Chiral ion exchangers based on quinine (QN) and quinidine (QD), namely Chiralpak QN-AX and QD-AX as anionic and ZWIX(+) and ZWIX(-) as zwitterionic ion exchanger chiral stationary phases (CSPs) have been investigated with respect to their retention and chiral resolution characteristics.
Trang 1journalhomepage:www.elsevier.com/locate/chroma
Dániel Tanácsa, Tímea Orosza, István Ilisza,∗, Antal Pétera, Wolfgang Lindnerb,∗
a Institute of Pharmaceutical Analysis, Interdisciplinary Excellence Centre, University of Szeged, H-6720 Szeged, Somogyi u 4, Hungary
b Department of Analytical Chemistry, University of Vienna, Währinger Strasse 38, 1090 Vienna, Austria
a r t i c l e i n f o
Article history:
Received 27 February 2021
Revised 22 April 2021
Accepted 25 April 2021
Available online 1 May 2021
Keywords:
High-performance liquid chromatography
Cinchona alkaloid-based weak
anion-exchangers and zwitterionic chiral
stationary phases
αapparent and enantiomer separations,
Solvent and additives effects
a b s t r a c t
Chiralionexchangersbasedonquinine(QN)andquinidine(QD),namelyChiralpakQN-AXandQD-AXas anionicandZWIX(+)andZWIX(-)aszwitterionicionexchangerchiralstationaryphases(CSPs)havebeen investigatedwithrespecttotheirretentionandchiralresolutioncharacteristics.Fortheevaluationofthe effectsofthecompositionofthepolarorganicbulksolventsofthemobilephase(MP)andthoseofthe organicacidandbaseadditivesactingasdisplacersnecessaryforaliquidchromatographicion-exchange process,racemic N-(3,5-dinitrobenzoyl)leucine and otherrelatedanalytes wereapplied.The mainaim wastoevaluatetheimpactoftheMPvariationsontheobserved,andthustheapparentenantioselectivity (α app), and the retentionfactor.Significant differences werefoundusing either polarprotic methanol (MeOH)orpolarnon-proticacetonitrile(MeCN)solventsincombinationwiththeacidandbaseadditives
ascounter-andco-ions.Itbecameclear,thatthechargedsitesofboththechiralselectorsoftheCSPs andtheanalytesgetspecificallysolvated,accompaniedbytheadsorptionofallMPcomponentsonthe CSP,therebybuildingastagnant“stationaryphaselayer” withacompositiondifferentfromthebulkMP ViaasystematicchangeoftheMPcomposition,trendsofresultingα appandretentionfactorshavebeen identifiedanddiscussed
Inadetailedsetofexperiments,theeffectoftheconcentrationoftheacidcomponentintheMP con-tainingMeOHorMeCNwasspecificallyinvestigated,withtheacidconsideredtobeadisplacerin anion-exchangetypechromatographicsystems
Surprisingly,allfourchiralcolumnsretainedandresolvedthetestedN-acyl-Leuanalyteswithα appvalues
upto21withinaretentionfactorwindowof0.03and 10withpureMeOH aseluent.However, using pureMeCNaseluent,analmostinfinite-longretentionoftheacidicanalytewasnoticedinallcases.We suggestthattheratherdifferentthicknessofthesolvationshellsgeneratedbyMeOH orMeCNaround thecharged/chargeablesitesofthechiralselectordetermineseventuallythestrengthoftheelectrostatic selector–selectandinteractions
As acontrol experiment we included the non-chiral N-acylglycine derivatives as analyte inall cases
to support the interpretations with respect to the contribution of the enantioselective and non-enantioselectiveretentionfactorincrementsasapartoftheobservedα app
© 2021 The Author(s) Published by Elsevier B.V ThisisanopenaccessarticleundertheCCBYlicense(http://creativecommons.org/licenses/by/4.0/)
1 Introduction
Various liquid chromatographic enantiomer and diastereomer
separation concepts have reached a high analytical and
prepara-tive standard over the years as exemplifiedby diversededicated
∗ Corresponding authors
E-mail addresses: ilisz.istvan@szte.hu (I Ilisz), wolfgang.lindner@univie.ac.at (W
Lindner)
reviewarticles[1–9].Besidethefocusonrelevantapplications, in-vestigationsandinterpretationsrelatedtotheunderlying enantios-electivemolecularrecognitionmechanismsandadsorptionmodels havealsobeenundertakenanddescribedindetail[10–13]
Ageneralized view,wherethe enantiomer resolutionis based
on the formation of intermediate molecule associates between thechiral selector(SO)moietyandtheindividual enantiomers of the chiral analytes, the selectands [(R)-SA and (S)-SA] does not pay the necessary attention to the actual situation of a wetted
https://doi.org/10.1016/j.chroma.2021.462212
0021-9673/© 2021 The Author(s) Published by Elsevier B.V This is an open access article under the CC BY license ( http://creativecommons.org/licenses/by/4.0/ )
Trang 2chiral stationaryphase(CSP), wherethechemically modified
sur-faceofthesilicaparticlesisnotfullyhomogenous.Whatwe
actu-allyobserveasachromatogramistheresultofalloccurring
stere-ospecificandnon-stereospecificinteractionsofSO–SAmolecule
as-sociates, and additional interactions of SA with the imperfectly
derivatizedandwettedsilicasurface,including,e.g.,theremaining
freesilanolgroups.Inadditiontotheseconsiderations,the
confor-mational flexibilityofthechiralmotifaroundthebinding sitesof
the SOmoietyhas tobe takeninto considerationwhich,in turn,
will dependon thesolventenvironment ofthewettedCSP being
inequilibriumwithallcomponentsoftheMP
Theobserved(apparent)retentionfactork appofeachindividual
enantiomer(R)-SA(1)and(S)-SA(2)isthesumofenantioselective
(es)andnon-enantioselective(ns) adsorption phenomena
(includ-ing partition, when applicable), which can largely differ in their
magnitude [14–16] The apparent retention factors can be
writ-ten as k app1 = k es1 + k ns1 for the first-eluting enantiomer and
k app2 =k es2 + k ns2 forthesecond-eluting enantiomer ofachiral
analyte,wherethenonselectiveretentionfactors(k ns1andk ns2)are
identical
Theapparentenantioselectivityfactorαappistherefore:
∝app=k app2
Inliquidchromatographyanintegralpartoftheobserved
phe-nomena relates to the chemical structure of the SO motif,
in-cluding all the functional groups capable for hydrogen bonding,
electrostatic interaction,etc.,anditsconformational flexibilityand
thustothesolvatedCSP,whichisdirectlyassociatedwithall
mo-bile phase components andtheir physico-chemical characteristics
[17,18].TheSO–SAinteractionsencompassessentiallyelectrostatic,
hydrogen bonding, π–π andvander Waals (hydrophobic) forces,
whicharequitedifferentintheirmagnitude Ina simplifiedview,
solvents act as quasi competitors (displacers) to the overall
ac-tive SO–(R)-SA andSO–(S)-SA interactions Asa consequence,the
liquid-chromatographic process can be formulated by a stepwise
adsorption, desorption, and elution ofthe solvatedanalytes from
thesolvatedCSP.First,thereisade-solvationeventofthe
individ-ually solvated molecular entities (SO andSAs) duringthe
forma-tion ofthe two SO–(R)-SA andSO–(S)-SA associatesaccompanied
by theirsolvation,followedbyare-solvationoftheindividual
en-tities(theSOandtheSAs)asaconsequenceofthedissociationof
the diastereomeric associates The chemical characteristics ofthe
SOandSAaswellasthetypeofsolvents,acids,bases,saltsor
