Recapitulation of Human Neural Microenvironment Signatures in iPSC-Derived NPC 3D Differentiation

Joana Campainhas Bas os Licenciada em Biologia Humana Ionic Liquids in he De elopmen o No el Bioma e ials Disse ação pa a Ob enção do G au de Mes e em Bioquímica pa a a Saúde O ien ado : Ana Belén Pe ei o, In es igado a Auxilia do REQUIMTE, Faculdade de Ciências e Tecnologia da Uni e sidade No a de Lisboa Co-o ien ado : João Miguel Mendes de A aújo, In es igado Auxilia do REQUIMTE, Faculdade de Ciências e Tecnologia da Uni e sidade No a de Lisboa No emb o 2016 Joana Campainhas Bas os Licenciada em Biologia Humana Ionic Liquids in he De elopmen o No el Bioma e ials Disse ação pa a ob enção do G au de Mes e em Bioquímica pa a a Saúde O ien ado : Ana Belén Pe ei o, In es igado a Auxilia do REQUIMTE, Faculdade de Ciências e Tecnologia da Uni e sidade No a de Lisboa Co-o ien ado : João Miguel Mendes de A aújo, In es igado Auxilia do REQUIMTE, Faculdade de Ciências e Tecnologia da Uni e sidade No a de Lisboa Cons i uição do Jú i: P esiden e: Dou o Ped o Ma ias, In es igado P incipal do Ins i u o de Tecnologia Química e Biológica An ónio Xa ie da Uni e sidade No a de Lisboa A guen e: Dou o a Ka ina Shimizu, In es igado a do Ins i u o Supe io Técnico da Uni e sidade de Lisboa Vogais: Dou o a Ana Es e éz, In es igado a Auxilia do REQUIMTE, Faculdade de Ciências e Tecnologia da Uni e sidade No a de Lisboa Dou o a Ma ga ida A che , In es igado a P incipal do Ins i u o de Tecnologia Química e Biológica An ónio Xa ie da Uni e sidade No a de Lisboa Ins i u o Tecnológico de Química e Biológica An ónio Xa ie da Uni e sidade No a de Lisboa (ITQB), Oei as, Po ugal No emb o 2016 I Ionic Liquids in he De elopmen o No el Bioma e ials COPYRIGHT Joana Campainhas Bas os Ins i u o de Tecnologia Química e Biológica An ónio Xa ie Uni e sidade No a de Lisboa O Ins i u o de Tecnologia Química e Biológica An ónio Xa ie e a Uni e sidade No a de Lisboa êm o di ei o, pe pé uo e sem limi es geog á icos, de a qui a e publica es a disse ação a a és de exempla es imp essos ep oduzidos em papel ou de o ma digi al, ou po qualque ou o meio conhecido ou que enha a se in en ado, e de a di ulga a a és de eposi ó ios cien í icos e de admi i a sua cópia e dis ibuição com obje i os educacionais ou de in es igação, não come ciais, desde que seja dado c edi o ao au o e edi o . II III Aos meus pais, po me pe mi i em alcança odos os meus sonhos IV V Ag adecimen os Es e espaço é dedicado a odos os que, de alguma o ma con ibuí am pa a que es a disse ação osse ealizada. Não pudendo nomeá-los a odos, há alguns a quem não posso deixa de mani es a o meu ap eço e ag adecimen o since os. Em p imei o luga que o ag adece á minha o ien ado a, Dou o a Ana Belén Pe ei o e co-o ien ado Dou o João Miguel Mendes de A aújo, po me e em p opo cionado es a opo unidade. Exp esso ainda o meu p o undo ag adecimen o pelo apoio e disponibilidade mani es ada, con iança deposi ada e pa ilha de conhecimen o que con ibuí am decisi amen e pa a que es e abalho enha chegado a um bom e mo. Aos meus colegas do labo a ó io Fluídos Al e na i os pa a a Química Ve de po me e em acolhido e animado nos dias mais á duos de abalho. À Sa a Ca alho e à Fá ima Moscoso pelo companhei ismo e pala as amigas. Um ag adecimen o especial à Ma ga ida Fe ei a e à Nicole Viei a po pode semp e con a com o osso en usiasmo, aleg ia e paciência nos meus dias menos bons du an e es e ano. Gos a ia de ag adece ambém à P o esso a Dou o a Isabel Fonseca e Dou o a Isabel Pacheco pela disponibilidade dos seus labo a ó ios pa a ealização de algumas medidas espo ádicas expe imen ais. Ainda, um especial ob igada à Dou o a Manuela Dias pela ajuda imp escindí el nes a úl ima ase do meu abalho. Aos meus amigos, especialmen e à Ve a Mo ei a e à Inês Cab i a, ou in es e a en os aos meus desânimos e sucessos, que mesmo longe semp e me ajuda am mui o. Um mui o ob igada pelo apoio e pela alo ização semp e ão en usias a do meu abalho. À minha amília, uma pala a de econhecimen o mui o especial pelo amo incondicional e pela o ma como ao longo de odos es es anos, ão bem, me soube am ajuda . Aos meus pais o meu e e no ob igada po me p opo ciona em es a opo unidade de p og edi na ida p o issional. XII 2.4.1.1. The mal P ope ies ……………………………………………………………………. 24 2.4.1.2. The mophysical Cha ac e iza ion ……………………………………………………... 26 2.4.1.3. Walden-Plo …………………………………………………………………………… 30 2.4.2. Mix u es o Fluo ina ed Ionic Liquids and Wa e …………………………………………... 31 2.4.2.1. Liquid-liquid Equilib ia ……………………………………………………………….. 31 2.4.2.2. Sel -agg ega ion Beha iou …………………………………….……………………... 34 2.5. Conclusions ……………………………………………………………………………………. 38 2.6. Re e ences ……………………………………………………………………………………... 39 3. Pa i ion P ope ies o Fluo ina ed Ionic Liquids……………………………………………….. 43 3.1. In oduc ion ……………………………………………………………………........................ 45 3.2. Ma e ials ………………………………………………………………………………………. 47 3.3. Expe imen al P ocedu es …………………………………………………………………….. 48 3.3.1. Slow S i ing Me hod ……………………………………………………………………….. 48 3.4. Resul s and Discussion ………………………………………………………………………... 49 3.5. Conclusion …………………………………………………………………………………….. 52 3.6. Re e ences ……………………………………………………………………………………... 53 4. Kamle -Ta Sol a och omic Pa ame e s o Fluo ina ed Ionic Liquids ………………………. 55 4.1. In oduc ion …………………………………………………………….................................... 57 4.2. Ma e ials ………………………………………………………………………………………. 59 4.3. Expe imen al P ocedu es …………………………….............................................................. 61 4.3.1. Dye Solu ions and Samples P epa a ion ……………………………………………………. 61 4.3.2. UV-Vis Measu emen s ………………………………………………................................... 62 4.4. Resul s and Discussion ………………………………………………………………………... 62 4.4.1. Kamle -Ta Pa ame e s o Nea Fluo ina ed Ionic Liquids ……………............................... 62 4.4.1.1 In luence o he Imidazolium, Py idinium and Te abu ylammonium Ca ions…….….. 63 4.4.1.2. In luence o he Hyd ogena ed Alkyl Chain Leng h o he Ca ion ……………………. 64 4.4.1.3. In luence o he Fluo ina ed Chain Leng h o he Anion …………………………….. 66 XIII 4.4.2. Kamle -Ta Sol a och omic Pa ame e s o Aqueous Solu ions o Fluo ina ed Ionic Liquids…………………………………………………………………………………………….. 68 4.4.2.1. In luence o he Imidazolium, Py idinium and Te abu ylammonium Ca ion ………… 70 4.4.2.2. In luence o he Hyd ogena ed Alkyl Chain Leng h on he Imidazolium Ca ion …….. 71 4.4.2.3. In luence o he Fluo ina ed Chain Leng h on he Anion ……………………………... 72 4.5. Conclusion………………..……………………………………………………………………. 74 4.6. Re e ences ……………………………………………………………………………………... 75 5. Conclusions and Pe spec i es……….……………………………………………………………. 79 5.1. Conclusions …………………………………………………………………...……………….. 81 5.2. Pe spec i es …………………………………………………………………..……………….. 82 6. Scien i ic Communica ions…………….……………..................................................................... 83 6.1 Pape s………………………………………………………………….………………………... 85 6.2. Pos e s in Scien i ic Mee ings…………………….....……….................................................... 85 XIV XV Lis o Abb e ia u es R&D Resea ch and De elopmen IL Ionic Liquids FILs Fluo ina ed Ionic Liquids PFC Pe luo oca bons C Ca bon F Fluo C-F bonds Ca bon and Fluo Bonds F-Chains Fluo ina ed Chains O2 Oxygen CO2 Ca bon Dioxide CMC C i ical Micella Concen a ions ACV Acyclo i ETO E odolac pH Po encial o Hyd ogen MMT Mon mo illoni e MAA Me hhac ylic Acid PDA Polydopamine DOX Doxo ubicin MWTT Mic owa e The mal The apy ECD Elec ochemical de ec o CE Capilla y elec opho esis UV-Vis Ul a iole isible Abs Abso bance AFMC Analy e Focusing by Micelle Collapse DNA Deoxy ibonucleic Acid LLE Liquid-Liquid Equilib ium CTAB Ce yl ime hylammonium B omide -AuNPs Fluo ina ed Gold Nanopa icles siRNA In e e ing Ribonucleic Acid TEM T ansmission Elec on Mic oscopy NMR Nuclea Magne ic Resonance XVI KF Ka l Fishe TGA The mog a ime ic Analysis TA The mal Analysis DSC Di e en ial Scanning Calo ime e D Demal (1 g equi alen o solu e dissol ed in 1dm3 sol en ) KCl Po assium Chlo ide API Ac i e Pha maceu ical Ing edien s ADME Abso p ion, Dis ibu ion, Me abolism and Exc e ion HPLC High-Pe o mance Liquid Ch oma og aphy HBD Hyd ogen Bond Dona ion HBA Hyd ogen Bond Accep ance KT Kamle -Ta DCM Diclo ome hane RSD Rela i e S anda d De ia ion dsDNA Double-s and DNA ssDNA Single-s anded H-bonding Hyd ogen bonding N-H Ni ogen hyd ogen bond C-H Ca bon hyd ogen bond XVII Lis o Symbols [CnC1Im]+ Imidazolium-based Ils (n, numbe o me hylene g oups) [C2C1py]+ Py idinium-based ILs [C1CO2]- Ace a e anion [(CH3O)2PO2]- Dime hylphospha e anion [C1C1Im][(CH3O)2PO2] Dime hylimidazole dime hylphospha e [C4C1Im][PF6] 1-Bu yl-3-me hylimidazolium hexa luo ophospha e [C16C1Im]B 1-Hexadecyl-3-me hyllimidazolium b omide [C4C1Im]B 1-Bu yl-3-me hylimidazolium b omide [C8C1Im][PF6] 1-Oc yl-3-me hylimidazolium hexa luo ophospha e Cº Celsius [C8C1Im] B 1-Oc yl-3-me hylimidazolium b omide [C4C1Im][PF6] 1-Bu yl-3-me hylimidazolium hexa luo ophospha e [C2C1Im] [(CH3)2PO4)] 1-E hyl-3-me hylimidazolium dime hylphospha e [C10POHIM] B 1-(1,2-Dihyd oxyp opyl)-3-decylimidazolium b omide [C16POHIM] B 1-(1,2-Dihyd oxyp opyl)-3-hexadecylimidazolium b omide [C2C1Im] Cl 1-E hyl-3-me hylimidazolium chlo ide [C2C1Im][BF4] 1-E hyl-3-me hylimidazolium e a luo obo a e [C2C1Im] [NT 2] 1-E hyl-3-me hylimidazolium bis( i luo ome hylsul onyl)imide [NT 2]- Bis( i luo ome hylsul onyl)imide anion [BF4]- e a luo obo a e anion [PF6]- hexa luo ophospha e anion Cl- Chlo ide anion [C6C1Im] Cl 1-Hexyl-3-me hylimidazolium chlo ide [C4C1Im][BF4] 1-Bu yl-3me hylimidazolium e a luo obo a e [C1C2py][C4F9SO3] 1-E hyl-3-me hylpy idinium pe luo obu anesul ona e [C4C1Im][C4F9SO3] 1-bu yl-3me hylimidazolium pe luo obu anesul ona e [C10C1Im][C4F9SO3] 1-decyl-3-me hylimidazolium pe luo obu anesul ona e [C4F9SO3]- Pe luo obu anesul ona e anion [C8F17SO3]- Pe luo ooc anesul ona e anion XVIII [N4444]+ Te abu ylammonium based-ILs K Kel in ppm Pa s pe million Tonse onse empe a u e Ts a s a ing empe a u e Tdec decomposi ion empe a u e Tm Mel ing empe a u e [C2C1Im][C4F9SO3] 1-e hyl-3-me hylimidazolium pe luo obu anesul ona e [C8C1Im][C4F9SO3] 1-bu yl-3-me hylimidazolium pe luo obu anesul ona e [C6C1Im][C4F9SO3] 1-hexyl-3-me hylimidazolium pe luo obu anesul ona e [C12C1Im][C4F9SO3] 1-dodecyl-3-me hylimidazolium pe luo obu anesul ona e ρ Densi y η Viscosi y  Fluidi y nD Re ac i e Index k Ionic Conduc i i y T Tempe a u e αe Elec onic pola izabili y Rm Mola pola izabili y NA A ogad o’s numbe ε0 Vacuum pe mi i i y Vm Mola ee olume Mw Molecula weigh a Sphe ical molecula adius m F ee olume 𝛬 Mola conduc i i y 𝐶 Cons an 𝑤𝑤𝑎𝑡𝑒𝑟 1 Wa e mass ac ion on FIL ich-phase 𝑤𝐹𝐼𝐿 1 FIL mass ac ion on FIL ich-phase 𝑤wa e 2 Wa e mass ac ion on wa e ich-phase XIX 𝑤FIL 2 FIL mass ac ion on wa e ich-phase αCMC Deg ee o ioniza ion o he agg ega es βCMC Deg ee o coun e ion binding o coun e ions condensed on he micella in e ace ΔG0agg S anda d ee ene gy o he agg ega ion p ocess RT Uni e sal gas cons an and absolu e empe a u e 𝑙𝑛 𝑥𝐶𝑀𝐶 Nepe ian loga i hm o c i ical micella concen a ion exp essed in mole ac ion π Pi ʎmax Maximum abso p ion wa eleng h  Hyd ogen bond acidi y / hyd ogen bond dona ion   Hyd ogen bond basici y / hyd ogen bond accep ance   Dipola i y/pola izabili y   Maximum abso p ion wa eleng h o he sol a och omic dyes 𝐸𝑇 30, Pola i y scale based only on Reicha d ’s dye w % Weigh pe cen age [C2C1Im][CF3SO3] 1-E hyl-3-me hylimidazolium i luo ome hanesul ona e [C4C1Im][CF3SO3] 1-Bu yl-3-me hylimidazolium i luo ome hanesul ona e [C2C1py][C4F9SO3] 1-E hyl-3-me hylpy idinium pe luo obu anesul ona e [C2C1Im][C4F9SO3] 1-E hyl-3-me hylimidazolium pe luo obu anesul ona e [C6C1Im][C4F9SO3] 1-Hexyl-3-me hylimidazolium pe luo obu anesul ona e [C8C1Im][C4F9SO3] 1-Oc yl-3-me hylimidazolium pe luo obu anesul ona e [N1112OH][C4F9SO3] Choline pe luo obu anesul ona e [N4444][C4F9SO3] Te abu ylammonium pe luo obu anesul ona e [N4444][C8F17SO3] Te abu ylammonium pe luo oc anesul ona e [C2C1Im][C1CO2] 1-E hyl-3-me hylimidazolium ace a e [C2C1py] B 1-E hyl-3-me hylpy idinium b omide [C2C1Im] Cl 1-E hyl-3-me hylimidazolium chlo ide [N1112OH] Cl Choline chlo ide [N1112OH]+ Choline-based ILs XX XXI Lis o Figu es 1.In oduc ion Page Figu e 1.1. Mos common ca ions s uc u es o ILs: a) py olidinium; b) imidazolium; c) pipe idinium; d) py idinium; e) ammonium and ) choline whe e R could be a hyd ogena ed chain, a luo ina ed chain o a unc ional g oup……………………………………………… 4 Figu e 1.2. Mos common anion s uc u es o ILs: a) chlo ide; b) b omide; c) e a luo obo a e; d) hexa luo ophospha e; e) ni a e; ) ace a e; g) dicyanamide; h) dime hylphosphona e i) me hylphosphona e j) e hylphosphona e; k) i luo ome hanesul ona e and l) bis( i luo ome hylsul onyl)imide………………………… 4 Figu e 1.3. Rele an applica ions o ionic liquids as bioma e ials in chemis y, chemical enginee ing, bio echnology and pha maceu ics indus ies…………………………………… 5 Figu e 1.4. Applica ion a eas o imidazolium-based su ac an s…………………………….. 7 Figu e 1.5. Schema ic illus a ion o he use o IL-PDA-DOX nanopa icles o combined chemo he apy wi h MWTT…………………………………………………………………… 8 Figu e 1.6. T anspo , elease, and accumula ion o analy es by micelle collapse…………… 9 Figu e 1.7. Ad an ages o using ILs o biomolecula p ocesses…………………………….. 9 Figu e 1.8. Schema ic illus a ion o sel -assembly o phy ase enzyme (Aspe gillus nige phy ase) in [C4C1Im][BF4], leading o phy ase nanosphe es and pla inum–phy ase nanosphe e. Cu cumin was loaded on bo h samples and was en apped in inne hyd ophobic domains o p o ein empla e…………………………………………………………………... 11 2. Cha ac e iza ion o Fluo ina ed Ionic Liquids: The mophysical, Phase Equilib ia and Agg ega ion Beha iou Figu e 2.1. a) TA ins umen Model TGA Q50 used o he measu emen s and b) TA Ins umen Model DSC Q200 used o he measu emen s…………………………………….. 