Phase behaviour of the pseudo-ternary system carbon dioxide + ethanol + fish oil at high pressures

1 Phase beha iou o he pseudo- e na y sys em ca bon dioxide + e hanol + ish oil a high p essu es Rod igo Melgosa, Mª Te esa Sanz*, Ángela G. Solaesa, Sag a io Bel án Depa men o Bio echnology and Food Science (Chemical Enginee ing Sec ion) Uni e si y o Bu gos, Pza. Misael Bañuelos s/n 09001 Bu gos, Spain *Co esponding au ho . Tel.: +34-947258810; Fax: +34-947258831; e-mail: e sa[email p o ec ed] ABSTRACT This wo k p o ides expe imen al luid phase equilib ium da a o he pseudo- e na y mix u e CO2 + e hanol + ish oil, a sys em o in e es in pha maceu ical and ood-indus y applica ions such as he p oduc ion o omega-3-en iched lipid de i a i es a mild, non-oxida i e condi ions. Expe imen al ie-lines we e ob ained by means o an analy ical iso he mal me hod wi h eci cula ion o he apou phase. Measu emen s we e ca ied ou in he empe a u e ange 323.15 K-343.15 K and a p essu es om 10 MPa o 30 MPa. The Peng-Robinson equa ion o s a e coupled wi h he con en ional an de Waals mixing ules wi h wo adjus able pa ame e s was used o expe imen al da a co ela ion. Keywo ds Phase equilib ium, Supe c i ical ca bon dioxide, E hanol, Fish oil, The modynamic modelling, Peng-Robinson equa ion o s a e. 2 1. INTRODUCTION Dense ca bon dioxide is expec ed o play an impo an ole as a eac ion medium in ecologically iendly p ocessing. Enzyme-ca alysed e hanolysis o lipid sou ces in supe c i ical ca bon dioxide (SC-CO2) o CO2-expanded media can be used o imp o e he p oduc ion o lipid de i a i es, including concen a es o omega 3 polyunsa u a ed a y acids (n-3 PUFAs) om ish oil [1,2]. Howe e , he ela i ely low solubili y o he eac an s in SC- CO2 limi s he eac ion pe o mance in his medium; hus, a CO2-expanded media is p e e ed o minimise mass ans e limi a ions [3]. Unde s anding he phase beha iou o he e hanol + ish oil subs a e mix u e wi h CO2 would help in he selec ion o adequa e e hanolysis condi ions (p essu e, empe a u e, e hanol- o-oil a io, and amoun o dissol ed CO2 in he eac ion mix u e). This knowledge can be also ex ended o o he po en ial applica ions and may be o in e es in he ish oil indus y, since SC-CO2 o CO2-expanded media can be used h oughou all he n-3 PUFA concen a ion p ocess, including he supe c i ical ex ac ion o he ish oil [4], he e ining s ep [5], he sepa a ion and ac iona ion o he eac ion p oduc s [6,7], o he o mula ion o he inal p oduc by means o pa icle o ma ion echniques [8]. Expe imen al da a ela ed wi h high-p essu e phase equilib ium o he bina y sys em CO2 + e hanol a e ex ensi ely epo ed in he li e a u e [9–14]. Besides, e na y and highe sys ems comp ising CO2 and pu e iglyce ides, o he lipid de i a i es and hei mix u es ha e been also in es iga ed [15]. Howe e , only a ew da a ega ding he phase equilib ium o pseudo- e na y mix u es o CO2, e hanol, and edible oils can be ound in he li e a u e, and hese da a a e usually ela ed o he solubili y o he lipid compounds in CO2 wi h e hanol as a co-sol en [16]. Se e al publica ions ha e p e iously epo ed luid phase equilib ium o pseudo- e na y mix u es o CO2 + e hanol + ege able oils. Howe e , o ou knowledge, his is he i s wo k 3 dealing wi h oils ich in n-3 PUFAs om animal sou