A Simple Method for the Quantification of Free Isocyanates on the Surface

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Article A Simple Method for the Quantiﬁcation of Free Isocyanates on the Surface of Cellulose Nanocrystals upon Carbamation using Toluene Diisocyanate

Hatem Abushammala

Fraunhofer Institute for Wood Research (WKI), Bienroder Weg 54E, 38108 Braunschweig, Germany; [email protected]; Tel.: +49-5312155409 Received: 9 May 2019; Accepted: 4 June 2019; Published: 13 June 2019

Abstract: In many reports, cellulose and nanocellulose have been carbamated using 2,4-toluene diisocyanate (2,4-TDI) to allow the grafting of molecules or polymers onto their surfaces. Such a process usually involves the reaction of the more reactive isocyanate group of TDI (para-NCO) selectively with a hydroxyl group from the cellulose surface, followed by the reaction of the free isocyanate (ortho-NCO) with a desired molecule. After the first step, it is not possible, using elemental analysis, to determine the amount of ortho-NCO on the cellulosic surface, as an ideal para/ortho selectivity is difficult to obtain. This paper presents a simple method for the quantification of ortho-NCOs on the surface of cellulose nanocrystals upon TDI-based carbamation. It relies on the pH increase upon a complete hydrolysis of ortho-NCOs to amine groups using acidified dimethylsulfoxide. The method was found to be accurate and valid for a degree of substitution of up to 20%.

Keywords: cellulose; nanocellulose; toluene diisocyanate; isocyanate; carbamation

1. Introduction The surface modification of cellulose and nanocellulose is often used to significantly increase their potential. A wide range of functionalities have been placed on cellulose surfaces through simple modifications such as acetylation, oxidation, and carboxymethylation or through a more-complicated grafting of polymers such as polycaprolactone and poly(ethylene glycol) [1–8]. In some cases, chemical linkers were needed to bind certain functionalities to nanocellulose; among these are aromatic diisocyanates [9,10]. 2,4-toluene diisocyanate (2,4-TDI, or TDI) is one of the most commonly used isocyanates for the production of polyurethanes [11]. It is a very useful linker to bind different chemicals together due to the reported difference in reactivity of its two isocyanate groups; o-NCO and p-NCO. It has been reported that o-NCO is 5–10 times less reactive than p-NCO due to the stearic hindrance from the neighboring methyl group [12,13]. A typical TDI-based binding process includes 1) the carbamation of a certain chemical (cellulose in this case) using the more reactive p-NCO of TDI followed by 2) the carbamation of a desired chemical to the cellulose surface using the free o-NCO (Figure1). TDI was used for the first time with cellulose in 1977 to bind it to certain biomolecules [14]. Before being applied for the first time on cellulose nanocrystals (CNCs) [15], it was used to graft certain polymers on the surface of starch nanocrystals [16,17]. Since then, TDI has been used more frequently for grafting polymers on the surface of CNCs [18–21]. However, most of these studies have not reported the amount of TDI on the CNC surface after the first carbamation reaction, i.e., degree of carbamation [4]. The degree of carbamation is mainly determined using elemental analysis, which relies on the increase in the nitrogen/carbon ratio (N/C) upon carbamation. Still, elemental analysis does not provide an accurate information about the amount of o-NCO available on the CNC surface after the first carbamation as the calculations that follow elemental analysis assume an ideal p-NCO/o-NCO

Surfaces 2019, 2, 444–454; doi:10.3390/surfaces2020032 www.mdpi.com/journal/surfaces Surfaces 2019, 2 445 selectivity, which means no o-NCO reacts at all at that stage (no crosslinking). This assumption is certainly invalid, as it is known that a certain amount of o-NCO still reacts, reducing the eﬃciency of carbamation [22]. Here, a simple method is developed for the determination of the free isocyanates of TDI-carbamated CNCs using a pH meter. It relies on the pH increase upon hydrolyzing the free isocyanates on the CNC surface to amine groups. Such a method could be useful in optimizing carbamationSurfaces 2019, 2 reactionsFOR PEER REVIEW and to accurately proceed with any later grafting of desired chemicals. 2

FigureFigure 1. 1.The The functionalizationfunctionalization ofof cellulosecellulose with a desireddesired chemical using 2,4-TDI 2,4-TDI as as a a linker. linker.