21 Figu e 2.2. a) SVM 300 An on Paa used o iscosi y measu emen s and b) DMA 5000 An on Paa densime e used o densi y measu emen s……………………………………… 22 Figu e 2.3. a) ABBEMAT 500 An on Paa au oma ed e ac ome e and b) CDM210 Radiome e Analy ical………………………………………………………………………... 23 Figu e 2.4. Cells used o liquid-liquid equilib ium measu emen s whe e a u bidi y ansi ion can be obse ed, om a) an immiscible sample o b) a miscible one, as well as he c) expe imen al se up. conduc ime e ……………………………………………………………. 24 Figu e 2.5. Decomposi ion empe a u es e sus mel ing empe a u es o he luo ina ed ionic liquids in his s udy, [C4C1Im][C4F9SO3] and [C10C1Im][C4F9SO3], and hose ound on li e a u e o he same amily………………………………………………………………….. 25 2 Ionic Liquids in he De elopmen o No el Bioma e ials In oduc ion 3 1.1 Gene al con ex The pha maceu ical indus y is acing a g owing p essu e due o en i onmen al issues, pa en expi a ions, leng hening o d ug-de elopmen cycles and mo e demanding egula o y equi emen s (1). Thus, pha maceu ical R&D is mo e han e e ocusing on a ew new molecula compounds o enhance clinical e icacy a low cos (2). The possibili y o p oducing inno a i e medicinal ma e ials “by design” b ough an inc easing numbe o compounds possessing low aqueous solubili y and limi ed bioa ailabili y (1). To o e come hese es ain s pha maceu ical indus ies a e pu suing new s a egies, such as, de elopmen o p od ugs (3), emulsions using su ac an componen s (4), micella sys ems (5,6), solid dispe sions (7) and c ys al enginee ing (8). Ionic liquids (ILs) ha e eme ged as a new class o ma e ials ha ha e conside able po en ial o p o ide ad ances in pha maceu ical applica ions (9–11). Thei negligible apo p essu e a ambien condi ions, low su ace ension, high he mal, chemical and elec ochemical s abili y, and widely unable p ope ies such as iscosi y, densi y, pola i y, hyd ophobici y and sol en miscibili y a e some in e es ing p ope ies o hese compounds (8). These ema kable cha ac e is ics a e a esul o he innume ous possible combina ions be ween ILs cons i u i e ions (10). Due o hei su ac an beha iou , ILs can o m micelles which can be used as d ug-deli e y de ices o spa ingly wa e -soluble d ugs (12). In he pas decades, a emp s o use ILs o enhance bioa ailabili y o d ug molecules ha e been made (5,11,13,14). Fluo ina ed anions such as bis( i luo ome hylsul onyl)imide, hexa luo ophospha e, o e a luo obo a e ha e been equen ly cha ac e ized and explo ed o se e al biomedical applica ions (15). Howe e , ew wo ks ha e ocused on he luo ina ed ionic liquids (FILs) he e cha ac e ized, wi h a anionic luo ina ed alkyl chain o a leas ou ca bons (16). One o he majo in e es in FILs is ha hey combine he bes p ope ies o pe luo oca bon compounds (PFCs) wi h hose ones o ionic liquids (15). Then, FILs can be use ul in a eas we e PFCs a e applied such as o oxygen he apeu ics o enhance oxygen anspo , pulmona y d ug deli e y de ices wi h dispe sions o d ug mic opa icles and mic oemulsions, gel mic oemulsions o de mal d ug deli e y, and also luo ina ed mic ocon aine s o gene al d ug deli e y (17,18). The main ocus o his wo k is he de elopmen o luo ina ed ionic liquids o biomedical applica ions in o de o esol e some o he abo emen ioned d awbacks o he pha maceu ical indus y. To design he mos sui able FILs as no el and imp o ed ma e ials o pha maceu ical applica ions, such as d ug deli e y sys ems, he he mophysical and pa i ion p ope ies, solu ion and sel -agg ega ion beha iou we e e alua ed. Fu he mo e, he hyd ogen-bonding abili y and pola izabili y we e also s udied o add ess he ele ance o designing unc ionalized (so-called ask-speci ic) ILs ha ha e been syn hesized wi h a pa icula applica ion in mind, e.g. dissolu ion o he apeu ic molecules. Ionic Liquids in he De elopmen o No el Bioma e ials In oduc ion 4 1.2 Ionic Liquids Ionic liquids a e sal s, en i ely cons i u ed o ions whe e ca ions a e o ganic while anions can be o ganic o ino ganic (8). The mos common amilies o ca ions and anions used o biomedical applica ions a e shown in Figu e 1.1. and Figu e 1.2., espec i ely (19,20). Figu e 1.1. Mos common ca ions s uc u es o ILs: a) py olidinium; b) imidazolium; c) pipe idinium; d) py idinium; e) ammonium and ) choline whe e R could be a hyd ogena ed chain, a luo ina ed chain o a unc ional g oup. Figu e 1.2. Mos common anion s uc u es o ILs: a) chlo ide; b) b omide; c) e a luo obo a e; d) hexa luo ophospha e; e) ni a e; ) ace a e; g) dicyanamide; h) dime hylphosphona e i) me hylphosphona e j) e hylphosphona e; k) i luo ome hanesul ona e and l) bis( i luo ome hylsul onyl)imide. Ionic Liquids in he De elopmen o No el Bioma e ials In oduc ion 5 The possibili y o manage he inal cha ac e is ics h ough he di e se combina ion o ca ions and anions make his compounds sui able o nume ous applica ions (21). Nowadays, ILs a e used as eac ion media o o ganic ans o ma ions, sepa a ions and ex ac ions (10) in nano echnology, bio echnology (20) and enginee ing p ocesses (22). In he biomedical ield, ILs ha e p o en o be use ul as sol en s o enhance solubili y o poo wa e soluble d ugs, and hus o be used as d ug deli e y de ice (8,23,24). In Figu e 1.3. a e ep esen ed some o he ele an applica ions whe e ILs ha e been ex ensi ely s udied. Figu e 1.3. Rele an applica ions o ionic liquids as bioma e ials in chemis y, chemical enginee ing, bio echnology and pha maceu ics indus ies. 1.2.1 Fluo ina ed Ionic Liquids Fluo ina ed ionic liquids a e de ined as ionic liquids wi h luo ine ags equal o longe han ou ca bon a oms (15). The in e es in hese compounds is ha hey can combine he bes p ope ies o ILs wi h hose ones o pe luo oca bons compounds, such as, excep ional chemical and biological ine ness, low su ace ension and e ac i e index, ex eme su ace ac i i y, high densi y and excellen sp eading cha ac e is ics (17,25). In oducing luo ine a oms o he IL’s composi ion can gene a e no el beha iou and an unma ched pe o mance in se e al p ocesses in ol ing biomolecules (16,26–28). The high ioniza ion po en ial, high elec onega i i y and low pola izabili y a e some o he a ibu es ha poin ed ou he elemen luo ine (29). The he mal and chemical s abili y o F-chains a e due o he s eng h o he C-F bonds, he s onges single bond in o ganic chemis y (30). Addi ionally, esis ance o F-chains o me abolism could be use ul in enzyma ic ma e ials (27). IONIC LIQUIDS Chemis y O ganic syn hesis and nanoma e ial syn hesis Chemical enginee ing Sepa a ion, ex a ion and ca aly ic eac ions Pha maceu ics D ug o mula ion, d ug deli e y and o mula ion o APIs Bio echnology Bioca alysis and bio uel p oduc ion Ionic Liquids in he De elopmen o No el Bioma e ials In oduc ion 6 One o he mos ema kable cha ac e is ics o FILs is hei amphiphilic beha iou which enhance hei sel -agg ega ion (31). Due o he luo ina ed componen s he e is a mo e o ganized space (a molecula , nanome e and mic ome e scales) inc easing he hyd ophobic seg ega ion e ec and making hem sui able su ac an s (32). P e ious wo ks shown ha he c i ical micella concen a ion (CMC) alues o sho chain luo ina ed su ac an s a e analogous o hose o hyd ogena ed su ac an s wi h longe chain leng hs as will be u he discussed (31). Al hough luo ina ed compounds ha e been ex ensi ely in es iga ed (18), no signi ican s udies using FILs o biomedical applica ions ha e been ca ied ou . Mo eo e , a comp ehensi e in es iga ion o FILs p ope ies is needed o e alua e he applica ion o hese ema kable ILs. 1.2.2. Applica ions o Ionic Liquids as Bioma e ials One o he mos challenging p oblems aced by he pha maceu ical indus y is he limi ed aqueous solubili y p esen in 20-30% o he pha maceu ical compounds which en ails a e y poo bioa ailabili y (33). The e o e, o inc ease d ugs solubili y se e al app oaches ha e been desc ibed such as he use o liquid and gel mic oemulsions , sal o ma ion and coa ing sys ems (8). Ionic liquids we e ound o play a special ole in he pha maceu ical indus y (10). As unable chemicals hey a e ideal candida es o inco po a ing a ious unc ional g oups, including biologically ac i e subs ances (8,15). An impo an se ies o ionic liquids based on he 1-alkyl-3-me hylimidazole [CnC1Im]+ ca ion, whe e n is he ca bon numbe in he alkyl g oup, we e ex ensi ely in es iga ed (34). These imidazolium-based ILs can ha e amphiphilic beha iou depending on he alkyl side chain, such as ca ionic su ac an s (34). Due o hei hyd ophobic chains and pola imidazolium g oups hey ha e been called “su ac an -like” ionic liquids (35). In he pas decades, imidazolium-based ILs we e explo ed o se e al pha maceu ical and indus ial applica ions as illus a ed in Figu e 1.4.. The i s wo k ha uses IL-based mic oemulsions as d ug deli e y de ices was epo ed in 2010 by Moni uzzanan e al. (36). In his s udy, he solubili y o acyclo i (ACV), a poo wa e and o ganic soluble an i i al, was es ed in [C1-4C1Im]+ imidazolium-based ILs combined wi h se e al anions. I was obse ed ha ACV was only soluble on IL wi h ace a e ([C1CO2]-) and dime hylphospha e ([(CH3O)2PO2]) anions. Despi e he enhanced solubili y, u he es s o ep esen anspo o ACV h ough skin we e pe o med and no signi ican esul s we e ound. The e o e, dime hylimidazole dime hylphospha e ([C1C1Im][(CH3O)2PO2])) was es ed as a co-sol en o AVC mic oemulsion oge he wi h Tween-80 and Span-20 comme cial su ac an s. The esul s show ha he skin pe meabili y was inc eased by se e al o de s o magni ude compa ed o esul s ob ained o he c eam cu en ly a ailable on ma ke (37). Since hen, ionic liquids ha e been s udied as addi i es in se e al o mula ions as well as enhance s o d ug solubili y. Ionic Liquids in he De elopmen o No el Bioma e ials In oduc ion 7 Goindi e al. used 1-bu yl-3-me hylimidazolium hexa luo ophospha e ([C4C1Im][PF6]) oge he wi h Tween 80 and e hanol o c ea e a ype gel mic oemulsion o a opical deli e y o e odolac (ETO) (38). ETO 1 demons a e poo o al bioa ailabili y in which gas oin es inal and ca dio ascula side e ec s a e no iced (39). To o e come hese side e ec s and ob ain high d ug concen a ion a he a ge si e, opical applica ion o d ug seems o be an ideal ou e o adminis a ion (38). Thus, he au ho s compa ed he esul s ob ained om he gel- ype mic oemulsion wi h he a ailable comme cial o mula ion (P oxym gel®) (38). The esul s showed a d op in g anuloma issue weigh in mice up o 20 %w wi h he IL- o mula ions. In addi ion, a dec ease in he numbe o leukocy es on he a ec ed a ea whe e obse ed (7800 o IL o mula ions and 8333.33 o P oxym gel®) demons a ing he an i-in lamma o y e ec (38). The au ho s jus i ied he esul s on he basis o an enhanced pe mea ion o ETO h ough he IL (38). In addi ion, Wang e al. epo ed ha 1-hexadecyl-3-me hyllimidazolium b omide ([C16C1Im] B ) p esen ed mo e sui able esul s o p epa ing d ug-loading nanopa icle mic oemulsions in compa ison wi h con en ional su ac an s molecules (40). In his pape , nanopa icles we e loaded wi h me hylene blue (d ug model) and he IL enhanced encapsula ion and mic oemulsion s abiliza ion up o 2h. Mo eo e , a d ug elease p o ile was conduc ed and me hylene blue showed a elease p ocess almos up o 8 hou s wi h an 99.9% e iciency (40). Figu e 1.4. Applica ion a eas o imidazolium-based su ac an s. Adap ed om li e a u e (35). Taking in o accoun he use o ILs as nanoca ie s o d ug deli e y de ices, Mahkam e al. p epa ed a no el pH-sensi i e nanoca ie o colon speci ic d ug deli e y 2 (41). An imidazolium chlo ide based 1 E odolac (ETO) is a poo ly wa e soluble nons e oidal an i-in lamma o y (NSAID) used o elie e in lamma ion, swelling, s i ness, and pain associa ed wi h heuma oid a h i is and os eoa h i is (39) 2 Colon-speci ic d ug deli e y sys em is a so o pH sensi i e sys em ha p o ec s he d ugs om deg ada ion in he acidic medium o s omach and a e passing he acidic medium, hey elease con aining d ugs a he alkaline media o colon (pH 7.4) (41) Ionic Liquids in he De elopmen o No el Bioma e ials In oduc ion 8 IL was placed be ween mon mo illoni e (MMT) laye s and copolyme ized wi h me hac ylic acid (MAA) o encapsula e nap oxen (used o ea pain o in lamma ion). The inco po a ion o posi i e cha ges in o he amewo k o he nanocomposi es (IL++MMT+MAA) p omo ed a con olled d ug ealise by changes o he pH media. By inc easing he pH alue (pH 7.4), di usion o he hyd olysing agen s in o he ca ie is inc eased, he e o e he a e o hyd olysis inc eased (41). Tang and co-wo ke s ha e epo ed o he i s ime an amazing use o ILs as mic owa e sensi iza ion agen (42). By loading polydopamine (PDA) nanopa icles wi h 1-bu yl-3-me hylimidazolium hexa luo ophospha e ([C4C1Im] [PF6]) i was possible o enhance encapsula ion o doxo ubicin (DOX). Since [C4C1Im][PF6] is conside ed highly suscep ible o mic owa e i adia ion, a combined chemo he apy and mic owa e he mal he apy (MWTT) was p oposed o imp o e he deli e y e ec o he d ug o umou si es. The esul s showed ha IL-PDA-DOX nanopa icles can abla e DOX unde mic owa e adia ion inhibi ing e icien ly he umou size wi hou inducing any app eciable issue oxici y (42). In Figu e 1.5 is illus a ed he e ec o IL-PDA-DOX nanopa icles. Figu e 1.5. Schema ic illus a ion o he use o IL-PDA-DOX nanopa icles o combined chemo he apy wi h MWTT. Adap ed om li e a u e (42). Ionic liquids a e powe ul sol en s and elec ically conduc ing luids (10). They can be used as bioma e ials o enhance de ec ion o biomolecules h ough elec opho e ic echniques as desc ibed by Abd El-Hady e al. (43). In his wo k, an elec ochemical de ec o (ECD) based on 1-oc yl-3- me hylimidazolium hexa luo ophospha e ([C8C1Im][PF6]) was de eloped o imp o e de ec ion o i amins B2, B6 and C. This de ice, coupled wi h capilla y elec opho esis (CE) based on micelle collapse 3 (see Figu e 1.6), enhanced he micella encapsula ion o i amins and p e en ed i amin deg ada ion on he elec opho esis bu e /solu ion media. Thus, he sensi i i y o he me hod was subs an ially imp o ed up o 5000- old compa ed o con en ional CE coupled wi h UV- isible echniques (43). 