Investigation of robustness for supercritical fluid chromatography separation of peptides: Isocratic vs gradient mode

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Journal of Chromatography A

jou 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

Investigation of robustness for supercritical fluid chromatography separation of peptides: Isocratic vs gradient mode

MartinEnmark^a,b,EmelieGlenne^a,MarekLe´sko^a,c,AnnikaLangborgWeinmann^d, TomasLeek^e,KrzysztofKaczmarski^c,MagnusKlarqvist^d,JörgenSamuelsson^a,∗, TorgnyFornstedt^a,∗

aDepartmentofEngineeringandChemicalSciences,KarlstadUniversity,SE-65188Karlstad,Sweden

bPharmacognosy,DepartmentofMedicinalChemistry,UppsalaUniversity,BiomedicalCentre,Box574,SE-75123Uppsala,Sweden

cDepartmentofChemicalandProcessEngineering,RzeszówUniversityofTechnology,PL-35959Rzeszów,Poland

dEarlyProductDevelopment,PharmaceuticalSciences,IMEDBiotechUnit,AstraZeneca,Gothenburg,Sweden

eMedicinalChemistry,Respiratory,InflammationandAutoimmunity,IMEDBiotechUnit,AstraZeneca,Gothenburg,Sweden

a r t i c l e i n f o

Articlehistory:

Received27April2018

Receivedinrevisedform1July2018 Accepted5July2018

Availableonline10July2018

Keywords:

SFC Peptide Gramicidin Robustness Methodtransfer Water

a b s t r a c t

Weinvestigatedandcomparedtherobustnessofsupercriticalfluidchromatography(SFC)separations ofthepeptide gramicidin,usingeitherisocraticorgradientelution.Thiswas doneusingdesignof experimentsinadesignspaceofco-solventfraction,watermassfractioninco-solvent,pressure,and temperature.Thedensityoftheeluent(CO2-MeOH-H2O)wasexperimentallydeterminedusingaCorio- lismassflowmetertocalculatethevolumetricflowraterequiredbythedesign.Forbothretentionmodels, themostimportantfactorwasthetotalco-solventfractionandwatermassfractioninco-solvent.Com- paringtheelutionmodes,wefoundthatgradientelutionwasmorethanthreetimesmorerobustthan isocraticelution.Wealsoobservedarelationshipbetweenthesensitivitytochangesandthegradient steepnessandusedthistodrawgeneralconclusionsbeyondthestudiedexperimentalsystem.

Totesttherobustnessinapracticalcontext,boththeisocraticandgradientseparationsweretransferred toanotherlaboratory.Thegradientelutionwashighlyreproduciblebetweenlaboratories,whereasthe isocraticsystemwasnot.Usingmeasurementsoftheactualoperationalconditions(notthesetsystem conditions),theisocraticdeviationwasquantitativelyexplainedusingtheretentionmodel.Thefindings indicatethebenefitsofusinggradientelutioninSFCaswellastheimportanceofmeasuringtheactual operationalconditionstobeabletoexplainobserveddifferencesbetweenlaboratorieswhenconducting methodtransfer.

©2018TheAuthors.PublishedbyElsevierB.V.ThisisanopenaccessarticleundertheCCBYlicense (http://creativecommons.org/licenses/by/4.0/).

1. Introduction

The separation of therapeutic peptides has long been an important application area for chromatography, particularly reversed-phaseliquidchromatography(RPLC)[1].Withgrowing interest in supercritical fluid chromatography (SFC) for analyz- ingandpurifyingsmallmolecules(i.e.molecularweights<1kD) [2,3],severalauthorsfrombothacademiaandindustryhavealso startedtoinvestigatehowSFCcouldbeusedtoanalyzeandpurify peptides[4–13]. Whilethequality-by-design(QbD)paradigmis firmlyestablishedinliquidchromatography[14],itisnotsimilarly

∗ Correspondingauthors.

E-mailaddresses:Jorgen.Samuelsson@kau.se(J.Samuelsson), Torgny.Fornstedt@kau.se(T.Fornstedt).

establishedinSFC,probablybecauseSFCislessrobustthanliquid chromatography[3].Somestudieshaveinvestigatedtherobust- nessofSFCseparationmethodsinthecontextofmethodtransfer andbyinvestigatingtherobustnessinadesignspace[15].