neu-traladditivesandtheircomposition intheMPmayplaya signifi-cantroleinbuildingupanenvironmentonthesurface,whichwe assigninthefollowingasthesolvatedCSPlayer.ThesolvatedCSP maybeconsideredasathin“layer” onthemodifiedsurface,which maygive rise toany additionalnon-stereoselective partition-type retention causing increments of the retention of the SAs As al-ready mentioned, thetype ofthe solvents and the resulting sol-vation status ofall interaction sites of the SO andSAs will also affectthe compositionof the various conformers ofthe SO moi-etyaswellwhich,naturally,dependsonitschemicalstructure.For small or polymeric type SO molecules it may be quite different, butitisadynamicprocess,wherebyinducedfitphenomenaofSO upontheapproachingandcomplexformationswiththeSAhasto
beconsidered[19]
Inthepresentpaper,thecontributionsofdiverseMP composi-tionsonfourCinchonaalkaloid-basedCSPsareevaluated.Namely, utilizationofChiralpakQN-AXandQD-AXCSPsemployedaschiral anion exchangers [20], and zwitterionic ion exchangers ZWIX(+) andZWIX(-)CSPsused asanion-exchangertype “chiral columns” [21]fortheresolutionofthreeacidicracemicN-acyltype leucine (Leu) derivatives are discussed In these experiments, N-tagged glycine(Gly)derivativesservedasnon-chiralreferencecompounds forthediscussion ofthemolecule-dependentretentionand stere-oselectivity characteristics Fig 1A and 1B schematically depict the most prominent interactions of the employed SOs, as eluci-dated earlier by diverse studies [18,20–29] In such representa-tions,neitherconformationalnorsolvationeffectsareusually con-sidered.However, becauseofsolvationissuesdiscussedabove,we intended to depict, at least schematically, the status of solvated CSPsandSAs,butomittingtheeventuallypresentco-and counter-ions inthe stagnant mobile phase layer close to theCSP surface and within the pores (Fig 2) As shown previously, these types
ofionexchangersshowexcellentenantioselectivity,particularlyfor
N-(3,5-dinitrobenzoyl)(DNB) derivatizedaminoacids[19–23] Be-tween theπ-acidicDNB-groupandthe π-basicchinolineresidue
oftheQN/QDselectormoiety,strongintermolecularπ–π interac-tion canbe formed incooperationwith dominanthydrogen sup-ported Coulomb attraction and strong hydrogen bonding of the amide groups ofthe SOs andSAs (Fig 1A and1B), thus leading
torelativelylargeαappvalues
Inthefirstpartofthisstudy,weaimedtoelucidatethe individ-ualcontributionofthetwopolarsolvents,namely,proticmethanol (MeOH)andnon-protic acetonitrile(MeCN), together witha con-stantamountofformicacid(FA)andtriethylamine(TEA)asmobile
Fig 1 Molecular structures of chiral selectors (SOs), scheme of intermolecular interactions between the chiral selectors (SOs) and the chiral selectands (SAs): A , Scheme for
QN-AX and QD-AX type CSPs; B , Scheme for ZWIX( + ) and ZWIX(-) type CSPs; C , Molecular structure of analytes including their molecular volume ( ˚A 3 ) and pK a values
Trang 3Fig 2 Scheme of the status of solvated SOs and of solvated SAs A , Solvation layer developing on QN-AX and QD-AX type CSPs; B , Solvation layers developing on ZWIX( + )
and ZWIX(-) type CSPs; C , Effect of size of solvation shell on multiple SO–SA interactions
phase additives The composition of freely mixable MeOH/MeCN
wasgradually changed from100/0 to 0/100 (v/v), whilethe
con-centration of the MP additives was kept constant The questions
to be answered were twofold:to which extentdo the two polar
solvents and their composition have an effect on (i) the overall
retention ofthe individual enantiomers ofthe investigatedacidic
analytes, andon(ii) theapparent enantioselectivity(αapp).Inthe
secondpartofthispaper,wediscusstheeffectofvariousamounts
of acidandbaseadditives appliedin 100%MeOHor 100%MeCN
asbulksolventwithfocusingon(i)theobservedretention
charac-teristics and(ii)the apparentenantioselectivities Mechanistically,
it wasassumedthat the well-establishedstoichiometric
displace-ment model [27,30,31] remains applicable for the description of
the retentioncharacteristicsoftheinvestigatedacidic analyteson
thestudiedCSPsundertheappliedconditions
2 Experimental
2.1 Chemicals and reagents
N-(3,5-Dinitrobenzoyl)glycine (DNB-Gly), N
-(3,5-dinitrobenzoyl)-D,L- and L-alanine (DNB-Ala), N
-(3,5-dinitrobenzoyl)-D,L- and L-leucine (DNB-Leu) were home-made
according to a standard protocol N-Benzoylglycine (Bz-Gly),
N-benzoyl-D,L-leucine (Bz-Leu), N-acetylglycine (N-Ac-Gly), and
N-acetyl-D,L- andL-leucine (N-Ac-Leu) were from TCI (Eschborn,
Germany)(forstructuresseeFig.1C)
MeCN, MeOH, tetrahydrofuran (THF) ofHPLC grade,and TEA,
FA, aceticacid(AcOH)ofanalyticalreagentgradewerepurchased
from VWR International (Radnor, PA, USA) Ultrapure water was
obtainedfromUltrapure WaterSystem, PuranityTUUV/UF (VWR
Internationalbvba,Leuven,Belgium)
2.2 Apparatus and chromatography
Chromatographicmeasurementswereperformedona1100
Se-riesHPLCsystemfromAgilentTechnologies(Waldbronn,Germany)
consistingofasolventdegasser,apump,anautosampler,acolumn
thermostat, a multi-wavelength UV–Vis detector, and a
corona-charged aerosol detector fromESA Biosciences, Inc (Chelmsford,
MA, USA) Data acquisition and analysis were carried out with
ChemStation chromatographic data software from Agilent
Tech-nologies
The commercially available Cinchona alkaloid-based Chiralpak ZWIX(+)TMandZWIX(-)TMcolumns(150× 3.0mmI.D.,3-μm par-ticlesize) andChiralpak QN-AX andQD-AX (150 × 3.0 mm I.D., 5-μmparticlesize)weregiftsfromChiralTechnologiesEurope (Il-lkirch,France).Dead-time(t 0)ofthecolumnswasmeasuredby in-jectionofacetonedissolvedinmethanol
3 Results and discussions
3.1 Gradual exchange of MeOH and MeCN as bulk solvent at constant MP additive compositions
As a standard protocol for the elution and enantiomer sepa-ration of N-acyl-amino acids on the chiral anion exchanger
QN-AXandQD-AX andalsothe ZWIX(+) andZWIX(-)columns,it is common to use organic acids and bases (forming organic salts)
as additives, which act as counter-ions and displacers, respec-tively, dissolved in the MP It is expected that the concentration
of thecounter-ion, in the formof a deprotonated acid, regulates theretention,butshouldnottakemuchpartintheoverallSO–SA moleculerecognitionmechanismassymbolizedinFig.1Aand1B Such insensitivity towards thetype ofpolarorganic solventswas largelyassumed
ForthezwitterionicCSPs,hereconsideredasanionexchangers,
a similar concept holds, that is, one has to account for an addi-tional, more or less strong, intramolecular counter-ion effect via
thestoichiometricamountofthe deprotonatedsulfonic acid moi-etyofthezwitterionicselectormotif(seeFig.1Aand1B)
In the present study the salt of TEA and FA well-soluble in MeOH and MeCN has been employed to fulfill the operational modus ofionexchangers Insuch anon-aqueousmedium the or-ganicsalt(ionpair)formationtakesplaceviaaprotontransfer re-actionbetweentheacidtothenon-protonatedbase.Atfirstglance, theacid-typeanalytes(SAs),inourcasetheDNB-Leu-OHandthe others (Fig 1C), will thus get retained via a hydrogen supported ion-pairformationbetweentheprotonatedbasicsiteoftheSO(i.e., thequinuclidinescaffoldoftheQNandQDmoiety)andthe depro-tonatedSA.Thestrengthofthiselectrostaticallydriveninteraction dependson the pKa values of the acidic andbasic sitesand the sizeofsolvatedchargedsites,respectively.Inthiscontextitshould
benotedthatthenegativelychargeddeprotonatedstatemolecules aredifferentlysolvatedbytheproticMeOHandtheaproticMeCN
Trang 4than in water resulting in a significant shift in pKa values [32].
