A novel poly(4-vinylpyridine) based stationary phase was investigated for its performance under supercritical fluid chromatography (SFC) mode. Due to its unique structure, this stationary phase has high molecular planarity recognition ability for aromatic samples possessing the same number of aromatic rings and -electrons.
Trang 1jou rn al h om ep a g e : w w w e l s e v i e r c o m / l o c a t e / c h r o m a
DAICEL Corporation, CPI Company, Life Science Development Center, Innovation Park, 1239, Shinzaike, Aboshi-ku, Himeji, Hyogo, 671-1283, Japan
a r t i c l e i n f o
Article history:
Received 31 May 2018
Received in revised form 7 August 2018
Accepted 16 August 2018
Available online 23 August 2018
Keywords:
Supercritical fluid chromatography
Stationary phase
Ligand
Selector
Polymer
Poly(4-vinylpyridine)
a b s t r a c t
Anovelpoly(4-vinylpyridine)basedstationaryphasewasinvestigatedforitsperformanceunder super-criticalfluidchromatography(SFC)mode.Duetoitsuniquestructure,thisstationaryphasehashigh molecularplanarityrecognitionabilityforaromaticsamplespossessingthesamenumberofaromatic ringsand-electrons.Takingadvantageoftheplanarityrecognitionabilityobserved,separationsof structurallysimilarpolycyclicaromatichydrocarbonsandsteroidswereachieved.Thisnovel station-aryphaseaffordedgoodpeaksymmetryforbothacidicandbasicactivepharmaceuticalingredientseven whenexcludingtheuseofadditivessuchasacids,bases,andsalts.Thesefindingsmaybeattributedtothe polymericpyridylgroupscovalently-attachedonsilicagel,whichwilleffectivelyshieldtheundesirable interactionbetweenresidualsilanolgroupsonthesurfaceandtheanalytes.Moreover,thepropertiesof pyridylgroupontheselectorcanbereversiblytunedtocationicpyridiniumformbyelutingtrifluoroacetic acidcontainingmodifier.Columnrobustnesstowardcycledurabilitytestingwasalsoconfirmed
©2018TheAuthor(s).PublishedbyElsevierB.V.ThisisanopenaccessarticleundertheCCBYlicense
(http://creativecommons.org/licenses/by/4.0/)
1 Introduction
Supercritical fluid chromatography (SFC) is increasing inuse
in the analytical and preparative separation field [1 SFC uses
supercritical orsubcritical mobile phasesconsisting of
pressur-izedcarbondioxide(CO2),usuallymixedwithamiscibleorganic
solvent(e.g.an alcohol).Thistechnologyhasmajor advantages
overmoreconventionalliquidchromatography(HPLC)orgas
chro-matography(GC), becauseit has a low viscosityallowing high
diffusivitiesandlimitedpressuredrop.Therefore,highflowrates
can beappliedwithout losing efficiency [2–8] In addition,the
“green”aspectisasignificantmotivationforSFCbecauseCO2isa
nontoxicrecycledmaterialandgeneratesnowastedisposalissues
Thehigh-throughputpotentialtogetherwithecologicaladvantages
contributetomakingSFCattractivetechnologyforawiderangeof
applications,notonlyforchiral[9–15],butalsointheachiralfield
[16–30]
TheretentionandseparationmechanismsinSFCarelikelyto
dependon a combination of both mobilephase and stationary
phase(SP)[5 AvarietyofSPsarecurrentlyavailableforusein
SFCmode.Mostofthesephaseshavebeendevelopedinand
trans-ferredfromthecommerciallyavailableportfoliosofHPLCSPs(e.g
∗ Corresponding author.
E-mail address: kn nagai@jp.daicel.com (K Nagai).
reversephase,normalphase,and/orHILIC).Inparallel,thereare someactivitiestodevelopnovelSPsspecificallydesignedforSFC use[31].OneofthemostrecognizedSPdedicatedtoachiralSFC separationis2-ethylpyridine(2-EP)bondedsilicaphase.This2-EP
SPaffordsgoodpeakshapesespeciallyforbasiccompounds, with-outanyadditiveinthemobilephase[32].OthernovelSPsforSFC havebeendevelopedbyacademicandindustrygroups[33–38] Most of the SPs used for achiral SFC separations are com-posedoflow-molecular-weightselectorscovalentlybondedonto
a solidsupport,usuallysilicagel.Polymertypeselectorswould
beexpected tointeractwithanalytesby utilizing multipleand cooperativemechanismsandinadditionpossesshighdurability However,onlyaverylimitednumberofexampleshavebeen intro-ducedthatutilizepolymer-basedligandsforachiralSFCseparation [36]
Basedontheexperienceofourresearchteaminthepolymeric field,werecentlydevelopedanovelpoly(butyleneterephthalate) basedcolumn,whichexhibiteduniquemolecularrecognition abil-itytogetherwithhighrobustnessincycledurabilitytests[38].We consideredthat thesefeatures maybeattributedtothe associ-atedmacromoleculareffectandasaresultwedecidedtodevelop variouspolymer-basedSPsandtoevaluatetheirperformance Forthedesignofthenovelpolymerstationaryphaseseries,we attemptedtoprepareseveralpolymersbased onthe ethylpyri-dine moiety, mainlyasthe commercialphasescontaining such synthonareconsideredasbenchmarksformanyresearchers.In
https://doi.org/10.1016/j.chroma.2018.08.038
0021-9673/© 2018 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 2120 K Nagai et al / J Chromatogr A 1572 (2018) 119–127
Fig 1.Structure of poly(4-vinylpyridine) or P4VP-based selector of the column
DCpak P4VP.