ces, such as ish oil. Geana and S eine [17] epo ed luid phase equilib ium da a o he pseudo- e na y sys em CO2 + e hanol + apeseed oil in he empe a u e ange 313 K-353 K and a p essu es om 6 MPa o 12 MPa, sa is ac o ily co ela ing he phase beha iou wi h he Peng-Robinson equa ion o s a e (PR EoS) [18] coupled wi h he con en ional an de Waals mixing ules wi h wo adjus able pa ame e s ( dW2). Ndiaye e al. [19] s udied he luid phase equilib ia o bina y and e na y mix u es in ol ing CO2, e hanol, soybean oil, cas o oil, and hei a y acid e hyl es e s. The pseudo- e na y sys em CO2 + e hanol + cas o oil was s udied a ixed e hanol- o-oil a ios, empe a u es anging om 313.15 K o 343.15 K and p essu es om 2.13 MPa o 27.13 MPa. Expe imen al da a we e co ela ed bo h wi h PR EoS dW2 and he S a is ical Associa ing Fluid Theo y (SAFT) [20] wi h one bina y in e ac ion pa ame e . Among hese wo models, he au ho s conside ed ha SAFT EoS desc ibed be e he phase beha iou o he pseudo- e na y sys em, ye hey poin ed ou a some de ia ions om hei expe imen al esul s, such as he o e -p edic ion o he cloud poin p essu e a high e hanol a ios [20]. He nández e al. [21] in es iga ed he luid phase equilib ium beha iou o he pseudo- e na y mix u e CO2 + e hanol + sun lowe oil a wo di e en condi ions o empe a u e and p essu e (313.15 K and 13 MPa; 333.15 K and 20 MPa). A g oup con ibu ion equa ion o s a e (GC EoS) [22] was used o co ela e he expe imen al da a. Two di e en se s o pa ame e s we e adop ed o he in e ac ion be ween he iglyce ide and he alcohol g oups, one o hem co esponding o he Liquid + Liquid (L1+L2) 2-phase egion, and he o he o he Vapou + Liquid (V+L2) 2- phase egion. Mo e ecen ly, Dalmolin e al. [23] s udied he phase ansi ions in he sys em CO2 + e hanol + apeseed oil, a empe a u es in he ange 313.15 K-343.15 K and p essu es up o 22.53 MPa. They ound a 3-phase egion wi h a Vapou + Liquid + Liquid (V+L1+L2) phase ansi ion ha occu ed a highe p essu es when inc easing empe a u e, and sa is ac o ily explained hei expe imen al esul s wi h he PR EoS dW2 model. 4 In his wo k, he phase beha iou o he pseudo- e na y mix u e CO2 + e hanol + ish oil in he empe a u e ange om 323.15 K o 343.15 K and p essu es om 10 MPa o 30 MPa has been de e mined by means o an analy ical iso he mal me hod wi h eci cula ion o he apou phase (AnTVci , as desc ibed by Doh n and B unne [24]). The main goal o he s udy in ol es a con ibu ion owa ds unde s anding he phase beha iou o sys ems con aining CO2, e hanol, and oils ich in n-3 PUFAs. The knowledge ob ained will be use ul in he de elopmen o applica ions in ol ing hese pseudo- e na y mix u es, such as he p e iously men ioned enzyma ic eac ions, supe c i ical ex ac ion and ac iona ion, and pa icle o ma ion echniques. 2. EXPERIMENTAL 2.1. Ma e ials Fish oil was p o ided by AFAMSA S.A. (Pon e ed a, Spain) being a mix u e o una (Thunnus sp.) and sa dine (Sa dina pilcha dus) e ined oils. The a y acid p o ile and ee a y acid con en o he ish oil ha e been de e mined acco ding o AOCS me hods [25]. The a y acid p o ile has been p e iously epo ed [26] and is also p o ided in Table 1. F ee a y acid con en was ound o be 0.29 ± 0.04 % oleic acid. Densi y o ish oil was also measu ed in an An on Paa DMA 5000 ins umen , inding alues