Thesmallbut growingbody ofstudiestreating theSFC sep- aration of peptides [4–13] has investigated a limited number of peptides, for example, gramicidin D [6,12,13], leucine- enkephalin[4–6,10],methionine-enkephalin[4–6,10],angiotensin I [4], angiotensin II [4–6], cyclosporin analogs [7], beta- methylphenylalanine [11], oxytocin [10], bradykinin [4,10], Pro-Leu-Glyamide[4],sauvagine[4],leupeptide[4],urotensinII [4],sulfomycin[8],cyclicpeptides[16],andcustomacidicandbasic linearuncappedpeptides[9].

Most studies have used traditional liquid chromatographic stationary phases suchas silica [7,7,9], diol [8,9], C18 [4,9], 2- ethylpyridine[4,5,9], cyano [6], and various chiral phases [11],

https://doi.org/10.1016/j.chroma.2018.07.029

0021-9673/©2018TheAuthors.PublishedbyElsevierB.V.ThisisanopenaccessarticleundertheCCBYlicense(http://creativecommons.org/licenses/by/4.0/).

Table1

Propertiesofthegramicidinisoformspartiallyseparatedinthestudy.

Gramicidinspecies formyl-X-Gly-Ala-Leu- Ala-Val-Val-Trp-Leu-Y- Leu-Trp-Leu-Trp- ethanolamine

Mw[gmol⁻¹] X Y Specifiedpurity

Val-A 1881 Val Trp 80–85%^*

Ile-A 1895 Ile Trp Unknown^**

Val-B 1842 Val Phe 6–7%^*

Ile-B 1856 Ile Phe Unknown^**

Val-C 1858 Val Tyr 5–14%^*

Ille-C 1872 Ile Tyr Unknown^**

* Specifiedbyvendor.

** Notspecifiedbyvendor.

as well as polymer-based phases such as divinylbenzene [10]

or poly(styrene–divinylbenzene) [12,13]. Eluents are typically CO2 modified with acetonitrile/water [5,8], acetonitrile [4,11], methanol[4,6–9,11],ethanol[7,11],andisopropylalcohol[7,11]

towhichacidicorbasicadditivessuchastrifluoroaceticacid(TFA) [4,5,9],2,2,2-trifluoroethanol(TFE)[6],ammoniumacetate[4,6], aceticacid[6],andisopropylamine[6,8]areadded.Severalstudies haveinvestigatedthemodificationofco-solventswithwater,and founditsadditionnecessarytoachieveresolutionortoimprove peakshape[4,6,9].Moststudieshaveusedgradientelution,but somehavealsoinvestigatedtheisocraticelutionmode[11].

Duetothesmallchemicalspaceinvestigated,itisdifficultto drawgeneralconclusionsastothefeasibilityofusingSFCforpep- tideanalysisandpurification.However,severalofthementioned studiesdid investigatethe effects of thestationary phase, elu- ent,andotheroperationalconditions,suchasbackpressureand temperature[7].Clearly,amechanisticunderstandingofpeptide separationinSFCislackingcomparedwithourunderstandingof themuchmorematureRPLCtechnique[17–19].

Robustness is “a measure of ... [an analytical procedure’s]

capacitytoremainunaffectedbysmall,butdeliberatevariationsin methodparametersandprovidesanindicationofitsreliabilitydur- ingnormalusage”[20].Itiswellknownthatdeliberatevariations inoperatingconditionsinSFCcangreatlyaffectseparation[21,22]

whichisanadvantageofSFCascomparedtoLCforimprovingselec- tivity;however,thisalsoaffecttherobustness.However,itisless knownandunderstoodthatunintentionalvariationscanalsohave amajorimpact,forexample,whenoperatingSFCinhighlycom- pressibleregionsorwhengeneralretentionmechanismsarepoorly understood.Studyingtherobustnessofseparationsconductedin thehighco-solventregimeofSFC,technicallyinsubcriticalcondi- tions[23]cangivevaluableinsightintoareastypicallynotstudied inSFCwhere theeluent ismoreLClikebecausethefluidcom- pressibilitydecreaseswithincreasingco-solventintheeluent.Most studiesindicatethatworkingwithalargefractionofco-solventis necessarytoelutepeptides.

Beyazetal. [24] systematically studiedtheeffects of differ- ent instrumental and operating conditions on the precision of retentiontimesforalargesetofsoluteselutedonC18usingace- tonitrile/buffer/water.Theyconcluded,forexample,thatisocratic elutionwasmoresensitivethanwasgradientelutionwhenstudy- ingtheeffectsofvariationinthemobilephasecomposition.No similarinvestigationhasbeendoneforSFC.