thisperspective,movingfromamonomerictoapolymeric
selec-tor,wewereexpectingthatthemolecularrecognitionabilitymay
beimprovedbymultipleconcertedinteractionsbetweenthemore
abundantpolymericpyridylligandinteractionswiththeanalyte
sample.Similartotherecentlyreportedpoly(butylene
terephtha-late)selector,these polymertype selectorswereanticipated to
displayahighdurabilityasthepolymerlayeronsilicagelshould
effectivelyshieldanyundesirablechemicalinteraction
TheHPLCseparationbehaviorofpyridinecontainingpolymerSP
hasbeenstudiedbyIhara[39–41].ThisSPshowedgoodselectivity
particularlyforplanaranddisk-likearomaticmoleculesinreverse
andnormalphaseHPLCmodes.Theinvestigationsofthepolymeric
phaseinHPLCmodewastheobjectiveofthiswork
Inanearlierstudy,wefocusedonnovelvinylpyridinepolymers
andrelated vinylheteroaromaticpolymers,and evaluatedtheir
performanceinSFCmode.We testedvariouspoly(vinylpyridine
isomers),including poly(2-vinylpyridine), poly(3-vinylpyridine),
and poly(4-vinylpyridine), together with poly(vinylimidazole)
[42,43].TheseSPsaffordeddistinctivemolecularrecognition
abili-ties,particularlyforstructurally-similarisomericsamples.Among
them,poly(4-vinylpyridine)(P4VP)SPwasfoundtoprovide
bet-termolecularshaperecognitionperformance(Fig.1).Thepresent
articlefocusesonthisP4VPcolumnanddescribesitsseparation
behaviorbyusingvarioussamples.Basedontheseresults,its
char-acteristicsandsuitablechromatographicconditionsarediscussed
2 Materials and methods
2.1 Chemicals
ThemodifierusedinthisstudywasJapaneseIndustrialStandard
specialgrademethanol(MeOH)obtainedfromNacalaiTesqueInc
(Kyoto,Japan).Carbondioxideofindustrialgrade(over99.5%)was
purchasedfromTatsumiIndustryCo.,Ltd.(Hyogo,Japan)
Ammo-niumformatewasobtainedfromWakoPureChemicalIndustries
(Osaka,Japan)
o-Terphenyl,triphenylene,anthracene,phenanthrene,pyrene,
chrysene, perylene, theobromine, trans-cinnamic acid,
3-phenylphenol, adenine, and diethylamine were purchased
from Tokyo Chemical Industry Co (Tokyo, Japan)
1,3,5-Tri-tert-butylbenzene, 2-acetylanthracene, 9-acetylanthracene,
3-acetylphenanthrene,9-acetylphenanthrene,paraxanthine,
feno-prophen,ketoprofen,naproxen,alprenolol,propranolol,atenolol,
pindolol,andcyanocobalaminwerepurchasedfromSigma-Aldrich
Corporation(St.Louis,MO,USA).Trifluoroaceticacid(TFA),
naph-thaceneanddexamethasonewerepurchasedfromNacalaiTesque
Inc.(Kyoto, Japan).2-Propanol(IPA), prednisone,estrone,
pred-nisolone,estradiol, estriol, caffeine, theophylline, nicotinamide,
andpyridoxinewerepurchasedfromWakoPureChemical
Indus-tries(Osaka,Japan).N-hexane(nHex)waspurchasedfromKanto
ChemicalCo.(Tokyo,Japan)
2.2 Instrumentationandchromatographicconditions DCpak P4VP column (initially launched as DCpak SFC-B), sized 150mm×4.6mm(i.d.), wassupplied byDAICEL Corpora-tion (Tokyo, Japan) This selector is composed of immobilized P4VP on 5 m silica particle (N.B also available on 3 m)
A Silica 2-ethylpyridine (2-EP) column of 5 m particle, sized
150mm×4.6mm(i.d.),waspurchasedfromWatersCorporation (Milford,MA,USA).TheSFCinstrumentusedinthisstudyis
Nexera-UC supplied by Shimadzu Corporation(Kyoto, Japan) equipped withaCO2pump,amodifierpump,avacuumdegasser,acolumn oven,amultiplewavelengthUVdetector,andautomatedback pres-sureregulator(ABPR).LabSolutionssoftware(V5.89)wasusedfor systemcontrolanddataacquisition.Chromatographicconditions, suchasmodifier,columntemperature,ABPRpressure,totalflow rate,detectionwavelength, sampleconcentration,and injection volumeweredescribedineachfigure,respectively
2.3 Dataanalysis Relativeretentionfactor(k)andseparationfactor(˛)were cal-culatedwiththeequationsbelow
where V is the elution volume of an analyte and V0 is the columnvoidvolume.V0wasestimatedbyinjecting 1,3,5-tri-tert-butylbenzeneasanon-retainedmarker.k1andk2inEq.(ii)arethe retentionfactorsofthefirstandsecondelutedpeaks,respectively
3 Results and discussion
3.1 Planarityrecognitionofaromatics Consideringthestructurefeaturesofthepoly(4-vinylpyridine)
SP,itwasexpectedtointeractwithplanararomaticsamplesasa resultofthemultiplearomaticpyridylunitscovalentlyattached
onsilicagel.Non-planaro-terphenyl(1)andplanartriphenylene
(2)withthesamenumberofaromaticringsand-electronswill providedetailed perception ofthe planarityrecognitionability, becausetheyhavebeenconsideredasindicatorformolecular pla-narityrecognitioninHPLC[44,45]andSFC[46].Fig.2Ashowsthe SFCchromatogramof1and2byusingP4VP,whenitsperformance wascomparedwithcommerciallyavailable2-EPSPunderisocratic conditions.Theretentiontimeofnon-planar1wasalmostidentical forthenewselectorand2-EP,whilethatofplanarsample2 signif-icantlyincreasedforP4VP.TheseparationfactorforP4VPselector between1and2(˛:k2/k1)reached30.6,whereasthatobtained
by2-EP is 4.4 Thisresult indicatesthat-electron rich planar
2couldstronglyinteractwithvinylpyridinepolymerselectorvia
–interaction
Takingadvantageofthishighplanarityrecognitionability, com-merciallyavailablepolycyclicaromatichydrocarbons(PAHs)were analyzed.Fig.3showstheSFCchromatogramofeightPAHs(2–9)
undergradientcondition.Eightpeakswerewellseparatedonthe P4VPcolumn.Ofparticularnoteisthatanthracene(4)and phenan-threne(5)havethesamemolecularweightandsimilarmolecular sizeandpolarity.Therefore,thesetwocompoundscannotbe dis-tinguishedbyMSdetector,whichmeanthattheonlymethodto separate4and5mustbebycolumnseparation.Thisnew selec-torachievedabaselineresolutionfor4and5.Ontheotherhand, whenthesecompoundswereanalyzedby2-EPSPunderthesame condition,4and 5 co-eluted Theslightadjustmentof gradient conditionswasnecessarytoseparate4and5inisocraticmode, immediatelyaftereluting4and5,alineargradientprogramstarted
Trang 3Fig 2.SFC chromatograms of o-terphenyl and triphenylene on (A) DCpak P4VP and
(B) 2-EP SPs Modifier, MeOH (isocratic condition, 3%); temperature, 40◦C; ABPR,
15 MPa; total flow rate, 3 mL/min; UV detection, 254 nm; sample concentration,
0.3 mg/mL in nHex/IPA = 9/1; injection volume, 1 L.
