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Herzi, Nejia and Camy, Séverine and Bouajila, Jalloul and Destrac,
Philippe and Romdhane, Mehrez and Condoret, Jean-Stéphane
Supercritical CO2 extraction of Tetraclinis articulata: Chemical
composition, antioxidant activity and mathematical modeling. (2013) The
Journal of Supercritical Fluids, vol. 82. pp. 72-82.
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Supercritical
CO
2
extraction
of
Tetraclinis
articulata:
Chemical
composition,
antioxidant
activity
and
mathematical
modeling
Nejia
Herzi
a,b,
Séverine
Camy
b,∗,
Jalloul
Bouajila
c,∗∗,
Philippe
Destrac
b,
Mehrez
Romdhane
a,
Jean-Stéphane
Condoret
baUnitéderechercheMACS,ENIG,RoutedeMédenine,6029Gabès,Tunisia
bUniversitédeToulouse,INPT,UPS,LaboratoiredeGénieChimiqueUMRCNRS5503,4,AlléeEmileMonso,F-31030Toulouse,France
cUniversitédeToulouse,LaboratoiredesInteractionsMoléculairesetRéactivitéChimiqueetPhotochimiqueUMRCNRS5623,UniversitéPaul-Sabatier,118
routedeNarbonne,F-31062Toulouse,France
Keywords:
Supercriticalcarbondioxideextraction Tetraclinisarticulata
Essentialoil Antioxidantactivity Modeling
a
b
s
t
r
a
c
t
OperatingconditionsforextractionfromtheleavesofTetraclinisarticulatausingsupercriticalcarbon diox-ide(SCCO2)werestudiedtofocusonthefeasibilityofobtainingvolatileandnonvolatilefractionsthrough
theuseofdifferentextractionpressures(90,280and1000bar).Inaddition,influenceoftemperature, staticpretreatmentanddynamicextractiondurations,particlesizeandCO2flowratewereinvestigated.
AllextractswereanalyzedbyGC–FID/MSandtheirantioxidantactivitywasmeasuredusingABTS•+and
DPPH•methods.Conventionalhydrodistillation(HD)wasalsoperformedforcomparison.AthighCO 2
pressure(280and1000bar),theamountofphenolicsintheextractswashigher(respectively102.03 and267.90GAEmg/g)thanforHDandsupercriticalfluidextraction(SFE)at90bar(respectively8.89and 9.70GAEmg/g).Correlatively,highantioxidantactivitywasfoundforhighpressureSFE.Surprisingly,for extractsobtainedbySFEat90bar,despiteverylowphenoliccontent,significantantioxidantactivitywas observed,whileessentialoilobtainedbyHD,whichpresentedalsolowphenoliccontent,exhibitedlow antioxidantactivity.
Physicalaspectswereonlyinvestigatedforthelowpressuresupercriticalextraction(90bar)process. Qualitativeassessmentofkineticcurvestogetherwiththeirmodelingrevealedthattheextraction pro-cesswasmainlylimitedbythethermodynamicequilibriumofeasilyaccessiblesolutesbutwhereaxial dispersionwassignificant.Fromthisresultasimpleextrapolationprocedurewasproposed.
1. Introduction
Inthelast20years,researchuponnaturalsubstancessuchas aromaticandmedicinalplantshasconcentratedontwoprimary areas:identificationoftheirbiologicalactivity(suchasantioxidant activity)anddevelopmentofnovelextractionmethods insuring betterqualityandeco-friendlyoperation.Inthiswork,the Tetra-clinisarticulataspeciewillbeinvestigatedinrespecttoboththese aspects.
T.articulatabelongstothefamilyCupressaceaeandisanative plantofthesouth-westernMediterranean,mainlyNorthAfrica.Itis animportantelementoftheMaghrebvegetation(Morocco, Alge-riaandTunisia).ThisplantisawidelydistributedtreeinNorth Africawhereitcoversatotalareaof10,000km2 [1].T.articulata
∗ Correspondingauthor.Tel.:+33534323713. ∗∗ Correspondingauthor.Tel:+33562256885.
E-mailaddresses:severine.camy@ensiacet.fr(S.Camy), jalloul.bouajila@univ-tlse3.fr(J.Bouajila).
isknownasahighqualityhealthfoodwhichiscommonlyusedin Tunisiantraditionalmedicine.Ithasbeenfoundtohave pharmaco-logicaleffects,includingantioxidantactivitythatmayexplainthe benefitsintreatingcirculatorydisordersperceivedfromtheuse ofthisherboverthecenturies.Itismainlyusedagainstchildhood fevers,respiratoryandintestinalinfections,stomachpain,diabetes andhypertension[2].Therefore,itisinterestingtofindaneffective methodforisolationofbioactivecompoundsfromT.articulata.
Itisknownthattheisolationofnaturalantioxidantsisdifficult becausemostofthemareheat-sensitiveandvolatileinsteam,and canbethereforedestroyedwhenanonadaptedextractionprocess is used [3]. Traditionally, extraction of bioactive compounds fromplantsisperformedbysteamdistillation,hydrodistillation (HD)ororganicsolventextraction[4,5].Usingthesetechniques, long extraction time, toxic solvent residues, labor-intensive operation and degradation of thermo-sensitive compounds are usuallyobserved.Suchdisadvantagescanbeavoidedbyusingthe supercriticalcarbondioxide(SCCO2)technique[6,7].Indeed,this
technique,moregenerallyreferredassupercriticalfluidextraction (SFE), should improve the volatile oil quality by avoiding any
thermal stresstothevolatilecomponent. Thesolventpowerof SCCO2 isdirectlyrelated toits density.So, choiceof thevalues
of operating pressure and temperatureis a critical step in the development of an efficient process. These operating variables determinethevalueofthedensityandconsequentlytheextract yieldand composition, and,therefore, thefunctionalproperties [8,9] of theextract. Also, other factorssuch asduration ofthe staticanddynamicperiodsofextraction,particlesizeandsolvent flowratecaninfluencetheprocessperformance[10],andmustbe investigated.
Intheliterature,toourknowledge,veryfewstudieswere per-formedonT.articulata.Asanexample,Rachedetal.[11]presented resultsobtainedfromaplantfromAlgeriaharvestedinApril2008. But,up tonow, nostudy ontheSFE extractionof T. articulata fromTunisiahasbeenreportedintheliterature.However,very numerousworkshavestudiedtheextractionofvolatileoilfrom otherplantsusingSCCO2[6,7,12,13].Forexample,extractionofthe
volatileoilfromSalviaofficinalishasbeenstudiedbyseveralauthors (Reverchon et al.[14],Aleksovski and Sovová[15],Glisic et al. [16])whousedthesameextractionconditionstorecovervolatile oils(pressuresbetween80and130bar,temperaturesbetween40 and60◦Candsolventflowratesbetween5and30g/min
approxi-mately).OnlythemethodproposedbyReverchonetal.[14]differed fromthefactthattheycarriedouttheseparationoftheextracts usingtwoseparatorsatdifferentconditions,whichyieldedabsence ofwaxesinthecollectedvolatileextract.