Theaimofthisstudyistoinvestigateandcomparetherobust- nessofpeptideseparationsconductedunderisocraticandgradient conditions in SFC. As a model compound, we studied the lin- earunchargedpentapeptidegramicidinseparatedonapH-stable hybridsilicacolumnusinganeluentcontainingCO2,water,and methanol.Therobustnesswasinvestigatedbyevaluatingthevari- ationin theretentionfactor usingdesign ofexperiments (DoE) by perturbing the most important operational conditions, i.e.

varyingthetotal orinitialgradientfractionofco-solvent,water massfractioninco-solvent,pressure,andtemperature.Secondly, simulationsbasedontheexperimentaldata,butwithdifferentsen- sitivitiestotheperturbations,wereperformedinordertoreveal howtherobustnesswouldvaryforhypotheticalsolutesingradi- entseparationsofdifferentgradientslopes.Finally,thepractical consequencesoftheobserveddifferencesinrobustnessbetween gradientandisocraticseparationswerequantifiedbytransferring theisocraticandgradientmethodstoadifferentlaboratory.

2. Materialandmethods

2.1. Chemicals

The mobile phase consisted of CO2 (99.99%) from AGA Gas AB(Lidingö,Sweden),HPLC-grademethanolfromVWR(Radnor, PA, USA), and water with conductivity of 18.2Mcm from a Milli-QPlus185waterpurificationsystemfromMerckMillipore (Darmstadt,Germany).Gramicidin(CAS#1405-97-6)fromBacillus aneurinolyticuswasobtainedfromSigma-Aldrich(St.Louis,MO, USA).Thislinearpeptidehasthesequenceformyl-X-Gly-Ala-Leu- Ala-Val-Val-Trp-Leu-Y-Leu-Trp-Leu-Trp-ethanolamine, where X canbeeitherValorIleandYeitherTrp(GramicidinA),Phe(Gram- icidinB),orTyr(GramicidinC)[25],andisthereforereferredto ascomprisingtheX–Yisoformsofgramicidin(Table1).AllGram- icidinsamplesweredissolvedinneatMeOHtoaconcentrationof 1mgmL^–1.

2.2. Instrumentation

Inthisstudy,twodifferentanalyticalSFCsystems,ofthesame modelandmanufacturerwereused,butattwodifferentlocations:

KarlstadUniversity(denotedLaboratory1)andatAstraZenecain Gothenburg(Laboratory2).TheLaboratory1systemwasaWaters UPC²(WatersCorporation,Milford,MA,USA)equippedwithaPDA detector.TheLaboratory 2systemwasalso aWaters UPC² but connectedviaapassivesplitter(UPC²MSsplitter)toaPDAdetec- toranda Waterssingle-quadrupolemassspectrometer(Waters SQD2)usingelectrosprayionizationinpositivemode.Bothselected ionmonitoringandscan mode(800–1000m/z;833Das⁻¹)were used,respectively.InLaboratory1theUPC²instrumenthadasin- glestackconfigurationfrombottomtotopofpump,autosampler, convergencemanager(backpressureregulator),columnmanager andPDAdetector.InLaboratory2,theUPC²inLaboratory2had atwo-stackconfigurationwithpump,autosampler,convergence managerinthefirststackandmake-uppump,columnmanager andPDAdetectorinthesecondstack.Theinnerdiameterofthe stainlesssteeland PEEKtubingfrominjectortocolumntoPDA toconvergencemanagerwas0.18mmatbothlaboratoriesexcept fromthesplittertoconvergencemanageratLaboratory2werethe innerdiameterwas0.25mm.ThePDAflowcellvolumewas8.4␮L atbothlaboratories.

Themobilephaseflowtothemassspectrometerwassplitwith apassivesplitteranddilutedwitha0.2mLmin^–1mixtureof95/5%

(v/v)methanol/10mMammoniumformate.Theextracolumnvol- umewas measuredfrom the retention time of an injectionof 1mgmL^–1gramicidinwithazero-dead-volumeunioninplaceof thecolumninbothsystems.Thedifferencewascompensatedfor incomparisonsbetweenthetwosystems.Pressurewasmeasured usingtwomodelEJX530Aabsolutepressuretransmitters(Yoko- gawaElectricCorporation,Tokyo,Japan)connectedtothecolumn inletandoutletusingatee.AdataloggerfromPicoTechnology(St.

Neots,UK)wasusedtorecordthepressure.