3 Analy e ocusing echnique achie ed by micelle collapse. The CE/AFMC me hod is based on he p esence o a sample zone con ained he micelles- o ming agen wi h highe conduc i i y a he beginning o he un. Due o concen a ion di e ence a micelle dilu ion zone is o med. Subsequen ly, micelles we e collapsed and he analy e is accumula ed (43). Ionic Liquids in he De elopmen o No el Bioma e ials In oduc ion 9 In addi ion, Abd El-Hady and co-wo ke s also es ed he use o ILs o s abilize i amins in biological samples in which 1-oc yl-3-me hylimidazolium b omide ([C8C1Im] B ) showed he bes esul . The samples we e iable up o 5 days unde lab s o age condi ions (25ºC, ligh and ai oxygen) wi hou he need o leng hy and ex ensi e p e ea men (43). Figu e 1.6. T anspo , elease, and accumula ion o analy es by micelle collapse. Adap ed om li e a u e (44). The echnological u ili y o biomolecules can also be signi ican ly enhanced by using ILs a he han o he o ganic sol en s o aqueous eac ion media due o he IL’s unique sol en cha ac e is ics (10). In Figu e 1.7. a e ep esen ed some o he ad an ages o using ILs o biomolecula p ocesses in con as wi h o he o ganic sol en s. Figu e 1.7. Ad an ages o using ILs o biomolecula p ocesses (20). ADVANTAGES OF USING ILS FOR BIOMOLECULAR PROCESSES  Oppo uni y o design pa icula biop ocesses due o ailo -made and inimi able physical, chemical and biological p ope ies o ILs.  In se e al cases, biomolecules ha e become mo e soluble and s able (bo h ope a ionally and he mally) in p esence o ILs.  Easy eco e ies o sub ac , p oduc s and ILs a e bioca alys s and bio ans o ma ion  P e en sel -agg ega ion o biomolecules du ing solubilisa ion p ocess  Enhance solubili y in o insoluble/spa ingly soluble sub ac s in ILs p omo ing biomolecula eac ions  Enhance immobiliza ion o biomolecules in highly iscous ILs o biochemical p ocesses Ionic Liquids in he De elopmen o No el Bioma e ials In oduc ion 10 Fo ins ance, DNA can be ex ac ed om biological samples using he phenol/chlo o o m me hod whe e he p o eins a e dissol ed in he o ganic medium (phenol/chlo o o m) while DNA emains in he aqueous solu ion (45). Howe e , his ype o p ocesses can in ol e oxic o ganic compounds and ha e se e al ime-consuming s eps (45). Recen ly, a numbe o s udies epo ed he use o ILs o sepa a ion and ex ac ion o nuclei acids, especially DNA (46,47). A p e ious s udy employed he 1-bu yl-3- me hylimidazolium hexa luo ophospha e ([C4C1Im][PF6]) IL o di ec ex ac ion o DNA om aqueous solu ion using he liquid-liquid ex ac ion echnique (LLE) (48). The in e ac ions be ween he imidazolium ca ion alkyl chain and he phospha e g oups o he DNA we e deemed esponsible o he DNA/IL binding and he e icien ex ac ion and pu i ica ion (48). Ga cía e al. also es ed se e al ILs o p oduce a as and e icien s a egy o he ex ac ion o DNA di ec ly om maize (49). The esul s sugges ed ha 1-e hyl-3-me hylimidazolium dime hylphospha e ([C2C1Im] [(CH3)2PO4)]) was he mos e icien ex ac ion sys em wi h a yield anging om 696.7-721.2 μg DNA / g sample (49). These esul s imp o e he common a ailable me hod based on he su ac an ce yl ime hylammonium b omide (CTAB) wi h a yield be ween 252.3 and 256.2 μg DNA / g sample. Mo eo e , his echnique demons a ed imesa ings o app oxima ely 3h and he DNA ex ac ed was s able when s o ed a oom empe a u e o 10 days (49). The hyd ogen bonding in e ac ions be ween ILs and DNA we e conside ed an impo an pa ame e when ILs a e used as sol en in ex ac ion p ocess (49,50). Addi ionally, Li e al. s udied wo speci ically designed ILs, namely 1-(1,2-dihyd oxyp opyl)-3- decylimidazolium b omide ([C10POHIM] B ) and 1-(1,2-dihyd oxyp opyl)-3-hexadecylimidazolium b omide ([C16POHIM] B ) which con ain wo hyd oxyl g oups capable o o ming hyd ogen bonds (45). Conside ing he ex ac ion e iciency, [C10POHIM] B and [C16POHIM] B show he mos sui able yields (52.4% and 95.2%, espec i ely) when compa ed wi h o he ILs con aining simila s uc u es bu lacking hyd oxyl unc ional g oups (45). Thus, he impo ance o he alkyl chain o he IL o DNA biding was also p o ed in his wo k (45). Fo success ully design o IL i is c ucial o mee he equi emen s o he pa icula ask. Fo ins ance, in IL-based ex ac ion p ocesses, he numbe o ca bons p esen in he alkyl chain is aken in o conside a ion since i in luences he abili y o o m biphasic sys ems (51). The ac i i y and s abili y o enzymes, such as lipases, p o eases, alcohol dehyd ogenases and oxido educ ases, ha e been e alua ed using ILs as media (20). The pola i y, ca ion/anion na u e, alkyl chain leng h, hyd ophobici y and iscosi y a e some o he main ac o s ha can a ec he enzyme pe o mance (52). Rega ding he anion, hey can ha e a p o ound e ec on he s abili y and ac i i y o enzymes due o hei high hyd ogen bond o ming capabili ies which s ongly in e ac wi h p o eins and can cause con o ma ional changes (20). No i omi e al. compa ed he e ec o a ious anions on he ac i i y o lysozyme and ound ha i had lowe ac i i y in [C2C1Im] Cl a he han [C2C1Im][BF4] and [C2C1Im] [NT 2] (53). In his case, i was sugges ed ha luo ina ed anions can p omo e s abili y o his Ionic Liquids in he De elopmen o No el Bioma e ials In oduc ion 11 enzyme (53). In ac , some wo ks ha e epo ed ha enzymes a e mos ac i e in a p esence o a hyd ophobic anion, such as [BF4]- and [PF6]-. On he o he hand, he mos deac i a ing enzyma ic eac ions a e ound when a e used ILs con aining anions capable o b eaking hyd ogen bonds, such as Cl- (20). Machado and Sa ai a in es iga ed he e ec o he ca ion chain leng h o alkyl-imidazolium based ILs (e hyl-, bu yl-, and hexyl-imidazolium based ILs) on he enzyme ac i i y o ho se adish pe oxidase(54) and ound ha 1-hexyl-3-me hylimidazolium chlo ide ([C6C1Im] Cl) lowe ed he pe oxidase enzyme ac i i y. Co ela ing he enzyme s abili y and nanoca ie s, Soni e al. epo ed he inhe en an i umo e iciency o sel -assembled phy ase enzyme nanosphe es (55). This e iciency can be enhanced wi h he use o pla inum nanopa icles loaded wi h he an icance d ug cu cumin. In his s udy, he 1-bu yl-3- me hylimidazolium e a luo obo a e IL ([C4C1Im][BF4]) was used o o m he apeu ically ac i e phy ase nanosphe es (55). Figu e 1.8. illus a es he sel -assembled phy ase nanosphe es p omo ed by he IL. Figu e 1.8. Schema ic illus a ion o sel -assembly o phy ase enzyme (Aspe gillus nige phy ase) in [C4C1Im][BF4], leading o phy ase nanosphe es and pla inum–phy ase nanosphe e. Cu cumin was loaded on bo h samples and was en apped in inne hyd ophobic domains o p o ein empla e. Adap ed om li e a u e (55). Al hough he numbe o publica ions ega ding ILs has g own exponen ially, he e a e s ill nume ous unexplo ed applica ions o FILs amily. Fluo oca bons o luo ina ed amphiphiles ha e been widely s udied o hei applica ion in oxygen he apeu ics, pulmona y d ug deli e y sys ems, gel emulsions and micelle ype con aine s o d ug deli e y de ices (17,18). Recen ly, i was pa en ed (56) he use o luo opolyme -based emulsions o he in a enous deli e y o luo ina ed ola ile anaes he ics. This ype o emulsion enhanced he capabili y o deli e ing and eleasing con olled amoun s o luo ina ed 18 Ionic Liquids in he De elopmen o No el Bioma e ials Cha ac e iza ion o Fluo ina ed Ionic Liquids: The mophysical, Phase Equilib ia and Agg ega ion Beha iou 19 2.1 In oduc ion One o he mos ou s anding ad an age o ionic liquids, and hus o luo ina ed ionic liquids, is he possibili y o uning he inal cha ac e is ics h ough in ini e combina ions be ween ca ions and anions. This manipula ion allows o con ol se e al cha ac e is ics such as he mal and he mophysical p ope ies, solubili y, hyd ophobici y and oxici y (1). As he in e es in ionic liquids inc ease exponen ially, he demand o a comp ehensi e in es iga ion o hei physicochemical p ope ies is clea . In o de o e alua e he ad an ages o luo ina ed ionic liquids, he modynamic, he mophysical and he mal cha ac e iza ion, liquid-liquid equilib ia, as well as he sel -agg ega ion beha iou in aqueous solu ion we e ob ained o imidazolium-based ionic liquids wi h he pe luo obu anesul ona e anion. De e mina ion o densi y and iscosi y could headligh FILs anspo p ope ies (2). On he o he hand, iscosi y can es ima e he FIL ease o mo emen h ough di e en sol en s, also called luidi y (3). Addi ionally, ionic conduc i i y ela ed wi h FIL concen a ion may p edic he o ma ion o agg ega es (4). The ela ionship be ween conduc i i ies and iscosi y, known as ionici y, is also impo an o cha ac e ize pu e FILs (5). Fo ins ance, pe luo ina ed compounds (PFC) p o ed o be use ul in se e al biomedical applica ions such as a i icial blood subs i u es and in i o gas ca ie s (6). Since luo ina ed ionic liquids sha e some common cha ac e is ics wi h PFC (1), a compa ison be ween hese wo compounds is done when possible. The sel -agg ega ion beha iou o some FILs in aqueous solu ions induces he o ma ion o sel - assembled s uc u es ha can be used ad an ageously in he deli e y o di e en he apeu ic compounds (4). Fo example, d ug molecules exhibi ing poo solubili y due o hei high molecula weigh s and s uc u e complexi y ha lead o low bioa ailabili y (7). Acco dingly, designing no el excipien s wi h uneable physicochemical p ope ies and capable o o ming micelles o mic oemulsions is p omising. Wi h his aim in mind, a solubili y and sel -agg ega ion beha iou analysis o imidazolium-based luo ina ed ILs was conduc ed. In conclusion, he da a ob ained in his chap e will allow he c i ical e alua ion o FILs he mophysical p ope ies, and solu ion beha iou , acknowledging hei use as bioma e ials such as gas ca ie compounds, su ac an o mic oemulsions o nanos uc u ed d ug deli e y componen s (8). 2.2 Ma e ials 1-Bu yl-3-me hylimidazolium pe luo obu anesul ona e, ≥98% mass ac ion pu i y; and 1- decyl-3-me hylimidazolium pe luo obu anesul ona e, ≥98% mass ac ion pu i y; we e syn he ized in ou lab acco ding o he ion exchange esin me hod (9). 1-Hexyl-3-me hylimidazolium pe luo obu anesul ona e, >99% mass ac ion pu i y; 1-oc yl-3-me hylimidazolium Ionic Liquids in he De elopmen o No el Bioma e ials Cha ac e iza ion o Fluo ina ed Ionic Liquids: The mophysical, Phase Equilib ia and Agg ega ion Beha iou 20 pe luo obu anesul ona e, >99% mass ac ion pu i y and 1-dodecyl-3-me hylimidazolium pe luo obu anesul ona e, >98% mass ac ion pu i y we e acqui ed om IoLiTec. S uc u es and ac onyms a e lis ed in Table 2.1 In o de o con i m he pu i y o he compounds 1H and 19F NMR we e pe o med. The compounds we e i s d ied unde a 3·10-2 To acuum wi h a con inuous s i ing a 323.15 K o a leas 48h o educe ola ile impu i ies. The wa e con en o FILs used in his wo k was de e mined using Ka l Fishe Table 2.1. Designa ion and chemical s uc u e o each ionic liquid used along his s udy. IL designa ion Chemical s uc u e 1-Bu hyl-3-me hylimidazolium pe luo obu anesul ona e [C4C1Im][C4F9SO3] 1-Hexyl-3-me hylimidazolium pe luo obu anesul ona e [C6C1Im][C4F9SO3] 1-Oc yl-3-me hylimidazolium pe luo obu anesul ona e [C8C1Im][C4F9SO3] 1-Decyl-3-me hylimidazolium pe luo obu anesul ona e [C10C1Im][C4F9SO3] 1-Dodecyl-3-me hylimidazolium pe luo obu anesul ona e [C12C1Im][C4F9SO3] Ionic Liquids in he De elopmen o No el Bioma e ials Cha ac e iza ion o Fluo ina ed Ionic Liquids: The mophysical, Phase Equilib ia and Agg ega ion Beha iou 21 coulome ic i a ion me hod (Me ohm 831 KF Coulome e ) and i was less han 100 ppm. Milli-Q ul apu e wa e (Milli-Q In eg al Wa e Pu i ica ion Sys em) was used when necessa y h oughou he wo k. 2.3 Expe imen al P ocedu es 2.3.1 Nea Fluo ina ed Ionic Liquids 2.3.1.1. The mal P ope ies The he mal s abili ies o he FILs we e de e mined using a he mog a ime ic analysis (TGA), using he TA Ins umen model Q50 ep esen ed in Figu e 2.1. a). Samples o FILs we e loaded in an aluminium pan and hea ed wi h a ni ogen low a e o 60 ml·min–1 un il comple e he mal deg ada ion using a scan a e o 1 K·min–1. TA Uni e sal Analysis (so wa e e sion 4.4) was used o de e mina e he onse empe a u e (Tonse ), s a ing empe a u e (Ts a ) and decomposi ion empe a u e (Tdec) which co espond o he empe a u es a which he baseline slope changed du ing hea ing, he weigh loss was less han 1%, and he weigh loss was 50%, espec i ely. Duplica es we e measu ed and he unce ain y o hese empe a u es is ± 3 K. Mel ing poin s we e de e mined using a Di e en ial Scanning Calo ime e (DSC), TA Ins umen model Q200 (see Figu e 2.1. b)), calib a ed wi h an Indium s anda d. In hese expe imen s, samples o FILs we e loaded in an aluminum pan and con inuously pu ged wi h 50 ml∙min-1 o ine ni ogen gas. Nex , he samples we e cooled o 183.15 K, empe ed du ing 30 min, and hea ed o 313.15 K. This p ocess was epea ed h ee imes a di e en a es (10 K·min-1, 5 K·min-1 and 1 K·min-1). The ob ained alues om he second and subsequen cycles we e ep oducible a he same a e. Duplica es we e measu ed and he unce ain y o he mel ing empe a u e is ±2K Figu e 2.1. a) TA ins umen Model TGA Q50 used o he measu emen s and b) TA Ins umen Model DSC Q200 used o he measu emen s. a) b) Ionic Liquids in he De elopmen o No el Bioma e ials Cha ac e iza ion o Fluo ina ed Ionic Liquids: The mophysical, Phase Equilib ia and Agg ega ion Beha iou 22 2.3.1.2. Viscosi y and Densi y Measu emen s Measu emen s o dynamic iscosi y we e ca ied ou using an au oma ed SVM 3000 An on Paa o a ional S abinge iscome e (see Figu e 2.2. a)) ope a ing a a mosphe ic p essu e in he empe a u e ange 288.15 o 358.15 K. The equipmen uses Pel ie elemen s o as and e icien he mos abili y wi h a empe a u e unce ain y o ± 0.02 K. Duplica es we e measu ed and he epo ed da a a e a e age alues wi h a maximum ela i e s anda d de ia ion o 1%. The unce ain y o he expe imen al alues, aking in o accoun he pu i y and handling o he samples is es ima ed o be 2%. Densi y was measu ed wi h an An on Paa ib a ing ube densime e , model DMA 5000 (see Figu e 2.2. b)) ope a ing a a mosphe ic p essu e and in he empe a u e ange 288.15 K o 358.15 K. The e ec o iscosi y was conside ed in all expe imen s using he in e nal calib a ion. The empe a u e was con olled by se e al Pel ie uni s and he cell was embedded in a ca i y inside a me allic block. This equipmen allowed a empe a u e s abili y o 0.002 K. Duplica es we e measu ed and he epea abili y and expanded unce ain y o he densi y is be e han 5·10-5 and ± 3·10-4 g·cm-3, espec i ely. Figu e 2.2. a) An on Paa SVM 300 used o iscosi y measu emen s and b) An on Paa DMA 5000 densime e used o densi y measu emen s. 2.3.1.3. Re ac i e Index Measu emen s An An on Paa ABBEMAT 500 au oma ic e ac ome e wi h a esolu ion o ± 10-6 was used o measu e he e ac i e index. The equipmen was calib a ed using Millipo e quali y wa e and e achlo oe hylene p o ided by he supplie . Duplica es we e measu ed and he epo ed esul s ha e an unce ain y o ± 4·10-5. The appa a us is ep esen ed in Figu e 2.3. a). 