Theobjectiveofthepresentworkwastoevaluatetheextracting capacityofSCCO2toobtainT.articulatavolatileoilwithpossibly
differentpropertiesandtoinvestigatetheeffectoftheextraction conditions.Itissuspectedthatthevolatilefractioncouldbe selec-tivelyisolatedatlowpressureextraction,wherebythepressures between100and1000barwouldbemorefavorablefor obtain-ingfractionscontainingheaviercompoundswithhighantioxidant characteristics.Tochecktheseassumptions,extractionswere per-formedatpressures90,280and1000barandtemperaturesequal to40and60◦C.CO
2flowratewasvariedbetween5and20g/min,
anddifferentmeanparticlesizessoasdifferentextraction dura-tionsweretested.Inthisworkcomparisonwithextractsobtained byHDwasalsoproposed.
On account of importance of SFE for industrial applica-tion,extrapolationof laboratoryexperimentaldata isnecessary and requires extensive knowledge of the physical phenomena occurringduringthecourseoftheextraction.Thentheuseof math-ematicalmodelsallowsgivingtheextrapolationrules.Especially, forextrapolationpurposes,itisofgreatinteresttoidentifywhether theextractionprocessislimitedbymasstransferphenomenaor bythermodynamics.Suchinformationcanbeobtainedfromthe studyofextractionkineticsandtheirmodelingusingmathematical modelsproposedintheliterature[17].
2. Materialsandmethods 2.1. Plantmaterial
LeavesfromcultivatedplantsofT.articulatawerecollectedby handduringOctober2010(inthemorning)fromthesame loca-tion,Korbos,locatedintheregionofCapBonat60kilometersfrom Tunis(latitudes36.82/36◦49′44′′andlongitudes10.59/10◦35′36′′),
Tunisia.Harvestedmaterialwasdriedintheair,protectedagainst directsunlight,untilaconstantweightwasachieved.Theaverage particlesizeofT.articulata,wasobtainedaftersievingofgrinded leaves(usinglaboratoryknifegrinder).Themoisturecontentofthe air-driedplantmaterial,determinedbytheKarlFischer volumet-rictitration,was9%(w/w).Theefficiencyofthedifferentextraction processesisassessedusingtheextractionyield,definedasthemass
ofcollectedextractdividedbyinitialmassofwetplantmaterial(9%, w/wmoisturecontent).
2.2. Chemicals
Allchemicalswereofanalyticalreagentgrade.Allreagentswere purchased from Sigma–Aldrich–Fluka (Saint-Quentin, France). Commercialcarbondioxide(99.99%purity)waspurchasedfrom AirLiquide(Bordeaux,France) fortheextractionofvolatileand nonvolatilecomponentsbySFEprocess.
2.3. Supercriticalfluidextraction(SFE)
Supercriticalextractionswerecarriedoutusingtwodifferent devices.ThefirstonetermedSF200extractionpilotunit,from Sepa-rexChimiefine,(France)isdescribedelsewhere[18,19].Briefly,this apparatusiscomposedofa113mL(16cmheightand3cminternal diameter)tubularextractor(300barmax)withacascadeofthree 15-mLcyclonicseparatorsconnectedtotheextractoroutlet.
Inthepresentstudy,the113mLstainlesssteelextractorwas loadedwith50gofdriedT.articulataleaveswithanaverageparticle sizeof1.5±0.3mm.Thesystemwasoperatedatatemperature between40and60◦Candpressureof90barforvolatilefraction
extraction.PureCO2 waspassedintothecellwiththeflowrate
keptbetween5and20g/mininallruns.
Thesecondapparatus,termedA21(SFE1000bar),from Sepa-rexChimiefine,France,iscomposedofa63mLtubularextractor whichcanbeoperatedupto1000barand200◦C.Onlyone
sepa-ratorisconnectedtotheextractoroutlet.Pressureintheextractor isadjustedbyabackpressureregulator.Inthiswork,theextractor wasloadedwith28gofdriedT.articulataleaveswithanaverage particlesizeof1.5±0.3mm.Thesystemwasoperatedat280and 1000bar,CO2flowrateof20g/minandtwotemperatures,60and
40◦C.
Densityofthesolidphasehasbeenexperimentallydetermined andisequaltos=815kg/m3.Thebedvoidfractionwasestimated
byε=1−a/s=0.4575wheresolidapparentdensityisobtained
witha=m0/Vextr(a=442kg/m3)wherem0istheinitialmassof
plantmaterial. 2.4. Hydrodistillation
AconventionalmethodofHDwascarriedouttocomparethe extractionperformanceswithSFE.Thisset-upwascomposedofa 2-Lboiler,acondenserandameasuringtubewithastopcock.Areturn tubefortheaqueouspartofthedistillateallowedthecohobation techniquetobeused.DriedleavesofT.articulata(100g),groundat diameter1.5mm,wereplacedinaClevengerapparatusandmixed withdistilledwaterataratioof1/10(plantmaterial/water).After 180min(correspondingtothedurationwhennomoreessential oilwasobtainedatthecondenser),theessentialoilwascollected, driedoveranhydroussodiumsulfateandstoredat4◦Cinthedark
untilused.
2.5. Chemicalcomponentsanalysis:GC–FIDandGC–MS
Quantitative and qualitative analysis of the volatile oil was carried out by gas chromatography–flame ionization detection (GC–FID)andgaschromatography–massspectrometry(GC–MS). Gaschromatography analyseswereperformedona VarianStar 3400 Cx chromatograph (Les Ullis, France) fitted with a DB-5MSfusedsilicacapillarycolumn(5%phenylmethylpolysyloxane, 30m×0.25mm,filmthickness0.25mm).Chromatographic condi-tionswereatemperaturerisefrom60◦Cto260◦Cwithagradientof
5◦C/minandthen15minat260◦C.Asecondgradientof40◦C/min
Fig.1.SFEyieldofvolatileoilfromT.articulataatWCO2=20g/min,P=90barandT=40
◦
C.(a)Kineticsoftheextractionprocess,(b)influenceofstaticperiodduration (followedby30minofdynamicextraction)and(c)influenceofdynamicextractionduration(withastaticperiodof10min).
analysispurposes,thevolatileoilwasdissolvedinpetroleumether. Samples(1mL)wereinjectedinthesplitmodeataratioof1:10. Helium(purity99.999%)wasusedasthecarriergasat1mL/min. Theinjectorwasoperatedat200◦C.Themassspectrometer
(Var-ianSaturnGC-MS-MS4D)wasadjustedforanemissioncurrentof 10mAandelectronmultipliervoltagebetween1400and1500V. Thetemperatureofthetrapwas150◦Candthatofthetransferline
was170◦C.Massscanningwasfrom40to650amu.
Compoundswereidentified (i)bycomparisonoftheir reten-tionindex(RI),obtainedonanonpolarDB-5MScolumnrelative toC5-C24n-alkanes,withthose providedintheliterature,(ii)by
comparisonoftheirmassspectrawiththoserecordedinNIST08 (NationalInstituteofStandardsandTechnology)andreportedin publishedarticles,(iii)byco-injectionofavailablereference com-pounds.Thesampleswereanalyzedinduplicate.Thepercentage compositionofthevolatileoilwascalculatedbythenormalization methodfromtheGCpeakareas,assumingidenticalmassresponse factorsforallcompounds.Resultswerecalculatedasmeanvalues aftertwo injectionsofvolatileoil,withoutusingcorrection fac-tors.Theidentificationisonlymadeforthevolatilecompounds. Especially,forSFEextracts,somenonvolatilecompoundscannotbe identified.Thedetectionlimitis0.1mg/Lwhilethequantification limitis1mg/L.