These aspects willbe takenintoconsideration duringthe
discus-sionoftheexperimentalresults.Elutionisenforcedbytheamount
ofcounter-ionssolvatedintheMPdependingontheir
characteris-tics (pKa,size, polarity).All associationanddissociationeventsof
ion-exchanger type reactions are inequilibrium;that is, a higher
concentration ofa particulardisplacer (counter-ion)should result
in a reduced retention followingthe stoichiometric displacement
model[33].Asexpected,theconcentrationoftheacidandthebase
determinestheconcentrationofthecounter-ion.Inthecaseof
us-ing onlythe free acid(FAorAcOH)or freeacidinexcess toTEA
intheMP,anadditionaldisplacementeffectbytheacidinexcess
maybeexpected(seelater).Nevertheless,freeFAandAcOHneed
tobe classifiedasproticsolventcomponentsincombinationwith
MeOH orMeCN andinthis waytheymay alsocontribute tothe
overallchromatographicresults
Assumingthat theacidadditive intheMPisinexcesstoboth
the ion-paired TEA and the quinuclidine moieties of the SOs, it
can getadsorbedontothesurfaceofthesolvatedCSPanditisin
equilibriumwiththestagnantmobilephasewithinthepores.Due
to theproton transferreactionof thefree acidintheMP,
specif-ically, inthe solvated CSP layer, towards the protonizable site of
the SO, it will also act as a displacer thus enforcing elution of
the ionically bound SAs Assuming a proton transfer equilibrium,
the free acid exists in a higher concentration in both the
stag-nant MPandthe solvatedCSP layer Consequently,itshould lead
toareductionoftheretentionfactor,similartothatformulatedfor
thecommonstoichiometricdisplacementmodelshownpreviously
forweak acidicSAs[34].Thiswaywe envisionedtogeta handle
ontheunderlyingmobilephase inducingretentionandmolecular
recognitionprinciples
Based on thediscussion detailedabove, displacement(ion
ex-change) maybe affected by both the solventcomposition ofthe
solvation shell and its thickness, where the charged sites of the
SO and SA will depend on the organic solvent type of the MP
Naturally, this applies for all ionizable molecules of the system
However, as the MP contains either free FA or FA plus TEA as
components, these entries may additionallytake part in the
for-mation andcompositionofthe“entiresolvationshell” of the
sol-vated CSP due to the diverse, but overlaid adsorption equilibria
Inanycase,thestrengthofthelong-rangeelectrostaticforces
be-tween the SO(+) andSA(−) sites will in essencebe affected by
the thickness of the solvation shell (Fig 2C) It is assumed that
the charged sites of both the SO and the SAs are fully solvated
and their status will be directly correlated to the retention
fac-tors particularlyaffectedby Coulombinteractions.However, since
we have to consider also additional SO–SA non-stereoselective
and stereoselective interactions in enantioselective ion-exchange
chromatography, several effects occurring simultaneously will be
involved
The whole set of chromatographic data, generated in these
broadly concertedexperimentsof thesolvent exchangeseries for
the DNB-LeuandDNB-Gly aswell asfor Ac-LeuandAc-Gly
ana-lytes,aresummarizedfortheQN-AX,QD-AX,ZWIX(+),and
ZWIX(-) columnsina comparablewayinTables S1–S4asSupporting
In-formation.Inaddition totheMeCN-basedexperiments,we
inves-tigatedtheeffectofthepolarnon-proticTHFassolventinvarious
combinationswithMeOHinacomparablefashion(TablesS1Dand
S2D)
Intentionally, we also tried to replace MeOH with the much
morepolarH2OincombinationwithvariousamountsofMeCNto
explore the differencesof thetype of protic solvents inthe
con-textofthepresentstudy(theresultsaresummarizedinTablesS1E
andS2E).Basedonthesedata,weattemptedtodrawconclusions
with focus(i) on the interpretation ofthe observed strongshifts
ofretentionfactors and(ii)on theshiftoftheobservedapparent
enantioselectivityαappvalues.Theoutcomeofthesediverse exper-imentswillbediscussedinmoredetailinthefollowing
3.1.1 Behavior of QN-AX and QD-AX columns employing polar organic MP variants
Itshouldbenotedhere,thatQN-AXEandQD-AXEcolumns be-havepseudo-enantiomericallytoeachother,althoughtheyare ac-tuallyindiastereomericrelation,whichimpliesthattheelution or-der of the resolved analytesswitch accordingly[35].The experi-mentaldata wheretheMeOH/MeCNratio wasgradually changed from100/0to0/100(v/v)andtheMPadditivesFA/TEAfrom50/25
to25/25and50/0,areillustratedinFig.3andaresummarizedin theTables ofSupplementaryInformation.Wecan clearlynoticea characteristicU-shapedprofile ofall retentionfactors ofDNB-Gly andDNB-Leuenantiomerswithamorepronouncedshapingofthe morestronglyretainedDNB-Leu enantiomer.Thistrend,however,
isnotparalleledwiththeexperimentalαappvalues,whichare al-ways the highestwith pure MeOH and decrease withincreasing MeCN ratiosinamoreorlesslinearfashion.The magnitudesare divergent fortheQN-AX andthe diastereomeric QD-AXcolumns Namely,fortheQD-AXcolumn,a decreaseofαapp fromabout20 forMeOH to about 16for MeCN is significantly lesspronounced than that for the QN-AX column, where a change from 18 to 9 canbenoticed.Note,thatinthelattercasesFA waspresentinan excess
InspectingmorecloselythedatadepictedinFig.3,several addi-tionalsignificantobservationscanbedrawn.Attheminimaofthe U-shapedcurvesataround40/60MeOH/MeCNeluentcomposition, the solvation shells of the charged sites seem to be the largest assuming the electrostaticallydriven SO–SA interactionbeing the mostdominantone.Letusemphasizehere,thattheactual compo-sitionofMeOHandMeCN inthesolvationshells,inparticular,in thecaseoftheCSP,mightbedifferentfromthebulkcomposition Thistrendismorepronounced forthesecond-eluting enantiomer andappliesessentiallyforall threeMPadditivecompositions, in-cludingtheFA/TEA(50/0 v/v)case Asafurtherproofofthis con-cept,weundertook asortoftitrationexperimentbyselectingthe solventcomposition MeOH/MeCN(60/40v/v)andaddingonly dif-ferent amounts ofFA (from 25 to 100 mM)to the mobile phase
inordertoreveal, whetherornot thedisplacementmodel raised earlierholdsforfreeFAasdisplacer.Theobtainedresults(listedin TableS5,anddepictedinFig.S1)fitperfectlytothemodel.Under theseconditions,freeFAintheMPactsasaperfectdisplacer,but
it isnot completelyclear whetherthe varied amountsof free FA areadsorbedviasecondaryequilibriainthesolvationlayerofthe solvatedCSP.Additionalconsiderationswillbediscussedlater
