobilephasepH. SelectivityofIonizable
25. OptimizingSeparation
Figure2:EffectofadditionofacidicandbasicmodifierstomobilephasepH.
SelectivityofIonizable
Analytes
Additionofacids,whichareprotondonors,willincreasethehydrogenionconcentration,thusloweringthepH. Additionofbases,
26. EffectofTemperatureon
whichareprotonacceptors,willreducethehydrogenionconcentration,thusraisingthepH.
ReversedPhaseHPLC
ThemobilephasepHcanbeusedtoinfluencethechargestateofionizablespeciesinsolution. Theextentofanalyteionizationcan
27. Glossary
beusedtoaffectretentionandselectivity.
28. References
Assessment
AcidBaseChemistry
Whenworkingwithionizableanalytes,itisimportanttoconsiderthepHofthemobilephasetooptimizeseparation;therefore,
knowledgeoftheanalyteisofutmostimportance. KnowledgeofthefunctionalgroupchemistryandtheassociatedpK a ofananalyte
willallowfortuningofthepHofthemobilephase.
Allacidsandbaseshaveaunique“ionizationconstant”(K a ),whichspecifiesthedegreetowhichthespeciesionizesinaqueous
solution. Thegreatertheionization,thegreatertheinfluenceofthespeciesonthehydrogenionconcentration,andthus,the
“stronger”theacidorbase.
Forexample,astrongacidwillcompletelyionize,creatingahighhydrogenionconcentration,andaverylowpHinthesolution.
Conversely,astrongbasewillcompletelyionizebyremovinghydrogenionsfromthesolution,thus,loweringthehydrogenion
concentrationandraisingthepHofthesolutiontoaveryhighvalue. Weakeracidsandbaseswillionizetoalesserdegreeand,
therefore,havelesseffectonchangingthepH.
Brønstedwasthefirsttodemonstratetheadvantageofexpressingtheionizationofbothacidsandbasesusingthesamescale. He
madeanimportantdistinctionbetweenstrongandweakacidsandbases:
StrongacidsandbasesarecompletelyionizedoverthepHrange0­14
WeakacidsandbasesareincompletelyionizedwithinthepHrange0­14
Whenanacidorbaseisdissolvedinwatertheequilibriashownareestablished(Equation1and4).
Theequilibriumconstants(dissociationconstants,K a andK b )aregivenbyEquation2and5. Thepartialacid(orbase)dissociation
(ionization)constantisdefinedasthenegativelogarithmoftheequilibriumcoefficientoftheneutralandchargedformsofa
compound(Equation3and6). ThisallowstheproportionofneutralandchargedspeciesatanypHtobecalculated,aswellasthe
basicoracidicpropertiesofthecompoundtobedefined. ItisveryimportanttonotetheextentofionizationatdifferentpHvalues
aroundtheanalytepK a .
AstrongbasewillhavealargeK b andexistsinequilibriumwithitsconjugateacidwhichisaweakacidwithasmallK a . The
converseistrueforacidicspecies. Allacid­basereactionsinaqueoussolutionscanbeviewedfromthestandpointoftheconjugate
acidformlosingaprotontoformtheconjugatebase.WhenthisisdonepK a canalwaysbeusedincalculationsandK b orpK b isnot
required,makingcomparisonsofacidicandbasicmoleculesmuchmorefacile.
ForAcids
ForBases
Remember:
ThestrongertheacidthesmallerthepK a
ThestrongerthebasethelargerthepK a
ThepHcorrespondingtothepointatwhichthetwoformsoftheanalyte(ionizedandnon­ionized)arepresentinequal
concentrations(i.e.theanalyteis50%ionized)isthecalledthepKavalue. Eachionizablefunctionalgroupontheanalytemolecule
willhaveitsownpK a value. SincethepK a representsanequilibriumvalue,whenthepHisequaltothepK a theanalytewillbe
rapidlyconvertingbetweentheionizedandnon­ionizedform,whichcanresultinpoorpeakshapeinHPLC.
AscanbeseenfromFigure3thepK a givesanindicationofthestrengthoftheacidorthebase(relativetoitsconjugateacidorbase),
however,itcannotbedecidedfromthepK a ifthemoleculeisanacidorabase,thefunctionalgroupscontainedwithinthemolecule
mustbeknown. Forexample,thepK a ofaspirinanddiazepamareverysimilar(3.5and3.3respectively). Aspirinisaweakacidasit
containsacidiccarboxylicacidgroups,whilediazepamisaweakbaseasitcontainsbasicnitrogenfunctionalgroups.

2/14/2018

The Theory Of HPLC ‐ Reverse phase chromatography ‐ Reversed Phase HPLC of Ionizable Samples

Ibuprofen,p K a 4.9 Aspirin,p K a 3.5 Diazepam,p K a 3.3 Amphetamine,p K a

Ibuprofen,pK a 4.9

Ibuprofen,p K a 4.9 Aspirin,p K a 3.5 Diazepam,p K a 3.3 Amphetamine,p K a 9.8

Aspirin,pK a 3.5

Ibuprofen,p K a 4.9 Aspirin,p K a 3.5 Diazepam,p K a 3.3 Amphetamine,p K a 9.8

Diazepam,pK a 3.3

K a 4.9 Aspirin,p K a 3.5 Diazepam,p K a 3.3 Amphetamine,p K a 9.8 Figure3:

Amphetamine,pK a 9.8

Figure3: Ibuprofen,aspirin,diazepam,andamphetamine. pK a valuesareshownforeachmolecule. Acidicgroupsareshowninred

andbasicgroupsareshowninblue.

Table1detailsthepK a rangeofseveralcommonfunctionalgroups.

FunctionalGroup

pK a range(H 2 O)

Carboxylicacids

0.65­4.76

Alcohols

8.4­24.0

Amines

4.7­38

Amides

18.2­26.6*

Imides

8.30­17.9*

Hydrocarbons

15­53

Esters

11­24.5

Ketones

7.7­32.4*

Ethers

22.85­49

Table1:pK a rangesforselectedfunctionalgroups. *=ValuesinDMSO. 6

EachmoleculewillhaveaspecificpKaandthereareseveralresourceswhichdetailphysicochemicaldataofknowncompounds.

Websites

Books

CRCHandbookofChemistryandPhysics 4

Lange’sHandbookofChemistry 3

Itisimportanttorealizethatthetwoformsofionizableanalytemoleculesgivedifferentretentioncharacteristics. Theionizedformis muchmorepolar,anditsretentioninreversedphaseHPLCismuchlower(shorterretentiontime(t R ),smallerretentionfactor(k)).

Thisbehaviorisexpected,asthemorepolaranalytehasahigheraffinityforthemobilephaseandmovesmorequicklythroughthe column. Theconverseistrueofthenon­ionizedformasitismuchmorehydrophobic,relativetotheionizedform.

2/14/2018

The Theory Of HPLC ‐ Reverse phase chromatography ‐ Reversed Phase HPLC of Ionizable Samples

FurtherinformationonfunctionalgroupchemistrycanbefoundinourSamplePrepChannel>

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