TDI was used for the first time with cellulose in 1977 to bind it to certain biomolecules [14]. The hydrolysis of isocyanates is a topic of strong interest. Due to health and environmental Before being applied for the first time on cellulose nanocrystals (CNCs) [15], it was used to graft concerns, environmentalists have investigated the fate of commonly used isocyanates in water and soil certain polymers on the surface of starch nanocrystals [16,17]. Since then, TDI has been used more by studying their hydrolysis kinetics [23]. Both aromatic and aliphatic isocyanates get hydrolyzed in frequently for grafting polymers on the surface of CNCs [18–21]. However, most of these studies have aqueous solutions to their corresponding amines, which readily react with neighboring isocyanates not reported the amount of TDI on the CNC surface after the first carbamation reaction, i.e. degree of forming a polyurea. This side reaction can be fully hindered in acidic media due to the instant carbamation [4]. The degree of carbamation is mainly determined using elemental analysis, which protonationrelies on the of increase the generated in the nitrogen/carbon amine groups [ratio24]. The(N/C) hydrolysis upon carbamation. in acidic media Still, elemental is, however, analysis slow, butdoes can not be stronglyprovide an catalyzed accurate by information the addition about of organic the amount solvents of such o-NCO as dimethylsulfoxide available on the CNC (DMSO) surface [25]. Moreover,after the suchfirst organiccarbamation solvent as unlike the calculations water can to that a good follow extent elemental disperse carbamatedanalysis assume CNCs. an This ideal work, p- therefore,NCO/o-NCO proposes selectivity, acidiﬁed which DMSO means as an no organic o-NCO system reacts that at canall at disperse that stage TDI-carbamated (no crosslinking). CNCs This and hydrolyzeassumption their is certainly free isocyanates invalid, toas amineit is known groups that in ordera certain to quantifyamount of them o-NCO using still a pH-meter. reacts, reducing the efficiency of carbamation [22]. Here, a simple method is developed for the determination of the 2. Materials and Methods free isocyanates of TDI-carbamated CNCs using a pH meter. It relies on the pH increase upon 2.1.hydrolyzing Materials the free isocyanates on the CNC surface to amine groups. Such a method could be useful in optimizing carbamation reactions and to accurately proceed with any later grafting of desired chemicals.Cellulose nanocrystals suspension (10.4 % w/w) was purchased from the University of Maine, which was prepared using the sulfuric acid method. Acetone ( 99%), Toluene ( 99%), DMSO ( 99%), The hydrolysis of isocyanates is a topic of strong interest.≥ Due to health≥ and environmental≥ trimethylamine (TEA) ( 99%), and hydrochloric acid 37% were purchased from VWR (Darmstadt, concerns, environmentalists≥ have investigated the fate of commonly used isocyanates in water and Germany). 2,4-toluene diisocyanate ( 98%), o-toluene isocyanate ( 98%), p-toluene isocyanate ( 98%), soil by studying their hydrolysis kinetics≥ [23]. Both aromatic and aliphatic≥ isocyanates get hydrolyzed≥ and o-toluidine ( 99%) were purchased from TCI Chemicals (Eschborn, Germany) and were stored in aqueous solutions≥ to their corresponding amines, which readily react with neighboring isocyanates informing sealed bottlesa polyurea. in the This fridge. side DMSO, reaction TEA, can and be toluenefully hindered were stored in acidic over media A4 molecular due to sieve,the instant while acetoneprotonation was stored of the undergenerated A3 molecular amine groups sieve. [24]. Both The molecular hydrolysis sieves in acidic were purchasedmedia is, however, from Carl slow, Roth (Karlsruhe,but can be Germany)strongly catalyzed and regenerated by the addition before of use. organic solvents such as dimethylsulfoxide (DMSO) [25]. Moreover, such organic solvent unlike water can to a good extent disperse carbamated CNCs. 2.2. Optimization of Acidiﬁed DMSO as Hydrolysis Medium This work, therefore, proposes acidified DMSO as an organic system that can disperse TDI- carbamatedTwo milliliters CNCs and of HCl hydrolyze solutions their of varyingfree isocyanates molarities to (0.1–10amine groups M) was in transferred order to quantify to 100 mLthem of DMSOusing a and pH-meter. mixed for an hour. The pH was recorded using a SevenEasy S20-KS pH meter equipped with an InLab®Routine Pro electrode (Mettler Toledo, Giessen, Germany). All measurements were recorded2. Materials in duplicate and Methods at a temperature of 25 0.5 C. ± ◦ 2.1. Materials Cellulose nanocrystals suspension (10.4 % w/w) was purchased from the University of Maine, which was prepared using the sulfuric acid method. Acetone (≥99%), Toluene (≥99%), DMSO (≥99%), trimethylamine (TEA) (≥99%), and hydrochloric acid 37% were purchased from VWR (Darmstadt, Germany). 2,4-toluene diisocyanate (≥98%), o-toluene isocyanate (≥98%), p-toluene isocyanate (≥98%), and o-toluidine (≥99%) were purchased from TCI Chemicals (Eschborn, Germany) and were stored in sealed bottles in the fridge. DMSO, TEA, and toluene were stored over A4 molecular sieve, while acetone was stored under A3 molecular sieve. Both molecular sieves were purchased from Carl Roth (Karlsruhe, Germany) and regenerated before use. Surfaces 2019, 2 446