o ρ323.15 K = 906.53 kg·m-3 and ρ343.15 K = 895.83 kg·m-3 (u(ρ) = ± 0.05 kg·m-3). Absolu e e hanol (0.999 mass ac ion) was pu chased om Me ck KGaA. Ca bon dioxide (0.999 mass ac ion in he liquid phase) was supplied by Ai Liquide S.A. (Spain). Compounds ha e been used as p o ided by he manu ac u e s wi hou u he pu i ica ion. 5 The po en ial eac i i y o ish oil in con ac wi h e hanol could lead o some ex en o ans- es e i ica ion and eac ion p oduc s could be o med du ing phase equilib ia measu emen s, mainly a y acid e hyl es e s (FAEE). Al hough eac ion in he absence o ca alys s is e y slow, he p esence o FAEE and o he in e media e componen s was analysed a e phase equilib ia measu emen s by NP-HPLC. Ch oma og aphic me hod is epo ed elsewhe e [27]. FAEE con en was ound less han 0.001 mole ac ion and i was conside ed no o a ec he phase equilib ia. Hid olysis o he ish oil was also e alua ed by means o he ee a y acid con en [25], inding a maximum inc ease up o 0.81 ± 0.05 % oleic acid a he highes s udied empe a u e (343.15 K). Fa y acid p o ile was also de e mined a e phase equilib ia measu emen s, inding no signi ican changes compa ed o he ini ial p o ile (Table 1). Addi ionally, since ish oil is ich in polyunsa u a ed a y acids (Table 1) i is e y p one o oxida ion. The e o e p ima y oxida ion was e alua ed by means o pe oxide alue analysis [28] be o e and a e phase equilib ia measu emen s. Al hough PV sligh ly inc eased om 2.0 ± 0.2 meq O2/kg o 3.5 ± 0.1 meq O2/kg, oxida ion p oduc s a e mino componen s p esen in small amoun s ha would no a ec he phase equilib ia o he sys em. 2.2. Appa a us and p ocedu e A schema ic diag am o he high-p essu e appa a us used o luid phase equilib ium measu emen s is shown in Figu e 1. I was buil by Eu o echnica GmbH (Ge many) and consis s o an equilib ium cell made o s ainless s eel (SS-316) and equipped wi h a sapphi e window o obse ing he con en o he cell du ing measu emen s. In e nal olume o he cell anges om 40 o 70 mL, adjus able h ough a manual sc ew pis on. The cell includes a p essu e ansduce and an imme sed he mocouple. Bo h o hem calib a ed and connec ed o a Da a Acquisi ion Sys em (DAS). The equipmen was placed inside an o en ha allowed empe a u e con ol o he sys em. Mixing o he componen s o he sys em was achie ed by 6 con inuously aking he apou phase and passing i back in o he equilib ium cell h ough he liquid phase by means o a gea pump (Mic opump IDEX). A 750 μL loop ha could be isola ed by means o a 6 way al e (VICI) was placed in he eci cula ion pa h o sampling he apou phase wi h minimal equilib ium dis u bance. Besides, a mic o-me e ing al e was connec ed o he bo om o he equilib ium cell h ough a 1/16” capilla y o sampling he hea y phase. P essu e d op occu ing when sampling he liquid phase was compensa ed by educing he olume o he cell h ough he manual sc ew pis on. Maximum speci ica ions o he appa a us a e p = 32 MPa and T = 393 K. A ypical expe imen wi h he high-p essu e a iable- olume iew cell began wi h he p e- hea ing o he sys em up o he desi ed empe a u e. When he empe a u e was achie ed, he equilib ium cell was gen ly pu ged wi h low p essu e CO2 o sweep he esidual ai inside he cell. Immedia ely a e wa ds, known olumes o ish oil and e hanol we e in oduced