An entirely differentand even more pronounced trend is no-ticed, when exchanging MeCN with the polar non-protic THF as bulk solvent, asdepicted in Fig S2 Respective data are summa-rizedinTableS1D.Bothretentionfactorsandαappvaluesreduced stronglyandcontinuouslywithincreasingTHFcontents.In partic-ular,αapp reducedtoone third,whichis muchmorepronounced thanthatfoundwithMeCNassolvent.THFobviouslysolvateswell all charged sites of the ion-exchange type CSP and the SA, but alsodisruptstheadditionalSO–SAinteractionsmorestronglythan MeOHorMeCNdoes
Replacing the protic MeOH with the even stronger polar wa-terandcarryingoutasimilar solvent-exchangeprotocolasinthe otherexperimentstowards100%MeCNwithpurewaterassolvent,
an infinitively high retention of the analyteswas obtained Con-sequently, we could start the solvent-exchange experiments only withamixtureofH2O/MeCN(70/30v/v).Thedataaresummarized
inTables S1EandS2EandFig.S3.Asan outcome ofthis investi-gation,itbecameevident thatintheoverallretention characteris-ticsthehydrophobic, multiplevanderWaals-typeSO–SA interac-tion incrementshaveto be takenintoaccount in additionto the
Trang 5Fig. 3 Effect of MeOH/MeCN ratio on k and αvalues of DNB-Leu enantiomers and k values of DNB-Gly-on the QN-AX and on QD-AX type CSP Chromatographic conditions: column, QN-AX and QD-AX; mobile phase, MeOH/MeCN (100/0 – 0/100 v/v ) containing 50 mM FA and 25 mM TEA; 50 mM FA; and 25 mM FA and 25 mM TEA; flow rate, 0.6 ml min −1 ; detection, 254 nm; temperature, 25 °C; elution sequence on QN-AX ( D < L ), on QD-AX ( L < D )
electrostatically driven ion pair formation Furthermore,it is
ob-vious, that variation of the solvation shells of the SAs and SO
causedby waterhampers stronglytheoverallshapeofthe
acces-sible binding grooves of the SOresulting in a reduction ofαapp
TheminimaoftheU-shapedcurvesisataround30/70H2O/MeCN
(v/v)(Fig.S3).Interestingly,athighwatercontent,αappisquitelow
on the QN-AX CSP and steadily increases when shifting towards
100%MeCN.Incontrast,inthecaseofQD-AXCSP,wedidnot
no-tice sucha strongdependencyofαapp.Valuesstayaround 11–12,
whereasfortheQN-AXcolumn,αappshiftedfrom2.8toabout10
Mechanistically,thestereodiscriminationcanmarkedlybeaffected
inonecasebythepresenceofwaterasastronghydrogen-bonding
typesolvent,whereasitismuchlessofanissuefortheothercase
As afurthercontrol experiment,the impactofthetypeofthe
N-tagginggroupsofLeuandGlyontheoverallretentionand
enan-tioselectivity characteristics wasinvestigated The results of
ana-lyzing Ac-Leu andAc-Gly (instead ofDNB-Leu andDNB-Gly thus
avoiding the strong π-stacking retention-causing increment), are
depictedinFig.S4 andrelatedchromatographicdataare
summa-rizedintheTablesS1FandS2FfortheQN-AXandQD-AXcolumns,
respectively.Whatcanimmediatelybeseenaretheper sestrongly
reducedretentionfactorsforAc-LeuandAc-Glyaccompaniedwith
much smaller αapp values In pure MeCN, retention is increased
compared to that in pure MeOH, while αapp decreased strongly
What is worth mentioning isa still reasonableenantioselectivity
Obviously,it isnotashighasforDNB-Leu,since thepronounced
drivingπ–π-stackingincrementisabsentintheseanalytes[19]
3.1.2 Behavior of the ZWIX(+ and ZWIX(-) columns with polar
organic MP variants
Conceptually similar to the QN-AX and QD-AX columns, a
smaller set of experiments with the gradual exchange of MeOH
withMeCNwasalsocarriedout,applyingFA/TEAratiosof50/25, 50/0,and25/25asthebasisforasystematiccomparison Respec-tive data are summarized in Tables S3A, S3B, S3C and S4A, S4B, S4CforZWIX(+) andZWIX(-)columns,respectively,andthey are graphicallydepicted inFig.4 Althoughthere isa structural sim-ilarityof thetwo series ofchiralSOs because ofthe QN andQD motifsofSOs,theenantioselectivitywillnotbethesamefor DNB-Leu.Thereasonisthatthe tert-butyl-carbamoylgroup ofthe
QN-AX and QD-AX selectors represents an optimum structural scaf-fold around the enantioselective binding pocket, as it has been observed for the resolution of the DNB-Leu enantiomers, which could not be realized to the same extent with the ZWIX SOs [36,37] Consequently, the αapp values are actually about half as highas those seen for the pure methanolic conditions (compare data of Table S1A and S3A as well as S2A and S4A and Figs 3 and4)
As expected, U-shaped retention factor curves have been ob-served, but the retention factors are significantly shorter for the puremethanolicconditionscomparedtotheMeCNcontaining elu-ents,whichmaybeasignforasomewhatchangedsolvationstatus
ofthe ZWIXSOs comparedto thoseobserved onthe QN-AXand QD-AXcolumns.The retentioncharacteristicsinpureMeOH com-paredto those found underpure MeCN conditionsfavor the lat-teronefortheZWIXphases;however,theextentwasunexpected Theoverall enantioselectivityαapp valuesofthe ZWIX(+) column drop significantly when shifting from MeOH towards MeCN, but obviouslyitgoesfirstthroughanoticeablemaximumwhenmixed solvents are used The retention factors ofDNB-Leu enantiomers aresimilarfortheZWIX(+) andQN-AXcolumnswithpureMeCN
at FA/TEA (50/25 v/v) conditions, whereas for the ZWIX(-) col-umn, they aremuch lower thanthose onthe comparableQD-AX column
Trang 6Fig. 4 Effect of MeOH/MeCN ratio on k and αvalues of DNB-Leu enantiomers and k values of DNB-Gly-on ZWIX( + ) TM and ZWIX(-) TM CSPs Chromatographic conditions: column, ZWIX( + ) TM and ZWIX(-) TM ; mobile phase, MeOH/MeCN (100/0 – 0/100 v/v ) containing 50 mM FA and 25 mM TEA; 50 mM FA; and 25 mM FA; flow rate, 0.6 ml
min −1 ; detection, 254 nm; temperature, 25 °C; elution sequence on ZWIX( + ) TM ( D < L ), on ZWIX(-) TM ( L < D )
The change ofthe αapp values betweenZWIX(+) andZWIX(-)
columnsdoesnotfullycorrelateswiththechangeofαappbetween
the QN-AX andQD-AXcolumns, whichagain isa signof
notice-able differencesof solvent(solvation) effectson the two pairs of
chiral columns.Itbecomesclearlyevident thatthe twoMP
addi-tivecompositionsofeitherFA/TEA50/25(v/v)or25/0(v/v)do
sig-nificantlydivergewithregardstotheαappvalues,althoughthe
ab-solute amountofexcessFA (25mM)issimilar.It appliesforboth
ZWIXcolumnsandgivesahintthat possiblytheFA/TEAsaltalso
getsadsorbedintothesolvatedCSPlayerthusgivingrisetoan
ad-ditionalmodulationofthe overallαapp.Thatis,theentire
molec-ularrecognitionprocessbecomesevenmorecomplexthan
antici-pated
Alongthislineitisastonishingthattheretentionfactorof
DNB-Glyisevenhigherthanthoseforthebetterfitting(retained)