2.3. pH Profile of o-Toluidine and o-Toluene Isocyanate in Acidified DMSO Two milliliters of 2 M HCl was transferred to 100 mL of DMSO. A certain amount of o-toluidine or o-toluene isocyanate (ca. 20 mg) was transferred dropwise to the acidified DMSO using a syringe. The pH value was recorded after 15 min using a SevenEasy S20-KS pH meter equipped with an InLab®Routine Pro electrode (Mettler Toledo, Giessen, Germany). Similar amounts were added every 15 min until a total amount of ca. 6 mmol was added. All measurements were recorded at a temperature of 25 0.5 C. ± ◦ 2.4. Carbamation of Cellulose Nanocrystals using TDI The CNCs were carbamated following the method of Habibi after minor modifications [15]. 9.6 g of 10.4% CNC suspension (equivalent to 1.0 g of dried CNCs (6.2 mmol)) was solvent-exchanged to anhydrous toluene using a washing/precipitation procedure starting with anhydrous acetone (three times) then anhydrous toluene (twice). The precipitation was performed by centrifugation using a Sigma 3-16P centrifuge (g-force of 4472, 5000 rpm for 30 min) (Sigma Laborzentrifugen, Osterode am Harz, Germany). After final washing, the precipitated CNCs were transferred to a 100 mL round-bottom flask using 50 mL of anhydrous toluene. 1.1 g of 2,4-TDI (6.2 mmol) and 1.0 mL of triethylamine (TEA) as catalyst were added to the reaction flask. The reaction proceeded at 35, 55, or 75 ◦C for 24 h in a moisture-free environment (under nitrogen). The reaction mixture was then centrifuged to collect the carbamated CNCs from the unreacted TDI and TEA. The carbamated CNCs were then washed once with anhydrous toluene and twice with anhydrous DMSO before transferred to acidified DMSO for hydrolysis and quantification of free isocyanates. For characterization, the carbamated CNCs were not washed with DMSO, but only with toluene (three times), and then dried overnight at 50 ◦C under vacuum.

2.5. Hydrolysis of the Carbamated CNCs for the Determination of Free Isocyanates Degree of Substitution (DSNCO) One hundred milliliters of DMSO was transferred to a 200 mL beaker before being acidiﬁed to a pH of 2.47 0.01 using 2 mL of 2 M HCl. The carbamated CNCs, after washed with DMSO, were ± transferred to the beaker. The pH started to increase because of isocyanate hydrolysis to amine groups. The hydrolysis was complete when no further increase in pH was observed (a maximum of 1 h). All pH measurements were recorded at a temperature of 25 0.5 C. The hydrolysis was performed in ± ◦ duplicate to assure reproducibility. The amount of free isocyanates (DSNCO) available on the surface of the carbamated CNCs (moles of NCO/moles of CNC hydroxyl groups*100%) was determined based on the ﬁnal pH value.

2.6. Determination of the Degree of Carbamation (DSTDI) using SEM-EDX The elemental analysis (C, N, O, S, Na) of the CNCs, before and after carbamation, was determined using the scanning electron microscope (SEM) ZEISS GeminiSEM Crossbeam 340 (ZEISS, Oberkochen, Germany) equipped with the energy dispersive X-ray (EDX) detector X-MaxN (Oxford Instruments, Abingdon, UK) and operating at a voltage of 10 kV. The samples in the powder form were pressed in a FT-IR-KBr mold using a mild force to obtain discs of smooth surfaces. The degree of carbamation (moles of TDI/moles of CNC hydroxyl groups*100%) was determined based on the increase in the nitrogen/carbon ratio (see the Supplementary Materials). The results were conﬁrmed using the elemental analyzer VarioMICRO V3.1.1 CHNS-Modus (Elementar Analysensysteme, Langenselbold, Germany) and using the mass yield of the carbamation reaction (see Supplementary Materials).