2.6. Determinationoftotalphenoliccontent
The phenolic contents of extracts were determined by the Folin–Ciocalteumethod[20]. Adiluted solutionof each extract (0.5mL) was mixed with the Folin–Ciocalteu reagent (0.2M, 2.5mL).Thismixturewaskeptatroomtemperaturefor5minand thenasodiumcarbonatesolution(75g/Linwater,2mL)wasadded.
After1hofincubation,theabsorbancewasmeasuredat765nm againstwaterblank.Astandardcalibrationcurvewasplottedusing gallicacid(0–300mg/L).TheresultswereexpressedasmgofGallic AcidEquivalent(GAE)/kgofplantmaterial.
2.7. DPPH•freeradicalscavengingactivity
Antioxidantscavengingactivitywasdeterminedusingthe 1,1-diphenyl-2-picrylhydrazyl free radical (DPPH•)as described by
Blois[21]withsomemodifications;variousdilutionsofthetest materials(ascorbicacidorextracts,1.5mL) weremixed witha 0.2mM methanolicDPPH• solution (1.5mL).After 30min
incu-bationperiod at25◦C, theabsorbanceat 520nm wasrecorded
asA(sample).Acontrolexperimentwasalsocarriedoutby
apply-ingthesame proceduretoa solutionwithoutthetest material and the absorbance was recorded (A(blank)). The free radical
scavenging activity of each solution was then calculated as percentageinhibitionaccordingtothefollowingequation:% inhi-bition=100×[(A(blank)−A(sample))/A(blank)]
ExtractantioxidantactivitywasexpressedasIC50,definedasthe
concentrationofthetestmaterialrequiredtocausea50%decrease ininitialDPPH•concentration.Valueswereestimatedusinglinear
regression.Ascorbicacidwasusedasareference. 2.8. ABTS•+radicalscavenging
The radical scavenging capacity of the samples for the ABTS•+ (2,2′-azinobis-3-ethylbenzo-thiazoline-6-sulfonate) was
determinedasdescribedbyReetal.[22].ABTS•+wasgeneratedby
mixinga7mMsolutionofABTS•+atpH7.4(5mMNaH
2PO4,5mM
(finalconcentration)andstoredinthedarkatroomtemperature for16hbeforeuse.Themixturewasdilutedwithwatertogive anabsorbanceof0.70±0.02unitsat734nmusing spectrophoto-metry.Foreachsample,adilutedmethanolsolutionofthesample (100mL)wasallowedtoreactwithfreshABTS•+solution(900mL),
andtheabsorbancewasmeasured6minafterinitialmixing. Ascor-bicacidwasusedasareferenceandthefreeradicalscavenging capacitywasexpressedbyIC50(mg/L)values,whichrepresentsthe
concentrationrequiredtoscavenge50%ofABTS•+.Thefreeradical
scavengingcapacityIC50wasdeterminedusingthesameequation
asusedpreviouslyfortheDPPH•method.
2.9. Statisticalanalysis
Resultsofantioxidantactivityandtotalphenoliccontentwere expressedasmeans±standard deviationsoftriplicate measure-ments.Theconfidence limitswere setatP<0.05. Dataanalysis procedure(ANOVA)wasperformedinordertoassessthedata. 3. Resultsanddiscussion
3.1. InfluenceofparametersforSFE 3.1.1. Influenceofextractionduration
Anexampleofkineticsoftheextractionprocessat90barand 40◦CispresentedinFig.1(a).Theglobalextractionyieldincreases
linearlyuntilabout30minutesandthenaplateauisreached.The initiallinearshapeofthecurvesuggeststhatextractionislimited bythesolubilityofvolatileoilinCO2 orbysolid–fluid
equilib-rium;internaldiffusionlimitationseemsnottobepresentinthese operatingconditions.
Weuseda dynamicextractionmethodwhere theextraction durationisactuallyconstitutedofaperiodofstaticpretreatment (i.e.,noCO2flowrate)followedbyadynamicperiod(i.e.,with
con-stantCO2flowrate).Thestaticpretreatmentallowsequilibration
ofthesolidandthefluidanddoesnotrequireCO2flow,andalso
allowsSCCO2dissolvingintotheoilpresentinsecretorystructures
oftheplanttissue(cells,cavitiesorducts),previouslyopenedby grinding,asdescribedbyStameni ´cetal.[23].
Inthefirstpartofthisstudy,theeffectofstaticpretreatment onSFEefficiencywasstudied.Threestaticpretreatmentdurations (5,10and15min),followedby30mindynamicextraction,were employed.Thepressureandtemperaturewerefixedat90barand 40◦Cwithparticlediameter1.5mm.Experimentalglobalyieldsare
presentedonFig.1(b).Itisseenthatincreasingthestaticextraction period,from5to10min,increasedextractionefficiencyofvolatile oilbymore than40%.However,increasingthestaticextraction durationfrom10to15minhadminimaleffectontheextraction efficiency.Therefore,inallexperiments,10minofstatic pretreat-mentwasused.
Effectofdynamicextractiondurationonextractability ofthe naturalsubstanceswasinvestigatedwhilekeepingother parame-tersfixed.Thedynamicmethodusingthreedurations(20,30,and 60min)wastestedwith10minstaticpretreatmentduration.The pressure,temperatureandCO2 flow ratewere90bar,40◦C and
20g/minforallexperiments.
Fig.1(c) shows how extended dynamic extractiondurations increasetheefficiencyoftheprocess.However,thedependence betweenrecoveryandextractiondurationgraduallybecomesflat. Thereforea dynamicmethodwith30minwaschosenin subse-quentextractions.
3.1.2. Influenceoftemperature
TheinfluenceoftemperatureontheextractionyieldofT. artic-ulataat three temperaturelevels,40, 50and 60◦C isshown in
Fig.2.Ahightemperatureislikelytoimprovemasstransferrates
Fig.2. InfluenceofthetemperatureontheT.articulatavolatileoilyieldfor1.5mm particlesasafunctionoftheextractiontimeatP=90barandWCO2=20g/min.
andthuskineticsofextraction.Neverthelessatlowpressure,the density of CO2 is very sensitive to temperature and decreases
withincreasingtemperature,leadingpossiblytoreducedsolvent powerofSCCO2dependingonthecompetitionbetweendensity
ofSCCO2andvolatilityofthesolute(retrogradesolubilityeffect)
[24].Thismightbethereasonoftheobserveddecreaseoftheyield (1.6–1.48mg/g)whentemperatureisincreasedfrom40to60◦C,as
seenonFig.2.SuchasimilarresultwasalsoobtainedbydaCruz Franciscoetal.[25]inthecaseoftheextractionofmonoterpenesof eucalyptusoil.Inaddition,itisverylikelythathigh-temperature extractionhaveadetrimentaleffectonthequalityoftheextract becauseofpossiblethermaldegradationreactions.Thepossibility toperformefficientextractionofvolatilecompoundsatlow tem-peratureisindeedoneofthemainadvantagesoftheSFEtechnology whencomparedtotraditionalHD.