Thetotal andco-solventmassflows weremeasureddirectly afterthemobilephasemixerandbetweentheco-solventpump

andtheventvalve,respectively,usingaminiCORI-FLOWM12low- flowCoriolismassflowmeter(BronkhorstHigh-TechB.V.,Ruurlo, Netherlands),hereafterdenoted“CFM.”Thecolumnsusedwerea 2.5-␮mKromasil SFC-2.5-XT(100×3.0mm)(AkzoNobel,Bohus, Sweden) and a 1.7-␮m Waters 2-picolylamine (100×3.0mm) (WatersCorporation).

2.3. Procedure

2.3.1. Designofexperiments

A three-level, four-factor, central composite face-centered experimentaldesignwiththreecenterpointswasusedtoinves- tigatehow the logarithmic valueof the retention factor ofthe Val-Aisoformofgramicidinvarieswithtotalco-solventfraction (MeOH+water),watermassfractioninco-solvent,pressure,and temperaturefortheisocraticandgradientelutions,respectively.

OnlytheKromasil SFC-2.5-XTcolumnwasinvestigated.Thelog transform of the retention factor wasused because the reten- tion generally hasa logarithmic relationship with the fraction ofco-solventusedintheseparation[26].Thecentralcomposite face-centeredexperimentaldesignmodelwasselectedinorderto achievegood predictivepowerinthedesign space[27]as well astoinvestigatepotentialquadratictermsandinteractionterms betweenfactors.Intheisocraticelutionexperiments,thetotalco- solventfractionwasidenticaltotheisocraticcomposition,andin thegradientelutionexperiments,thetotalco-solventfractionindi- catedtheconditionatthestart(andend)ofthegradient.Theset designwasasfollows:thetotalco-solventfractionduringtheiso- craticexperiments was30, 33,and 35 v/v%and theco-solvent gradientwas28.3–61.3,30.0–65.0, and31.7–68.7 v/v%in5min whentheretentiontimeswerefoundtobereasonable.Aftereach changeofeluentcomposition,thesystemwasequilibratedforat leastonehour.Thewatermassfractioninco-solventwas1.2,5, and8.7w/w%.Theset,backpressurewas110,130,and150bar.

Thetemperaturewas30,45,and60^◦C.Duetothenatureofmixing abinaryco-solvent[28,29]withCO₂,thesetvolumetricfraction ofco-solventwasnotusedbutratherthemeasuredmassfraction [30].The design wasrescaled for theactualandmeasuredval- uesoftheco-solventfraction,watercontent,andpressure.2␮L injectionsof1mgmL^–1 gramicidininneatMeOHweremadeat leastinduplicateforeachexperimentalcondition.Chromatograms wererecordedat220nm.Retentiontimeswereestimatedfrom peakapexandnormalizedtoretentionvolumesusingthemea- suredmassflowanddensity(seesection2.3.2).Thevoidtimewas obtainedfromtheinitialbaselinedisturbanceandwasalsonormal- izedtovoidvolume.Theaverageofallvoidvolumeswasusedin calculatingeachretentionfactor.Multiplelinearregressionsofthe log₁₀-transformedretentionfactorswereperformedusingMODDE 11(Umetrics,Umeå,Sweden)witha95%confidencelevelandnon- significantfactorsweremanuallyremoved.

2.3.2. Characterizingtheexperimentalconditions

Asfactorsfortheexperimentaldesign,thetotalco-solventmass fraction,watermassfractioninco-solvent,columntemperature, andaveragecolumnpressurewereused.Theco-solventfractions weremeasuredusingtheCFM.Thearithmeticmeanofthecolumn inletandoutletpressuresforeachisocraticandgradientcondition wasusedasthepressurefactor.Theinstrumentsettemperature wasusedasinputtotheexperimentaldesign,asseveralof our studieshaveindicatedthatourinstrumentsettemperatureisvery accurate[22,30].ThemassfractionofwaterinMeOH,takenfrom thegravimetricpreparationofco-solvents,wasusedasinputtothe experimentaldesign.

Tocalculatethevolumetricflowrate,thedensityoftheeluentis required.However,toourknowledgeitisimpossibletoaccurately calculatethedensityoftheternaryCO2-MeOH-H2Ofluidusedhere.