2.3.1.4. Ionic Conduc i i y Measu emen s Ionic conduc i i y measu emen s we e de e mined using a Radiome e Analy ical CDM210 conduc ime e in a jacke ed glass cell wi h a magne ic s i e as ep esen ed in Figu e 2.3. b). a) b) Ionic Liquids in he De elopmen o No el Bioma e ials Cha ac e iza ion o Fluo ina ed Ionic Liquids: The mophysical, Phase Equilib ia and Agg ega ion Beha iou 23 Figu e 2.3. a) An on Paa ABBEMAT 500 au oma ed e ac ome e and b) Radiome e Analy ical CDM210 conduc ime e . A wa e ba h was used o he mos a ize he cell and he empe a u e was measu ed by a pla inum esis ance he mome e coupled o a Kei hley 199 Sys em DMM/Scanne wi h an unce ain y o ± 0.01 K. To ensu e a secu e seal and o p e en humidi y, he sample was closed wi h d y ni ogen and sc ew caps. The calib a ion was pe o med a each empe a u e wi h 0.01 D KCl s anda d solu ions supplied by Radiome e Analy ical. The ionic conduc i i y alues we e alida ed as desc ibed p e iously (10). Duplica es we e measu ed and he epo ed da a a e he a e age alues wi h an unce ain y o ± 1% 2.3.2. Mix u es o Fluo ina ed Ionic Liquids and Wa e 2.3.2.1. Liquid-liquid Equilib ia In o de o de e mine he empe a u e-composi ion phase diag ams, he u bidi y o he bina y sys em FIL + wa e was analyzed using a isual me hod a a mosphe ic p essu e. Ini ial immiscible samples we e p epa ed di ec ly in Py ex glass cells wi h inco po a ed magne ic s i e s using an analy ical high-p ecision balance (unce ain y o ± 0.00002 g). Then, he cell was imme sed in a he mos a ic ba h (a mix u e o wa e and e hylene glycol was used as he mos a ic luid) and bo h mix u e and he mos a ic luid we e con inuously s i ed. The liquid-liquid phase ansi ion was accomplished by aking he empe a u e when he u bidi y o he sys em disappea ed. The empe a u e was measu ed using a ou -wi e pla inum esis ance P 100 he mome e coupled o a Kei hley 199 Sys em DMM/Scanne mul ime e (unce ain y o 0.01 K). The o e all unce ain y o he empe a u e ansi ions in his isual me hod was es ima ed o be ± 0.5 K and he unce ain y o he mix u e composi ion was ± 0.0001 in mass ac ion. Figu e 2.4. depic s (a) cell wi h isible u bidi y co esponding o immiscibili y, a (b) cell wi h o al miscibili y and (c) he expe imen al se up o pe o ming liquid-liquid equilib ia measu emen s a) b) Ionic Liquids in he De elopmen o No el Bioma e ials Cha ac e iza ion o Fluo ina ed Ionic Liquids: The mophysical, Phase Equilib ia and Agg ega ion Beha iou 24 Figu e 2.4. Cells used o liquid-liquid equilib ium measu emen s whe e a u bidi y ansi ion can be obse ed, om a) an immiscible sample o b) a miscible one, as well as he c) expe imen al se up. 2.3.2.2. C i ical Micella Concen a ion To de e mine he c i ical micella concen a ion (CMC), he Philips de ini ion was used (11). Acco ding o Philips e al., he concen a ion a which he e is a maximum change in g adien in he conduc i i y-concen a ion cu e co esponds o a CMC. The ionic conduc i i ies we e measu ed a 298.15 K and pe o med wi h a Radiome e Analy ical CDM210 conduc ime e using a CDC749 elec ode in a glass cell wi h a magne ic s i e (see Figu e 2.3. b)). The equipmen was calib a ed o each empe a u e by using a ce i ied 0.01 D KCl s anda d solu ion p o ided by Radiome e Analy ical. The cell was he mos a ized wi h a wa e ba h and he empe a u e was measu ed using a pla inum esis ance he mome e coupled o a Kei hley 199 sys em DMM/scanne (unce ain y o ±0.01 K). Each aqueous solu ion was added o he cell and s i ed o he expe imen al measu emen s. Conduc i i y was measu ed a leas h ee imes and he unce ain y o each sample was es ima ed o be 1%. 2.3.2.3. T ansmission Elec on Mic oscopy (TEM) The samples analyzed wi h mic oscopy we e placed on a 200 nesh coppe g id wi h 3 mm diame e using a o m a ilm. A il e pape was used o emo e he excess o aqueous solu ion. Solu ions we e d ied and measu ed using a Philips CM20 model wi h LaB6 ilamen , a a wo king ol age o 200 kV. These measu emen s we e ca ied ou by an ex e nal se ice (Uni e si y o Vigo). 2.4. Resul s and Discussion 2.4.1. Nea Fluo ina ed Ionic Liquids 2.4.1.1. The mal P ope ies Mel ing and decomposi ion empe a u es a e one o he mos ema kable p ope ies o ionic liquids which de e mine he liquid ange, and, consequen ly, hei ange o applica ions (1). Fo his a) b) c ) Ionic Liquids in he De elopmen o No el Bioma e ials Cha ac e iza ion o Fluo ina ed Ionic Liquids: The mophysical, Phase Equilib ia and Agg ega ion Beha iou 25 pu pose, [C4C1Im][C4F9SO3] and [C10C1Im][C4F9SO3] we e analyzed and in Table 2.2. a e epo ed he he mal p ope ies (onse empe a u e, Tonse , s a empe a u e, Ts a , decomposi ion empe a u e, Tdec, and mel ing empe a u e, Tm) o he luo ina ed ionic liquids s udied in his wo k. Table 2.2. The mal p ope ies o luo ina ed ionic liquids: s a ing empe a u e, Ts a , onse empe a u e, Tonse , decomposi ion empe a u e, Tdec, and mel ing empe a u e, Tm a 1 K·min-1. FIL Tm / K Ts a / K Tonse / K Tdec / K [C4C1Im][C4F9SO3] 286 554 638 670 [C10C1Im][C4F9SO3] 307 545 627 655 Expe imen al da a a e plo ed in Figu e 2.5. whe e he mel ing empe a u es speci y he empe a u e a which he compound becomes liquid and he decomposi ion empe a u es de e mine he uppe ope a ing empe a u e a which he luid can be used (3). Also, he ob ained esul s o he s udied FILs we e compa ed wi h he luo ina ed ionic liquids [C2C1Im][C4F9SO3], [C6C1Im][C4F9SO3], [C8C1Im][C4F9SO3] and [C12C1Im][C4F9SO3] om p e ious wo ks (1,3). Figu e 2.5. Decomposi ion empe a u es e sus mel ing empe a u es o he luo ina ed ionic liquids in his s udy, [C4C1Im][C4F9SO3] and [C10C1Im][C4F9SO3], and hose ound on li e a u e o he same amily (1,3). In o de o e alua e he use o hese luo ina ed ionic liquids o biomedical pu poses he mos ele an empe a u e is 310.15K, which is accep ed o be he a e age body empe a u e in humans (1). Wi h he a ailable da a, i is possible o analyse he e ec o he inc emen o he hyd ogena ed alkyl side chain leng h on he imidazolium-based luo ina ed ionic liquids wi h he pe luo obu anesul ona e anion, [C4F9SO3]-. Taking in o accoun he onse empe a u e alues, Tonse , no subs an ial di e ences we e ound. These high he mal s abili ies a e cha ac e is ic o hese compounds and an ad an age o se e al applica ions in he elec ochemical ield, o example as ba e y elec oly es (12). Compa ing he decomposi ion Mel ing Tempe a u e / K 280 290 300 310 320 Decomposi ion Tempe a u e / K 630 642 654 666 678 [C2C1Im][C4F9SO3] [C4C1Im][C4F9SO3] [C6C1Im][C4F9SO3] [C8C1Im][C4F9SO3] [C10C1Im][C4F9SO3] [C12C1Im][C4F9SO3] Tempe a u e = 310.15 K (37ºC) Ionic Liquids in he De elopmen o No el Bioma e ials Cha ac e iza ion o Fluo ina ed Ionic Liquids: The mophysical, Phase Equilib ia and Agg ega ion Beha iou 26 empe a u es (Tdec), co esponding o 50% o weigh loss o he compound, i is possible o obse e ha he inc emen o he hyd ogena ed side chain dec eases his empe a u e. When analyzed wi h o he FILs i leads o he ollowing end: [C4C1Im]+ ≈ [C2C1Im]+ ≈ [C6C1Im]+ > [C8C1Im]+ ≈ [C10C1Im]+ > [C12C1Im]+. The same end is obse ed o he mel ing empe a u es: [C4C1Im]+ ≈ [C2C1Im]+ ≈ [C6C1Im]+ > [C10C1Im]+ ≈ [C8C1Im]+ > [C12C1Im]+. These ends a e in ag eemen wi h he li e a u e, whe e he inc emen o he hyd ogena ed alkyl chain leng h is ela ed o an inc ease on he mel ing and dec ease on he decomposi ion empe a u es (1,3,5). 2.4.1.2. The mophysical Cha ac e iza ion The he mophysical p ope ies measu ed in his wo k, namely densi y, dynamic iscosi y, e ac i e index and ionic conduc i i y o he wo luo ina ed ionic liquids s udied in his hesis a e lis ed in Table 2.3. as unc ion o empe a u e. The densi ies, luidi ies (1 / dynamic iscosi y) and ionic conduc i i ies a e ep esen ed in Figu e 2.6., Figu e 2.7. and Figu e 2.8., espec i ely, oge he wi h da a o [C2C1Im][C4F9SO3], [C6C1Im][C4F9SO3], [C8C1Im][C4F9SO3] and [C12C1Im][C4F9SO3] aken om he li e a u e (1,3). Densi y and luidi y a e impo an pa ame e s o be analyzed when one wan o apply his no el compounds o biomedical applica ions, such as anspo and deli e y o espi a o y gases (13), o mula ion o d ug deli e y sys ems (14),among o he s (1). The ob ained densi y alues we e compa ed wi h [C2C1Im][C4F9SO3], [C6C1Im][C4F9SO3], [C8C1Im][C4F9SO3] and [C12C1Im][C4F9SO3] (1,3) in o de o obse e he e ec o he hyd ogena ed alkyl chain leng h o he imidazolium ca ion. In o de o e alua e he densi y o luo ina ed ionic liquids, he densi y o blood (1.05 g∙cm-3) was aken in o accoun (15). Addi ionally, since PFCs p o ed o be use ul in a ple ho a o biomedical uses as su ac an s o mic oemulsions and o d ug deli e y encapsula ions, he densi y o hese compounds (≈1.7 g∙cm-3) is also conside ed as e e ence alue (16). The bes candida es will be he luo ina ed ionic liquids wi h densi ies close o he alues men ioned abo e. Analysing he esul s o [C4C1Im][C4F9SO3] and [C10C1Im][C4F9SO3] i can be concluded ha in he ange o empe a u es s udied he inc ease in he hyd ogena ed alkyl side chain leng h leads o a dec ease o he densi ies: [C4C1Im][C4F9SO3] > [C10C1Im][C4F9SO3]. Compa ing he esul s wi h blood densi y a app oxima ely 310.15 K (a e age body empe a u e, 37ºC) he [C10C1Im][C4F9SO3] (≈1.29 g∙cm-3) is closes o 1.05 g∙cm-3. Howe e , while inc easing he hyd ogena ed chain leng h could be ad an ageous conce ning he blood low, i leads o an inc ease in lipophilici y (highe oxici y) as will be discussed in Chap e 3. Taking in o accoun he densi y o PFCs (≈1.7 g∙cm-3) (16), [C2C1Im][C4F9SO3] and [C4C1Im][C4F9SO3] p esen ed he closes alues ≈ 1.55 g∙cm-3 and ≈ 1.44 g∙cm-3 a 310.15 K, espec i ely. Consequen ly, hese luo ina ed ionic liquids could be p omising bioma e ials o eplace Ionic Liquids in he De elopmen o No el Bioma e ials Cha ac e iza ion o Fluo ina ed Ionic Liquids: The mophysical, Phase Equilib ia and Agg ega ion Beha iou 27 (ei he pa ially o o ally) PFCs, in hei dis inc use ul applica ions (e.g. d ug deli e y, con as ul asound imaging agen ) (6). Compa ing he ob ained esul s wi h hose ound in he li e a u e o he same amily o imidazolium- based ionic liquids (see Figu e 2.6), i is e i ied ha densi y dec eases wi h he inc ease o empe a u e and wi h he inc emen o he hyd ogena ed alkyl side chain o he imidazolium ca ion. These esul s a e in ag eemen wi h he li e a u e (1,3,5) Figu e 2.6. Densi y o he luo ina ed ionic liquids measu ed in his wo k and compa ison wi h [C2C1Im][C4F9SO3], [C6C1Im][C4F9SO3], [C8C1Im][C4F9SO3] and [C12C1Im][C4F9SO3] ound on li e a u e (1,3). The a e age blood iscosi y s ands be ween 1.1 o 1.35 mPa∙s, and so blood luidi y is in a ange o 0.74-0.91 mPa-1s-1 (17). The PFCs iscosi y a ied om 0.6 o 5.4 mPa∙s wi h a luidi y ange o 0.19- 1.67 mPa-1s-1 depending on he composi ion (18). In Figu e 2.7., i is possible o obse e luidi y dependence on empe a u e o [CnC1Im][C4F9SO3] (n=2; 4; 6; 8; 10; 12) FILs. A 310.15 K, conside ed o be he a e age human body empe a u e, luidi ies o all he FILs s and be ween 0.003-0.013 mPa- 1s-1. Howe e , wi h he inc ease in empe a u e he di e ences a e mo e p onounced. Compa ing he measu emen s o [C4C1Im][C4F9SO3] and [C10C1Im][C4F9SO3] i can be concluded ha he inc emen o he hyd ogena ed alkyl chain leng h leads o a dec ease in luidi y (3,5). Rela ing hese esul s wi h hose ound on he li e a u e o [C2C1Im][C4F9SO3], [C6C1Im][C4F9SO3], [C8C1Im][C4F9SO3] and [C12C1Im][C4F9SO3] he end become mo e ob ious (1,3). Despi e densi y and iscosi y alues (see Table 2.3.) being highe han ac ual blood physical p ope ies and so c ea ing an obs acle, addi ion o wa e may lead o a dec ease on bo h p ope ies (19). Rod íguez and B ennecke (19) s udied he densi y and iscosi y o aqueous solu ions o imidazolium-based ILs wi h he anions e hylsul a e, i luo oace a e and i luo ome hanesul ona e. Bo h p ope ies we e ound o dec ease wi h an inc ease in ei he empe a u e o in mola ac ion o wa e . 275 300 325 350 375 Densi y / g.cm-3 1.20 1.30 1.40 1.50 1.60 [C2C1Im][C4F9SO3] [C4C1Im][C4F9SO3] [C6C1Im][C4F9SO3] [C8C1Im][C4F9SO3] [C10C1Im][C4F9SO3] [C12C1Im][C4F9SO3] Tempe a u e T / K Ionic Liquids in he De elopmen o No el Bioma e ials Cha ac e iza ion o Fluo ina ed Ionic Liquids: The mophysical, Phase Equilib ia and Agg ega ion Beha iou 34 could lead o he unexpec ed beha iou o he aqueous- ich phase o he LLE phase diag am epo ed in his wo k. A close analysis ega ding he epo ed end is being conduc ed by ou esea ch g oup. Figu e 2.10. a) Full composi ion o he liquid-liquid phase diag am o bina y mix u es (FILs (1) + wa e (2)) in e ms o mass ac ion; ampli ied liquid-liquid phase diag am a he b) wa e ich-phase and c) a ≈335 K and he d) FIL ich-phase. 