3.1.3. Influenceofpressure
Fig.3presentstheinfluenceofpressureontheextractionyield of T. articulata leavesin SC-CO2 at three pressure levelsof 90,
280and 1000barfor 30minextractionduration (1.6, 19.2and 25.5g/kg,respectively).
TheSFEyieldofT.articulatawassignificantlyinfluencedbythe pressure,ascanbeseeninFig.3.Infact,increasingtheextraction pressurefrom90to280barincreasedtheyieldmorethan9times and,from90to1000bar,formorethan16times.ThehighCO2
densityathighpressureincreaseditssolventpowerandtherefore, moresubstanceswereextracted.Also,itcanbenoticedthat, espe-cially,theincreasefrom90to280barisveryefficientupon the yield.
High extraction pressure is likely tofavor the extractionof heavy and more polar compounds suchas phenolics, lipidsor waxes. Therefore, in thepresent study,the phenolic contentis expectedtoincreasewithincreasingpressure(280and1000bar).
Fig.3.InfluenceofthepressureontheT.articulataextractionyieldfor1.5mm particles,aCO2flowrateof20g/min,T=40◦Candextractionduration30min.
Fig.4.T.articulatavolatileoilyieldfor1.5mmparticlesatdifferentCO2flowrates
asafunctionoftheextractiontime(a)andafunctionofamountofCO2referredto
initialamountofplantmaterial(PM)(b)atT=40◦CandP=90bar.
Nevertheless, increasing the pressure obviously results in a decreaseoftheselectivityoftheextraction[26,27].
3.1.4. InfluenceofCO2flowrate
Asshownin Fig.4(a),forextractionat90barand 40◦C,the
CO2flowrateexhibitedapositiveandsignificanteffectontheT.
articulatavolatileoilextraction.Thisresultindicatesthat,forCO2
flowrateequalto5g/min,60minofextractionarenotsufficient to achieve complete extraction while 30min are sufficient at 20g/min.Also,atallflowrates,theinitialquasi-linearshapeofthe
Fig.5.T.articulatavolatileoilyieldfordifferentmeanparticlesizesataCO2flow
rateof20g/min,temperature40◦Candpressure90bar.
curvessuggeststhehypothesisthatthisSFEprocessissolubility orsolid–fluidequilibriumlimited.
WhenplottingtheyieldversustheamountofCO2(Fig.4(b)),
thecurvesmergeandthisconfirmstheoccurrenceofsolubilityor equilibriumlimitationfortheextractionprocess.Theslopeofthese curvesgavethesoluteconcentrationintheoutputfluid(C0)ata
valueof0.20×10−2g/gCO
2,avaluewhichisbelowthevalueof
sol-ubilityofusualterpenes(a-pineneforinstanceis1.051×10−2g/g
CO2atT=40◦CandP=90bar[28]).Whenhighmasstransfer
lim-itationisnotsuspected,this indicatesvery probableadsorption phenomenawhichlimittheextractionprocess.
3.1.5. Influenceofparticlesize
TheeffectofparticlesizeonextractionrateisshowninFig.5. Despitesomedispersionin theexperimentalpoints,thecurves aresimilar,andespeciallythesamefinalyieldisreachedafterthe same extractionduration (around30min) for allparticle sizes, indicatingsimilarkineticbehavior.Thisresultisinaccordancewith thesuspectedpredominantequilibriumlimitationwhichpredicts kineticsalmostindependentofinternalandexternalmasstransfer, andthereforeofparticlesize.Thelowimpactofparticlesizeon extractionkineticsistypicalofglandularstructurescorresponding tosecretoryducts[23,29],asitisthecaseforplantsofthe Aster-aceae,Apiaceae,Hypericaceae,PinaceaeorConiferaefamily.These ductsareopenedbygrindingandtheoiliseasilyaccessibleforCO2.
CO2dissolvesintheearlystageofextraction,leadingtoanincrease
ofthevolumeoftheoilthatwets thesurfaceoftheplant.This
Table1
Chemicalcomposition(GC–MS)andglobalyieldofT.articulataextractsobtainedbySFEatdifferentpressuresandbyHD. Typeofextractiontechnique
SFE HD
Pressure(bar) 90 280 1000 –
Temperature(◦C) 40 40 40 100
Flowrate(g/min) 20 20 20 0.02mL/s
Solvent CO2 CO2 CO2 Water
Extractiontime(min) 30 30 30 180
Globalyield(g/kg) 1.6 19.2 25.5 0.61 RI Compounds % % % % 906 Santolinatriene 0.85 0.39 931 Artemesiatriene 0.8 0.6 936 a-Pinene 31.32 24.9 940 Cumene 0.33 953 a-Fenchene 0.22 0.14 958 Camphene 0.49 0.45 967 b-Thujene 2.64 1.99 973 1-Ethyl-4-methylbenzene 0.43 990 Myrcene 0.17 0.48 1004 Pseudolimonene 0.26 0.37 1008 3-Carene 3.39 3.44 1012 a-Terpinene 0.12 990 Myrcene 0.17 0.48 1037 Limonene 0.16 0.18 1047 3-Methyl-1-decene 0.13 1068 Isoterpinolene 0.26 0.46 1077 Ethylmaltol 0.26 1079 Artemisiaalcohol 0.19 1089 a-Terpinolene 1108 cis-Thujone 0.22 0.35 1122 trans-2,8-Menthadienol 0.17 0.38 1128 a-Campholenal 3.44 0.1 0.43 3.84 1132 Isothujol 0.17 1148 Camphenehydrate 1.25 4.36 1157 Isoborneol 1159 b-Terpineol 0.18 0.73 1172 3,5,5-Trimethyl-1,4-cyclohexanedione 0.09 0.14 1.41 1193 Myrtenal 0.23 0.08 1200 Z-Dihydrocarvone 0.18 0.34 1232 cis-Carveol 0.15 1266 Linaloolacetate 18.18 1.72 21.44 1279 p-sec-Butylphenol 0.58 1318 (Z)-3-Hexenyl2-methyl-(E)-2-butenoate 0.38
1327 Trans-Pulegoneoxide[a] 0.91 0.31 1.53