Therefore,directdensitymeasurementusingtheCFMwaseval- uatedandperformed.Themainchallengewasthatthepressure andtemperatureinsidetheCoriolisflowcellmustbeidenticalto thoseinsidethecolumn.Thiswasachievedbyremovingthecol- umnandsettingtheback-pressureregulatorsothatthecolumn averagepressureswereachievedintheCFM.Thetemperaturewas adjustedbysimultaneouslyincreasingtheflowrateandthesetcol- umnoventemperatureuntilthedesiredtemperatureintheCFM wasobtainedandstabilized.TubingfromtheUPC²totheCFMwas insulatedtominimizeheatloss.

To plotcontourplots and calculate densities otherthan the experimentalmeasureddatapoints,seeSupplementaryDataTable S1; theexperimentaldatawerefitted toa second-ordermulti- polynomialequationwithinteractiontermsusingMODDE11.

Theaccuracyofthesedensitymeasurementswasfirstevalu- atedbycomparingtheoreticalandmeasureddensitiesusingpure CO₂atthreesetbackpressures(110,130,and150bar)andthree temperatures(30,45,and60^◦C)at3mLmin⁻¹.Thetheoreticalden- sitywascalculatedusingNISTReferenceFluidThermodynamicand TransportpropertiesDatabaseversion9.1(REFROP)[31]withthe measuredarithmeticmeanpressureandmeasuredtemperatureas inputs,seeSupplementaryDataTableS2.

Allpressureanddensitymeasurementswereconductedsepa- ratelytominimizeextracolumnvolumes.

2.3.3. Methodtransferexperiments

Thesame2.5-␮mKromasilSFC-2.5-XTusedfortheDoEinLab- oratory1wasinstalledinLaboratory2andthesamesetmethod conditionswereusedaswhenrunningtheexperimentaldesigns center-pointexperimentsintheisocraticandgradientelutions.The totalmassflow,co-solventmassflowandaveragecolumnpressure weredeterminedatbothsites.

3. Resultsanddiscussion

TheretentionbehaviorinSFCofthemainisoformofgramicidin, Val-A,wasinvestigatedintheisocraticandgradientelutionmodes usingamixtureofMeOHandwaterasco-solventsatdifferenttem- peraturesandpressures.Thegoalwastouseaquantitativemodel oftheretentionfactortocomparetherobustnessoftheseparation systemineitherelutionmodewithinthedefineddesign space.

Experimentaldatawerealsoextrapolatedtogivegeneralinsight intotherobustnessoftheisocraticand gradientelutionsepara- tionsystems.Tocalculatetheretentionvolumeintheexperimental space,theeluentdensitywasdeterminedusingtheCFM.Finally, theseparationsystemwastransferredtoadifferentlaboratoryto evaluatethepracticalimplicationsoftransferringamoreorless robustseparationsystem.

3.1. Retentioncharacteristicsofgramicidin

Thegoalofthescreeningwastofindasatisfactoryseparation systemandtofindsuitableboundariesfortheexperimentaldesign.

Initialscreeningofthechromatographicbehaviorofgramicidin anditsisoformswasdoneonhybridsilicaand2-picolylaminesta- tionaryphasesusingMeOH/waterastheco-solvent.Fig.1presents thechromatogramfroma2-␮Linjectionof1.0mgmL^–1gramicidin separatedonthehybridsilica(Fig.1a)and2-picolylamine(Fig.1b) columns.Frommassspectrometricdata,theretentionorderonthe hybridsilicaphasewasfoundtobeIle-B,Val-B,Ile-C/Ile-A,andVal- C/Val-A(Fig.1c)andonthe2-picolylaminephasetobeIle-B/Val-B, Ile-C/Val-C,Ile-A,andVal-A(Fig.1d).The2-picolylaminestation- aryphasemanagedtoresolveeacharomaticisoformbutnotthe aliphaticforms,exceptforIle-AandVal-A.Thehybridsilicasta- tionaryphase,ontheotherhand,managedtoresolvethealiphatic isoformsbutwaslessabletodifferentiatebetweenthearomatic

Fig.1. Analyticalinjectionsofgramicidin(a,c)onthehybridsilicacolumn(KromasilSFC-2.5-XT)using5.00-mingradientelutionof28–62v/v%at110barand60^◦Cand (b,d)onthe2-picolylaminecolumnusing5.00-mingradientelutionof23–57v/v%at110barand60^◦C.Toprowshows220-nmUVtracesof2-␮Linjectionsof1mgmL^–1 gramicidin.Bottomrowshowsselectiveiontracesofallgramicidinisoformsfor[M+2H]²⁺fragments.

forms.Theseresultsareasexpectedconsideringthenatureofthe hybridsilicaandthe2-picolylamineligand.Furtherstabilityexper- imentsusingthe2-picolylaminecolumnrevealedanon-reversible retentiondriftwhenvaryingtheamountofwater,sothiscolumn wasnotusedinfurtherstudies(datanotshown).