in o he cell by means o a bina y HPLC pump (Agilen 1200 Se ies). A ce ain amoun o CO2 was hen cha ged in o he cell by using a high-p essu e sy inge pump (ISCO 260D). The exac amoun s o ish oil and e hanol we e calcula ed using hei espec i e densi ies a oom empe a u e, whe eas he mass o CO2 cha ged in o he cell was measu ed by a Co iolis mass low me e (Rheonik RHE015). Once he cell was cha ged and he desi ed p essu e was adjus ed by ac ua ing he manual sc ew pis on, he gea pump was connec ed and eci cula ion o he apou phase was pe o med o a leas 2 h o acili a e he mixing o he componen s and i s dis ibu ion in he di e en phases o he sys em. The sys em was hen le o s and o ano he 2 h a cons an empe a u e and p essu e. Phase sepa a ion was isually e i ied h ough he sapphi e window and samples om he apou and liquid phases we e aken by he 6-way and he mic o-me e ing al e, espec i ely. P essu e a ia ions up o ±0.1 MPa we e obse ed du ing sampling, while empe a u e change was no de ec ed. O e all s anda d 7 unce ain ies in he equilib ium measu emen s we e u(p) = ± 0.15 MPa, u(T) = ± 0.1 K, and u(wi) = ± 0.005; being wi he mass ac ion o componen i. Samples we e decomp essed o a mosphe ic p essu e and eleased CO2 was measu ed by means o a he mal mass low me e (B onkho s F-110C). E hanol and ish oil we e sepa a ed om CO2 and collec ed in an ice-cooled glass ap. The amoun o each componen was de e mined by weighing he ials in a p ecision analy ical balance (accu a e o ± 0.0001 g) be o e and a e e apo a ion o e hanol a T = 373.15 K. 3. RESULTS AND DISCUSSION 3.1. Expe imen al da a In o de o check he eliabili y o he appa a us and expe imen al p ocedu e, p elimina y measu emen s o he bina y sys em CO2 + e hanol we e ca ied ou . Expe imen al high- p essu e phase equilib ia da a o he CO2 + e hanol bina y sys em a e summa ised in Table 2. The esul s ob ained we e compa ed wi h da a aken om li e a u e [9,12], inding a good ag eemen (Figu e 2). Expe imen al high-p essu e phase equilib ium da a o he pseudo- e na y sys em CO2 + e hanol + ish oil a e lis ed in Tables 3-5. Due o he la ge di e ences in molecula weigh o he componen s o he sys em, composi ions a e exp essed in e ms o mass ac ion ins ead o mole ac ion. Expe imen al esul s a T = 323.15 K showed wo di e en 2-phase egions o he wo p essu es in es iga ed (10 MPa and 30 MPa). In one o hem, wo liquid phases could be dis inguished, being he ligh and hea y phases ich wi h e hanol and oil, espec i ely 8 (L1+L2). In he o he 2-phase egion, a ligh apou phase ich wi h CO2 and a hea y liquid phase ich wi h ish oil (V+L2) we e obse ed. Homogeneous monophasic mix u es we e isually and analy ically e i ied, samples aken om he op and bo om o he equilib ium cell we e simila wi h di e ences smalle han he expe imen al unce ain y. In he case o he phase equilib ium a T = 343.15 K and p = 10 MPa, h ee 2-phase egions (L1+L2, V+L1, and V+L2) and a 3-phase egion (V+L1+L2) we e obse ed. The appea ance o he V+L1 egion is consis en wi h he published phase equilib ium da a o he bina y sys em CO2 + e hanol a T = 343.15 K [9,12]. Resul s ob ained o he bina y e hanol + ish oil ie-lines a T = 323.15 K, and p essu es o 10 MPa and 30 MPa (Figu e 3) a e simila o hose ob ained by Bucio e al. in p e ious wo ks a