2.7. Characterization of the Carbamated and Hydrolyzed CNCs using FT-IR Vacuum-dried samples of the CNCs, before and after carbamation and after hydrolysis, were characterized using Tensor 27 FT-IR spectrometer (Bruker, Billerica, Massachusetts, USA) in the Surfaces 2019, 2 FOR PEER REVIEW 4 Surfaces 2019, 2 447 Langenselbold, Germany) and using the mass yield of the carbamation reaction (see supplementary materials). 1 1 750–40002.7. cm Characterization− range (resolution of the Carbamat ofed 2 and cm −Hydrolyzed) using CNCs the ATR using transmission FT-IR mode. The spectra were obtained as the average of 64 scans and processed using OPUS 6.5 software. Vacuum-dried samples of the CNCs, before and after carbamation and after hydrolysis, were 2.8. Determinationcharacterized of using CNC Tensor Thickness 27 FT-IR using spectrometer Atomic Force (Bruker, Microscopy Billerica, Massachusetts, USA) in the 750– 4000 cm−1 range (resolution of 2 cm−1) using the ATR transmission mode. The spectra were obtained 4 A dropas the ofaverage a diluted of 64 suspensionscans and processed (10− %) using of theOPUS original 6.5 software. CNCs in water, prior to solvent exchange, was deposited on a fresh mica surface and kept to air-dry overnight. The surface was imaged in the 2.8. Determination of CNC Thickness using Atomic Force Microscopy tapping mode using the atomic force microscope Agilent 5500 (Keysight Technologies, Santa Rosa, California, USA).A drop Theof a diluted silicon suspension tips PPP-NCH (10−4 %) (Nanoandmore, of the original CNCs Wetzler, in water, Germany) prior to solvent were exchange, used which had was deposited on a fresh mica surface and kept to air-dry overnight. The surface1 was imaged in the a resonance frequency of ca. 350 kHz and a spring constant of ca. 50 N.m− . The CNC thickness was tapping mode using the atomic force microscope Agilent 5500 (Keysight Technologies, Santa Rosa, determinedCalifornia, using USA). Gwyddion The silicon software tips PPP-NCH (version (Nan 2.26)oandmore, using a Wetzler, sample sizeGermany) of 100 were particles. used which had a resonance frequency of ca. 350 kHz and a spring constant of ca. 50 N.m−1. The CNC thickness 3. Resultswas anddetermined Discussion using Gwyddion software (version 2.26) using a sample size of 100 particles.

3.1. Method3. Results Development and Discussion

3.1.1. Optimization3.1. Method Development of the Hydrolysis Medium

Three3.1.1. reactions Optimization take of placethe Hydrolysis during Medium the hydrolysis of isocyanates: (1) the reaction of isocyanate with water to form carbamic acid; (2) spontaneous decomposition of carbamic acid into amine; and Three reactions take place during the hydrolysis of isocyanates: 1) the reaction of isocyanate with (3) a side-reactionwater to form of carbamic the generated acid, 2) spontaneous amine with decomp not-yetosition hydrolyzed of carbamic isocyanates acid into amine, forming and a3) polyureaa (Figure2side-reaction). The third of reactionthe generated can amine be fully with blocked not-yet hydrolyzed if the generated isocyanates amines forming get a polyurea instantly (Figure protonated, which can2). The be successfullythird reaction achievedcan be fully by blocked performing if the generated the hydrolysis amines inget acidicinstantly medium protonated, [24]: which can be successfully achieved by performing the hydrolysis in acidic medium [24]:

FigureFigure 2. The 2. mechanismThe mechanism of isocyanateof isocyanate hydrolysishydrolysis and and the the possible possible formation formation of polyurea. of polyurea. In order to develop a simple pH-based method for determining the amount of free isocyanates In(o-NCO) order to on develop the surface a simple of CNCs pH-based upon carbamation method using for determining 2,4-TDI, a complete the amount hydrolysis of of free the isocyanates free (o-NCO)isocyanates on the surfaceshould be of achieved CNCs uponwithout carbamation any side reactions. using I propose 2,4-TDI, in this a complete work the hydrolysisuse of HCl- of the acidified DMSO as an efficient hydrolysis medium, as I believe that 1) the water from the HCl solution free isocyanates should be achieved without any side reactions. I propose in this work the use of will hydrolyze the isocyanates into amines, 2) HCl can then keep the generated amines protonated HCl-acidifiedpreventing DMSO them asfrom an ereactingfficient with hydrolysis not-yet hydr medium,olyzed asisocyanates, I believe and that 3) (1) DMSO the water prevents from the the HCl solutionaggregation will hydrolyze of the carbamated the isocyanates CNCs and into accelerates amines; the (2) hydrolysis HCl can [25]. then It is keepworth thementioning generated here amines protonatedthat preventingHCl-acidified them DMSO from has reactingbeen used with before not-yet but for hydrolyzed different purposes isocyanates; [26,27]. and (3) DMSO prevents the aggregationDMSO of can the be carbamated easily acidified CNCs using and any accelerates concentration the hydrolysisor volume of [25 HCl.]. It isHowever, worth mentioningit is here thatimportant HCl-acidified to use a DMSOminimum has volume been usedof HCl before solution but as forthe dicarbamatedfferent purposes CNCs, unlike [26,27 neat]. CNCs, are not water-dispersible. Based on few trials, 2 mL of any HCl concentration were found enough to DMSO can be easily acidified using any concentration or volume of HCl. However, it is important acidify 100 mL of DMSO. The impact of HCl concentration on the acidity of the resulting DMSO was to use athen minimum investigated volume (Figure of 3).HCl The pH solution was found as theto significantly carbamated decrease CNCs, by the unlike addition neat of CNCs,2 mL of are not water-dispersible. Based on few trials, 2 mL of any HCl concentration were found enough to acidify 100 mL of DMSO. The impact of HCl concentration on the acidity of the resulting DMSO was then investigated (Figure3). The pH was found to significantly decrease by the addition of 2 mL of low concentrations of HCl (0.1 M–2 M) but almost reach a plateau using concentrations higher than 2 M. As a sensitive method should show a significant pH change in response to a minimal amount of analyte (isocyanate groups here), HCl concentrations higher than 2 M were rejected. At the same time, it is important to have a high proton concentration to protonate a maximum amount of the generated amines upon isocyanate hydrolysis to allow the determination of relatively high amounts of free Surfaces 2019, 2 FOR PEER REVIEW 5

low concentrations of HCl (0.1 M–2 M) but almost reach a plateau using concentrations higher than Surfaces2 M.2019 As ,a2 sensitive method should show a significant pH change in response to a minimal amount of448 analyte (isocyanate groups here), HCl concentrations higher than 2 M were rejected. At the same time, it is important to have a high proton concentration to protonate a maximum amount of the isocyanates.generated amines Therefore, upon HCl isocyanate concentration hydrolysis of 2 to M a seemedllow the todetermination be optimum. of 2 relatively mL of 2M high HCl amounts (4 mmol) canof acidify free isocyanates. DMSO to Therefore, a pH of 2.47 HCl concentration0.01. It is important of 2 M seemed to mention to be optimum. here that the2 mL activity of 2M HCl of H (4+ in ± thismmol) system can is acidify low (activity DMSO to coe a ﬃpHcient of 2.47 of ca.± 0.01. 0.07) It is as important DMSO represents to mention the here major that componentthe activity of of H this+ hydrolysisin this system medium. is low That (activity is the coefficient reason why of ca. the 0.07) mmoles as DMSO of H represents+ calculated the from major the component measured of pH this are signiﬁcantlyhydrolysis lessmedium. than theThat theoretical is the reason amount why the of 4mmoles mmol (Figureof H+ calculated3). from the measured pH are significantly less than the theoretical amount of 4 mmol (Figure 3).