2.4.2.2. Sel -agg ega ion Beha iou Fluo ina ed ionic liquids a e amphiphilic molecules and a e cons i u ed by a pola head and a hyd ophobic ail (34). In aqueous media, FILs can sel -assembly in di e en o agg ega e s uc u es o hide hei hyd ophobic moie ies and expose he hyd ophilic ones. Some common o ms o agg ega es a e ubules, esicles, ibbons and helices (4). Fo ins ance, his abili y o FILs o c ea e o ganized nanos uc u es could be use ul in he de elopmen o no el d ug deli e y sys ems whe e a d ug wi h low solubili y could be encapsula ed in one o hese s uc u es and di ec ed o he a ge (14). Addi ionally, hei su ac an beha iou could be also use ul o acili a e he o ma ion and s abiliza ion o mic oemulsions o enhanced biomolecule solubili y (29,36). Measu emen s o ionic conduc i i y a e gene ally implemen ed in he s udy o ionic micella solu ions. The p esence o micelles can dec ease he mobili y and as consequence a slope on conduc i i ies could be no iced (11). Thus, ionic conduc i i y measu emen s o [C4C1Im][C4F9SO3] and w FIL 0.0 0.2 0.4 0.6 0.8 1.0 T/ K 294 308 322 336 350 364 [C4C1Im] [C4F9SO3] [C6C1Im] [C4F9SO ] [C8C1Im] [C4F9SO3] [C10C1Im] [C4F9SO3] [C12C1Im] [C4F9SO3] w FIL 0.00 0.02 0.04 0.06 0.08 0.10 T/ K 294 308 322 336 350 364 w FIL 0.75 0.80 0.85 0.90 0.95 1.00 T/ K 294 308 322 336 350 364 n in [CnC1Im][C4F9SO3] 4 6 8 10 12 wFIL 0.012 0.016 0.020 0.024 0.028 C4 C12 C10 C8 C6 C4 C6 C8 C10 C12 C12 C10 C8 C6 C4 a) b) d) c) Ionic Liquids in he De elopmen o No el Bioma e ials Cha ac e iza ion o Fluo ina ed Ionic Liquids: The mophysical, Phase Equilib ia and Agg ega ion Beha iou 35 [C10C1Im][C4F9SO3] we e pe o med a 298.15 K, 308.15 K and 318.15 K. Figu e 2.11. illus a e hese measu emen s a 298.15 K o [C10C1Im][C4F9SO3], whe e he expe imen al conduc i i ies a e depic ed agains FIL concen a ion. This change in beha iou is explained by Philips de ini ion, whe e he c i ical micella concen a ion (CMC) is he concen a ion co esponding o he maximum change in he g adien o he solu ion p ope y, ionic conduc i i y, e sus concen a ion cu e (11). The CMC alues can be de e mined using hese plo s. Figu e 2.11. Concen a ion dependence o he ionic conduc i i y o [C10C1Im][C4F9SO3] in aqueous solu ion a 298.15 K a he CMC. The dashed line shows a maximum change in he g adien o ionic conduc i i y as a unc ion o FIL concen a ion, ob ained second de i a i e. In his wo k, he ionic conduc i i y measu emen s we e ca ied ou a 298.15, 308.15 and 318.15 K and he CMC alues calcula ed om hese plo s a e epo ed in Table 2.5 and compa ed wi h he CMC o FILs om same amily measu ed a 298.15 K (4,30). Figu e 2.12 illus a es he beha iou o he FILs s udied in his wo k as well as o [C2C1Im][C4F9SO3], [C6C1Im][C4F9SO3], [C8C1Im][C4F9SO3] and [C12C1Im][C4F9SO3] (4,30). Taking in o accoun hese expe imen al esul s, i can be concluded ha he inc emen o he hyd ogena ed alkyl side-chain o he imidazolium ca ion is di ec ly ela ed o lowe CMC alues, as expec ed (30). The CMC is in ima ely ela ed o he su ac an beha iou o a compound: a lowe CMC leads o an inc ease o su ac an beha iou (1). The comme cial pe luo oca bon su ac an s such as sodium pe luo ooc anoa e and ammonium pe luo ooc anoa e ha e CMC alues o ≈30 mmol∙Kg (37–39) , app oxima ely 30 imes highe han some FILs, such as [C10C1Im][C4F9SO3]. This ad an ageous cha ac e is ic could be use ul o subs i u ing ( o ally o pa ially) he PFCs in mic oemulsion o mula ions whe e he main pu pose is hei su ac an ac ion (40). FIL Concen a ion / (mmol.Kg-1) 0.00 0.46 0.92 1.38 1.84 2.30 Conduc i i y/ (mS.cm-1) 0.00 0.02 0.04 0.06 0.08 0.10 Concen a ion dependence o he ionic conduc i i y be o e CMC Concen a ion dependence o he ionic conduc i i y a e CMC Second De i a i e Ionic Liquids in he De elopmen o No el Bioma e ials Cha ac e iza ion o Fluo ina ed Ionic Liquids: The mophysical, Phase Equilib ia and Agg ega ion Beha iou 36 Table 2.5. Values o c i ical micella concen a ion, CMC, a di e en empe a u es o FILs in his wo k and compa ison wi h alues ound on li e a u e * a 298.15 K (4,30). T/K FILs CMC (mmol/ Kg) 298.15 [C2C1Im][C4F9SO3] 14.55* [C4C1Im][C4F9SO3] 12.17 [C6C1Im][C4F9SO3] 8.22* [C8C1Im][C4F9SO3] 2.41* [C10C1Im][C4F9SO3] 1.29 [C12C1Im][C4F9SO3] 0.01* 308.15 [C4C1Im][C4F9SO3] 11.36 [C10C1Im][C4F9SO3] 1.21 318.15 [C4C1Im][C4F9SO3] 11.31 [C10C1Im][C4F9SO3] 1.14 Figu e 2.12. Compa ison o he CMCs alues o he luo ina ed ionic liquids s udied in his wo k a 298.15 K and hose ound in he li e a u e (4,30) o he same empe a u e. The deg ee o ioniza ion o he agg ega es, αCMC, is also a pa ame e o be e alua ed. I is ela ed wi h he ac ion o cha ges o su ac an ions in he micelle neu alized by micelle-bound coun e ions. The ions o hese su ac an s a e comple ely dissocia ed in aqueous solu ions. Howe e , hey a e pa ly associa ed wi h coun e ions when o ming hei agg ega es (4). The deg ee o ioniza ion o he agg ega es can be calcula ed om he a io o he slopes o he linea agmen s abo e and below he CMC concen a ion (4). These alues a e p esen ed in Table 2.6. o he s udied [CnC1Im][C4F9SO3] (n = 2, 4, 6, 8, 10, 12) FILs. A lowe alue o αCMC indica es a be e packed micelle (4). Fu he mo e, he deg ee o ioniza ion o he agg ega es is mo e p onouced a lowe empe a u es. On he o he hand, CMC (mmol/ Kg) 0 1 2 3 8 10 12 14 16 [C2C1Im][C4F9SO3] [C4C1Im][C4F9SO3] [C6C1Im][C4F9SO3] [C8C1Im][C4F9SO3] [C10C1Im][C4F9SO3] [C12C1Im][C4F9SO3] Ionic Liquids in he De elopmen o No el Bioma e ials Cha ac e iza ion o Fluo ina ed Ionic Liquids: The mophysical, Phase Equilib ia and Agg ega ion Beha iou 37 he deg ee o coun e ion binding o coun e ions condensed on he micella in e ace, βCMC, can be de e mined hough he ela ionship (4): 𝜷𝑪𝑴𝑪 = 𝟏−𝜶𝑪𝑴𝑪 Equa ion 2.5. This pa ame e , βCMC, is ela ed o he size o he agg ega e, he cha ge densi y a he agg ega e su ace, and he hyd ophobic na u e o he coun e ions (4). FILs wi h long chains can o m bulkie agg ega e s uc u es, compa a i ely o smalle FILs ([C2C1Im][C4F9SO3]) (30). Then, he numbe o ions pe agg ega e and he olume pe su ac an ion in he case o [C10C1Im][C4F9SO3] a e highe . Mo eo e , he pola headg oups o hese su ac an s a e packed mo e closely and hey a e neu alized by a la ge ac ion o hei coun e ions (30). Fo he [CnC1Im][C4F9SO3] he αCMC dec eases and βCMC inc eases wi h he inc ease o he hyd ogena ed alkyl chain up o a decyl (n=10). Howe e , [C12C1Im][C4F9SO3] does no ollow his end. In o de o unde s and hese esul s, a comp ehensi e s udy needs o be done o e alua e he αCMC and βCMC alues o he imidazolium-based FILs up o a dodecyl. Table 2.6. Deg ee o ioniza ion o he agg ega es, αCMC, deg ee o coun e ion binding o ac ion o coun e ions condensed on he micella in e ace, βCMC, and s anda d ee ene gy o he agg ega ion p ocess, ΔG0agg, measu ed o FILs in his wo k a 298.15 K and compa ed wi h hose ound on li e a u e* o he same amily (4,30). FILs αCMC  βCMC  ΔG0agg [C2C1Im][C4F9SO3]* 0.79 0.21 -24.7 [C4C1Im][C4F9SO3] 0.89 0.11 -23.2 [C6C1Im][C4F9SO3]* 0.63 0.37 -29.9 [C8C1Im][C4F9SO3]* 0.38 0.62 -40.3 [C10C1Im][C4F9SO3] 0.35 0.65 -43.8 [C12C1Im][C4F9SO3]* 0.73 0.27 -41.7 The s anda d ee ene gy o he agg ega ion p ocess (ΔG0agg, mmol∙Kg-1) was also calcula ed aking in o accoun he pseudophase model o micelliza ion (43): ∆𝑮𝒂𝒈𝒈 𝟎=𝑹𝑻(𝟏+𝜷𝑪𝑴𝑪)𝒍𝒏𝒙𝑪𝑴𝑪 Equa ion 2.6. whe e he R and T a e he uni e sal gas cons an and absolu e empe a u e, espec i ely. The 𝒙𝑪𝑴𝑪 is he c i ical micella concen a ion exp essed in mole ac ion. Table 2.6. lis s s anda d ee ene gies o he agg ega ion p ocess o he FILs in his s udy and includes alues o o he FILs aken om he li e a u e (4,30). The s anda d Gibbs ee ene gy o agg ega ion, ΔG0agg, quan i ies he ee ene gy di e ence pe mole be ween FIL monome s in aqueous solu ions and in he agg ega es o med (30). Nega i e alues o his pa ame e indica e spon aneous agg ega ion o hese su ac an s. Conside ing he ΔG0agg alues he e epo ed, he agg ega ion p ocess is spon aneous as indica ed om he nega i e Ionic Liquids in he De elopmen o No el Bioma e ials Cha ac e iza ion o Fluo ina ed Ionic Liquids: The mophysical, Phase Equilib ia and Agg ega ion Beha iou 38 alues. Addi ionally, an inc ease on he hyd ogena ed alkyl side chain len gh leads o an inc ease o he agg ega ion p ocess as he ΔG0agg alues become mo e nega i e (30). In o de o be e unde s and he sel -agg ega ion beha iou o hese FILs based on he pe luo obu anesul ona e anion, TEM images we e ob ained o he i s ime o [C6C1Im][C4F9SO3], [C8C1Im][C4F9SO3] and [C12C1Im][C4F9SO3] (see Figu e 2.13). The sel -assembly o [C2C1Im][C4F9SO3] (Figu e 2.13 a)) was p e iously published (4) and used o compa ison. To ensu e he micelle o ma ion, he concen a ions used in he aqueous solu ions we e 2-4 imes highe han he CMC alues p e iously calcula ed. Figu e 2.13. TEM images o a) [C2C1Im][C4F9SO3] a a concen a ion 2.5 imes highe han CMC (4), b) [C6C1Im] [C4F9SO3] a a concen a ion 2 imes highe han CMC, c) [C8C1Im] [C4F9SO3] a a concen a ion 4 imes highe han CMC and d) [C12C1Im] [C4F9SO3] a a concen a ion 4 imes highe han CMC. The scales o images a e 200 nm, 0.5 m, 0.5 m and 1 m, espec i ely. Analyzing he esul s, i is possible o see ha agg ega es size inc eases wi h he inc emen o he hyd ogena ed alkyl chain leng h. The [C2C1Im][C4F9SO3] and [C6C1Im] [C4F9SO3] yield a mo e sphe ical micelle shape whe eas [C8C1Im][C4F9SO3] and [C12C1Im] [C4F9SO3] seem o o m micelles g owing om sphe ical o globula . 2.5. Conclusions The main objec i e o his chap e was o cha ac e ize he [C4C1Im][C4F9SO3] and [C10C1Im] [C4F9SO3] FILs in o de o expand he knowledge on he pe luo obu anesul ona e-based FILs, and o a) b) c) d) Ionic Liquids in he De elopmen o No el Bioma e ials Cha ac e iza ion o Fluo ina ed Ionic Liquids: The mophysical, Phase Equilib ia and Agg ega ion Beha iou 39 assess hei mos ema kable ea u es in he selec ion o he mos sui able FIL o biomedical applica ions. The mal and he mophysical p ope ies, as well as hei beha iou on aqueous solu ions we e s udied. The esul s we e compa ed wi h o he membe s o he [CnC1Im][C4F9SO3] amily o unde s and how he inc emen o he hyd ogena ed side-chain o he imidazolium ca ion (n=2, 4, 6, 8, 10, and 12) could in luence he beha iou o hese FILs. Taking in o accoun he he mal p ope ies, bo h [C4C1Im][C4F9SO3] and [C10C1Im] [C4F9SO3] showed a good he mal s abili y and highe decomposi ion empe a u es. Then, hey can be liquid in a la ge ange o empe a u es and ye no decompose. Mo eo e , he mel ing empe a u es inc eased wi h he inc emen o he hyd ogena ed alkyl side-chain leng h and he opposi e beha iou was obse ed o he decomposi ion empe a u es, as expec ed. The [C4C1Im][C4F9SO3] showed o be a be e op ion in compa ison wi h [C10C1Im][C4F9SO3] when analysing he he mophysical and he mochemical p ope ies he e epo ed. In he ange o empe a u es s udied, he inc ease in he hyd ogena ed alkyl side-chain leng h lead o a dec ease on densi ies and luidi y, being he [C4C1Im][C4F9SO3] wi h a densi y (≈1.44 g∙cm-3) and luidi y (0.01-0.05 mPa-1∙s-1) close o PFCs 1.7 g∙cm-3 and 0.19-1.67 mPa- 1∙s-1, espec i ely. Mo eo e , conduc i i ies p o en o be be e o luo ina ed ionic liquids wi h sho hyd ogena ed alkyl side chain leng hs showing highe ionici y. The agg ega ion beha iou was he e e alua ed by measu emen s o CMC and cha ac e ized by elec on mic oscopy. The FILs wi h long hyd ogena ed chains ha e smalle CMC and o m mo e s able and o ganized micelles. These sel -assemblies could in luence he solubili y o FILs in aqueous solu ion since hey ha e dis inc ea angemen s depending on he hyd ogena ed chain leng h. These indings co obo a e he esul s om LLE in which he FILs solubili y in he wa e phase appea s o be closely linked o he hyd ogena ed chain leng h: up o [C6C1Im]+ / [C8C1Im]+ he solubili y inc eases and hen a u he inc ease on he alkyl chain leads o a inc emen on he solubili y. 2.6. Re e ences (1) Pe ei o A. B. e al. Fluo ina ed Ionic Liquids: P ope ies and Applica ions. ACS Sus ain. Chem. Eng. 2013, 1, 427–439. (2) Ta iq, M.; Fo e, P. A. S.; Gomes, M. F. C.; Lopes, J. N. C; Rebelo, L. P. N. Densi ies and Re ac i e Indices o Imidazolium- and Phosphonium-based Ionic Liquids: E ec o Tempe a u e, Alkyl Chain Leng h, and Anion. J. Chem. The modyn. 2009, 41, 790–798. (3) Viei a, N. S. M. e al. 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Ionic Liquids in he De elopmen o No el Bioma e ials Cha ac e iza ion o Fluo ina ed Ionic Liquids: The mophysical, Phase Equilib ia and Agg ega ion Beha iou 42 43 3.Pa i ion P ope ies o Fluo ina ed Ionic Liquids Ionic Liquids in he De elopmen o No el Bioma e ials Pa i ion P ope ies o Fluo ina ed Ionic Liquids 50 In Figu e 3.3. i is possible o obse e he ends o he Kow alues wi h he s udied ange o FILs concen a ions. In all cases no subs an ial di e ences we e ound. Then i can be concluded ha o he ange o concen a ions he ein s udied, Kow is concen a ion independen and can be de e mined. Figu e 3.3. Va ia ion o Kow wi h concen a ion o he s udied FILs. The in luence o he alkyl side chain leng h o he imidazolium ca ion on he Kow alue is also an impo an pa ame e o ake in o conside a ion. Thus, he [CnC1Im] [C4F9SO3] amily (n=2, 4, 6 and 8) was s udied. Taking he analysis o he esul s in o conside a ion (see Figu e 3.4.), i is possible o e i y ha he inc emen o he alkyl side chain p o ides an inc ease on Kow alues. By de ini ion, i he Kow is ≥ 1 (Log Kow > 0) i is expec ed ha all he es compound emain in he oc anol-phase (see Equa ion 3.1) demons a ing a lipophilic beha iou (12). Thus, a end ega ding lipophilici y o he es compounds could be made, since alkyl chains highe han an oc yl (n=8) could be conside ed lipophilic. Figu e 3.4. In luence o he imidazolium hyd ogena ed side-chain leng h on Kow alues. A change om [C2C1Im][C4F9SO3] o [C8C1Im][C4F9SO3] leads o an inc ease o lipophilici y wi h a Kow app oxima ely 6 imes highe . This beha iou is explained since alkyl chain leng h o he ca ionic Concen a ion (mol/L) 0.00 5.00e-4 1.00e-3 1.50e-3 2.00e-3 Log Kow -1.8 -0.9 0.0 0.9 1.8 [C2C1Im][C4F9SO3] [C4C1Im][C4F9SO3] [C6C1Im][C4F9SO3] [C8C1Im][C4F9SO3] [C2C1py][C4F9SO3] [C2C1Im][C8F17SO3] Log Kow -1.6 -1.0 -0.4 0.2 0.8 Lipophilic Hyd ophilic [C2C1Im][C4F9SO3] [C4C1Im][C4F9SO3] [C6C1Im][C4F9SO3] [C8C1Im][C4F9SO3] Ionic Liquids in he De elopmen o No el Bioma e ials Pa i ion P ope ies o Fluo ina ed Ionic Liquids 51 ing c ea es s ong an de Waals in e ac ions be ween he alkyl g oup o he FIL and he hyd ophobic pa o 1-oc anol (11,18). These esul s a e consis en wi h he indings o o he au ho s o con en ional ILs wi h luo ina ed anions such as bis( i luo ome hylsul onyl)imide, [NT 2]-, (20) hexa luo ophospha