1356 a-Cubebene 1.11 0.47 0.26 1370 Cyclosativene 0.72 0.16 1383 b-Bourbonene 1.74 0.44 1400 g-Caryophyllene 4.16 2.05 2.53 1409 Aromadendrene 1435 a-trans-Bergamotene 1.29 0.59 1.04 1444 a-Caryophyllene 0.13 1455 a-Patchoulene 0.48 0.18 1461 Alloaromadendrene 7.55 1.41 1.14 1474 g-Muurolene 0.35 1479 g-Curcumene 0.58 0.43 1494 a-Zingiberene 2.14 2.38 0.53 1501 a-Amorphene 1.39 1.64 1516 a-Selinene 0.32 1534 incisol 0.17 0.59 1557 Caryophyllenealcohol 1.06 1568 Caryophylleneoxide 1.82 2.45 4.24 1588 b-Oplopenone 0.38 1594 Cedrenol 0.48 1.03 2 1613 1-epi-Cubenol 0.52 2.12 2.83 1619 trans-Isolongifolanone 0.42 1626 g-Eudesmol 0.58 2.56 1642 Spathulenol 0.43 0.77 2.37 1660 14-Hydroxy-9-epi-(E)-caryophyllene 0.14 0.5 0.97 1673 Cedr-8-en-13-ol 0.23 1678 cis-Alpha-santalol 0.44 1.24 1.74 1717 (Z)-2-Heptadecenea 0.22 1761 1,10-Dihydronootkatone 0.88 0.08 1928 16-Hexadecanolide 0.43 0.15 1962 Palmiticacid 2.98 36.19 1991 Manoyloxide 0.13 0.26 2008 Kaur-16-ene 0.14 0.1
Table1(Continued)
Typeofextractiontechnique
SFE HD 2053 abietatriene 0.32 2095 Heneicosane 0.4 3.66 0.09 2135 1R-pimaral 0.13 6.09 0.27 2143 Cubitene 0.22 2187 CembreneAketone 0.25 1.37 0.14 2216 15-Ripperten-3alpha-ol 0.41 2.11 0.52 2223 trans-Totarol 0.12 2274 Larixola 2.17 2288 2-Methyltricosane 10.29 2311 trans-Totarol 4.85 0.05 0.07 2368 androst-5-en-17-ol,4,4dimethyl 2.28 2.49 2383 Podocarp-7-en-3-one,13a-methyl-13-vinyl 7.77 25.53 2473 Docosylacetate 14.4 1.35 2922 Abieticacid 0.31 2925 ni 1.05 2930 Podocarp-8(14)-en-15al,13a-methyl-13-vinyl 10.79 ni(M=410) 0.73 ni(M=552) 2.14 ni(M=618) 8.96 ni(M=296) 7.34 7.98 Numberofcompounds 56 29 14 56 Monoterpenehydrocarbons 40.73 0 0 34 Monoterpeneoxygenated 6.92 0.41 0.43 12.36 Sesquiterpeneshydrocarbons 21.48 6.9 0 8.83 Sesquiterpenesoxygenated 5.64 8.99 0 18.41 Others 21.14 67.55 97.71 25.03 Total 96.24 83.85 98.14 98.63
ni,notidentified.
aTentativeidentificationsupportedbygoodmatchofmassspectrum.
hypothesis could explain the predominance of equilibrium phenomenaascomparedtointernalmasstransferintheextraction process.
3.1.6. EffectofSCCO2extractiononthestructureofvegetal
material
Optical microscope or Scanning Electron Microscope (SEM) analysesofthematerialweredonetovisualizeitsstructureand theeffectoftheextractionprocessuponthisstructure.Theinitial structureofthegroundleavesfromT.articulata,observedbySEM, isshowninFig.6(a).FromcomparisonwithFig.6(b),itisseenthat SFEhadalmostnoeffectonthestructureoftheplant,atleaston avisualpointofview.Moreover,SEMimagestendtoconfirmthe hypothesisofsecretoryducts,sincetheplantisactuallymadeofa coreofinterconnectedchannelssurroundedbyadensebark. 3.2. CharacterizationofextractsandcomparisonwithHD 3.2.1. Chemicalcompositionoftheextracts
TheglobalyieldandtheresultsoftheGC–MSanalysisofvolatile oilobtainedfromT. articulata by SFEand HD are presented in Table1.
Asexpected,therewasasignificantdifferenceintheextraction yieldsbetweentheextractsobtainedusingHDandSFEatthethree pressures.Extractionathighpressurewasfoundtobemore effi-cientthanotherconditionsstudiedinextractingtheantioxidant componentspresentintheT.articulataleaves.
Althoughatotalof86compoundswereidentified,forthesake ofsimplicity,thediscussionhasbeenrestrictedtothemajor com-ponentsidentifiedforeachmethod.Thesemajorcomponentsare presentedinTable2,whereatotalof28componentswitha per-centagehigherthan2%aregathered.Resultsarepresentedinterms ofrelativeproportion(%ofareaofthepeakinthechromatograms) andamountinmg/kgofextract.CompoundsappearinTable2inthe orderofdecreasingvolatility.Althoughthiscouldleadto approx-imateresults,becauseterpenesarethemaincomponents,ithas
beenconsideredherethatpalmiticacid,abieticacidandterpenes havesimilarresponsefactor.
Whencomparingcompositionofessentialoil(HD)andvolatile oilobtainedbySFEat90bar,nogreatdifferenceswereobserved anda-pineneandlinaloolacetatearepredominantintheextracts obtainedwiththesemethods.However,thecompoundsinextracts obtainedbySFEat280and1000bararesignificantlydifferent com-paredtothoseobtainedbyHDorSFEat90bar.
ItappearsthatthehigherthepressureofSFE,thelowerthe numberofdetectedcompounds,whichinthatcasecorresponds toheavycompounds.For example,only 14volatilecompounds havebeendetectedbyGC–MSintheextractobtainedbySFEat 1000bar(only10havebeenreportedinTable2).Notealsothat, duetothegaschromatographymethod,onlyhighmolecularmass compoundswithretentionindex(RI)valuegreaterthan1900were detected.Ontheotherside,theessentialoil(HD),volatileoilofSFE (at90bar)andextractfromSFEat280barwerecomposedof56,56 and29compounds,respectively.Thisresultissurprisingbecause highpressureoperationallowsextractingmorecompoundsdueto theincreasedsolventpowerofSCCO2.Indeed,thisresultsfroman
“artifact”oftherecoverysystemoftheA21(SFE1000bar)apparatus whichwasnotveryeffectiveoratleastincorrectlyused.This appa-ratushasonlyoneseparator(converselytoSF200extractionwhich has3separatorsinseries).Experimentsat280and1000bar(where volatilecomponentswerenotdetected)weredoneonA21 appara-tuswhileexperimentsat90barweredoneontheSF200extraction (wherethesevolatilecomponentswererecoveredanddetected). SoitisveryprobablethattheuniqueseparatorofA21hasfavored theselectivelossofthevolatilecomponentsthatobviouslywere extractedathighpressurebutnotrecoveredinthelowpressure recoveryzone.Thisexplainsthatlowmolecularmasscompounds donotappearinthecomposition.