ThelongeraspectratioofthePAHanalyzedresultedinashorter
retentiontime.Wiseetal.proposedlength-to-breadth(L/B)ratio
fordescribingtwodimensionalaspectratioofPAH,andthesmaller
L/Bratioindicatesthedisk-likemolecule[47].Indeed,L/Bratioof
naphthacene(7),chrysene(8),andtriphenylene(2)whichhavethe
samenumberofaromaticringsand-electronsis1.89,1.72,and
1.12,respectively.Theelutionorderof7, 8,and2is7<8<2,which
istheinverseofL/Bratio.Thesetendenciesarealmostidenticalfor
similarstationaryphaseusedinHPLCmode[41]
Similartotheseparationof non-substitutedPAH separation,
regioselectiveacetylated anthracene(10, 12) andphenanthrene
(11, 13)werealsoexaminedunderisocraticconditions.Although
thesesampleshavealmostthesamemolecularsizeandpolarity,
theP4VPselectorcanrecognizetheslightstructuraldifferencesand
thefourpeakswerewellresolvedasshowninFig.4
Fromtheseresults,thenewcolumnshowsexcellentplanarity
recognitionandmolecularshaperecognitionofvariousaromatic
isomersandPAHs.CombiningP4VPcolumnwithsupercriticalfluid
extraction(SFE)andsubsequentSFCtechniquewouldenableusto
analyzetheresidualPAHinsoilandatmosphereetc[21]
3.2 Separationofsampleswithdifferentstructuralfeatures
Inspiredbytheinterestingplanarityandmolecularshape
recog-nition,othertypeofplanarsamplefamiliese.g.steroidsmixtures
(14–19)wereanalyzedunderisocraticconditions.Fig.5showsthe
SFCchromatogramofsixstructurallyrelatedsteroids.In
particu-lar,prednisone(14),prednisolone(16),anddexamethasone(18)
Fig 3.SFC chromatogram of eight polycyclic aromatic hydrocarbons on (A) DCpak P4VP and (B) 2-EP SPs Inset shows magnified chromatogram Modifier, MeOH (gra-dient condition); temperature, 40 ◦ C; ABPR, 15 MPa; total flow rate, 3 mL/min; UV detection, 254 nm; sample concentration, 0.1 mg/mL (except9),0.3 mg/mL(9)in nHex/IPA = 9/1; injection volume, 1 L The gradient started with 3% of MeOH, after
3 min hold at 3% of MeOH, linear gradient ramped up to 38% of MeOH over 14 min, followed by 1 min hold at 38% of MeOH, then returned to 3% of MeOH over 2 min, followed by 1 min hold at 3% of MeOH.
have almostsame skeleton withsome slightdifferences in the substituents, and thereforetheyare difficulttoseparate.When investigatingthesesamplesbyusing2-EPSPunderthesame con-ditions,theywereco-eluting.Wenotethattheconditionsapplied wereonlytocomparetheselectivityinexactlyidenticalconditions
Abetterseparationmightbepossiblefor2-EPbyusingdifferent gradientcondition.Thedirectseparationofsuchsteroidmixturesin SFCmodewouldhavesignificanceinsteroidprofiling,aspotential biomarkers[48]oralsoinanti-dopingcontrol
Theanalysisofcaffeine(20)anditsdemethylatedderivatives, theophylline(21),theobromine(22)and paraxanthine(23)was alsoinvestigated.Fig.6showstheSFCchromatogramofthe mix-ture.Goodpeakresolutionwithsymmetricalpeakswasobserved forthesepolaranalytesaswellasforless-polarPAHderivativesand steroids.Thelongerretentionof21–23than20maybeattributed
tothehydrogenbondinginteractionsbetweenthedemethylated protonoftheanalytesandtheprotonacceptorbehaviorofP4VP selector
Trang 4122 K Nagai et al / J Chromatogr A 1572 (2018) 119–127
Fig 4.SFC chromatogram of acetylated anthracene and phenanthrene on DCpak
P4VP SP Modifier, MeOH (isocratic condition, 3%); temperature, 40◦C; ABPR,
15 MPa; total flow rate, 3 mL/min; UV detection, 254 nm; sample concentration,
0.15 mg/mL in nHex/IPA = 9/1; injection volume, 1 L.