e, [PF6]-, and e a luo obo a e, [BF4]- (21) as well as o ionic liquids in gene al (12, 14). Mo eo e , Ropel e al. epo ed ha solubili y o imidazolium based ionic liquids in alcohols inc eases wi h alkyl chain leng h (18). The dis ibu ion and posi ion o he luo ine a oms, as well as he amoun o luo ina ion, a e known o a ec he lipophilic beha iou (22). The e o e, he e ec o changing a pe luo obu anesul ona e, [C4F9SO3]-, o a pe luo oc anesul ona e anion, [C8F17SO3]-, was analysed. The inc ease o he luo ina ed chain leng h o he anion p omo es an ex eme dec ease o he hyd ophilici y. The Kow alue o he [C8F17SO3]-based FIL is app oxima ely 16 imes highe han he [C4F9SO3]-based FIL, as shown in Figu e 3.5.. Figu e 3.5. In luence o he luo ina ed chain leng h on Kow alues. These ends a e consis en wi h he li e a u e and ollow an empi ical ule which dic a es ha a luo ine con en ep esen ing ca. 60% o he o al molecula weigh can in luence he hyd ophilici y beha iou o he compound (23). In his case, he [C2C1Im][C4F9SO3] exhibi ed 41.6 %w and [C2C1Im][C8F17SO3] 52.9 %w . Pu se e al. epo ed ha a oma ic luo ina ion, pe /poly luo ina ion and luo ina ion adjacen o a oms wi h π-bonds (as p esen on he imidazolium ca ion) exponen ially inc eases lipophilici y (24). This beha iou is due o he excellen o e lap be ween he luo ine 2s o 2p o bi als wi h he co esponding o bi als on ca bon, making he C–F bond highly non-pola izable, he eby con ibu ing o inc eased lipophilici y (24). In addi ion, he Kow o 1-bu yl-3-me hyilimidazolium i luo ome hylsul ona e [C4C1Im][CF3SO3] was no measu ed in his wo k bu can be ound in he li e a u e wi h a alue o 0.20 (20). Thus, compa ing ou measu emen s o [C4C1Im][C4F9SO3], wi h a Kow o 0.43, he same end can be seen. The o e me hyla ion and luo ina ion o he ca ion enhances FIL solubili y in lipophilic cons i uen s which will allow hei en y in cells (10,22). Log Kow -1.6 -1.0 -0.4 0.2 0.8 [C2C1Im][C4F9SO3] [C2C1Im][C8F17SO3] Lipophilic Hyd ophilic Ionic Liquids in he De elopmen o No el Bioma e ials Pa i ion P ope ies o Fluo ina ed Ionic Liquids 52 The las s udy was ca ied ou in o de o unde s and how he ca ionic ing p esen in FILs can in luence he hyd ophilici y. Conside ing [C4F9SO3]-based FILs, he imidazolium ([C2C1Im]+) and py idinium ([C2C1py]+) ca ions we e compa ed. Bo h ionic liquids p esen a highly hyd ophilic beha iou (see Figu e 3.6.). In he li e a u e, he same end o imidazolium and py idinium-based ILs was obse ed (20,21). Mon albán e al. mesu ed he Kow o [C4C1py][BF6] and [C4C1Im][BF6] (21) and Lee e al. o [C6C1py][NT 2] and [C6C1Im][NT 2](20). Bo h au ho s demons a ed ha he Kow o imidazolium-based ILs a e sligh ly highe when compa ed o py idinium. Figu e 3.6. E alua ion o Kow alues o imidazolium and py idinium [C4F9SO3]-based FILs. 3.5 Conclusion Compounds wi h low Kow can p e en bioaccumula ion on human cells and ensu e a sui able ci cula ion o he compounds in he blood s eam (10). Thus, [C2C1Im][C4F9SO3] and [C2C1py][C4F9SO3] wi h Kow ≤ 0.3 a e he mos sui able FILs o he de elopmen o a d ug coa ing de ice. Fo ins ance, using hose FILs d ugs a e deli e ed in o he issues bu he compounds a e kep in he blood s eam. On he o he hand, i an API is conjuga ed wi h a FIL a high Kow ([C2C1Im][C8F17SO3] o [C8C1Im][C4F9SO3]) is equi ed. In his way, he FIL enhance he pe mi i i y o he d ug hough cellula memb anes (6). Fo he [CnC1Im][C4F9SO3] amily, a u he inc ease o he alkyl chain leng h (up o an oc yl, n=8), FILs concen a ions in oc anol a e highe sugges ing a lipophilic beha iou . Conce ning he anionic luo ina ed chain, when compa ing [C4F9SO3]- and [C8F17SO3]- anions he Kow alues we e app oxima ely 15 imes highe . Thus, by inc easing luo ina ion on he FIL anion lipophilici y is enhanced. Finally, bo h sho alkyl chain leng h FILs based on imidazolium and py idinium wi h a pe luo obu anesul ona e anion p esen ed a highly hyd ophilic beha iou . Log Kow -1.6 -1.0 -0.4 0.2 0.8 Lypophilic Hyd ophilic [C2C1Im][C4F9SO3] [C2C1py][C4F9SO3] Ionic Liquids in he De elopmen o No el Bioma e ials Pa i ion P ope ies o Fluo ina ed Ionic Liquids 53 3.6 Re e ences (1) Lipinski, C. A.; Lomba do, F.; Dominy, B. W.; Feeney, P. J. Expe imen al and Compu a ional App oaches o Es ima e Solubili y and Pe meabili y in D ug Disco e y and De elopmen Se ings. Ad . D ug. Deli . Re . 2012, 64, 4-17. (2) Mizuuch, H.; Jai ely, S.; Mu dan, S.; Flo ence, A.T. Room Tempe a u e Ionic Liquids and Thei Mix u es: Po en ial Pha maceu ical Sol en s. Pha m. Sci., 2008, 33, 326–331. (3) Moni uzzaman, M.; Kamiya, N.; Go o, M. Ionic Liquid Based Mic oemulsion Wi h Pha maceu ically Accep ed Componen s: Fo mula ion and Po en ial Applica ions J. Colloid In e ace Sci., 2010, 352, 136–142. (4) Moni uzzaman, M.; Tamu a, M.; Taha a, Y; Kamiya, N.; Go o, M. Ionic Liquid-in-Oil Mic oemulsion as a Po en ial Ca ie o Spa ingly Soluble D ug: Cha ac e iza ion and Cy o oxici y E alua ion. In . J. Pha m., 2010, 400, 243–250. (5) Jai ely, V.; Ka a as, A.; Flo ence, A. T. Wa e -immiscible Room Tempe a u e Ionic Liquids (RTILs) as D ug Rese oi s o Con olled Release. In . J. Pha m., 2008, 354, 168–173. (6) A aújo J. M. M.; Flo indo, C.; Pe ei o, A. B.; Viei a, N. M. S.; Ma ias, A. A.; Dua e, C. M. M.; Rebelo, L. N. R.; Ma ucho, I. M. Cholinium-based Ionic Liquids wi h Pha maceu ically Ac i e Anions RSC Ad ., 2014, 4, 28126–28132. (7) Flo indo, C. e al. E alua ion o Solubili y and Pa i ion P ope ies o Ampicillin-based Ionic Liquids. In . J. Pha m. 2013, 456, 553–559. (8) Fe az, R.; B anco, L. C. B.; Ma ucho, I. M. De elopmen o No el Ionic Liquids Based on Ampicillin. Med. Chem. Commun., 2012, 3, 494-497. (9) Boe hling, R. S.; Mackay, D.; Lyman, W. J. Handbook o p ope y es ima ion me hods o chemicals: en i onmen al and heal h sciences; 2000, CRC p ess. (10) Finizio, A.; Vighi, M.; Sand oni, D. De e mina ion o n-Oc anol/Wa e Pa i ionCoe icien (Kow) o Pes icide: C i ical Re iew and Compa ison o Me hods. Chemosphe e. 1997; 34; 131–161. (11) Jain, P.; Kuma , A. Concen a ion-Dependen Appa en Pa i ion Coe icien s o Ionic Liquids Possessing E hyl- and Bi-sulpha e Anions. Phys. Chem. Chem. 2016, 18, 1105–13. (12) Lee, S. H.; Lee. S. B. Oc anol/Wa e Pa i ion Coe icien s o Ionic Liquids. J. Chem. Technol. Bio echnol. 2009, 84, 202–207. (13) Kdd mann, T.; Rei h, D.; A nold, A. Why he Pa i ion Coe icien o Ionic Liquids Is Concen a ion-Dependen . J. Phys. Chem. B. 2013, 117, 10711−10718. (14) Kah, M.; B own, C. D. LogD: Lipophilici y o Ionisable Compounds. Chemosphe e. 2008, 72, 1401–1408. (15) Poole, S. K.; Poole, C. F. Sepa a ion Me hods o Es ima ing Oc anol–Wa e Pa i ion Coe icien . J. Ch oma og . B. 2003, 797, 3–19. (16) Ing am, T.; Rich e , U.; Mehling, T.; Smi no a. I. Modelling o pH Dependen n-Oc anol/Wa e Pa i ion Coe icien s o Ionizable Pha maceu icals. Fluid Phase Equilib. 2011, 305, 197–203. (17) B uijn, J.; Busse , F.; Seinen, W.; He mens, J. De e mina ion o Oc anol/Wa e Pa i ion Ionic Liquids in he De elopmen o No el Bioma e ials Pa i ion P ope ies o Fluo ina ed Ionic Liquids 54 Coe icien s o Hyd ophobic O ganic Chemicals wi h he “Slow-s i ing” Me hod. En i on. Toxicol. Chem. 1989, 8, 499–512. (18) Ropel, L.; Bel èze, L. S.; Aki, S. N. V. K.; S ad he , M. A.; B ennecke, J. F. Oc anol–Wa e Pa i ion Coe icien s o Imidazolium-based Ionic Liquids. G een Chem. 2005, 7, 83–90. (19) Fukumo o, K.; Yoshizawa, M.; Ohno, H. Room Tempe a u e Ionic Liquids om 20 Na u al Amino Acids. J. Ame . Chem. Soc. 2005, 127, 2398–9. (20) Lee, B. S.; Lin, S. T. A p io i Pedic ion o he Oc anol-Wa e Pa i ion Coe icien (Kow) o Ionic Liquids. Fluid. Phase. Equilib. 2014, 363, 233–8. (21) Mon albán, M. G.; Collado-González M.; T igo, R.; Díaz, F. G. B.; Víllo a, G. Expe imen al Measu emen s o Oc anol-Wa e Pa i ion Coe icien s o Ionic Liquids. J. Ad . Chem. Eng. 2015, 5. (22) Jeschke, P. The Unique Role o Fluo ine in he Design o Ac i e Ing edien s o Mode n C op P o ec ion. Chem. Bio. Chem. 2004, 5, 570–89. (23) Ojogun, V.; Knu son, B. L.; Vyas, S.; Lehmle , H. J. Fluo ophilici y o Alkyl and Poly luo oalkyl Nico inic Acid Es e P od ugs. J. Fluo . Chem. 2010, 131, 784–790. (24) Pu se , S.; Moo e, P. R.; Swallow, S.; Gou e neu , V. Fluo ine in Medicinal Chemis y. Chem. Soc. Re . 2008, 37, 320–330. 55 4.Kamle -Ta Sol a och omic Pa ame e s o Fluo ina ed Ionic Liquids 56 Ionic Liquids in he De elopmen o No el Bioma e ials Kamle -Ta Sol a och omic Pa ame e s o Fluo ina ed Ionic Liquids 57 4.1 In oduc ion Recen ly, ionic liquids a e becoming o pa icula in e es as new and highly e icien sol en s o co-sol en s o biomolecule-based applica ions due o hei unique cha ac e is ics and biocompa ibili y (1). Using ILs can d ama ically enhance solubili y and s abili y o p o eins (2), DNA (3) and enzymes (4). The key cha ac e is ics o a sol en a e hose ha de e mine how i will in e ac wi h po en ial solu es. Pola i y scales ha e been used o desc ibe he a ini y o solu es o molecula sol en s as well as ionic liquids (5). One o he mos used pola i y scales was de ised by Kamle and Ta in 1975 (6). The Kamle -Ta me hod is based on he compa ison o he e ec s on he UV- is spec a o se s o dyes. Th ee pola i y pa ame e s ha e been p oposed: he dipola i y/pola izabili y e ec s (  ); he hyd ogen bond acidi y (  ), ha is, hyd ogen bond dona ion (HBD) abili y; and he hyd ogen bond basici y (  ), ha is, hyd ogen bond accep ance (HBA) o elec on pai dona ion abili y o o m a coo dina ion bond (6). I should be highligh ed ha he hyd ogen bond accep o s eng h o an IL is domina ed by he anion while he hyd ogen bond dono abili y is essen ially con olled by he ca ion (7). Howe e , ca ion-anion in e ac ions could in luence he IL’s abili y o hyd ogen bonding (7). In addi ion, he Kamle -Ta desc ip o s p o ed o be empe a u e dependen (8,9). The hyd ogen bond accep o capaci y,  , can be measu ed by compa ison o ʎmax alues o a homomo phic pai o sol a och omic dyes such as 4-ni oaniline/N,N-die hyl-4-ni oaniline. In Figu e 4.1 a e illus a ed he 4-ni oaniline (Figu e 4.1a)) and he N,N-die hyl-4-ni oaniline (Figu e 4.1b)). In ed is ep esen ed he chemical bond ha is he o igin o he sol a och omic ansi ion (10). Figu e 4.1. Chemical s uc u es o a) 4-ni oaniline and b) N,N-die hyl-4-ni oaniline. The ed line a he ni o oxygen ep esen he sol a och omic ansi ion. Measu emen s o he in amolecula cha ge- ans e  →  * by he 4-ni oaniline and N,N-die hyl-4- ni oaniline ʎmax can deduce he elemen o wa eleng h change due o hyd ogen bonding (10). In a) b) Ionic Liquids in he De elopmen o No el Bioma e ials Kamle -Ta Sol a och omic Pa ame e s o Fluo ina ed Ionic Liquids 58 Equa ion 4.1., he ela ionship be ween he maximum abso p ion wa eleng h o he sol a och omic dyes (𝒗 ) in kilo Kyse s (cm-1), and he maximum abso p ion wa eleng h o he es compound (ʎmax) in nanome e s (nm) is ep esen ed (8). 𝒗 =𝟏𝟎𝟕 ʎ𝒎𝒂𝒙 Equa ion 4.1. The hyd ogen bond accep o abili y (β) can be calcula ed by Equa ion 4.2. (8). 𝜷 = 𝟏.𝟎𝟑𝟓𝝂 𝑵−𝑵−𝒅𝒊𝒆𝒕𝒉𝒚𝒍−𝟒−𝒏𝒊𝒕𝒓𝒐𝒂𝒏𝒊𝒍𝒊𝒏𝒆+𝟐.𝟔𝟒−𝝂 𝟒−𝒏𝒊𝒕𝒓𝒐𝒂𝒏𝒊𝒍𝒊𝒏𝒆 𝟐.𝟖𝟎 Equa ion 4.2. The me hod used o de e mine he hyd ogen bond dona ion capaci y (  ) is simila . The sol a och omic dyes used in his calcula ion a e Reicha d ’s dye (2,6-Diphenyl-4-(2,4,6- iphenyl-1- py idinio)phenola e) and 4-ni oaniline. In his case hey ac as a non-HBD solu e. In Figu e 4.2. is illus a ed he chemical s uc u e o Reicha d ’s dye as well as he chemical bond ha is he o igin o he sol a och omic ansi ion (10). Figu e 4.2. a) Chemical s uc u e o Reicha d ’s dye and b) he sol a och omic ansi ion in ed. Reicha d ’s dye exhibi s a la ge sol a och omic ange. The in amolecula cha ge ans e π → π* is obse ed a ound 500 nm, om ʎmax = 453 nm in wa e (pola sol en ) o ʎmax = 925 nm in hexane (nonpola sol en ) (10). The 𝐸𝑇 30 is a pola i y scale based only on Reicha d ’s dye and p oposed by Dim o h e al. o calcula e he α pa ame e o he Kamle -Ta me hod as shown in Equa ion 4.3. and Equa ion 4.4. (8,10). 𝑬𝑻 𝟑𝟎 =𝟐𝟖𝟓𝟗𝟐 ʎ𝒎𝒂𝒙 Equa ion 4.3. 𝜶 = 𝟎. 𝟎𝟔𝟒𝟗𝑬𝑻 𝟑𝟎 − 𝟐. 𝟎𝟑 − 𝟎. 𝟕𝟐𝝅∗ Equa ion 4.4. a) b) Ionic Liquids in he De elopmen o No el Bioma e ials Kamle -Ta Sol a och omic Pa ame e s o Fluo ina ed Ionic Liquids 59 The dipola i y/pola izabili y e alua ion is accomplished by he  *-scale. The  * pa ame e is only dependen on one dye, N-N-die hyl-4-ni oaniline, and i is calcula ed by Equa ion 4.5. (8). 𝝅∗= 𝟎. 𝟑𝟏𝟒(𝟐𝟕.