In volatileoils obtained by HD and SFE at 90bar,the main constituents were a-pinene (24.90–31.32%), linalool acetate (21.44–18.18%), alloaromadendrene (1.14–7.55%), camphene hydrate(4.36–1.25%)andg-caryophyllene(2.53–4.16%).Themain
Table2
ChemicalcompositionobtainedbyGC–MS(abundance>2%)andglobalyieldofT.articulataextractsobtainedbySFEatdifferentpressuresandbyHD. Typeofextractiontechnique
SFE HD
Flowrate(g/min) 20 20 20 0.02mL/s
Temperature(◦C) 40 40 40 100
Pressure 90 280 1000
-Solvent CO2 CO2 CO2 Water
Extractiontime(min) 30 30 30 180
Globalyield(mg/kg) 1.63 19.2 25.9 0.61 RI Compounds % mg/kg % mg/kg % mg/kg % mg/kg 936 a-Pinene 31.32 511 24.9 152 967 b-Thujene 2.64 43 1.99 12 1004 Pseudolimonene 0.26 4 0.37 2 1008 3-Carene 3.39 55 3.44 21 1128 a-Campholenal 3.44 56 0.1 19 0.43 111 3.84 23 1148 Camphenehydrate 1.25 20 4.36 27 1266 Linaloolacetate 18.18 296 1.72 330 21.44 131 1400 g-Caryophyllene 4.16 68 2.05 394 2.53 15 1461 Alloaromadendrene 7.55 123 1.41 271 1.14 7 1494 a-Zingiberene 2.14 35 2.38 457 0.53 3 1568 Caryophylleneoxide 1.82 30 2.45 470 4.24 26 1613 1-epi-Cubenol 0.52 8 2.12 407 2.83 17 1626 g-Eudesmol 0.58 9 2.56 16 1642 Spathulenol 0.43 7 0.77 148 2.37 14 1962 Palmiticacid 2.98 572 36.19 9373 2095 Heneicosane 0.4 7 3.66 703 0.09 1 2135 1R-pimaral 0.13 2 6.09 1169 0.27 2 2216 15-Ripperten-3alpha-ol 0.41 7 2.11 405 0.52 3 2274 Larixola 2.17 417 2288 2-Methyltricosane 10.29 1976 2311 trans-Totarol 4.85 931 0.05 13 0.07 2368 Androst-5-en-17-ol,4,4dimethyl 2.28 438 2.49 645 2383 Podocarp-7-en-3-one,13a-methyl-13-vinyl 7.77 1492 25.53 6612 2473 Docosylacetate 14.4 2765 1.35 350 2930 Podocarp-8(14)-en-15al,13a-methyl-13-vinyl 10.79 2795 ni(M=552) 2.14 554 ni(M=618) 8.96 2321 ni(M=296) 7.34 1409 7.98 2067 Total 78.62 1282 76.94 14,772 95.91 24,841 77.49 473
ni,notidentified.
aTentativeidentificationsupportedbygoodmatchofmassspectrum.
compoundsofextractsobtainedbySFEat280and1000barwere palmitic acid (2.98–36.19%), docosyl acetate (14.40–1.35%), podocarp-7-en-3-one, 13a-methyl-13-vinyl (7.77–25.53%), 2-methyltricosane (10.29–0%), podocarp-8(14)-en-15al, 13a-methyl-13-vinyl(0–10.79%)and1R-pimaral(6.09–0%).Also,some compounds with high molecular mass were detected but not identified.Forinstance,theamountofsomeoftheseunidentified compounds was significant in SFE extract at 1000bar (8.96% (M=618g/mol)and7.98%(M=296g/mol)).
On the other hand, important differencebetween the com-poundsofeachextractscanbeseenintermofquantity(mg/kg). Itisthecaseforexampleofa-pinenewhichwasidentifiedasthe majorcompoundinHDandSFEat90bar,wherequantities deter-minedinHDandSFEextracts,were152mg/kg(for24.9%ofglobal oil)and511mg/kg(for31.31%ofglobaloil)respectively.
Itisinterestingtocomparetheseresultswiththefewstudies thathavebeenconductedontheessentialoilsofT.articulata.Ben HadjAhmedetal.[30]havestudiedessentialoilfromT.articulata plantsharvestedin theregionofSnedGafsa(Tunisia)andhave onlyreportedaboutantioxidantactivityoftheessentialoiland notaboutitschemicalcomposition.Tekaya-Karouietal.[31]have workedonT.articulataplantsharvestedintheregionofZaghouas (Tunisia)andhaveshownthatchemicalcompositionwasdifferent, dependingonthepartoftheplant.Intheessentialoilobtained from branches, themajor compounds were camphene (43.2%), Z-muurolène(29.0%)and p-cymene-8-ol(10.4%) whileessential oilfrom theroots was richer in camphene(70.2%). A study of Barreroetal.[32]uponthechemicalcompositionofessentialoil
fromleavesandwoodofT.articulataplantsharvestedintheregion ofAmsaTéouan(Morocco)hasshownthattheleaveswererich inbornylacetate(16.5%),camphor(19.1%)andborneol(9.6%)and thattheessentialoilfromthewoodwasrichincedrol(28.2%)and 1,7-di-epi-cedrol(17.9%).TheresultsreportedbyAitigri[33]have shownthatessentialoilfromwoodofT.articulataplantsharvested in the region of Rabat (Morocco) were rich in carvacrol and a-cedrol.AnotherstudyofZriraetal.[34]uponT.articulataplants fromtheregionofKhemissetandAoulouz(Morocco)hasreported thepredominant presence ofcarvacrol (21.3–36.4%), a-cedrene (10.1–13.1%),cedrol(1–7.3%)andterpinen-4-ol(2.8–6%).Inastudy fromBuhagiaretal.[35],18compoundswereidentifiedinaerial partsofT.articulataplants,wherea-pinene,limonene,camphor andbornylacetatewerethemaincompounds.Fromthissurvey,it isseenthatthechemicalcompositionsofT.articulataessentialoils isverydependentontheharvestingplaceandonthespecificpart oftheplant.Tothebestofourknowledge,nostudyuponchemical compositionofSFEextractsofT.articulatahasyetbeenproposed. 3.2.2. Phenolicscontentandantioxidantactivityoftheextracts
Theconcentrationofphenolicsintheextracts,expressedas Gal-lic AcidEquivalent(GAE) isshown inTable3.Asexpected,the amountofphenoliccompoundsintheSFEextractsathighpressure wasthehighest(102.03±4.57and267.90±8.06mgGAE/gplant materialfor280barand1000bar,respectively),followedbySFEat 90bar9.70±0.57andHD8.89±0.16mgGAE/gdryplantmaterial. TheresultsfromTable3showthat,althoughthequantityof phenolicsislowandalmostidenticalfortheHDessentialoiland
Table3
TotalphenolicscontentandantioxidantactivityofT.articulataextractsobtainedby SFEatdifferentpressuresandbyHD.
Samples Phenolics (GAEmg/g) DPPH ABTS GAEmg/g ± IC50 ± IC50 ± SFE90bar 9.70 0.57 146.02 1.99 40.91 0.53 SFE280bar 102.03 4.57 120.21 3.65 33.55 0.40 SFE1000bar 267.90 8.06 108.16 3.07 29.77 1.30 HD 8.89 0.16 3681.49 69.33 324.45 14.21
GAE,GallicAcidEquivalent.