ThisnovelSPcontainsthebasicpoly(4-vinylpyridine)moiety
andonemayguessthatacidicsampleswouldbestronglyretained
and/orwouldshowtailingpeaksonit Inorder toconfirm this
point, we tested propionic acidnonsteroidal anti-inflammatory
drugs(NSAIDs),fenoprofen (24),ketoprofen(25),andnaproxen
(26).Fig.7showstheSFCchromatogramofthreeNSAIDsunder
iso-craticconditionswithoutanyadditives,whichgavethreeresolved
peaks.Surprisingly,theirpeakshapeswererelativelysymmetric,
withpeak symmetryfactors(Ps)for 24of1.11,1.15for25, and
1.19for26.Theseresultsindicatethatthepyridinepolymer
lig-andmustefficientlyshieldtheundesirableinteractionsbetween
analytesandresidualsilanolgroupsonSP
3.3 Effectofadditives
IntheSFCfield,itiscommontouseadditivesasathird
com-ponentinthemobilephase,suchasacids,basesandsalts.They
improve the peak shapes and/or increase the solubility in the
mobilephaseespeciallyforpolaranalytes[5,31,49].Basicadditives
areoftenusedforbasicsamples;acidicadditivesforacids,butother
combinationsarealsopossible.Thecurrenttrendforbothacidic
andbasicsampleanalysisistousevolatilesalts,suchas
ammo-niumformateandammoniumacetate.Theseadditivesareoften
usedwhenseparatingAPIsinSFCinanalyticalaswellaspreparative
applications,becausemanyAPIsbearpolarand/orionizablegroups
whichcaneasilyinteractwiththeresidualsilanolgroups,andoften
resultindeficientpeakshapes(leading,tailing,andasymmetric
peaks)[31] However,ifa SPcouldafford goodpeaksymmetry
withoutanyadditive,itwouldbeconsideredadvantageous.The
absenceofadditiveswouldbeapositivefeatureforpreparative
applications(nosaltstoberecuperatedtogetherwiththe
prod-uct),butalsoforanalyticalUVdetection(thehighUVabsorptionof
Fig 5. SFC chromatogram of six steroids on (A) DCpak P4VP and (B) 2-EP SPs Modifier, MeOH (isocratic condition, 30%); temperature, 40◦C; ABPR, 15 MPa; total flow rate, 3 mL/min; UV detection, 225 nm; sample concentration, 0.33 mg/mL in nHex/IPA = 1/1; injection volume, 2 L.
Fig 6.SFC chromatogram of caffeine, theophylline, theobromine, and paraxanthine
on DCpak P4VP SP Modifier, MeOH (isocratic condition, 5%); temperature, 40 ◦ C; ABPR, 15 MPa; total flow rate, 3 mL/min; UV detection, 225 nm; sample concentra-tion, 0.2 mg/mL in MeOH/IPA = 1/1; injection volume, 2 L.
Trang 5Fig 7.SFC chromatogram of nonsteroidal anti-inflammatory drugs on DCpak P4VP
SP Modifier, MeOH (isocratic condition, 10%); temperature, 40◦C; ABPR, 15 MPa;
total flow rate, 3 mL/min; UV detection, 210 nm; sample concentration, 0.2 mg/mL
in IPA; injection volume, 2 L.
ammoniumformateorammoniumacetateespeciallyingradient
conditionssometimesleadstounstablebaselines)
Accordingly, we analyzed four-adrenergic blocking agents
(-blockers)(27–30)inthepresenceandabsenceofammonium
formate,tocomparethechromatographicperformanceofthetwo
SPs.Fig.8AshowstheSFCchromatogramoffour-blockersunder
gradientconditionsobtainedbyusingthe2-EPcolumnwithout
anyadditive.Ingeneral,forsuchbasicsamples,theirpeakswere broadenedwithoutanyadditives.However,whenanappropriate additive(e.g.20mMofammoniumformate)wasused,thepeak shapesweresubstantiallyimproved(Fig.8B)
Asexpected,theP4VPcolumncanproducerelatively symmet-ricalpeaksevenintheabsenceofanyadditive(Fig.8C).Asfor2-EP, aftertheadditionofsalts,peakswerefurthersharpened(Fig.8D) ThefactthattheP4VPcolumncouldgainsharppeakswithout anyadditivesforthesebasicAPIsmaybeattributedtothe poly-mericligandeffect.ThecovalentlybondedP4VPchainsmayspread
ontheporoussilicagelsurfaceandwillcontributetoreducethe undesirableinteractionsbetweentheresidualsilanolgroupsand thebasicanalytes
3.4 Effectofconditioningwithdifferentadditive
Asthenewselectorconsistsofbasicpoly(4-vinylpyridine) moi-eties,itcanbeenvisagedtoformacationicpyridiniumformby reactionwithstrongacidssuchastrifluoroaceticacid(TFA)and
beconvertedtoaquaternizedamphiphilicsaltformbyreaction withthecorrespondingalkylhalide.Recently,IharaandTakafuji reportedamphiphilicpoly(N-alkylpyridiniumsalt)basedHPLCSPs throughquaternizationreactions Theirseparationmodecanbe easilytunedbychangingtheN-alkylsidechainlength[50,51] The protonated pyridinium salt effect was investigated for theP4VPphasebypassingthroughTFA-containingmodifier.We selectedneutral(2, 13),acidic(31, 32)andbasicsamples(21, 34)
forthistestunderisocraticconditions
Fig 8. SFC chromatograms of -blockers (A, C) in the absence or (B, D) presence of ammonium formate on (A, B) 2-EP and (C, D) DCpak P4VP SPs Modifier, MeOH (gradient condition); temperature, 40◦C; ABPR, 15 MPa; total flow rate, 3 mL/min; UV detection, (A, C) 220 or (B, D) 280 nm; sample concentration, 0.1 mg/mL in MeOH; injection volume, 2 L The gradient started with 10% of modifier, after 1 min hold at 10% of modifier, linear gradient ramped up to 35% of modifier over 10 min, followed by 2 min
Trang 6124 K Nagai et al / J Chromatogr A 1572 (2018) 119–127
Fig 9. (A–E) SFC chromatograms and (F) retention factor dependent of neutral, acidic, and basic samples on DCpak P4VP SP by using various modifier under isocratic condition (10%) Before each analysis, the testing modifier was eluted for more than 30 min for equilibration Each modifier was, (A) MeOH, (B) MeOH/TFA = 100/1, (C) MeOH, (D) MeOH/DEA = 100/1, and (E) MeOH For detail, please see text Temperature, 40 ◦ C; ABPR, 15 MPa; total flow rate, 3 mL/min; UV detection, 254 nm; sample concentration, 0.13 mg/mL in nHex/IPA = 1/1; injection volume, 2 L.