he same empe a u e and a mosphe ic p essu e [26], indica ing ha , in he ange in es iga ed, p essu e does no signi ican ly a ec phase equilib ium o his bina y mix u e. The composi ion o he wo liquid phases in he L1+L2 egion became mo e simila as mo e CO2 is dissol ed. The same was ue o he V+L2 egion, whe e he composi ions o he apou and liquid phases in equilib ium end o me ge wi h inc easing amoun s o dissol ed e hanol. A simila end has been ound o o he pseudo- e na y mix u es o CO2 + e hanol + na u al lipids, such as cas o oil [19] and sun lowe oil [21]. The homogeneous monophasic egion a p = 30 MPa appea s o be sligh ly la ge , al hough no s ong e ec o p essu e on he phase beha iou o he mix u e a T = 323.15 K can be obse ed. F om he phase diag am a T = 343.15 K and p = 10 MPa (Figu e 4), i can be obse ed ha , s a ing om he bina y sides o he phase diag am, inc easing amoun s o he hi d componen made he ie-lines o he 2-phase egions app oach he sides o he 3-phase egion iangle. Inside his 3-phase egion, heo e ical mix u es spli in a V+L1+L2 sys em, each phase wi h a composi ion de ined by he e ices o he iangle. 9 Compa ing he phase beha iou a T = 343.15 K, p = 10 MPa (Figu e 4) wi h he 323.15 K iso he m a he same p essu e (Figu e 3a), i is no iceable ha he o me p esen s a lowe amoun o CO2 dissol ed in he liquid phase (V+L2 egion), p obably because o he empe a u e-d i en inc ease in he CO2 apou p essu e. On he con a y, i can be obse ed ha aising empe a u e om 323.15 o 343.15 K inc eases he solubili y o ish oil in e hanol (L1+L2 egion) om nea 0.075 mass ac ion up o 0.35 mass ac ion, ye adding CO2 has a sligh de-en aining e ec a 343.15 K since he mo e CO2 is dissol ed, he wide he ie-lines become. 3.2. Da a co ela ion In his wo k, ish oil has been ea ed as a pseudo-componen , assuming ha he di e en iacylglyce ols p esen in he ish oil beha e in a simila way, which has been p e iously e i ied [26]. Expe imen al phase equilib ium da a o he pseudo- e na y mix u e we e co ela ed wi h he Peng-Robinson equa ion o s a e (PR EoS) in combina ion wi h he con en ional an de Waals mixing ules wi h wo adjus able pa ame e s ( dW2). The PR EoS, was used as o iginally de ined by Peng and Robinson [18]: b)b( b) ( a(T) b T p−++ − − =R (1) whe e he pa ame e a is ela ed o he in e molecula a ac i e o ces and b o he size o he molecules. 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The modyn. 105 (2017) 385–395. 21 Table 1. Fa y acid composi ion o he ish oil [26]. Fa y acid % w . my is ic C14:0 3.8 palmi ic C16:0 21.0 palmi oleic C16:1n-7 6.1 s ea ic C18:0 6.0 oleic C18:1n-9 18.4 accenic C18:1n-7 3.0 linoleic cis (LA) C18:2n-6 2.4 α-linolenic (ALA) C18:3n-3 0.7 s ea idonic C18:4n-3 0.9 eicosenoic C20:1n-9 2.1 eicosa ienoic C20:3n-3 2.0 eicosapen aenoic (EPA) C20:5n-3 6.9 docosapen aenoic (DPA) C22:5n-3 1.8 docosahexaenoic (DHA) C22:6n-3 24.9 S anda d unce ain ies a e u(pe cen age) = ± 0.5 22 Table 2. Vapou + Liquid phase composi ions (mole ac ion) o he bina y sys em CO2 (1) + e hanol (2). p / MPa x 1 y 1 p / MPa x 1 y 1 T = 323.15 K T = 343.15 K 4.67 0.2647 0.9840 7.01 0.2930 0.9679 5.12 0.2771 0.9859 8.05 0.3785 0.9656 5.34 0.3058 0.9819 9.04 0.4260 0.9614 5.78 0.3128 0.9837 9.98 0.4968 0.9484 7.07 0.4128 0.9814 10.98 0.6130 0.9432 