SFEextract at90bar, theirantioxidantactivityis different.The SFEextractat90bar(IC50(mg/ml)=40.91±0.53)isabout8times
moreactivethanessentialoil(IC50(mg/ml)=324.45±14.21).These
resultssuggestthattheSFEvolatileoilfromT.articulata,ismore concentratedwithantioxidantsandcouldbeusedasapotential sourceofnaturalantioxidantswithpossibleapplicationsinfood systems.Thepresenceofantioxidantsinthis plantisconfirmed byapreviouspublication[11]wherephenolics andantioxidant activityofAlgerianT.articulataleavesextractweredetermined. Theseauthors showedthat extract ofT. articulata fromAlgeria obtainedbyheatrefluxwithdistilledwaterexhibitedgood antiox-idantactivity(IC50(mg/ml)=9.519±0.521andhightotalphenolic content(mgGAEg−1)=206.187±16.612).AlthoughGC–MSisable
toidentifysomeof thesemolecules,theywerenot detectedin ourcase.Thereforethehighmeasuredantioxidantactivitycanbe explainedbythepresenceofhighmolecularweightphenolics (tan-nins,anthocyanins,etc.)whichcannotbedetectedusingGC–MS.
Anotherexplanationcouldbetheoccurrenceoflowquantities ofhighlyactivephenoliccompounds.ForexampleHarishNayaka etal.[36]reportedthat,fromvaluesofIC50obtainedforphenolic
acidextractsofswallowroots,vanillicacidwas45timeslessactive thangallicacid.
3.3. Mathematicalmodeling
3.3.1. Preliminaryanalysisofthephysicalphenomena
Because vegetal matter is very diverse, numerous models describingsupercriticalextractionprocessareavailableinthe liter-ature,fromthesimpleempiricalonestothemostcomplexthatare abletohandleallphysicalphenomenaoccurringduringextraction. Thechoiceisalwaysdifficultandthemostcomplexmodelmight notbethemostusefulbecauseitisassociatedwiththeestimation ofnumerousparameters, whilesomeofthemmayhavea neg-ligibleinfluence.Moreovertheircomplexitypreventssometimes fromeasyunderstandingwhichisnecessarytoselecttheoptimal conditionsfortheextractionprocess,especiallywhenscale-upis envisaged.So,qualitativeinterpretationofkineticcurvestogether withtheuseofeasy-to-implementsimplifiedmodelsisvery use-ful.Especially,qualitativestudyoftheextractioncurvesatdifferent flow-ratesanddifferentparticlesizesisveryinstructive.Suchan approach hasbeenrecently recommended bySovová [17] in a recentpaperwhichprovidesasimplifiedmethodbasedontheuse ofcharacteristictimesforallinvolvedphysicalprocesses. Compari-sonofthesecharacteristictimesgreatlyhelpsfordiscriminatingthe prominentparameters.However,caremustbetakentoinsurethat, whenscalingup,thechoiceoftheseparametersremainspertinent. We haverestricted ourapproach tovolatile oilsupercritical extraction and therefore the modeling is proposed for results obtainedat90barand40◦C.
In the case presented here, from the overlapping of curves of Fig. 4(b), as indicated in Section 3.1.4, a limitation by the adsorption phenomenon is strongly suspected. In this case, when external mass transferis not involved, estimation of the
adsorptioncoefficientisobtainedfromtheinitialslopeofFig.4b. Thehypothesisofnegligibleexternalmasstransferlimitationcan beassessed by computingexternal mass transfer characteristic timetf[17].Externalmasstransfercharacteristictimetfdepends
on kf (external mass transfer coefficient), which is the
vol-ume/surfaceratiooftheparticle(=R/3forsphericalparticles)and ε,thevoidfractionoftheparticlebed.
tf =1ε −ε kf (1) t1= G Km·q′·(1−exp(−1/f)) (2) Theextractiontimet1correspondingtotheendofthelinearpart
oftheextractioncurveisthusafunctionofq′whichistheCO 2
flow-ratereferredtothemassofsolid,G,thefractionofeasilyaccessible cells,andKmwhichisthesolutepartitioncoefficient(kgplant/kg
solvent)definedasC=Km·q,whereCistheconcentrationofvolatile
oilinthefluidphase(gsolute/gCO2)andqistheconcentrationof
volatileoilinthesolidphase(gsolute/gplant).f,istheratioof
theexternaltransfercharacteristictimetftotheresidencetimetr.
Thislatterisgivenby: tr=
q′ (3)
where istheratiobetweeninitialCO2massandsolidmassinthe
extractor.
To providea quantitativeassessment of these characteristic times,extractionat thehigher flow-rate,20g/min,for assumed spherical 1.5mm diameterparticles, is consideredbecause this caseisthemostlikelytoexhibitinfluenceofexternalmass trans-fer.Consideringavoidfractionε=0.457,thecharacteristic time for external transfertf is computedat 1.05susing the
correla-tion of Tan et al. [37] for the evaluation of kf (which yielded
kf=1.61×10−4ms−1)andtrisequalto75s.Thevalueoftfbeing
lowascomparedtotrconfirmsthatexternalmasstransfer
resis-tancecanbeneglected.Thisresultmakesitpossibletouseasimple equilibriummodelforthelinearpartoftheextractioncurve.Such anapproachwasproposedbyReis-Vascoetal.[38]forthe extrac-tionofpennyroyalessentialoil.Visually,fromFig.4(a),thevalue G=0.7fortheaccessiblefractionisestimated,correspondingtothe endofthelinearpartofthecurveswhichoccursatt1=750s.
Theinterpretation of thecurved final partof the extraction curvesistrickier.Inafirstapproach,assuggestedbySovová,this partwouldcorrespondtoaninternalmasstransferlimitedprocess, concerningtheextractionofthenonaccessiblepartofthesolute. Ifthiswastrue,finalpartsofcurvesofFig.4(b)(whichrelatesthe extractionyieldtothemassofCO2used)shouldexhibitinfluence
oftheflowrate,whilethisisnotthecasebecausecurvesoverlapup totheendofextraction.Inaddition,aconfirmationofthis hypoth-esisisfoundwhenattemptingtofittheexperimentaldatawiththe simplifiedmodelforplug-flowwithadsorptiongiveninSovová’s paper[17]: e=xu−(1−G)xuexp
−t−t1 tcomb,i fort≥t1 (4)wherexuistheinitialsolutecontentofthesolidbeforeextraction
(itisdeterminedfromthevalueofthefinalyieldoftheextraction curvesatavaluexu=1.6×10−2g/g).Thecombinedcharacteristic
timeofmasstransfertcomb,iisobtained,asafunctionoftf,Kmandti
(internalmasstransfercharacteristictime)thatinvolvesthe effec-tiveinternaleffectivediffusioncoefficientDe.Thesecharacteristic
timesaregivenby: ti=
R2
tcomb,i=ti+
tf
·Km (6)
Whenusingthisapproachforthefinalpartoftheexperimental curvesobtainedatdifferentCO2flow-rates,parameter
identifica-tionyieldeddifferentvaluesoftheinternaldiffusioncoefficientDe
foreachflow-rate,whilethisparametershouldnotbeaffectedby theflow-rate.