Fig.9A showstheSFC chromatogramofthesix sample
mix-ture with MeOH as modifier These samples were eluted as
relativelysymmetricpeaks.AfterpassingaTFA-containing
mod-ifier(MeOH/TFA=100/1,(v/v))for more than30min.,thesame
sample mixturewas injected Fig.9B shows thecorresponding
chromatogramandFig.9Ftheretentionfactor(k)dependenceon
modifiercomposition.Theirelutionorderdramaticallychanged
Acidicsamples(31, 32)elutedfaster thanintheinitialanalysis,
whereasbasictheophylline(21)hardlychangeditsretentiontime
andmorebasicadenine(34)elutedsignificantlylater.Surprisingly,
neutralsamples(2, 13)alsoelutedfasterthanintheoriginal
anal-ysis.However,byusingMeOHagainasamodifierduring30min.,
retentiontimeofallanalyteswerelikelytorecovertheoriginal
pro-file(Fig.9C).Wethenusedadiethylamine(DEA)-containingMeOH
(MeOH/DEA=100/1, (v/v)) as a modifier, and thesame
experi-mentwasconducted.However,theelutionorderhardlychanged
(Fig.9D).AfterpassingMeOHasamodifieragain,theelutionalmost
revertedtothatofthefirstinjection(Fig.9E)
Basedontheseresults, weproposedthefollowing retention
mechanism.Asdiscussedintheprevioussection,thepyridylgroups
onthepolymersidechainseffectivelymaskthesilanolgroupson
silicagelsurface,whichleadtoashieldingofundesirable
interac-tionsbetweensilanolsandanalytes.WhenonlyMeOHwasusedas
modifier,theacidicsamples(e.g.31),caninteractwiththeSPvia
acid-baseinteraction,whilesuchaninteractionbetweenSPand basicanalytes(e.g.34)shouldnotbeexpected(Fig.10A).Forthis reason,theelutionof34mightbefasterthantheoneof31.When
aTFA-containingMeOHwasusedasamodifier,thepyridylgroups
onthesidechainareprotonated(Fig.10B).ContrarytoFig.10A,the protonatedSPand34caninteract.Wenotethatthepyridylgroups
intheSParemorepronetoprotonatethan34becausepyridineis morebasicthan34basedonthepKavaluesofthecorresponding conjugateacids[52,53].Hence,theelutionofbasic34wasslower thanacidic31.Thereasonwhytheretentiontimeofneutral sam-plesdecreasedunderacidicconditionsisstillunclear.Weenvisage thattheelectrondensityof thepyridylring intheSPmightbe decreasedbyprotonation,orMeOHmaysolvatetheprotonated sidechains,whichwillinterferewiththeSPandneutralanalyte interaction.Insuchacase,itmayaffecttheretentionbehaviorof neutralsamplesinacidicconditions.AftergraduallypassingMeOH
asmodifieragain,theprotonatedSPsidechainsgradually depro-tonatedtobeintheiroriginalstate.Therefore,theinitialretention behaviorgraduallyrecovered.DEAseemstohavelesseffectonthe retentionofthesesamples
Asdemonstratedhere,theelutionorderofthisselectorcanbe reverselytunedthroughacidmediatedpyridiniumsaltformation
Trang 7Fig 10.Postulated retention mechanism of DCpak P4VP in neutral conditions (A) and acidic conditions (B).
Fig 11.Cycle dependent SFC chromatograms of water soluble vitamins on (A) 2-EP and (B) DCpak P4VP SPs Modifier; MeOH (isocratic condition, 25%); temperature, 40 ◦ C; ABPR, 15 MPa; total flow rate, 3 mL/min; UV detection, 230 nm; sample concentration, 0.2 mg/mL in MeOH; injection volume,2 L.
3.5 Cycledurabilitytests
Asmentionedintheintroduction,polymer-typeselectorswere
expectedtoshowgood durabilityas weestimatethat
undesir-ableattackmaybeinterfered withthepolymerligandlayeron
theSPsurface.Recently,weconfirmedthecolumnrobustnessof
poly(butyleneterephthalate)selectorbyarangeofcycledurability
testing[38]
Inordertoinvestigatethedurabilityof anewP4VPcolumn,
threewatersolublevitamins(WSVs),nicotinamide(vitaminB3),
pyridoxine(vitaminB6),andcyanocobalamin(vitaminB12)were selected
Fig 11A shows the cycle-dependent SFC chromatograms of threeWSVsbyusing2-EPcolumnunderisocraticconditions.For thefirstinjection,threepeakswerewellseparatedanda character-isticlongretentionwasobservedforvitaminB12.However,asthe cyclepassed,theretentiontimegraduallydecreasedforvitaminB3
andvitaminB6,andsharplydecreasedforvitaminB12.Fig.11Aalso showsthechromatogramsafter20,41,60,and80cycles.The reten-tiontimecontinuouslydecreasedandthatofvitaminB12reduced
tolessthanhalfoftheoriginaltime.Fairchildetal.proposedthat
Trang 8126 K Nagai et al / J Chromatogr A 1572 (2018) 119–127
Fig 12. Cycle versus retention factor (k) of (A) vitamin B 3 , (B) vitamin B 6 , and (C) vitamin B 12 by using DCpak P4VP and 2-EP columns Experimental condition is same as Fig 11
silyletherformationbyacondensationreactionbetweensilanols
andMeOHusedasamodifierisamajorcontributiontoretention
variationovertimeinSFCmode[54]
WeinvestigatedthesamecycletestfortheP4VPcase(Fig.11B)
Incontrastto2-EPcase,theretentiontimeofthesesamplesdid
notchange after20, 40, 60,and 80 cycles Fig.12 shows cycle
versustheirretentionfactors(k).Wenotethatthesystematiccycle
investigationherereportedwasrunover80sequentialcyclesin
brand-newcolumn,howevertheP4VPcolumnsusedinthisstudy
togenerateallexperimentaldatareportedwereperiodicallytested
withthestandardsamplesandconfirmedthedurabilityandthe
stabilityoverseveralmonths
4 Conclusion
AnovelP4VPbasedcolumnwasdesignedanditsperformance
wasevaluatedunderSFCconditions.ThisSPshowedunique
molec-ularshape recognitionforplanarmoleculessuchasstructurally
related polycyclic aromatichydrocarbons and steroid mixtures