7.69 0.4795 0.9739 11.50 0.6910 0.9060 7.96 0.5069 0.9764 8.16 0.5455 0.9754 8.44 0.6348 0.9740 8.46 0.6279 0.9671 8.51 0.6535 0.9679 8.69 0.6875 0.9630 S anda d unce ain ies a e u(p) = ± 0.15 MPa, u(T) = ± 0.1 K, and u(w i ) = ± 0.005 23 Table 3. Liquid + Liquid (L1+L2) and Vapou + Liquid (V+L2) phase composi ions (weigh ac ion) o he pseudo- e na y sys em CO2 (1) + e hanol (2) + ish oil (3) a T = 323.15 K and p = 10 MPa. hea y phase ligh phase ype o phase eq. w1 w2 w3 w1 w2 w3 0.0000 0.2652 0.7348 0.0000 0.9256 0.0744 L1+L2 0.0423 0.2836 0.6741 0.0420 0.8692 0.0888 L1+L2 0.0761 0.2966 0.6273 0.0780 0.7957 0.1263 L1+L2 0.1012 0.3087 0.5901 0.1016 0.7638 0.1346 L1+L2 0.1696 0.3898 0.4406 0.1710 0.6176 0.2114 L1+L2 0.2247 0.0000 0.7754 0.9868 0.0000 0.0132 V+L2 0.2440 0.0272 0.7288 0.9580 0.0280 0.0140 V+L2 0.2776 0.0747 0.6478 0.9003 0.0751 0.0246 V+L2 0.3330 0.1181 0.5490 0.7924 0.1657 0.0419 V+L2 0.3622 0.1541 0.4837 0.7332 0.2193 0.0476 V+L2 0.3730 0.1710 0.4560 0.6949 0.2525 0.0526 V+L2 0.3804 0.1841 0.4355 0.6207 0.3010 0.0783 V+L2 0.2026 0.2224 0.5751 0.2024 0.2222 0.5754 homogeneous 0.2745 0.4691 0.2564 0.2747 0.4689 0.2564 homogeneous 0.3039 0.2838 0.4023 0.3030 0.2844 0.4026 homogeneous 0.4590 0.3640 0.1770 0.4593 0.3638 0.1769 homogeneous S anda d unce ain ies a e u(p) = ± 0.15 MPa, u(T) = ± 0.1 K, and u(wi) = ± 0.005 24 Table 4. Liquid + Liquid (L1+L2) and Vapou + Liquid (V+L2) phase composi ions (weigh ac ion) o he pseudo- e na y sys em CO2 (1) + e hanol (2) + ish oil (3) a T = 323.15 K and p = 30 MPa. hea y phase ligh phase ype o phase eq. w1 w2 w3 w1 w2 w3 0.0000 0.2219 0.7781 0.0000 0.9066 0.0934 L1+L2 0.0649 0.3010 0.6341 0.0771 0.8185 0.1044 L1+L2 0.1550 0.4288 0.4162 0.1520 0.6654 0.1826 L1+L2 0.2559 0.0000 0.7441 0.9832 0.0000 0.0168 V+L2 0.2843 0.0674 0.6484 0.9089 0.0639 0.0272 V+L2 0.3073 0.1174 0.5753 0.8588 0.1131 0.0281 V+L2 0.3404 0.1401 0.5194 0.8252 0.1417 0.0331 V+L2 0.3844 0.1666 0.4490 0.7079 0.2367 0.0555 V+L2 0.4922 0.2338 0.2740 0.6089 0.3036 0.0875 V+L2 0.2926 0.2224 0.4851 0.2928 0.2219 0.4853 homogeneous 0.2919 0.4499 0.2582 0.2902 0.4488 0.2610 homogeneous 0.4528 0.416 0.1312 0.4481 0.4163 0.1356 homogeneous 0.5219 0.3261 0.1520 0.5198 0.328 0.1522 homogeneous S anda d unce ain ies a e u(p) = ± 0.15 MPa, u(T) = ± 0.1 K, and u(wi) = ± 0.005 25 Table 5. Liquid + Liquid (L1+L2) and Vapou + Liquid (V+L1, V+L2) phase composi ions (weigh ac ion) o he pseudo- e na y sys em CO2 (1) + e hanol (2) + ish oil (3) a T = 343.15 K and p = 10 MPa. hea y phase ligh phase ype o phase eq. w1 w2 w3 w1 w2 w3 0.0000 0.2829 0.7171 0.0000 0.6497 0.3503 L1+L2 0.0363 0.2327 0.7310 0.0728 0.6418 0.2854 L1+L2 0.0618 0.2102 0.7280 0.1725 0.6291 0.1984 L1+L2 0.0890 0.1851 0.7259 0.3132 0.5677 0.1191 L1+L2 0.1157 0.1607 0.7236 0.4183 0.5088 0.0729 L1+L2 0.1624 0.1162 0.7214 0.4981 0.4199 0.0820 L1+L2 0.4854 0.5146 0.0000 0.9461 0.0539 0.0000 V+L1 0.4850 0.5014 0.0136 0.9239 0.0589 0.0172 V+L1 0.4870 0.4752 0.0378 0.9002 0.0680 0.0318 V+L1 0.1820 0.0000 0.8180 0.9796 0.0000 0.0204 V+L2 0.1843 0.0396 0.7761 0.9455 0.0325 0.0220 V+L2 0.1794 0.0717 0.7489 0.9143 0.0542 0.0315 V+L2 0.1814 0.0957 0.7229 0.9055 0.0607 0.0338 V+L2 S anda d unce ain ies a e u(p) = ± 0.15 MPa, u(T) = ± 0.1 K, and u(wi) = ± 0.005