Figure 3. The pH of 100 mL of DMSO upon the addition of 2 mL of HCl of varying concentrations. Figure 3. The pH of 100 mL of DMSO upon the addition of 2 mL of HCl of varying concentrations. The standard deviation of the pH values was not more than 0.01. The mmol of H+ is calculated from The standard deviation of the pH values was not more than 0.01. The mmol of H+ is calculated from the pH value ([H+] = 10-pH, mmol H+ = [H+] * Volume (100 mL)). + pH + + the pH value ([H ] = 10− , mmol H = [H ] * Volume (100 mL)). Based on the crystallographic results of Nishiyama, CNCs with a thickness of 5 nm are made of ca.Based 100 cellulose on the crystallographicchains (10 × 10), out results of which of Nishiyama, only 36 are CNCs on the with surface a thickness (36%) [28]. of 32 5 nmof these are made 36 ofsurface ca. 100 chains cellulose have chains half of (10 their hydroxyl10), out ofgroups which accessible only 36 on are the on surface the surface while (36%)the other [28 four]. 32 chains, of these × 36which surface are chains the corners have halfof the of crystal, their hydroxyl have all their groups hydroxyl accessible groups on on the the surface surface. while So CNCs the other with foura chains,thickness which of 5 are nm the have corners 20% of of their the crystal,hydroxyls have on allthe theirsurface hydroxyl (32%*50% groups + 4%*100% on the = surface.20%). To So obtain CNCs witha maximum a thickness DS of of 5 20%, nm have1 g of 20% CNCs of their(6.2 mmol) hydroxyls should on react the surface with 3.7 (32%*50% mmoles of+ TDI,4%*100% which= also20%). Tomeans obtain a a maximum maximum of DS 3.7 of mmol 20%, 1of g free of CNCs isocyanates (6.2 mmol) can exist should on such react CNC with su 3.7rface mmoles uponof a complete TDI, which alsosurface means carbamation. a maximum Therefore, of 3.7 mmol 4 mmoles of free isocyanatesof HCl are enough can exist to onprotonate such CNC all possible surface free upon isocyanate a complete surfacegroups carbamation. after being hydrolyzed. Therefore, 4 mmoles of HCl are enough to protonate all possible free isocyanate groups after being hydrolyzed. 3.1.2. pH Profiles of TDI Model Compounds in the Proposed Hydrolysis Medium 3.1.2. pHBefore Profiles studying of TDI the Model hydrolysis Compounds of isocyanates in the Proposedand carbamated Hydrolysis CNCs Medium in the acidified DMSO, it is Beforeimportant studying to have the an hydrolysis idea about of isocyanatesthe pH prof andile of carbamated the corresponding CNCs in aromatic the acidified amine DMSO, in this it is medium. Upon TDI-carbamation of CNCs and hydrolysis of o-NCO, the surface of a CNC can be important to have an idea about the pH profile of the corresponding aromatic amine in this medium. visualized as o-aminotoluene (or o-toluidine) molecules. Therefore, the pH of the acidified DMSO Upon TDI-carbamation of CNCs and hydrolysis of o-NCO, the surface of a CNC can be visualized as was studied upon the addition of o-toluidine (Figure 4). The results showed a typical acid/base o-aminotoluene (or o-toluidine) molecules. Therefore, the pH of the acidified DMSO was studied upon titration-like behavior starting with neutralizing the existing protons of HCl until an equivalent point the addition of o-toluidine (Figure4). The results showed a typical acid /base titration-like behavior is reached (upon the addition of ca. 425 mg o-Toluidine (or 4.0 mmol)) followed by a gradual increase startingin pH withby further neutralizing addition the of existingo-toluidine. protons The am ofount HCl untilof o-toluidine an equivalent (4.0 mmol) point needed is reached to reach (upon the the additionequivalent of ca. point 425 is mg in agreement o-Toluidine with (or the 4.0 amount mmol)) of followed HCl in DMSO by a gradual (2 mL of increase 2 M HCl in = pH4 mmol), by further i.e. additioneach toluidine of o-toluidine. molecule The was amount protonated. of o-toluidine (4.0 mmol) needed to reach the equivalent point is in agreement with the amount of HCl in DMSO (2 mL of 2 M HCl = 4 mmol), i.e., each toluidine molecule was protonated. For this method to succeed in determining the amount of isocyanates on the surface of CNCs upon carbamation, the acidified DMSO should efficiently hydrolyze the isocyanate groups (o-NCO) to amines in order to quantify them. Otherwise, this method would not be useful at all. After carbamation, the surface of CNCs can be visualized as o-toluene isocyanate molecules. Upon hydrolysis, o-toluene Surfaces 2019, 2 449 isocyanate becomes o-toluidine and both should, therefore, show similar pH profiles (Figure5). Until the equivalent point, o-toluene isocyanate had a similar pH profile to that for o-toluidine, i.e., both profiles showed similar pH values at equivalent molar amounts (a maximum standard deviation of 0.01). This confirms that acidified DMSO can efficiently hydrolyze isocyanates to amines. After the equivalent point, the pH profile of o-toluene isocyanate showed lower pH values, which is expected, as the generated amine groups at that stage tend to react with not-yet hydrolyzed isocyanate groups as they don’t get instantly protonated due to the absence of protons. This was evident by the appearance of white polyurea particles in the hydrolysis medium. In conclusion, it is possible using this procedure to quantify a maximum of ca. 4.0 mmol of aromatic isocyanates placed on the surface of CNCs Surfaces 2019, 2 FOR PEER REVIEW 6 (or a DSNCO of ca. 20% as mentioned earlier).

+ FigureFigure 4. 4.The The pH pH of of acidiﬁed acidified DMSO DMSO (blue (blue circles)circles) uponupon thethe additionaddition of o-toluidine. [H [H+] (green] (green + pH squares)squares) was was calculated calculated from from the the pH pH value value ([H ([H+] = 1010−−pH). ).