Addingwatertothemethanolco-solvent[32,33], wasfound tosignificantlyaffecttheretentionandpeakshapeinthecaseof gramicidin(Fig.2).Toinvestigatewhetheraddingwater tothe eluentresultedinacontinuousordiscontinuouschangeinreten- tionand/orpeakshape,thefirstinjectionswereperformedwith neatmethanolonnewcolumnsusingtheisocratic(Fig.2a,b)and gradient(Fig.2c,d)elutionmodes.Followingtheneatmethanol experiments,injectionsweredoneat1.2,5,and8.7w/w%water addedtotheco-solvent.WhilethesolubilityofwaterinneatCO₂in supercriticalconditionsisgenerallybelowamolarfractionof0.01 [34],itissignificantlyhigherwhenthewaterisaddedtogetherwith methanol[35].Byincreasingthewatercontentoftheeluent,the apparenttailingofthemainpeakdecreasesinsemi-analyticalcon- ditions(Fig.2a,c)andisconsiderablyreducedinsemi-overloaded conditionsinboththeisocraticandgradientelutions(Fig.2b,d).

Toconclude,wefoundtheretentiononthehybridsilicacol- umntobereproducible andabletoseparatealiphaticforms of gramicidin.Wealsofoundthatwaterreducedtheretentionfactor and considerably reduced the peak tailing, especially in semi- overloadedconditions.Addingwater totheeluentinthis range didnotinduceanydiscontinuousorunexpectedbehaviorsinthe retentionorpeakshape.

3.2. Measurementofdensitytoestimatevolumetricflow

ToevaluatetheestimationofdensityusingtheCFM,theden- sityofneat CO₂ wasmeasuredovertherange of 30–60^◦C and 134–175bar,inwhichtheCO₂densityvariesfrom530to871kg m⁻³(SupplementaryDataTableS2).Comparingthemeasuredand

calculated(REFPROP)densitiesshowedthattherelativedifference neverexceeded0.4%.ThisindicatesthatCFM shouldbeableto accuratelymeasuretheeluentdensity.

BecauselittleisknownofthepropertiesoftheCO₂-MeOH-H₂O eluentsusedhere,thedensitywasmeasuredatallexperimental conditions(SupplementaryDataTableS1).Thesedatawerethen fitted toa second-order multi-polynomial equationwith inter- actionterms tointerpolatedensitiesin otherconditions.It was possibletofindanacceptablecorrelation(R²=0.79atthe95%con- fidencelevel)betweenthefactorsandthemeasureddensity.

Fig.3a–cplotsthedensityvariationasafunctionoftempera- tureandpressureforaco-solventfractionof31.5w/w%with1.3 (a),5 (b),and 8.7 (c) w/w% water intheco-solvent. Ascanbe seen,thedensityvariesonlyslightlywithpressureandtemper- ature,andaddingwatertotheeluentonlyslightlyincreasesthe densityofthemobilephase.Thismeansthat,fromadensityper- spective,thesystemisratherinsensitivetochangesintemperature, pressure,andthefractionofwateraddedtotheeluent.Littleis knownofthesysteminvestigatedhere,sowe cancomparethe resultsusingacalculatedCO₂-MeOHmixturewithahighMeOH fractionintheeluent.ThedensityofaCO2-MeOHfluidatthecen- terpoint(68.5/31.5w/w%co-solventfraction,5w/w%H₂O,45^◦C, and163.3bar)wasmeasuredtobe844±8kgm⁻³,whileitwas calculatedtobe843kgm⁻³usingREFPROP,indicatingthatwater haslittleeffectonthedensityoftheeluent.

Fromthecorrelationof pressuretodensityatconstanttem- peratureand constant fractionsof co-solventandwater, it was alsopossibletodeterminehowdensityvariedinsidethecolumn duringaseparation.Fig.3dplotsthedensityvariationalongthe column assuminga linear pressuredrop at thecenter point in theexperimentaldesign.Thedensityalongthecolumnvariedby approximately1.5%fromcolumninlettocolumnoutlet(Fig.3d), meaningthatitisreasonabletousetheaveragedensitytodeter- minetheaveragevolumetricflowrate.