ThenewSPaffordedsymmetricpeaksforactivepharmaceutical
ingredientanalysisevenintheabsenceofanyadditives,e.g.acids,
bases, or salts,probably due tothe effective shield of residual
silanolsbythepolymericpyridineselector.Thesurfacechemical
propertiesofthenewcolumncanbeeasilyconvertedtocationic
pyridiniumformbyelutingTFAcontainingmodifiers,which
dra-maticallychangeelutionorderofacidic,basic,andeven neutral
analytes.Thissignificantelutionorderchangecanberecoveredto
theoriginalstatebypassingthroughDEAcontainingmodifier
Additionally,thecolumnperformancedidnotchangeasaresult
ofcycledurabilitytestingofwatersolublevitamins
Thepresentstudytogetherwiththatofanotherourpolymeric
SP[38,43,55]revealedthatoursyntheticpolymerbasedselector
wouldbeaversatiletoolinSFCanalysis.Furthermore,itcanbe
extendedinuseintopreparativefields.Furtherinvestigationsof
othersampleapplicationindifferentchromatographicmodesand
deepinsightofthisSPisnowinprogress
Acknowledgments
TheauthorswishtothankDr.PilarFrancoandTongZhangin
ChiralTechnologiesEuropeS.A.S forvaluablediscussions.The
authorsalsoappreciate Dr JosephM.Barendt and MsLorraine EvangelistainChiralTechnologies,Inc.andDr.BrianFreerinChiral TechnologiesEuropeS.A.SforEnglishgrammaticalcorrection Thisresearchdidnotreceiveanyspecificgrantfromfunding agenciesinthepublic,commercial,ornot-for-profitsectors
References
[1] C.F Poole (Ed.), Supercritical Fluid Chromatography (Handbooks in Separation Science), Elsevier, 2017.
[2] T.A Berger, Separation of polar solutes by packed column supercritical fluid chromatography, J Chromatogr A 785 (1997) 3–33, http://dx.doi.org/10 1016/S0021-9673(97)00849-2
[3] L.T Taylor, Supercritical fluid chromatography for the 21 st century, J Supercrit Fluids 47 (2009) 566–573, http://dx.doi.org/10.1016/j.supflu.2008 09.012
[4] G Guiochon, A Tarafder, Fundamental challenges and opportunities for preparative supercritical fluid chromatography, J Chromatogr A 1218 (2011) 1037–1114, http://dx.doi.org/10.1016/j.chroma.2010.12.047
[5] E Lesellier, C West, The many faces of packed column supercritical fluid chromatography - A critical review, J Chromatogr A 1382 (2015) 2–13846, http://dx.doi.org/10.1016/j.chroma.2014.12.083
[6] V Desfontaine, D Guillarme, E Francotte, L Nováková, Supercritical fluid chromatography in pharmaceutical analysis, J Pharm Biomed Anal 113 (2015) 56–71, http://dx.doi.org/10.1016/j.jpba.2015.03.007
[7] S Fekete, J.L Veuthey, D Guillarme, Comparison of the most recent chromatographic approaches applied for fast and high resolution separations: theory and practice, J Chromatogr A 1408 (2015) 1–14, http://dx.doi.org/10 1016/j.chroma.2015.07.014
[8] A Tarafder, Metamorphosis of supercritical fluid chromatography to SFC: an Overview, TrAC Trends Anal Chem 81 (2016) 3–10, http://dx.doi.org/10 1016/j.trac.2016.01.002
[9] G Terfloth, Enantioseparations in super- and subcritical fluid chromatography, J Chromatogr A 906 (2001) 301–307, http://dx.doi.org/10 1016/S0021-9673(00)00952-3
[10] G.B Cox, Enantioselective supercritical fluid chromatography using Daicel’s
“platinum series” polysaccharide-based columns, LC-GC North-Am Appl Noteb (2007) 31.
[11] L Miller, Preparative enantioseparations using supercritical fluid chromatography, J Chromatogr A 1250 (2012) 250–255, http://dx.doi.org/10 1016/j.chroma.2012.05.025
[12] P Franco, T Zhang, Common screening approaches for efficient analytical method development in LC and SFC on columns packed with immobilized polysaccharide-derived chiral stationary phases, in: G.K.E Scriba (Ed.), Chiral Sep Methods Protoc., 2nd ed., Humana Press, Totowa, NJ, 2013, pp 113–126, http://dx.doi.org/10.1007/978-1-62703-263-6
[13] J Lee, J.T Lee, W.L Watts, J Barendt, T.Q Yan, Y Huang, F Riley, M Hardink, J Bradow, P Franco, On the method development of immobilized
polysaccharide chiral stationary phases in supercritical fluid chromatography using an extended range of modifiers, J Chromatogr A 1374 (2014) 238–246, http://dx.doi.org/10.1016/j.chroma.2014.11.044
Trang 9[14] S Khater, M.A Lozac’h, I Adam, E Francotte, C West, Comparison of liquid
and supercritical fluid chromatography mobile phases for enantioselective
separations on polysaccharide stationary phases, J Chromatogr A 1467
(2016) 463–472, http://dx.doi.org/10.1016/j.chroma.2016.06.060
[15] K Zawatzky, M Biba, E.L Regalado, C.J Welch, MISER chiral supercritical fluid
chromatography for high throughput analysis of enantiopurity, J Chromatogr.
A 1429 (2016) 374–379, http://dx.doi.org/10.1016/j.chroma.2015.12.057
[16] D Thiébaut, Separations of petroleum products involving supercritical fluid
chromatography, J Chromatogr A 1252 (2012) 177–188, http://dx.doi.org/10.
1016/j.chroma.2012.06.074
[17] L.T Taylor, Packed column supercritical fluid chromatography of hydrophilic
analytes via water-rich modifiers, J Chromatogr A 1250 (2012) 196–204,
http://dx.doi.org/10.1016/j.chroma.2012.02.037
[18] K Ty´skiewicz, A D ˛ebczak, R Gieysztor, T Szymczak, E Rój, Determination of
fat- and water-soluble vitamins by supercritical fluid chromatography: a
review, J Sep Sci 41 (2018) 336–350, http://dx.doi.org/10.1002/jssc.