DNB-Leu enantiomer, which is different from the results obtained on
the QN-AX andQD-AX columns There are two factors
responsi-bleforthisbehavior:(i)thelongerretentionofDNB-Glyis
associ-atedwiththeoveralldecreaseofthesolventshellthicknessofthe
binding SOandSAinadditiontothe higherpKaofDNB-Gly(see
Fig 1C) and(ii) a geometrically (spatially) driven exclusion
phe-nomenontowards thebindingpocketinthecourseofthe
forma-tionofthetwodiastereomericSO-(R)-SA andSO-(S)-SAassociates
is undoubtedly involved.This phenomenon could alreadybe
par-tiallyinplaceforthemorestronglyretainedDNB-Leuenantiomer,
whereas aspatially drivenrepulsionwillcertainly bethecasefor
theweaklyretainedDNB-Leuenantiomer.Thisobservationisvalid
forbothZWIXcolumns.Fromastructuralpointofview,theZWIX
SOs provide less flexible of conformational freedom to adjust to
thebondedSAmoleculesduetotherelativelystrongandintrinsic
intramolecularCoulombinteraction(seeFig.1Aand1B)
3.2 Further unexpected observations of the investigated chiral ion exchangers
In thissubsection, the observed k 1 , k 2, andαapp values of N
acylamino acids obtained with pure MeOH or MeCN as mobile phasesolventsandFA orAcOHasacidandTEA asbaseadditives willbediscussedinthelightofadditionaladsorptionphenomena TherespectivechromatographicresultsaresummarizedinTables1 and2fortheQN-AXandtheQD-AXCSPsandinTables3and4for theZWIX(+) andZWIX(-)CSPs It is consideredthat the benzoyl (Bz)groupislesspolarandlessbulkythantheDNBgroup,andthe acetyl(Ac)groupisevenmorepolarandsmallerinsizewithonly
ashallowhydrophobicincrementforretention.TheDNBgroupof strong π-acidity can undergo a strong face-to-face π–π-stacking with the π-basic quinoline ring of the QN and QD moiety de-voidoftheBz-andAc-tags.Nevertheless,forallthese carbamate-type tags, the hydrogen bonding capacity with the SOs remains
an importantfactorashydrogen bonding isa directingforce and acts as essential asset for chiral discrimination of the studied SAs
3.2.1 Specific behavior of the QN-AX and QD-AX columns on MP changes
InspectingthedatapresentedinTables1and2,whichreferto the QN-AXandQD-AX columns,respectively, it becomesobvious that in pure MeOH and MeCN asbulk solvents and for the five
MPcompositionssignificantlydifferenteffectsbecomemanifested
As alreadylined out,DNB-Gly elutesalways muchlater than the weakly complexed and, consequently, weakly retained DNB-Leu enantiomers,butitelutesearlierthan themorestronglyretained DNB-L-Leu enantiomers This also holds for Bz-Leu, but not for
Trang 7Table 1
Comparison of chromatographic parameters, k 1 , α, and R S of racemic DNB-Leu, Bz-Leu, Ac-Leu , and DNB-Gly, Bz-Gly, Ac-Gly on QN-AX column operated with mobile phases
of either MeOH or MeCN and formic acid (FA) and triethylamine (TEA) additives in different ratios
Solvents + additives: MeOH/MeCN (100/0
v/v ) + FA/TEA (mM/mM)
Solvents + additives: MeOH/MeCN (0/100
v/v ) + FA/TEA (mM/mM)
Solvents + additives: MeOH/MeCN (100/0
v/v ) + FA/TEA (mM/mM) FA/TEA mM/mM)
k 1 D- DNB-
LeuDNB-Gly
k 2 L- DNB-
Leu- α app R S
k 1 D- DNB-
LeuDNB-Gly
k 2 L- DNB-
Leu- α app R S
k 1 Bz- LeuBz-Gly k 2 Bz-Leu- α app R S
(1) 50/0 4.16
17.0
75.1 –
18.0 –
43.0 –
5.49 26.5
54.6 –
9.95 –
35.7 –
1.06 2.68
2.80 –
2.64 –
9.85 – (2) 25/0 3.32
22.5
60.1 –
18.1 –
31.8 –
8.08 39.7
69.8 –
8.6 –
30.5 –
1.77 4.63
4.66 –
2.63 –
13.0 –
1.70
8.92 –
21.0 –
18.5 –
> 90
> 90
– –
– – – –
0.34 0.67
1.10 –
3.24 –
3.45 – (3) 0/0 0.33 §
1.03 §§
0.57 § –
1.72 § –
1.62 § –
> 90 §
> 90 §§
> 90 § –
– – – – (4) 50/25 2.20
14.4 37.9 –
17.2 –
40.8 –
3.10 12.6 29.9 –
9.65 –
19.2 –
1.18 2.68 3.15 –
2.67 –
15.4 – (5) 25/25 0.61
2.82
10.8 –
17.8 –
25.1 –
1.84 7.41
13.0 –
7.09 –
20.2 –
0.36 0.61
1.05 –
2.93 –
9.16 – Chromatographic conditions: columns, QN-AX ; mobile phase, MeOH/MeCN 100/0 and 0/100 ( v/v ) containing FA and TEA in various ratios; flow rate, 0.6 ml min –1 ; detection, 230–254 nm; temperature, ambient; dead time, t 0 , 3.31–3.55 min; k, α app , and R S values of Ac-Leu §and Ac-Gly § §, respectively
Table 2
Comparison of chromatographic parameters, k 1 , αand R S of racemic DNB-Leu, Bz-Leu, Ac-Leu , and DNB-Gly, Bz-Gly, Ac-Gly on QD-AX column operated with mobile phases
of either MeOH or MeCN and formic acid (FA) and triethylamine (TEA) additives in different ratios
Solvents + additives: MeOH/MeCN (100/0
v/v ) + FA/TEA (mM/mM) Solvents + additives: MeOH/MeCN (0/100 v/v ) + FA/TEA (mM/mM) Solvents + additives: MeOH/MeCN (100/0 v/v ) + FA/TEA (mM/mM) FA/TEA mM/mM)
k 1 L- DNB-
LeuDNB-Gly
k 2 D- DNB-
Leu- α app R S
k 1 L- DNB-
LeuDNB-Gly
k 2 D- DNB-
Leu- α app R S
k 1 Bz- LeuBz-Gly k 2 Bz-Leu- α app R S
(1) 50/0 3.92
28.4
81.2 –
20.7 –
39.2 –
3.42 27.5
54.5 –
15.7 –
32.7 –
0.67 2.07
2.14 –
3.20 –
13.0 – (2) 25/0 2.25
24.3
45.5 –
20.2 –
27.7 –
4.25 50.6
67.6 –
15.9 –
23.3 –
1.19 3.71
3.89 –
3.27 –
15.5 –
2.31
10.8 –
20.6 –
24.9 –
> 90
> 90
– –
– – – –
0.43 1.11
1.56 –
3.63 –
5.36 – (3) 0/0 0.45 §
1.72 §§
0.73 § –
1.62 § –
1.85 § –
> 90 §
> 90 §§
> 90 § –
– – – – (4) 50/25 1.45
11.8
29.6 –
20.4 –
37.6 –
2.66 15.6
42.3 –
15.9 –
36.8 –
0.83 2.36
2.72 –
3.28 –
14.3 – (5) 25/25 0.68
4.03
15.2 –
22.2 –
33.6 –
1.69 9.51
17.6 –
10.4 –
25.8 –
0.40 0.84
1.35 –
3.38 –
10.5 – Chromatographic conditions: columns, QD-AX ; mobile phase, MeOH/MeCN 100/0 or 0/100 ( v/v ) containing FA and TEA in various ratios; flow rate, 0.6 ml min –1 ; detection, 230–254 nm; temperature, ambient; dead time, t 0 , 3.36–3.47 min; k, α app , and R S values of Ac-Leu §and Ac-Gly §§ , respectively
Table 3
Comparison of chromatographic parameters, k 1 , αand R S of racemic DNB-Leu, Bz-Leu, Ac-Leu , and DNB-Gly, Bz-Gly, Ac-Gly on ZWIX( + ) column operated with mobile
phases of either MeOH or MeCN and formic acid (FA) and triethylamine (TEA) additives in different ratios
Solvents + additives: MeOH/MeCN (100/0
v/v ) + FA/TEA (mM/mM)
Solvents + additives: MeOH/MeCN (0/100
v/v ) + FA/TEA mM/mM)
Solvents + additives: MeOH/MeCN (100/0
v/v ) + FA/TEA mM/mM) or FA/TEA mM/mM)
k 1 D- DNB-
LeuDNB-Gly
k 2 L- DNB-
Leu- α app R S
k 1 D- DNB-
LeuDNB-Gly
k 2 L- DNB-
Leu- α app R S
k 1 Bz- LeuBz-Gly k 2 Bz-Leu- α app R S
(1) 50/0 0.28
2.16
1.64 –
5.86 –
10.4 –
1.08 4.99
2.90 –
2.67 –
7.97 –
0.09 0.21
0.09 –
1.00 –
0.00 – (2) 25/0 0.21
1.11 0.95 –
4.54 –
6.35 –
1.29 8.33 3.42 –
2.66 –
3.90 –
0.13 0.27 0.13 –
1.00 –
0.00 –
0.05
0.11 –
3.66 –
< 0.20
–
> 90