For this method to succeed in determining the amount of isocyanates on the surface of CNCs upon carbamation, the acidified DMSO should efficiently hydrolyze the isocyanate groups (o-NCO) to amines in order to quantify them. Otherwise, this method would not be useful at all. After carbamation, the surface of CNCs can be visualized as o-toluene isocyanate molecules. Upon hydrolysis, o-toluene isocyanate becomes o-toluidine and both should, therefore, show similar pH profiles (Figure 5). Until the equivalent point, o-toluene isocyanate had a similar pH profile to that for o-toluidine, i.e. both profiles showed similar pH values at equivalent molar amounts (a maximum standard deviation of 0.01). This confirms that acidified DMSO can efficiently hydrolyze isocyanates to amines. After the equivalent point, the pH profile of o-toluene isocyanate showed lower pH values, which is expected, as the generated amine groups at that stage tend to react with not-yet hydrolyzed isocyanate groups as they don’t get instantly protonated due to the absence of protons. This was evident by the appearance of white polyurea particles in the hydrolysis medium. In conclusion, it is possible using this procedure to quantify a maximum of ca. 4.0 mmol of aromatic isocyanates placed on the surface of CNCs (or a DSNCO of ca. 20% as mentioned earlier). Surfaces 2019, 2 450

Surfaces 2019, 2 FOR PEER REVIEW 7

Figure 5. The pH profile of acidified DMSO upon the addition of o-toluidine (blue circles) or o-toluene Figure 5.isocyanateThe pH (green profile squares). of acidified DMSO upon the addition of o-toluidine (blue circles) or o-toluene isocyanate (green squares). It is clear that acidified DMSO can efficiently hydrolyze aromatic isocyanates and that the pH Figure 5. The pH profile of acidified DMSO upon the addition of o-toluidine (blue circles) or o-toluene upon hydrolysis is directly linked to their amount: the higher the pH value after hydrolysis, the It is clearisocyanate that (green acidified squares). DMSO can efficiently hydrolyze aromatic isocyanates and that the pH higher the amount of isocyanates got hydrolyzed. The relationship until the equivalent point was upon hydrolysisbest fit as a is polynomial directly linked function to of their power amount: 4 (Figure the 6). higher Using thethis pHequation, value it after should hydrolysis, be possible the to higher It is clear that acidified DMSO can efficiently hydrolyze aromatic isocyanates and that the pH the amountquantify of isocyanates the amount of got free hydrolyzed. isocyanates on The CNC relationship surface upon until carbamation. the equivalent Finally, pointit is of wasa great best fit as upon hydrolysis is directly linked to their amount: the higher the pH value after hydrolysis, the a polynomialimportance function to check of powerif cellulose, 4 (Figure itself without6). Using free thisisocyanates, equation, could it shouldhave an beeffect possible on the pH to quantifyof the the higher the amount of isocyanates got hydrolyzed. The relationship until the equivalent point was amountacidified of free isocyanatesDMSO. For that on purpose, CNC surface CNCs were upon carb carbamation.amated using p-toluene Finally, itisocyanate is of a great (TDI importancewith no to best fit as a polynomial function of power 4 (Figure 6). Using this equation, it should be possible to check ifo-NCO) cellulose, so the itself resultant without CNCs free have isocyanates, no free isocya couldnates have at anall (blank effect onsample). the pH When of the added acidified to the DMSO. quantifyacidified the DMSO, amount no ofchange free isocyanatesin pH was detected. on CNC This surface confirms upon that carbamation. only free isocyanate Finally, groups, it is of when a great For that purpose, CNCs were carbamated using p-toluene isocyanate (TDI with no o-NCO) so the importancethey exist, to would check have if cellulose, an effect itself on the without pH of the free acidified isocyanates, DMSO. could have an effect on the pH of the resultantacidified CNCs DMSO. have For no that free purpose, isocyanates CNCs at were all (blankcarbamated sample). using When p-toluene added isocyanate to the (TDI acidified with no DMSO, no changeo-NCO) in so pH the was resultant detected. CNCs This have confirms no free that isocya onlynates free at isocyanate all (blank sample). groups, whenWhen theyadded exist, to the would haveacidified an effect DMSO, on the no pH change of the in acidified pH was detected. DMSO. This confirms that only free isocyanate groups, when they exist, would have an effect on the pH of the acidified DMSO.

Figure 6. Best-Fit for the relationship between pH value after hydrolysis and the molar amount of

isocyanates. DSNCO represents the moles of NCO groups divided by the moles of hydroxyl groups in a 1.0 g of CNCs (*100%).