201700598
[19] G.F Pirrone, R.M Mathew, A.A Makarov, F Bernardoni, A Klapars, R.
Hartman, J Limanto, E.L Regalado, Supercritical fluid
chromatography-photodiode array detection-electrospray ionization mass
spectrometry as a framework for impurity fate mapping in the development
and manufacture of drug substances, J Chromatogr B 1080 (2018) 42–49,
http://dx.doi.org/10.1016/j.jchromb.2018.02.006
[20] C West, E Lemasson, S Bertin, P Hennig, E Lesellier, Interest of
achiral-achiral tandem columns for impurity profiling of synthetic drugs with
supercritical fluid chromatography, J Chromatogr A 1534 (2018) 161–169,
http://dx.doi.org/10.1016/j.chroma.2017.12.061
[21] A.P Wicker, D.D Carlton, K Tanaka, M Nishimura, V Chen, T Ogura, W.
Hedgepeth, K.A Schug, On-line supercritical fluid extraction—supercritical
fluid chromatography-mass spectrometry of polycyclic aromatic
hydrocarbons in soil, J Chromatogr B 1086 (2018) 82–88, http://dx.doi.org/
10.1016/j.jchromb.2018.04.014
[22] O Petkovic, P Guibal, P Sassiat, J Vial, D Thiébaut, Active modulation in neat
carbon dioxide packed column comprehensive two-dimensional supercritical
fluid chromatography, J Chromatogr A 1536 (2018) 176–184, http://dx.doi.
org/10.1016/j.chroma.2017.08.063
[23] J.L Bernal, M.T Martín, L Toribio, Supercritical fluid chromatography in food
analysis, J Chromatogr A 1313 (2013) 24–36, http://dx.doi.org/10.1016/j.
chroma.2013.07.022
[24] K Taguchi, E Fukusaki, T Bamba, Simultaneous analysis for water- and
fat-soluble vitamins by a novel single chromatography technique unifying
supercritical fluid chromatography and liquid chromatography, J Chromatogr.
A 1362 (2014) 270–277, http://dx.doi.org/10.1016/j.chroma.2014.08.003
[25] T Yamada, Y Nagasawa, K Taguchi, E Fukusaki, T Bamba, 13 - polar lipid
profiling by supercritical fluid Chromatography/Mass spectrometry method,
in: M.U Ahmad, X Xu (Eds.), Polar Lipids, Elsevier, 2015, pp 439–462, http://
dx.doi.org/10.1016/B978-1-63067-044-3.50017-0
[26] E.L Regalado, C.J Welch, Separation of achiral analytes using supercritical
fluid chromatography with chiral stationary phases, TrAC Trends Anal Chem.
67 (2015) 74–81, http://dx.doi.org/10.1016/j.trac.2015.01.004
[27] C West, E Lemasson, S Bertin, P Hennig, E Lesellier, An improved
classification of stationary phases for ultra-high performance supercritical
fluid chromatography, J Chromatogr A 1440 (2016) 212–228, http://dx.doi.
org/10.1016/j.chroma.2016.02.052
[28] D.C Patel, M.F Wahab, D.W Armstrong, Z.S Breitbach, Advances in
high-throughput and high-efficiency chiral liquid chromatographic
separations, J Chromatogr A 1467 (2016) 2–18, http://dx.doi.org/10.1016/j.
chroma.2016.07.040
[29] A Grand-Guillaume Perrenoud, D Guillarme, J Boccard, J.L Veuthey, D.
Barron, S Moco, Ultra-high performance supercritical fluid chromatography
coupled with quadrupole-time-of-flight mass spectrometry as a performing
tool for bioactive analysis, J Chromatogr A 1450 (2016) 101–111, http://dx.
doi.org/10.1016/j.chroma.2016.04.053
[30] H Segawa, Y.T Iwata, T Yamamuro, K Kuwayama, K Tsujikawa, T Kanamori,
H Inoue, Differentiation of ring-substituted regioisomers of amphetamine
and methamphetamine by supercritical fluid chromatography, Drug Test.
Anal 9 (2017) 389–398, http://dx.doi.org/10.1002/dta.2040
[31] E Lemasson, S Bertin, C West, Use and practice of achiral and chiral
supercritical fluid chromatography in pharmaceutical analysis and
purification, J Sep Sci 39 (2016) 212–233, http://dx.doi.org/10.1002/jssc.
201501062
[32] A Grand-Guillaume Perrenoud, J Boccard, J.L Veuthey, D Guillarme, Analysis
of basic compounds by supercritical fluid chromatography: Attempts to
improve peak shape and maintain mass spectrometry compatibility, J.
Chromatogr A 1262 (2012) 205–213, http://dx.doi.org/10.1016/j.chroma.