𝟓𝟐 − 𝒗 𝑵,𝑵−𝒅𝒊𝒆𝒕𝒉𝒚𝒍−𝟒−𝒏𝒊𝒕𝒓𝒐𝒂𝒏𝒊𝒍𝒊𝒏𝒆) Equa ion 4.5. In his chap e , he hyd ogen bond acidi y (  ), hyd ogen bond basici y (  ) and dipola i y/pola izabili y (  *) we e i s calcula ed o cha ac e ize pu e luo ina ed ionic liquids. Fu he mo e, he e ec o wa e on he Kamle -Ta pa ame e s o FILs was e alua ed o add ess he ele ance o designing unc ionalized (so-called ask-speci ic) ILs ha ha e been syn hesized wi h a pa icula applica ion in mind. 4.2 Ma e ials 1-E hyl-3-me hylimidazolium i luo ome hanesul ona e, ≥ 99% mass ac ion pu i y; 1-bu yl- 3-me hylimidazolium i luo ome hanesul ona e, ≥ 99% mass ac ion pu i y; 1-e hyl-3- me hylpy idinium pe luo obu anesul ona e, >99% mass ac ion pu i y; 1-e hyl-3-me hylimidazolium pe luo obu anesul ona e >97% mass ac ion pu i y; 1-hexyl-3-me hylimidazolium pe luo obu anesul ona e, > 99% mass ac ion pu i y; 1-oc yl-3-me hylimidazolium pe luo obu anesul ona e, > 98% mass ac ion pu i y; choline pe luo obu anesul ona e, 97% mass ac ion pu i y; e abu ylammonium pe luo obu anesul ona e, 98% mass ac ion pu i y; e abu ylammonium pe luo ooc anesul ona e > 98% mass ac ion pu i y; 1-e hyl-3- me hylimidazolium ace a e, > 95 % mass ac ion pu i y; 1-e hyl-3-me hylpy idinium b omide, 99 % mass ac ion pu i y; 1-e hyl-3-me hylimidazolium chlo ide, ≥ 98 %; we e acqui ed om IoLiTec. Choline chlo ide, ≥ 98% mass ac ion pu i y was acqui ed om Sigma-Ald ich. The pu i y o all ionic liquids was e i ied by 1H, 13C and 19F NMR. P io o any use, all samples we e d ied unde a 3∙10-2 To acuum and igo ous s i ing o a leas 48 hou s o a oid ola ile impu i ies. Choline and ace a e- based ILs we e kep a 313.15 K and he emaining ILs a 323.15 K. The wa e con en was de e mined using Ka l Fiche coulome ic i a ion me hod (Me ohm 831 KF Coulome e ) o be less han 100 ppm. The s uc u es and ac onyms o he ionic liquids a e lis ed in Table 4.1. Table 4.1. Designa ion and chemical s uc u e o each ionic liquid used along his s udy. IL designa ion Chemical s uc u e 1-E hyl-3-me hylimidazolium i luo ome hanesul ona e [C2C1Im][CF3SO3] c Ionic Liquids in he De elopmen o No el Bioma e ials Kamle -Ta Sol a och omic Pa ame e s o Fluo ina ed Ionic Liquids 66 4.4.1.3. In luence o he Fluo ina ed Chain Leng h o he Anion I has been epo ed ha he anionic luo ina ed chain leng h has an in luence on he Kamle - Ta desc ip o s (18). A luo ina ed anionic g oup is capable o addi ional in e ac ions wi h pola componen s making hem sui able o enhanced solubili y o hyd ophobic bioac i e compounds (21). Fo ins ance, Ca doso and Micaelo (22) examined he molecula sol a ion o double-s and DNA (dsDNA) and single-s anded DNA (ssDNA) in nea ILs based on [BF4]- and [PF6]- anions. The au ho s ound ha ssDNA bases a e p e e en ially sol a ed by he anions ia hyd ogen bonding wi h he luo ine a oms. He ein, he dependence o he Kamle -Ta desc ip o s on he anion luo ina ed chain leng h in imidazolium ([CF3SO3]- and [C4F9SO3]-) and e abu ylammonium-based ([C4F9SO3]- and [C8F17SO3]-) FILs is e alua ed (see Figu e 4.7). The esul s a e lis ed in Table 4.2. Figu e 4.7. Kamle -Ta pa ame e s, hyd ogen-bond dono (α), hyd ogen-bond accep o (β), and dipola i y/pola izabili y (π*), measu ed a * 298.15 K o imidazolium-based ILs and a ** 323.15 K o e abu ylammonium-based ILs. Inc easing he luo ina ed chain leng h leads o an inc ease o  alues o bo h amilies being he imidazolium-based ILs he ones wi h a mo e p onounced abili y o dona e hyd ogen bonds. As men ioned abo e o ammonium-based ILs, he s abiliza ion o he dipola g ound s a e is mainly o igina ed om coulombic in e ac ions (17). Consequen ly, [N4444]+ is a poo H-bonding dono due o he lack o acidic hyd ogens compa ed o he imidazolium based ings (see Table 4.1). In e abu ylammonium-based ILs, he inc ease o luo ina ed chain leng h p oduces an inc ease on HBA. These esul s a e no su p ising because  alues o his amily mainly depend on he anion (17). Dipola i y/pola izabili y ( ) 0.0 0.2 0.4 0.6 0.8 1.0 1.2 Hyd ogen bond accepo ( ) 0.0 0.2 0.4 0.6 0.8 1.0 1.2 Hyd ogen bond dono ( ) 0.0 0.2 0.4 0.6 0.8 1.0 1.2 ** * ** ** * ** ** * *** ** [C2C1Im][CF3SO3] [C2C1Im][C4F3SO3] [N4444][C4F3SO3] [N4444][C8F17SO3] Ionic Liquids in he De elopmen o No el Bioma e ials Kamle -Ta Sol a och omic Pa ame e s o Fluo ina ed Ionic Liquids 67 The acidi y/basici y is p opo ional o he cha ge densi y o he compound and o he ela i e size- o- cha ge a io (7). The [C8F17SO3]- anion is la ge han he [C4F9SO3]- anion, which allows a highe dispe sion o he nega i e cha ges, p omo ing hyd ogen bonding wi h H+ a ailable on he media. Fo imidazolium-based FILs, i can be concluded ha HBA does no change by inc easing he luo ina ion o he anion. In ac , he -CF2 added o he anion co e only inc eases he acidi y main aining he FILs basici y. Imidazolium-based ILs wi h [NT 2]- and [PF6]- anions p o ed o be use ul o enhance he solubili y o biomolecules o se e al p ocesses (1). Thus, a compa ison o he α and β alues ob ained o he imidazolium-based ILs wi h [CF3SO3]- and [C4F9SO3]- anions is made in Figu e 4.8. Figu e 4.8. Kamle -Ta desc ip o s  and  o 1-e hyl-3-me hylimidazolium based ILs wi h luo ina ed anions. The  and  o [CF3SO3]- and [C4F9SO3]- we e ob ained in his wo k. The alues o *[PF6]- (16) and †[NT 2]- (20) we e ob ained om he li e a u e. In Figu e 4.8. an acidi y (  ) and basici y (  ) scale is ep esen ed. Analyzing he ILs o e all i is possible o obse e he possibili y o manage he inal cha ac e is ics o he compounds hough he di e se combina ion o he anions. The [C2C1Im][C4F9SO3] p o ed o be he mos acidic and basic in which hyd ogen bonding is mo e a o able. This esul could be use ul when add essing he design o unc ionalized ILs wi h a pa icula applica ion in mind. Fo ins ance, he hyd ogen bonding capaci y o nucleobases is esponsible o co ec DNA base-pai ing and s uc u e s abiliza ion (22). A aújo e al. (5) in es iga ed he sol a ion o nucleobases and concluded ha he ionic liquid mus be a good hyd ogen bond accep o and mode a e bond dono o dissol e he nucleobases. In addi ion, a high-HBA ionic liquid can s ongly coo dina e he HBD g oups o cellulose and igge solu e-sol en in e ac ions ha a e equi ed o cellulose dissolu ion (8). In some cases, a s ong hyd ogen bonding abili y is also ela ed o enzyme dena u a ion, which lowe s he enzyma ic Ionic Liquids in he De elopmen o No el Bioma e ials Kamle -Ta Sol a och omic Pa ame e s o Fluo ina ed Ionic Liquids 68 ac i i y (1). On he o he hand, in hyd ophobic ILs media (lowes  and  ) enzymes can e ain hei ac i i y and hei s abili y is enhanced in compa ison wi h adi ional sol en s (1). 4.4.2 Kamle -Ta Sol a och omic Pa ame e s o Aqueous Solu ions o Fluo ina ed Ionic Liquids Ionic liquids usually ha e high iscosi ies and high pola i ies due o hei cha ged s uc u es and ela i ely s ong anion-ca ion in e ac ions (2). Howe e , adding wa e in o hyd ophilic ionic liquids a dec ease in hei iscosi y is no iced (23). Ne e heless, IL + wa e mix u es can s ill exhibi sa is ac o y ex ac ion e iciency o pola bioac i e compounds (24). The hyd ogen-bond basici y (  ) i is one o he mos impo an pa ame e s e lec ing he hyd ogen bond accep ing abili y o he IL anion (7). Mos o he ele an p ope ies o ILs ega ding he solu e-sol en in e ac ion a e signi ican ly de e mined by he na u e o he anion a he han he ca ion (18). In ac , he  pa ame e is widely used o explain (and co ela es wi h) di e se p ope ies, such as sol a ion abili y and phase equilib ium beha io o ILs (25, 26). A good example o he applica ion o sol a och omic pola i y scales o ionic liquids a e he s udies ocused on he cellulose solubili y in 1,3-dialkylimidazolium-based ILs. The measu emen o Kamle - Ta pa ame e s showed ha ILs cha ac e ized by a high β pa ame e , indica ing an inc ease in he hyd ogen bonding accep ing capabili y o he anion, displayed inc eased cellulose solubili y (5). The IL 1-e hyl-3-me hylimidazolium ace a e ([C2C1Im][C1CO2] ep esen ed in Table 4.1), cha ac e ized by a high  pa ame e , has been inc easingly ecognized as a p omising sol en and can be ega ded as an enzyme- iendly co-sol en o bioca alysis as well as a good sol en o biomac omolecules such as cellulose and sube in, DNA, p o eins, small solu es such as ca bohyd a es, and nucleic acid bases and has signi ican abili y o sou gas seques a ion (27). Recen ly, Dohe y e al. (8) demons a ed ha he β pa ame e p o ides a measu e o he e iciency o ILs o se e as sol en in lignocellulosic biomass p e ea men . The ex ac ion powe o lignin and yields o e men able suga we e epo ed o inc ease among wi h he β alue. Mo eo e , hese au ho s epo ed ha he addi ion o wa e in o ILs has he g ea es impac on he β pa ame e (8) mainly due o he ela i ely poo hyd ogen bond basici y o wa e (  = 0.14–0.18) (2). Among ionic liquids, β displays he g ea es a ia ion compa ed o α and π* alues when wa e is added o he sys em (8). In his sec ion he Kamle -Ta desc ip o s we e measu ed o wa e mix u es wi h luo ina ed ionic liquids and con en ional ionic liquids (see Table 4.3). In his way, i could be assessed how IL+ wa e sys em can in luence he hyd ogen bond capaci y and pola izabili y. The main aim o his s udy is o design an IL ha do no loses he β p ope y in aqueous solu ions, add essing he design o unc ionalized (so-called ask-speci ic) ILs ha ha e been syn hesized wi h a pa icula applica ion in mind. Ionic Liquids in he De elopmen o No el Bioma e ials Kamle -Ta Sol a och omic Pa ame e s o Fluo ina ed Ionic Liquids 69 Table 4.3. Kamle -Ta pa ame e s hyd ogen-bond dono , α, hyd ogen-bond accep o , β, and dipola i y/pola izabili y, π*, de e mined o he aqueous solu ions wi h FILs and con en ional ILs a 298.15 K. Wa e (w %) α RSD (%) β RSD (%) π* RSD (%) Wa e (w %) α RSD (%) β RSD (%) π* RSD (%) [C2C1Im][CF3SO3] [C4C1Im][CF3SO3] Nea 0.62 3.59 0.48 1.25 1.02 0.09 Nea 0.62 3.87 0.48 0.80 1.02 0.09 12 0.74 1.79 0.45 6.74 1.10 1.68 10 0.72 0.19 0.45 0.77 1.08 0.18 31 0.84 0.63 0.39 3.55 1.19 0.35 30 0.78 0.47 0.40 0.59 1.17 0.01 50 0.85 0.29 0.35 2.11 1.25 0.08 50 0.77 0.06 0.36 0.31 1.22 0.01 69 0.80 0.66 0.29 0.59 1.32 0.08 70 0.75 0.03 0.26 4.95 1.31 0.26 [C2C1py][C4F9SO3] [C2C1Im][C4F9SO3] Nea 0.62 1.01 0.50 0.48 0.94 0.10 Nea 0.76 0.13 0.48 1.24 0.89 0.05 14 0.86 1.09 0.49 1.59 1.07 0.20 9 0.99 0.13 0.49 1.48 1.01 0.11 31 0.92 0.43 0.49 0.49 1.09 0.50 30 1.00 1.14 0.48 4.53 1.06 0.21 50 0.96 0.31 0.50 0.37 1.08 0.20 50 1.03 0.43 0.49 1.33 1.05 0.53 67 1.07 3.87 0.50 1.15 1.08 0.10 70 0.98 0.32 0.53 0.53 1.06 0.00 [N1112OH][C4F9SO3] [C2C1Im] C1CO2 10 1.14 0.21 0.42 0.50 1.05 0.32 11 0.49 0.44 0.91 0.15 1.11 0.00 30 1.14 1.02 0.46 8.53 1.06 0.42 22 0.58 0.76 0.67 0.65 1.22 0.17 50 1.14 0.13 0.46 1.83 1.05 0.53 44 0.53 0.14 0.49 1.47 1.31 0.08 69 1.13 0.37 0.48 2.09 1.04 0.21 70 0.82 0.09 0.37 4.25 1.34 0.08 [N1112OH] Cl [C2C1Im] Cl 24 0.95 0.51 0.48 0.23 1.21 0.61 25 0.52 0.01 0.55 1.69 1.27 0.16 32 0.94 0.04 0.40 0.30 1.24 0.00 30 0.51 0.01 0.47 1.25 1.31 0.07 36 0.96 0.79 0.38 2.95 1.25 0.08 35 0.66 0.96 0.50 1.24 1.31 0.31 51 0.97 0.04 0.30 0.40 1.29 0.00 52 0.69 0.09 0.37 1.65 1.35 0.06 71 0.94 0.26 0.22 0.71 1.32 0.26 72 0.80 0.25 0.20 2.01 1.40 0.24 [C2C1py] B 24 0.54 0.92 0.48 0.71 1.35 0.30 32 0.57 0.45 0.42 0.47 1.37 0.12 35 0.62 0.84 0.45 0.92 1.39 0.00 52 0.70 1.20 0.36 1.98 1.42 0.07 71 0.91 1.84 0.28 2.53 1.39 0.00 Ionic Liquids in he De elopmen o No el Bioma e ials Kamle -Ta Sol a och omic Pa ame e s o Fluo ina ed Ionic Liquids 70 4.4.2.1 In luence o he Imidazolium, Py idinium and Cholinium Ca ion In Figu e 4.9 i is possible o obse e he ends o [C2C1Im][C4F9SO3], [C2C1py][C4F9SO3] and [N1112OH][C4F9SO3] ega ding hei  ,  and  desc ip o s. [N1112OH][C4F9SO3] wi h a mel ing empe a u e o o 436 K (14), is solid a oom- empe a u e, hus measu ed o his nea FIL we e no accomplish. Figu e 4.9. Kamle -Ta pa ame e s, hyd ogen-bond dono (α), hyd ogen-bond accep o (β), and dipola i y/pola izabili y (π*), measu ed a oom- empe a u e o py idinium, imidazolium and choline- based FILs a di e en aqueous concen a ions a 298.15 K. The choline-based ILs ha e he highe  alues compa ed o imidazolium and py idinium. Tha esul is no su p ising since choline i sel has a pe manen dipole momen p omo ed by he non-symme ical s uc u e and, in pa icula due o i s e minal OH g oup, inc easing he acidi y (28). Then, [N1112OH][C4F9SO3] has an inc eased abili y o dona e hyd ogen bonds lowed by he imidazolium and py idinium-based FILs. Fo all FILs ep esen ed in Figu e 4.9, he  and π* alues inc ease only a low dilu ions and emain s able when inc easing he wa e con en . Thus, he addi ion o wa e in o nea FILs enhances hei HBD and pola izabili y. As discussed abo e, he β pa ame e is mainly de e mined by he o igin o he anion (18) and so no signi ican di e ences a e ound on HBA o he [C4F9SO3]- based FILs. In ac , no clea impac is obse ed in he  endency in opposi ion o con en ional ILs, whe e he addi ion o wa e has he g ea es impac on he HBA abili y (26). This beha io will be discussed in sec ion 4.4.2.3 o his chap e . Wa e (%) 020 40 60 80 Hyd ogen bond dono ( ) 0.1 0.4 0.8 1.1 1.5 Wa e (%) 020 40 60 80 Hyd ogen bond accep o ( ) 0.1 0.4 0.8 1.1 1.5 [C2C1py][C4FSO3] [C2C1Im][C4FSO3] [N1112OH][C4FSO3] Wa e (%) 020 40 60 80 Dipola i y/pola izabili y ( ) 0.1 0.4 0.8 1.1 1.5 Ionic Liquids in he De elopmen o No el Bioma e ials Kamle -Ta Sol a och omic Pa ame e s o Fluo ina ed Ionic Liquids 71 4.4.2.2 In luence o he Hyd ogena ed Alkyl Chain Leng h on he Imidazolium Ca ion In o de o e alua e he e ec o he hyd ogena ed alkyl chain leng h o he imidazolium ing, he Kamle -Ta desc ip o s o [C2C1Im][CF3SO3] and [C4C1Im][CF3SO3] we e measu ed and a e ep esen ed in Table 4.3. The ends o bo h FILs a e iden ical as can be seen in Figu e 4.10. Figu e 4.10. Kamle -Ta pa ame e s, hyd ogen-bond dono (α), hyd ogen-bond accep o (β), and dipola i y/pola izabili y (π*), measu ed a oom empe a u e wi h he inc ease o hyd ogena ed chain on imidazolium ca ion a di e en aqueous concen a ions a 298.15 K. Wi h he addi ion o wa e , he  alues inc eased un il p oximally 20 w % o wa e and hen emain ela i ely s able. On he o he hand, he π* alues a e d as ically inc eased in bo h cases indica ing a g ea e hyd ophilici y (20). The  pa ame e o bo h [C2C1Im][CF3SO3] and [C4C1Im][CF3SO3] dec eased wi h he addi ion o wa e in o he bulk. Fo ins ance, Cláudio e al. epo ed ha an in oduc ion o luo ina ed g oups om [CH3SO3]- o [CF3SO3]- leads o a dec ease in IL’s hyd ogen bond basici y o nea compounds (18). The low pola izabili y o he luo ina ed g oups and hei elec on wi hd awing e ec weaken he hyd ogen bonding abili y wi h he HBD o he sol a och omic p obe (18). This end can be ela ed o he poo HBD (2) o he wa e and he small alue o [CF3SO3]- based ILs anion. Ne e heless, compa ing hese esul s wi h hose ob ained o [C4F9SO3]- (see Table 4.3) i is no iced ha inc easing he luo ina ed chain e oke he   a ia ion. This beha io will be discussed in he nex sec ion. Wa e (%) 020 40 60 80 Hyd ogen bond dono ( 0.1 0.4 0.8 1.1 1.5 [C2C1Im][CF3SO3] [C4C1Im][CF3SO3] Wa e (%) 020 40 60 80 Hyd ogen bond accep o 0.1 0.4 0.8 1.1 1.5 Wa e (%) 020 40 60 80 Dipola i y/pola izabili y 0.1 0.4 0.8 1.1 1.5 Ionic Liquids in he De elopmen o No el Bioma e ials Kamle -Ta Sol a och omic Pa ame e s o Fluo ina ed Ionic Liquids 72 4.4.2.3 In luence o he Fluo ina ed Chain Leng h on he Anion The in luence o he anion will be discussed o he imidazolium, py idinium and cholinium- based ionic liquids. The p esence o a luo ina ed anion, and i s chain leng h, e sus con en ional anions (ace a e ([C1CO2]-), chlo ide (Cl-) and b omide (B -)) will be e alua ed ega ding he  ,  and  * pa ame e