Alltheseelementsindicatethatthefinalcurvatureofthe extrac-tioncurvescorrespondsmoreprobablytotheinfluenceofaxial dispersion(whichisnotaccountedforinthesimplifiedmodel). Eventually,thissuggestsanadsorptionlimitedextractionprocess where allthesoluteis accessiblewithnegligibleexternal mass transferlimitation,butwheresignificantaxialdispersionispresent. Thecomputation of thePeclet number,Pe=h·u/(DL·ε),withthe
relation of Funakuzuri [39] for theaxial dispersioncoefficient, yieldedPe=19whichisquitelowandascertainsthehypothesis ofanon-negligible axialdispersioninfluence(seedetailsbelow intheparagraphaboutmodeling).Thismakesitpossibletousea simplifiedmodelwhichindeedcorrespondstothefirstpartofthe Reis-Vasco’smodel,i.e.,differentialmassbalanceequations corre-spondingtothecaseofextractionlimitedbythesoluteadsorption anddescribingtheflowpatternasapistonflowwithaxial disper-sion[38].
3.3.2. Descriptionoftheequationsofthemodel
Massbalanceequationsinthatcasecorrespondtothefollowing assumptions:(1)thesuperficialvelocityofSCCO2,u,isconstant
duringtheprocess,(2)theprocessisisothermalandpressuredrop isneglected,(3) volatileoilisdescribedbyasinglecomponent, the“solute”and(4)alinearequilibriumrelationshipisassumed definedasC=Km·q.Thecorrespondingmodelequations,boundary
andinitialconditionsare(Reis-Vascoetal.)[38]: fε∂C ∂t +s(1−ε) ∂q ∂t =−fu ∂C ∂z +DLfε ∂2C ∂z2 (7) C=Km·q (8) t=0, C=C0 and xu=q0+ C0 z>0 (9) z=0, u εC−DL ∂C ∂z =0 t>0 (10) Z=L, ∂C ∂z =0 t>0 (11)
Eq.(7)canbere-writtenas:
1+(1−ε) ε s f 1 Km ∂C ∂t + u ε ∂C ∂z −DL ∂2C ∂z2 =0 (12)wheretisextractiontime,zistheaxialcoordinateofthe extrac-tionbed,C0istheconcentrationofvolatileoilinthefluidphaseat
thebeginningoftheextractionprocesswhichcorrespondstothe equilibriumwiththeinitialsolidphaseconcentrationq0,q0 and
C0beingrelatedbyEq.(9),εisthebedvoidfraction,sdensityof
vegetablematter,andDListheaxialdispersioncoefficient(m2/s).
ThesystemofequationswassolvedwiththeMatlab®software.
InitialconcentrationofsoluteinCO2,C0,wascomputedfromthe
initialslopeofFig.4(b)and,thevalueC0=0.2×10−2gsolute/gCO2
wasobtained.Equilibriumadsorption constantKm (=C0/q0)was
thencomputedatKm=0.14kgplant/kgCO2.
At90barand40◦C,densityandviscosityofCO
2wereevaluated
atf=448.45kg/m3andf=40.1×10−6Pas.Theaxialdispersion
Fig.7. T.articulatavolatileoilyieldfor1.5mmparticlessize,pressure90barand temperature40◦CatdifferentCO
2flowrateswithReis-Vascomodel(firstpartonly).
coefficientDLwasdeterminedusingthecorrelationproposedby
Funazukuri[39]: DL=1.317(ReSc)1.392
D12 ε (13) where Re=fudp f (14) Sc= f fD12 (15)The binary diffusion coefficient D12 was estimated by the
Wilke–ChangequationasproposedbyFunazukuri: D12=7.410−12
T
p
˚MCO2fV2bp0.6
(16) whereTisthetemperature(K),˚istheassociationfactorofthe solvent(˚=1 forCO2), Misthemolarmassofthesolventand
V2bpisthemolarvolumeofthesoluteatitsnormalboilingpoint
expressedincm3/mol.Asa-pineneisacompoundpresentinall
volatileextracts,its molarvolume V2bp=178.63×10−3m3/kmol
wasusedin(15)[17].AvalueofD12=1.7×10−8m2/swasobtained
for90bar,40◦C.Axialdispersioncoefficientwasfoundtobeequal
to1.9×10−5m2/sandPe=19inthecaseofCO
2flow-rateequalto
20g/min.
In Fig.7, experimentalcurves arecompared withcalculated yieldsusingthismodelandagoodagreementisobservedforthe threedifferentCO2flow-rates.Togetherwiththequalitative
anal-ysiswe have developed in the preliminaryanalysis,this result validatesthechoiceoftheproposedmodel.
3.3.3. Useofthemodelingforscaling-uptheextractionprocess Aswementioned,significantaxialdispersionwaspointedoutat laboratoryscale(Pe=19for20g/minCO2flow-rate).Nevertheless
itsinfluenceontheeffectivedurationforextractingthesoluteisnot verystrong.Forinstance,at20g/min,durationfortheobtaining of90%yield ispredictedat16minwiththeSovova’ssimplified model(whichneglectsaxialdispersion,t1obtainedbyEq.(2))while
ourmodeling,whichtakesintoaccountaxialdispersion,indicates avaluecloseto20min.Thisrathermoderatedifferencemakesit possibletoproposesimplifiedscale-upbymaintainingthespecific flow-rateq′ andtheratioheighttodiameteroftheextractor (5
inourcase).Withthisextrapolationrule,forinstancefora1000 foldgreatervegetalmasstoextract,simple computationresults ina113Lextractor(diameter0.3m,height1.6m)percolatedby
240kg/hofCO2(superficialvelocity10mm/s)inwhich90%yield
isobtainedafter16min.Inthiscase,itcanbecomputedthatthe Pecletnumbervalueisnow82,whichismorefavorablethaninthe laboratoryscaleextractor(wherePe=19)andindeedreinforcesthe validityofthissimpleextrapolationprocedure.
4. Conclusions
Recovery of bioactive compounds from T. articulata leaves was obtainedusing SCCO2 extraction. For volatile compounds,
optimalconditions were found to be90bar,40◦C, and 30min
duration. For non-volatile fraction, suitable conditions were found to be 1000bar, 40◦C and 30min duration. Also,
quan-titative assessment of the extract antioxidant power and the enrichment of antioxidant at different extraction conditions were performed. Interesting selectivity for compounds with highantioxidant activity(ABTS•+ essay (29.77±1.3, 33.55±0.4
and 40.91±0.53mg/L) and phenolic content (267.90±8.06, 102.03±4.57and9.70±0.57GAEmg/gmaterial))wereobserved for SFE extracts at different pressures (1000, 280, and 90bar), respectively. Conventional HD was tested and essential oils obtained by this technique revealed low antioxidant activity (324.5±14.21mg/L).
Also, different chemical compositions of the extracts were found,dependingontheSFEpressureextraction.Physicalaspects ofthelow pressuresupercritical extraction(90bar,40◦C)were
investigatedandmodelingoftheextractionkineticsusinga sim-plifiedformoftheReis-Vasco’smathematicalmodelprovedtobe adequate.Inourrangeofoperatingconditions,extractionofT. artic-ulatawasfoundtobegovernedbyadsorptionphenomenonand significantaxialdispersionwaspointedout.Thismadeitpossibleto proposesimpleextrapolationprocedure.Itisnoteworthythat pro-ductivitycanbeincreasedproportionallytoCO2flow-rate,aslong
asmasstransfereffectsremainnegligible.Also,adsorptionconstant canberenderedmorefavorablebyincreasingthesolventpowerof CO2 usinghigherpressurebuta correlativelossofselectivityis
expected. References
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