∗> 90 ∗ > 90
–
– –
1.32
∗ 1.98 ∗ 1.34
∗ –
n.d
–
0.85 – (3) 0/0 0.01 §
0.41 §§
0.06 § –
6.00 § –
0.57 § –
> 90 §
> 90 §§
> 90 § –
– – – – (4) 50/25 0.23
2.77
2.47 –
10.8 –
8.38 –
1.42 9.50
5.58 –
3.92 –
10.1 –
0.13 0.40
0.25 –
1.89 –
1.11 – (5) 25/25 0.15
1.10
1.39 –
9.25 –
9.80 –
1.63 6.70
4.89 –
3.00 –
10.3 –
1.55
∗ 1.78 ∗
1.65
∗ –
n.d
–
1.82 – Chromatographic conditions: columns, ZWIX( + ) TM ; mobile phase, MeOH/MeCN 100/0 and 0/100 ( v/v ) containing FA and TEA in various ratios; flow rate, 0.6 ml min –1 ; detection, 230–254 nm; temperature, ambient; dead time, t 0 , 1.52–1.64 min; k, α app and R S values of Ac-Leu §and Ac-Gly §§; respectively; ∗retention time (min) as peak elutes before t 0 ; n.d., not determined
Trang 8Table 4
Comparison of chromatographic parameters, k 1 , αand R S of racemic DNB-Leu, Bz-Leu, Ac-Leu , and DNB-Gly, Bz-Gly, Ac-Gly on ZWIX(-) column operated with mobile phases
of either MeOH or MeCN and formic acid (FA) and triethylamine (TEA) additives in different ratios
Solvents + additives: MeOH/MeCN (100/0
v/v ) + FA/TEA (mM/mM)
Solvents + additives: MeOH/MeCN (0/100
v/v ) + FA/TEA mM/mM)
Solvents + additives: MeOH/MeCN (100/0
v/v ) + FA/TEA mM/mM) FA/TEA mM/mM)
k 1 L- DNB-
LeuDNB-Gly
k 2 D- DNB-
Leu- α app R S
k 1 L- DNB-
LeuDNB-Gly
k 2 D- DNB-
Leu- α app R S
k 1 Bz- LeuBz-Gly k 2 Bz-Leu- α app R S
(1) 50/0 0.19
2.62
0.56 –
2.95 –
3.91 –
1.19 4.48
2.26 –
1.90 –
4.93 –
0.08 0.21
0.08 –
1.00 –
0.00 – (2) 25/0 0.24
1.36
0.63 –
2.67 –
4.08 –
1.36 7.80
2.48 –
1.82 –
2.80 –
0.11 0.20
0.11 –
1.00 –
0.00 –
∗ 0.33
1.60
∗ –
n.d
–
2.36 –
> 90
> 90
– –
– – – –
1.10
∗ 1.76 ∗ 1.10
∗ –
n.d
–
0.00 – (3) 0/0 0.03 §
0.17 §§
0.09 § –
3.31 § –
0.54 § –
> 90 §
> 90 §§
> 90 § –
– – – – (4) 50/25 0.24
2.37 1.50 –
6.24 –
9.84 –
1.18 14.5 6.67 –
5.65 –
11.7 –
0.17 0.38 0.17 –
1.00 –
0.00 – (5) 25/25 0.10
1.16
0.72 –
7.20 –
6.70 –
1.40 10.6
4.48 –
3.20 –
9.60 –
1.51
∗ 1.82 ∗ 1.54
∗ –
n.d
–
1.05 – Chromatographic conditions: columns, ZWIX(-) TM ; mobile phase, MeOH/MeCN 100/0 and 0/100 ( v/v ) containing FA and TEA in various ratios; flow rate, 0.6 ml min –1 ; detection, 230–254 nm; temperature, ambient; dead time, t 0 , 1.52–1.64 min; k, α app and R S values of N -Ac-Leu §and N -Ac-Gly §§, respectively; ∗ retention times (min) as peak elutes before t 0 ; n.d., not determined
Fig 5 Chromatograms of racemic-Bz-Leu ( A and B) on ZWIX( + ) TM and of racemic-DNB-Leu ( C ) on QN-AX CSP with 100% MeOH as bulk solvent Chromatographic conditions:
column, A and B, ZWIX( + ) TM and C , QN-AX; mobile phase MeOH 100% containing FA/TEA at molar ratios A, 50/25 mM/mM, B, 25/25 (mM/mM) and C , containing no
additives; flow rate, 0.6 ml min −1 ; detection, 254 nm; temperature, 25 °C
Ac-Leu.Ac-Glyisretainedsignificantly morestronglythanAc-Leu,
althoughithasshortretentiontimeswithoutFAandTEAadditives
[MP(3)].Theπ–π-stackingoftheDNB-tagissignificantlystronger
intheproticMeOHthaninthenon-proticMeCN,whichis
consid-ered toweakenintermolecularπ–π interactions Inother words,
MeCN couldbeclassifiedasaspecificdisrupterinthecaseofπ–
π-stacking,whichimpliesthatitsolvatestheπ-bindingsitestoa
certain extent.Ontheother hand,thepolarbutnon-protic MeCN
strengthens hydrogen-bondingeventsincontrast toprotic MeOH
In thepresentsituation,the carbamategroupsofSOandSAmay
play amoreprominentroleintheenantioselective intermolecular
interactions Inaddition,we shouldconsideratthispointFA asa
protic solvent,which may solvatethe acidic siteof the analytes
HydrogenbondingsitesofSOandSAmayalsobegettingsolvated
this way For the givenMP compositions [(1)-(5)], we have thus
a cascadeofcompetingbutoverlaid effectsonthestereoselective
andintermolecularSO–SAbinding,whichhampersthe
interpreta-tionofthediverseMPcompositioneffects
Unexpectedly, weobservedthatwithMeOHassolvent[MP(3)]
the DNB-Leuenantiomerscould beelutedfromaweak chiralion
exchangerwithoutanycounter-ionasdisplacerpresentintheMP
butwithroughlythesameαappaswithcounter-ionpresentinthe
MP, asdepicted inFig 5C In essence, all envisioned specific
as-sociationanddissociationprocessesinthecourseofthe
stereose-lectiveformationoftheSO–SAcomplexinMeOHaretakingplace
more orless simultaneously They occur in an equilibrium
fash-ionandtheelutionoftheanalytesfromtheCSPwithina reason-abletimeframewithpreservationoftheαappvaluesisapparently possible.Undertheseconditions, MeOHactsactually asan effec-tive displacer and weakens all active binding scenarios between
SOandSA.Forthisobservation,one mayalsorecall enantioselec-tive push-and-pull effects betweenthe solvated SO andSA part-ners,similar to those in ourprevious study aboutthe resolution
of zwitterionicanalytes on ZWIX phases without the useof any buffer system[28].The peak ofthe late-eluting enantiomerwith
a non-Gaussian shape hints that the adsorption/desorption steps are somewhat divergent from each other resulting in a strongly frontingpeak(Fig.5C) Althoughnoloadingexperimentsof DNB-Leu hasyetbeenmadeforsuch anion-exchangechromatographic systems,thisobservationcouldbeofvalueforpreparative applica-tionsasitenablesaneasy work-upofthecollectedsalt-freepeak fractions
Discussingfurtherthechromatographicresultsincontexttothe threeMPcompositionsdifferingonlyintheabsoluteamountofFA
inMeOH(intheabsenceofTEA),wenoticedastrongeffectofthe acid on the overall retention, butnot on enantioselectivity char-acteristics.WithMeOHasbulksolvent,theretentionsofDNB-Gly aremuchhigherinthepresenceofFA[MP(1)and(2)]than with-out FA [MP(3)] on both anion-exchanger CSPs (Tables 1 and 2) Moreover, theretention factors ofboth DNB-Leu enantiomers in-creasesharplywiththeincreasedFAcontent,whichisnot plausi-bleatthispoint.However,theαappvaluesremainroughlyconstant
Trang 9striking differenceisnoticedinthe absenceofFAwithextremely
large(infinitive)retentionfactorsofallanalytes.ForMP(2)the
re-tentionfactorsofDNB-GlyandD,L-DNB-Leuarehigherthanthose
forMP(1)
Inspecting nowthe resultsobtainedinthepresence ofTEA in
addition to FA in100% MeOH aseluent,where MP(4)containsa
25mMexcessofFA.Atfirstglance,thesecouldbecomparedwith
MP(2) assuming a stoichiometric saltformation between FA and
TEA InMP(4), theretentionfactors aredefinitivelylower than in
MP(2) without TEA MP(5) may also be comparable with MP(3)
in termsof excess ofFA The retentionfactors underMP(5)
con-ditions are somewhat higher than for MP(3)but, again, αapp