s. Focusing on he [C2C1Im]+ ca ion wi h con en ional anions, he ILs become mo e acidic (inc easing α) and he β dec eases wi h he addi ion o wa e (see Figu e 4.11), as expec ed om p e ious s udies (29). Figu e 4.11. Kamle -Ta pa ame e s, hyd ogen-bond dono (α), hyd ogen-bond accep o (β), and dipola i y/pola izabili y (π*), measu ed a oom o imidazolium-based ionic liquids wi h con en ional (C1CO2- and Cl-) and luo ina ed anions ([C4F9SO3]- and [CF3SO3]-) a di e en aqueous concen a ions a 298.15 K. Howe e , he luo ina ed anions (especially [C4F9SO3]-) p ese e hei basici y along wi h he inc eased wa e con en . These esul s could be explained by he a o able high elec onega i i y o he luo ine a om (F-) and by he s ong hyd ogen bond be ween C–F ··· H–O (2.4 kcal.mol-1), hal o he a e age O ··· H s eng h p esen on wa e molecules (21). Thus, he hyd ogen bonding o luo ina ed anions can be conside ed s able. Conce ning he π* alues, all ionic liquids p esen ed an excellen pola izabili y, highe han wa e (1.09) (8). In addi ion, [C2C1Im][C4F9SO3] e ains he same  * alue wi h he addi ion o wa e . As discussed abo e, high  alues a e ad an ageous o se e al p ocedu es in ol ing biomac omolecules (27). Ionic liquids based on he ace a e anion ([C1CO2]-) p o ed o be use ul o lignocellulose p e ea men (8,30) and o nucleobases sol a ion (5,27) due o hei s ong HBA abili y Wa e (%) 020 40 60 80 Hyd ogen bond dono ( ) 0.1 0.4 0.8 1.1 1.5 [C2C1Im][C4F9SO3] [C2C1Im][CF3SO3] [C2C1Im] C1CO2 [C2C1Im] Cl Wa e (%) 020 40 60 80 Hyd ogen bond accep o ( ) 0.1 0.4 0.8 1.1 1.5 Wa e (%) 020 40 60 80 Dipola i y/pola izabili y ( ) 0.1 0.4 0.8 1.1 1.5 Ionic Liquids in he De elopmen o No el Bioma e ials Kamle -Ta Sol a och omic Pa ame e s o Fluo ina ed Ionic Liquids 73 (see Table 4.3). Conce ning he ILs basici y (  ), he [C2C1Im][C1CO2] has highes  alues up o nea ly 50 w % wa e con en . Then, he [C2C1Im][C4F9SO3] demons a ed o be mo e ad an ageous wi h a highe  . Conce ning he wa e con en up o 50 w % he ollowing basici y scale acco ding o he anions could be es ablished: [C1CO2] > Cl ≈ [C4F9SO3] > [CF3SO3]. Howe e , be ween 50 w % and 80 w % he basici y end changes o: [C4F9SO3] > [C1CO2] > [CF3SO3] > Cl-. This esul shown he amazing abili y o ILs o “ une” cha ac e is ics acco ding o a pa icula applica ion in mind. Figu e 4.12 illus a e he in luence o a luo ina ed ([C4F9SO3]-) e sus con en ional (B -) anion o py idinium-based ILs in e ms o  ,  and  * desc ip o s. The b omide anion combined wi h he py idinium ca ion has being s udied o se e al biochemical p ocesses such as ex ac ion and sepa a ion o bioac i e compounds om plan s (31). Thus, unde s anding he hyd ogen bonding o his ILs will be bene icial o enhance ex ac ion and sepa a ion yields. Figu e 4.12. Kamle -Ta pa ame e s, hyd ogen-bond dono (α), hyd ogen-bond accep o (β), and dipola i y/pola izabili y (π*), measu ed a oom o py idinium-based ionic liquids wi h a con en ional (B -) and a luo ina ed anion ([C4F9SO3]-) a di e en aqueous concen a ions a 298.15 K. One ad an ageous cha ac e is ic o B - anions is hei highly hyd ophilici y which could inc ease ILs solubili y (32). Thus, he  * alues o [C2C1py] B - a e ema kably highe . Compa ing bo h HBA and HBD esul s, i is clea ha he luo ina ion o he anion p omo es a highe hyd ogen bonding abili y (see Figu e 4.12). Conce ning he  alues, a all wa e concen a ions he [C2C1py][C4F9SO3] is conside ed he mos acidic (inc eased  ). Rega dless he  desc ip o , he [C2C1py] B - a ≈ 25 w % has a basici y simila o [C2C1py][C4F9SO3] bu i d as ically declines wi h he u he addi ion o wa e . Wa e (%) 020 40 60 80 Hyd ogen bond dono ( ) 0.1 0.4 0.8 1.1 1.5 [C2C1py][C4F9SO3] [C2C1py]B Wa e (%) 020 40 60 80 Hyd ogen bond accep o ( ) 0.1 0.4 0.8 1.1 1.5 Wa e (%) 020 40 60 80 Dipola i y/pola izabili y ( ) 0.1 0.4 0.8 1.1 1.5 Ionic Liquids in he De elopmen o No el Bioma e ials Kamle -Ta Sol a och omic Pa ame e s o Fluo ina ed Ionic Liquids 74 This beha io could be explained by he wa e ’s s ong HBD which sol a e a B - anion a inc eased wa e concen a ions (22). The same beha io is epo ed o choline-based ILs. Fo [N1112OH][C4F9SO3] an highe acidi y and basici y is epo ed compa ed o [N1112OH] Cl- (see Figu e 4.13). Figu e 4.13. Kamle -Ta pa ame e s, hyd ogen-bond dono (α), hyd ogen-bond accep o (β), and dipola i y/pola izabili y (π*), measu ed a oom o choline-based ionic liquids wi h a con en ional (Cl-) and a luo ina ed anion ([C4F9SO3]-) a di e en aqueous concen a ions a 298.15 K. The  is highe o he choline con aining he luo ina ed anion. Rega dless he  pa ame e , un il ≈ 20 w % he [N1112OH] Cl- p esen ed a highe basici y. Then, wi h inc easing wa e concen a ions hei  alues dec ease linea ly. Addi ionally, Jingyi Hu e al. obse ed he same  beha io o chlo ide imidazolium-based ILs (33). On he o he hand, [N1112OH][C4F9SO3] main ained hei basici y ega dless adding wa e in o he bulk. The pola izabili y ep esen ed by he  * pa ame e is highe o Cl- choline- based ILs. Like B - py idinium-based IL (see Figu e 4.12), hose con aining he chlo ide anion ha e highe solubili y due o Cl- sol a ion by wa e . 4.5 Conclusion To da e, i is p o en ha ionic liquids can p o ide a new and powe ul pla o m o p o eins, enzymes and DNA enhanced solubili y, ac i i y and s abili y (1). In his chap e , he hyd ogen bonding and pola izabili y we e e alua ed o nea and aqueous solu ions o FILs. Wa e (%) 020 40 60 80 Hyd ogen bond dono ( ) 0.1 0.4 0.8 1.1 1.5 [N11112OH][C4F9SO3] [N11112OH] Cl Wa e (%) 020 40 60 80 Hyd ogen bond accep o ( ) 0.1 0.4 0.8 1.1 1.5 Wa e (%) 020 40 60 80 Dipola i y/pola izabili y ( ) 0.1 0.4 0.8 1.1 1.5 Ionic Liquids in he De elopmen o No el Bioma e ials Kamle -Ta Sol a och omic Pa ame e s o Fluo ina ed Ionic Liquids 75 F om he analysis o he Kamle -Ta desc ip o s o nea FILs, se e al conclusions could be made: i) imidazolium and py idinium-based FILs ha e simila hyd ogen bonding abili y a 323.15 K; ii) e abu ylammonium-based FILs achie ed he highe basici y and he lowes acidi y a 323.15 K; iii) he hyd ogena ed alkyl chain on he imidazolium ca ion has a small in luence on hyd ogen bonding o [CnC1Im CF3SO3] (n= 2; 4) and [CnC1Im C4F9SO3] (n= 2; 6; 8) a 298.15 K; i ) a u he inc ease on he luo ina ed chain on he anion p oduces highe HBD while p ese ing he HBA o imidazolium-based FILs; ) o e abu ylammonium-based FILs luo ina ion o he anion esul s in an inc ease o bo h  and  pa ame e s (highe acidi y and basici y). Finally, all he FILs p esen ed a highe hyd ophilic beha iou wi h  * alues close o pu e wa e . The wa e e ec on Kamle -Ta pa ame e s was e alua ed by assessing he  ,  and  a di e en concen a ions a oom- empe a u e. The main pu pose o his s udy was o ind an IL ha do no loses i s basici y (  ) in aqueous solu ions, an impo an p ope y ega ding he solu e-sol en in e ac ion. A se ies o imidazolium, py idinium and choline-based ILs wi h luo ina ed anions ([CF3SO3] and [C4F9SO3]) we e es ed and compa ed wi h con en ional ILs such as [C2C1Im] C1CO2, [C2C1Im] Cl, [C2C1py] B and [N1112OH] Cl. The analysis o he esul s sugges ed ha he inc easing luo ina ion on he anion ([CF3SO3]- e sus [C4F9SO3]-) p omo es ILs acidi y and a u he s abiliza ion o hei basici y. In ac , ILs wi h he pe luo obu anesul ona e anion do no lose hei basici y wi h inc eased wa e concen a ions. He ein, he hyd ogena ed alkyl chain leng h a ec s mainly he HBD abili y and does no change he HBA end o he [CnC1Im][CF3SO3] (n=2; 4) amily. Finally, he inc eased wa e con en p omo ed he pola izabili y o all ILs, inc easing hei hyd ophilici y. These o e all esul s u he s ess ha he IL ca ion and anion, o a la ge ex en , can be used o une he hyd ogen bonding and dipola i y/pola izabili y o ILs, depending on he desi ed applica ion. 4.6 Re e ences (1) Si ap agasam, M.; Moni uzzaman, M.; Go o, M. Recen Ad ances in Exploi ing Ionic Liquids o Biomolecules: Solubili y, S abili y and Applica ions. Bio echnol. J. 2016, 11, 1–14. (2) Jin, W.; e al. Enhanced Solubiliza ion and Ex ac ion o Hyd ophobic Bioac i e Compounds Using Wa e /Ionic Liquid Mix u es. G een Chem. 2016, 18, 3549–3557. (3) Zhao, H. DNA S abili y in Ionic Liquids and Deep Eu ec ic Sol en s J. Chem. Technol. Bio echnol. 2015, 90, 19–25. (4) Pa el, R.; Kuma i, M.; Khan, A. B.; Recen Ad ances in he Applica ions o Ionic Liquids in P o ein S abili y and Ac i i y: A Re iew. Appl. Biochem. Bio echnol. 2014, 172, 3701–3720. (5) A aújo, J. M. M.; Fe ei a, R., Ma ucho, I.M.; Rebelo, L. P. N. Sol a ion o Nucleobases in 1,3- Dialkylimidazolium Ace a e Ionic Liquids: NMR Spec oscopy Insigh s in o he Dissolu ion Mechanism. J. Phys. Chem. B. 2011, 115, 10739–10749. Ionic Liquids in he De elopmen o No el Bioma e ials Conclusions and Pe spec i es 82 The Kamle -Ta sol a och omic pa ame e s showed ha by a ca e ully selec ion o he FIL anion and ca ion, hyd ogen-bonding could be managed. Fo nea FILs, py idinium and imidazolium-based FILs p esen ed simila beha iou s. In addi ion, he inc ease in he ca ion hyd ogena ed alkyl chain leng h, o he [CnC1Im][C4F9SO3] (n=2, 6, 8) FILs p omo ed an inc ease on acidi y and a dec ease on basici y. Fo [CnC1Im][CF3SO3] FILs wi h n=2 and 4, no signi ican di e ences whe e no iced. The inc ease o he anionic luo ina ed chain leng h induced a mo e accen ua ed e ec on he e abu ylammonium-based FILs, inc easing bo h acidi y and basici y. Howe e , o he imidazolium-based FILs es ed he hyd ogen-bonding dona ion inc eased bu o he abili y o accep hyd ogen bonds no signi ican di e ences we e ound. Rega ding he e ec o wa e on he FIL basici y desc ip o , i was shown ha inc easing he anionic pe luo ina ed alkyl chain leng h ([CF3SO3]- e sus [C4F9SO3]-) p omo es ILs acidi y and a u he s abiliza ion o hei basici y when compa ed o con en ional ILs. No clea impac is obse ed in he basici y desc ip o o [C4F9SO3]-based ILs. This esul add ess he ele ance o designing unc ionalized (so-called ask-speci ic) ILs ha ha e been syn hesized wi h a pa icula applica ion in mind, e.g. dissolu ion o he apeu ic molecules. The o e all esul s u he s ess ha ILs cons i u i e ions, o some ex en , can be uned wi h se e al p ope ies depending on he desi ed applica ion. 5.2 Pe spec i es Du ing he pas decades he in e es in ionic liquids has inc eased, wi h applica ions in di e se a eas such as elec ochemis y, chemis y, nano echnology, bio echnology, and in enginee ing p ocesses, among o he s. The nume ous ad an ages o using ionic liquids as bioma e ials a e unques ionable. This ac makes hem e y a ac i e mainly in he biomedical ield whe e he use o bioma e ials o enhance clinical e icacy is e y challenging. The esul s ob ained in his wo k could help in he design o he mos sui able FIL o applica ion as su ac an s in biomedical applica ions. Besides, he luo ina ed domains p esen in emulsions can p omo e a highe solubili y o poo soluble d ug molecules and biomolecules such as enzymes, p o eins and DNA. Addi ionally, FILs a e a g eene al e na i e o subs i u e a m ul sol en s used nowadays in he pha maceu ical indus y o he ex ac ion, pu i ica ion and syn heses p ocesses o se e al biomolecules. As u u e wo k, he s udy o he in e ac ions be ween FILs and d ug molecules o biomolecules is equi ed. Also, d ugs and biomolecules solubili y in FILs will be needed o de e mine he e ec i eness o hese compounds as d ug deli e y de ices. Addi ionally, FIL solubili y should be de e mined in di e en biological luids such as gas ic and in es inal luid. The cellula iabili y in di e en human cell lines in o de o unde s and hei beha iou in he mos common ou es o adminis a ion (o al, in a enous and ansde mal) should be also s udied. Mo eo e , sel -agg ega ion p ope ies o FILs 83 6. Scien i ic Communica ions 84 Ionic Liquids in he De elopmen o No el Bioma e ials Scien i ic Communica ions 85 6.1 Pape s In p epa a ion: 1. “E alua ion o Cy o oxici y and Pa i ion P ope ies o Fluo ina ed Ionic Liquids”. Joana C. Bas os, Nicole M. Viei a, João M. M. A aújo, Sa a Nunes, Ana Ma ias, Isabel M. Ma ucho, Ca a ina M. M. Dua e, Ana B. Pe ei o, Luís P. N. Rebelo 2. “S udy o he In luence o Hyd ogena ed Alkyl Chain on Agg ega ion and Solubili y o Fluo ina ed Ionic Liquids using he So -SAFT Equa ion” . Fèlix Llo ell, Ana B. Pe ei o, João M. M. Aa aújo; Ma ía J. Pas o iza -Gallego, And eia S. S. San os, Joana C. Bas os , Luís P. N. Rebelo, Manuel M. Piñei o‖ and Lou des F. Vega 3. “Fluo ina ed Ionic Liquids–based Aqueous Biphasic Sys ems: S udy o Pola i y and Hyd ophobici y Scales” Joana C. Bas os, Sa a Ca alho, Ana B. Pe ei o, Luís P. N. Rebelo and João M. M. A aújo. 5.2 Pos e s in Scien i ic Mee ings 1. Au ho s: Joana C. Bas os, And eia S. S. San os, João M. M. A aújo, Luis Paulo N. Rebelo, Ana B. Pe ei o. Ti le: "De elopmen o Fluo ina ed Ionic Liquids o Biomedical Applica ions" Cong ess: 5 h Po uguese Young Chemis s Mee ing (5 h PYCheM) and 1s Eu opean Young Chemis s Mee ing (1s EYCheM) Place: Guima ães (Po ugal) Da e: Ap il, 2016 2. Au ho s: Joana C. Bas os, Nicole S. M. Viei a, Ana Ma ias, Ca a ina M. M. Dua e, João M. M. A aújo, Luís Paulo N. Rebelo, Ana B. Pe ei o Ti le: "E alua ing Fluo ina ed Ionic Liquids as No el D ug Deli e y Sys ems" Cong ess: 5 h Po uguese Young Chemis s Mee ing (5 h PYCheM) and 1s Eu opean Young Chemis s Mee ing (1s EYCheM) Place: Guima ães (Po ugal) Da e: Ap il, 2016 3. Nicole S. M. Viei a, J. C. Bas os, A. Ma ias, C. M. M. Dua e, João M. M. A aújo, Luís P. N. Rebelo, Ana B. Pe ei o Ti le: "Fluo ina ed Ionic Liquids o Biomedical Applica ion" Cong ess: 2nd EuCheMS Cong ess on G een and Sus ainable Chemis y Place: Lisbon (Po ugal) Da e: Oc obe , 2015 Ionic Liquids in he De elopmen o No el Bioma e ials Conclusions and Pe spec i es 86 4. Au ho s: N. S. M. Viei a, J. C. Bas os, J. M. M. A aújo, A. Ma ias, C. M. M. Dua e, Luís P. N. Rebelo, A. B. Pe ei o Ti le: "Cy o oxici y and Pa i ion Coe icien s o Fluo ina ed Ionic Liquids" Cong ess: Ibe oame ican Mee ing on Ionic Liquids- IMIL 2015 Place: Mad id (Spain) Da e: July, 2015