2012.08.091
[33] F.M Chou, W.T Wang, G.T Wei, Using subcritical/supercritical fluid
chromatography to separate acidic, basic, and neutral compounds over an
ionic liquid-functionalized stationary phase, J Chromatogr A 1216 (2009)
3594–3599, http://dx.doi.org/10.1016/j.chroma.2009.02.057
[34] J Smuts, E Wanigasekara, D.W Armstrong, Comparison of stationary phases
for packed column supercritical fluid chromatography based upon ionic liquid
motifs: A study of cation and anion effects, Anal Bioanal Chem 400 (2011)
435–447, http://dx.doi.org/10.1007/s00216-011-4767-z
[35] R McClain, M.H Hyun, Y Li, C.J Welch, Design, synthesis and evaluation of
separations, J Chromatogr A 1302 (2013) 163–173, http://dx.doi.org/10 1016/j.chroma.2013.06.038
[36] C.G.A da Silva, C.H Collins, E Lesellier, C West, Characterization of stationary phases based on polysiloxanes thermally immobilized onto silica and metalized silica using supercritical fluid chromatography with the solvation parameter model, J Chromatogr A 1315 (2013) 176–187, http://dx.doi.org/ 10.1016/j.chroma.2013.09.055
[37] M Dunkle, C West, A Pereira, S Van Der Plas, E Lesellier, Synthesis of stationary phases containing pyridine, phenol, aniline and morpholine via click chemistry and their characterization and evaluation in supercritical fluid chromatography, Sci Chromatogr 6 (2014) 85–103, http://dx.doi.org/10 4322/sc.2014.023
[38] K Nagai, T Shibata, S Shinkura, A Ohnishi, Poly(butylene terephthalate) based novel achiral stationary phase investigated under supercritical fluid chromatography conditions, J Chromatogr A 1549 (2018) 85–92, http://dx doi.org/10.1016/j.chroma.2018.03.032
[39] H Ihara, W Dong, T Mimaki, M Nishihara, T Sakurai, M Takafuji, S Nagaoka, Poly(4-Vinylpyridine) as Novel Organic Phase for RP-HPLC Unique Selectivity for Polycyclic Aromatic Hydrocarbons, J Liq Chromatogr Relat Technol 26 (2003) 2491–2503, http://dx.doi.org/10.1081/JLC-120023796
[40] H Ihara, M Fukui, T Mimaki, A Shundo, W Dong, M Derakhshan, T Sakurai,
M Takafuji, S Nagaoka, Poly(4-vinylpyridine) as a reagent with silanol-masking effect for silica and its specific selectivity for PAHs and dinitropyrenes in a reversed phase, Anal Chim Acta 548 (2005) 51–57, http:// dx.doi.org/10.1016/j.aca.2005.05.056
[41] U.G Gautam, A Shundo, M.P Gautam, M Takafuji, H Ihara, High retentivity and selectivity for polycyclic aromatic hydrocarbons with
poly(4-vinylpyridine)-grafted silica in normal-phase high-performance liquid chromatography, J Chromatogr A 1189 (2008) 77–82, http://dx.doi.org/10 1016/j.chroma.2007.12.018
[42] K Nagai, S Shinkura, Stationary Phase for Supercritical Fluid Chromatography, WO2016/152996, 2016.
[43] C West, E Lemasson, K Nagai, T Shibata, P Franco, S Bertin, P Hennig, E Lesellier, Synthesis and characterization of novel polymer-based pyridine stationary phases for supercritical fluid chromatography, Submitted (n.d.) [44] K Kimata, K Iwaguchi, S Onishi, K Jinno, R Eksteen, K Hosoya, M Araki, N Tanaka, Chromatographic characterization of silica C18 packing materials Correlation between a preparation method and retention behavior of stationary phase, J Chromatogr Sci 27 (1989) 721–728, http://dx.doi.org/10 1093/chromsci/27.12.721
[45] M Mifune, Y Mori, M Onoda, A Iwado, N Motohashi, J Haginaka, Y Saito, Separation characteristics of aminopropyl silica gels modified with copper-phthalocyanine as high performance liquid chromatography stationary phase, Anal Sci 14 (1998) 1127–1131, http://dx.doi.org/10.2116/ analsci.14.1127
[46] K Jinno, H Mae, Molecular planarity recognition of polycyclic aromatic hydrocarbons in supercritical fluid chromatography, J High Resolut Chromatogr 13 (1990) 512–515, http://dx.doi.org/10.1002/jhrc.1240130715 [47] S Wise, W.J Bonnett, F.R Guenther, W.E May, A relationship between reversed-phase C18 liquid chromatographic retention and the shape of polycyclic aromatic hydrocarbons, J Chromatogr Sci 19 (1981) 457–465, http://dx.doi.org/10.1093/chromsci/19.9.457
[48] J Teubel, B Wüst, C.G Schipke, O Peters, M.K Parr, Methods in endogenous steroid profiling – A comparison of gas chromatography mass spectrometry (GC–MS) with supercritical fluid chromatography tandem mass spectrometry (SFC-MS/MS), J Chromatogr A 1554 (2018) 101–116, http://dx.doi.org/10 1016/j.chroma.2018.04.035
[49] E Lemasson, S Bertin, P Hennig, H Boiteux, E Lesellier, C West, Development
of an achiral supercritical fluid chromatography method with ultraviolet absorbance and mass spectrometric detection for impurity profiling of drug candidates Part I: optimization of mobile phase composition, J Chromatogr.
A 1408 (2015) 217–226, http://dx.doi.org/10.1016/j.chroma.2015.07.037 [50] M Shahruzzaman, M Takafuji, H Ihara, Porous silica particles grafted with an amphiphilic side-chain polymer as a stationary phase in reversed-phase high-performance liquid chromatography, J Sep Sci 38 (2015) 2403–2413, http://dx.doi.org/10.1002/jssc.201500189
[51] M Shahruzzaman, M Takafuji, H Ihara, Tuning of separation mode using pyridinium salt-branched ionic polymer-grafted silica as stationary phase in HPLC, Chem Lett 45 (2016) 13–15, http://dx.doi.org/10.1246/cl.150890 [52] H.C Brown, D.H McDANIEL, O HÄFLIGER, Dissociation constants, in: E.A BRAUDE, F.C NACHOD (Eds.), Determ Org Struct by Phys Methods, Academic Press, New York, 1955,
http://dx.doi.org/10.1016/C2013-0-12413-0 , p iii.
[53] R.M.C Dawson, D.C Elliott, W.H Elliott, K.M Jones, Data for Biochemical Research, Oxford University Press, New York, 1959.
[54] J.N Fairchild, D.W Brousmiche, J.F Hill, M.F Morris, C.A Boissel, K.D Wyndham, Chromatographic evidence of silyl ether formation (SEF) in supercritical fluid chromatography, Anal Chem 87 (2015) 1735–1742, http:// dx.doi.org/10.1021/ac5035709
[55] M Douˇsa, 1H-tetrazole-5-amine immobilized on substituted polymer Gel/Silica as a new stationary phase for hydrophilic interaction chromatography, Chromatographia 81 (2018) 349–357, http://dx.doi.org/10 1007/s10337-017-3452-6