re-mains similar toall otherMP variants.Atthispoint,it shouldbe
highlighted that the investigated CSPsare heterogeneousin their
composition Furthermore, a good part of the remaining slightly
acidic silanol groups will be present, which eventually will
ad-sorb some ofthe basic TEA when exposed to such type ofMPs
Therefore, MP(5) maystill be considered slightly acidic dueto a
slight excess of FA The polar silica surface is known to adsorb
polar solvents(e.g.,water) strongly, that is,theadsorption ofthe
polar FA mayalsobe possible.Even thelesspolarAcOH maybe
adsorbed onto the remaining silanol groupsof the silica surface,
whichhasbeenchemicallymodifiedaccordingtotheinvestigated
CSPs
In100%MeCNasbulksolvent,asimilartrendcanbeseen,
al-thoughtheαappvaluesareabouthalfashighasthosefoundwith
100%MeOH,whichisagainpartiallyassignedtothestrong“bond
breaking” effectofMeCNinthecaseofthestrongπ–π-stackingof
theDNBandthequinuclidinegroups.Asacontrolexperiment,we
investigatedthebehaviorofBz-LeuandBz-Glyunder100%MeOH
conditions, asoutlined inTables 1 and2.Although the retention
factorsandtheαapp valuesaremuchlower, thesametrendcould
be found forall five MPvariants Finally,Ac-Leu andAc-Gly
em-ploying100%MeOH[MP(3)]werealsostudied.Inaccordancewith
the DNB-Leu experiment, Ac-Glyand Ac-Leuenantiomers are
re-tainedandenantioseparatedreasonably
We argued earlier that an increase inretention may be
asso-ciated (i) with a decrease in both the solvation shell of the SA
molecules andthe solvatedSO moieties and(ii) withan
adsorp-tionoftheexcessacidontothesurfaceofsilicaandthusintothe
solvated CSP layer We also argued that the free acidcan act as
a polardisplacer inthe course ofthe SO–SAinteractions
accord-ingtothestoichiometricdisplacementmodel,whichwasperfectly
attested for a MeOH/MeCN bulk solvent mixture of 40/60 (see
Fig.S1)
In polar aprotic MeCN, the retention factor decreases as the
amount ofdisplacer(in thiscaseFA)increases,whileforthe
po-lar protic MeOHthe situationis reversed,which appears tobe a
contradiction,andwehavetodealwithoverlaideffects,whichare
different in their directions FA is now considered to be a polar
protic solventbeingincompetitionwithMeOHassolvatingagent
leading, inessence, to a reduction ofboth the size of the
solva-tion shell andthethicknessof thelayer ofthe solvatedCSP.The
resultisanincreaseinretention,whichisstrikingwhencompared
to zeroFAMP(3)conditions.In otherwords, theFA seemstoget
adsorbedontotheCSPinusingbothMeOHandMeCNasbulk
sol-vents
Forbothsolventsituations,theαappremainsunaffectedbythe
amount of FA and, therefore, it contributes in the same
propor-tion to the retention factors k app1 andk app2 of the enantiomers
Hence,ifweputtheseresultsincontexttotheobservedresultsof
the MeOH/MeCN(40/60 v/v) solvent combination, wenotice that
theαapp isaround12,whichissignificantlylowerthanwithpure
MeOH(about18),buthigherthanwithpureMeCN(about9)used
asbulksolvents(Table1)
Fig. 6 Chromatographic data: retention factor ( k ) of DNB-Gly, and k 1 and αval- ues of DNB-Leu on QN-AX type CSP with 100% MeOH as bulk solvent containing FA/TEA or AcOH/TEA at different molar ratios Chromatographic conditions: analyte, DNB-Gly and DNB-Leu; column, QN-AX; mobile phase, MeOH containing FA/TEA
or AcOH/TEA at molar ratios 25/25, 50/25 and 50/0 (mM/mM); flow rate, 0.6 ml min −1 ; detection, 254 nm; temperature, 25 °C
Tostayconsistenttothedefinitionofαappfortheresolutionof DNB-LeuinMeOHasbulk solventcontainingMPadditives,itcan onlyhappenwhenthesumoftheMPeffectsiscontributingtothe non-stereoselectiveandthestereoselectiveSO–SAinteractionsina proportional manner.Anumberof overlaidandpartially compet-ingeffectsgeneratedbytheMP,whichessentially arereflectedin thesolvationshells oftheSOandSAmoieties,needtobe consid-ered when stereoselective SO–SA interaction mechanisms,driven
by molecular structure,are discussed Mostofthe time,such as-pectsare missingintheliterature,although some strikingresults haverecentlybeenpublishedsupportingthisview[38,39].In prin-ciple,onlyminuteamountsofMPadditivescaneventuallychange theαapp valuessignificantly,because oftheirselectiveadsorption anddistributioninthesolvatedCSPlayer.Conformationalchanges
ofthegivenSOmotifsmayevenresultinareversaloftheelution orderasshownforsomeexclusivecases[40]
Divergence from the stoichiometric displacement model dis-cussedabovecanalsobeseenfortheMPadditivecompositionFA plusTEA withMP(4)andMP(5) Wenoticeda similar,butstrong increase of the retention factorwith an excess of free FA It be-comesevidentthatthepresenceofbothTEAandFA/TEAsaltshas
amoderateeffectontheoverallretentionfactorsincomparisonto thesituation ofFA/TEA0/0and25/25 (mM/mM).There isonly a slightincreaseinthepresenceoftheFA/TEAsalt, whichindicates thatitmaynothaveaveryhighconcentrationinthesolvatedCSP Last but not least, we cross-evaluated a limited set of exper-iments with respect to the chromatographic effect generated by usingFA asacidcomponentinexchanging it withthelessacidic and lesspolar acetic acid (AcOH) as MP additive Here only the QN-AX CSP combination is investigated The results are summa-rizedin TableS6 andshould becompared todata withFA listed
inTableS5.InFig.6thedifferencescausedbyFA vsAcOHinthe absenceandpresenceofTEAasabaseadditiveareillustrated In-spectingfirstthesituationFA/TEAandAcOH/TEAwith50/0,50/25 and25/25(mM/mM)compositions,we identifysurprisingeffects DNB-Gly is retained with AcOH about four-times more strongly comparedto FA forthe50/0 (v/v) composite.A similartrend ap-pliesfortheDNB-Leuenantiomersaswell,althoughαappdropsfor theAcOHconditionto7.5comparedto18forFA.Obviously,inthe presenceofAcOH,thesolvationshellsaresmallerinsizeresulting
in a strongerCoulomb attractionand increasedretention factors
Ontheotherhand,theadsorbedAcOHbecomesmorestrongly in-volvedintheobservedmolecularrecognitionevents.Because DNB-Gly islesslipophilic thanthe DNB-Leu enantiomersand AcOHis more lipophilic than FA, this observationmakes sense However,
Trang 10a strongerbinding abilitywiththe CSP,theAcOH effectbecomes
contra-productivewithrespecttotheobservedenantioselectivity
TurningourattentiontotheeluentscontainingTEAinratiosof
50/25 and 25/25 (mM/mM), it is clear that the TEA reducesthe
effectoftheAcOHmoreasitdoesfortheFA resultinginshorter
retention factors.These findings are congruent withthe different
propertiesofthecombinedacidandTEAadditives
3.2.2 Specific behavior of the ZWIX(+ and ZWIX(-) columns on MP
changes
In a similar fashion as discussed above, all experimental
re-sultsobtainedinthecaseoftheZWIX(+)andZWIX(-)columnsare
summarizedinTables3and4.Theretentionfactorsofallanalytes
inMeOHarerathersmall,whereasinMeCNhigherretention
fac-tors were obtaineddue tothe lower solvationstrength ofMeCN
Inspecting the validity of the stoichiometric displacement model
for both zwitterionic columns, a clear discrepancy was found in
thecaseofMeOH.ApplyingFAasdisplacerin100%MeOHas
elu-ent, the retention increases with increased FA concentration In
MeCN assolvent,thisisnot thecase Thecontributionofthe
in-tramolecular ion-pairing effect (see Fig 1A and1B) is obviously
quitedifferentinMeOHandMeCN.Atthispointwehaveto
men-tion theunexpected andyetnotfullyunderstood chromatograms
describing the resolution of rac.-Bz-Leu on ZWIX(+) column
us-ing eitherMP(4)orMP(5)(see Fig.5Aand5B).With100%MeOH
and 50 mM/25 mM FA/TEA (i.e., in an excess of FA),the weakly
bound enantiomerofBz-Leuelutedaftert0,whilewithoutexcess
FA (25 mM/25 mM FA/TEA), it elutes before t0 (compare Fig 5A
and 5B) This stereoisomer isquasi repelled from a full entrance
into the pores of the solvated CSP, which is due to the peculiar
compositionofthestagnantMPinthepores.Toelucidatethis
phe-nomenon, furtherstudiesare currentlyundertaken Similartothe
case of the anion exchangers, the retention factors of the acidic
analytesin pure MeCN areextremely high, which definitively
re-sults from the poor solvation of both the SAs and the SO unit,
thus enforcinga verystrong SO–SACoulombicattraction
Follow-ing thesame trendaswe haveseenforthe QN-/QD-AXcolumns
withMeCNasbulksolvent,adropofαappofaboutafactoroftwo
compared to methanolic conditions wasmeasured, which has to
be attributedtothedifferentspatialenvironmentaroundthe
car-bamoylgroup(compareFig.1Aand1B)oftheSOandSAmoieties
4 Conclusions
Systematicliquidchromatographicexperimentshavebeen
car-ried out withtwo sets ofweak chiral anionandzwitterionic ion
exchangers based on immobilizedCinchonaalkaloid quinine(QN)
andquinidine(QD)type chiralselector (SO)motifsandrespective
chiralstationaryphases(CSPs).Thesewereappliedincombination
withtheuseofpolarorganicmobilephase (MP)invarying
com-positionsandN-acyl-leucineandN-acyl-glycineaschiraland
non-chiralweaklyacidicanalytes(selectands,SAs)
In our approach,we employed formicacid (FA) oracetic acid
(AcOH) asacid MP additive acting asdisplacer orcounter-ion to
control the ion exchange-based elution process of the acid-type
analytes, andtriethylamine(TEA)asbasefortheformation of
or-ganicsaltswiththeacidsactingaspositivelychargedco-ions.Free
acidswithouttheadditionofabaseinthenon-aqueousMPcould
alsoserveasdisplacerinfulfillingtheconceptofliquid
chromato-graphic“ionexchange” systems
As coreresultsofallexperimentswe cancollectthefollowing
observations:
(a) MeOH,asa protic polarsolvent,enableswell thesolvation
of the polarcarboxylic acid group of an acid-type analyte
but also of the given SO unit and thus of the entire CSP ThisincludestherespectivesaltsoftheacidswithbothTEA andthequinuclidinegroupoftheSOs.Forthegivenprobes, the carbamoyl groups classified as hydrogen donating and acceptingsites,may alsobe solvated.The retention factors arereasonable,buttheyencompassawidewindow
(b) MeCN,asanon-proticsolvent,lacksasolvationpowerofthe polarandchargeable sitesofSA andSOresultinginstrong CoulombattractionbetweentheSO(+)andSAs(–)leadingto extremely longretentiontimesboth ontheQN/QD-AX and theZWIX(+)/(−)typeionexchangers
(c) Viamixed MeOH/MeCNbulk solventcompositions, the re-tentionfactorscanbeadjustedwell,butenantioselectivities are compromised.They are generallylower in MeCN com-paredtothoseinMeOHduetotheweakenedπ–π stacking-typeinteractionsbetweentherespectivesitesofSOandSA The composition of the solvation shell of the CSP may be differentin comparisonto that ofthe bulk MP,because of theselectiveadsorption phenomenaeventually relatedalso
to the immobilization chemistry of the SO onto the silica surfaceandthestructuredheterogeneityofaCSP
(d) Asexpected,thecommoncounter-iondriven stoichiometric displacementmodelapplicableforion-exchange chromatog-raphyiscommonlyinplace.Itappliesalsoinsomecasesfor theuseofafreeaciddissolvedinMeOHthusalsoactingas
adisplacer.However,therehavebeenremarkableexceptions discussedaccordinglyinthiscontribution
(e) The highly polar FA is apparently well adsorbed onto the surfaceofthe CSP,thus changing its overall property As a consequence,retentionoftheanalytescanincreasewiththe concentrationofFAintheMPleadingtoastrongdivergence
of the stoichiometric displacement model Nevertheless, in the caseof free FA as MP additive in MeOH,retention in-creased but αapp kept constant Similar experiments with MeCNasbulksolventwillnotbepossible(seeentryb) (f) With pure MeOH as bulk solvent without additives in the
MP,it is possible to retain and resolve the probed N -acyl-Leuacidsonchiralionexchangersduetodiverseoverlaidas wellascompetingSO–SAassociationanddissociationevents duringthechromatographicprocess
(g) WhenexchangingfreeFAwithless-polarAcOH,another un-expectedbutstillplausibleeffectwasnoticed.AcOHsolvates theSAmoietiesapparentlytoalesserextentandduetothe reducedsize ofthe solvation shell,the retention factor in-creasesstrongly forDNB-Gly andDNB-Leu enantiomers In contrastto the three differentFA conditions forwhich the
αapp stays roughly constant, the use ofAcOH brings about
amarked change,which cansurpass theαappvaluesofFA The more lipophilic AcOH andits competition with MeOH
assolventwillcertainly changethecompositionofthe sol-vated CSP layer which, to some extent, maymodulate the conformationalflexibilityoftheSOunit
Although the MP components are non-chiral, they contribute
quasidirectlytotheobservedenantioselectivityofasolvatedCSP ThesolventsandtheadditivesoftheMPwillaffectthe conforma-tionoftheSOsand,consequently, theaccessibilityofthe stereos-elective binding sites (groves) approachedby the chiralanalytes Establishingthe highestαapp valuesin LC fora given CSP is not easy,sinceonealwaysdealswithanumberofoverlaidadsorption andsolventeffects,whicharedifficulttode-convolute
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