FigureFigure 6. Best-Fit 6. Best-Fit for for the the relationship relationship between between pH value after after hydrolysis hydrolysis and and the themolar molar amount amount of of isocyanates. DSNCO represents the moles of NCO groups divided by the moles of hydroxyl groups in isocyanates. DS represents the moles of NCO groups divided by the moles of hydroxyl groups in a 1.0 g of CNCsNCO (*100%). a 1.0 g of CNCs (*100%). Surfaces 2019, 2 451

Surfaces 2019, 2 FOR PEER REVIEW 8

3.2. Quantification of the Free Isocyanates of TDI-Carbamated CNCs (DSNCO) 3.2. Quantification of the Free Isocyanates of TDI-Carbamated CNCs (DSNCO) Now that the method is developed, it is time to apply it on carbamated CNC samples. CNCs of Now that the method is developed, it is time to apply it on carbamated CNC samples. CNCs of a thickness of 7.0 nm (determined using AFM) were carbamated at 35, 55, and 75 ◦C following the a thickness of 7.0 nm (determined using AFM) were carbamated at 35, 55, and 75 °C following the method of Habibi in order to have an initial idea about the effect of temperature on p-NCO/o-NCO method of Habibi in order to have an initial idea about the effect of temperature on p-NCO/o-NCO reactivity [15]. Upon carbamation, the CNCs were collected, transferred to the hydrolysis medium, reactivity [15]. Upon carbamation, the CNCs were collected, transferred to the hydrolysis medium, and the final pH was determined. and the final pH was determined. The carbamation of CNCs and the later hydrolysis of the free isocyanates can be confirmed using The carbamation of CNCs and the later hydrolysis of the free isocyanates can be confirmed using FT-IR (Figure7) by monitoring the appearance and disappearance of NCO asymmetric vibration bands FT-IR (Figure 7) by monitoring the appearance and disappearance of NCO asymmetric vibration around 2320 and 2370 cm 1, the urethane carbonyl stretching vibration band at 1720 cm 1, and the − −1 − −-1 bands around 2320 and 2370 cm , the urethane1 carbonyl stretching vibration band at 1720 cm , and bands of aromatic ring at 1520 and 1620 cm− [29]. The original CNCs showed the typical glycosidic the bands of aromatic ring at 1520 and 1620 cm−1 [29]. The original CNCs showed the typical vibration bands ca 1100 cm 1, the C-H stretching band at 2880 cm 1, and the O-H stretching band at − −1 − −1 glycosidic1 vibration bands ca 1100 cm , the C-H stretching band at 2880 cm , and the O-H stretching 3350 cm− . After carbamation, the vibration bands of isocyanate, urethane carbonyl, and aromatic band at 3350 cm−1. After carbamation, the vibration bands of isocyanate, urethane carbonyl, and ring can be seen as the 2,4-TDI molecules reacted with the CNCs. After hydrolysis, the NCO vibration aromatic ring can be seen as 1the 2,4-TDI molecules reacted with the CNCs. After hydrolysis, the NCO bands at 2320 and 2370 cm− expectedly disappeared as the NCO groups were hydrolyzed to amines. vibration bands at 2320 and 2370 cm−1 expectedly disappeared as the NCO groups were hydrolyzed It was, however, not possible to monitor the N-H stretching vibration band of the generated amine to amines. It was, 1however, not possible to monitor the N-H stretching vibration band of the groups at 3350 cm− as it overlaps with the O-H stretching band of the CNCs. generated amine groups at 3350 cm−1 as it overlaps with the O-H stretching band of the CNCs.

Figure 7. FT-IRFT-IR spectra of the CNCs before and after ca carbamationrbamation using 2,4-TDI and after hydrolysis using acidified acidiﬁed DMSO.

When a molecule of TDI reacts through its para-NCO with a hydroxyl group on the CNC surface, it is often assumed that o-NCO will stay free and will not be involved in any side reactions. This assumption is based on the reported 5–10 times difference in p-NCO/o-NCO reactivity, which is attributed to the steric hindrance imposed by the methyl group of TDI on the o-NCO [12,13]. However, in reality, this assumption is inaccurate as a significant amount of o-NCO still reacts minimizing the efficiency of the TDI-carbamation of CNCs and reducing the amount of desired molecules that can be grafted on the CNC surface at a later stage. This hypothesis can be confirmed by measuring the amount of free isocyanate groups available on the CNC surface after TDI-based carbamation (DSNCO) and comparing it to the actual amount of TDI reacted with CNCs (DSTDI). The degree of substitution of isocyanates (DSNCO) was calculated using the final pH value upon hydrolysis:

4 3 2 mmol NCO 24.3pH +290.3pH 1302.0pH +2598.7pH 1946.0 = = − − − DSNCO mmol CNC Hydroxyls 3 (mass CNCs/162) 100% (1) ∗ ∗

On the other hand, the degree of substitution of TDI (DSTDI) can be determined using EDX as a function of the increase in nitrogen/carbon molar ratio (N/C) (Please see the Supplementary Materials for more details about the estimation of DSTDI using EDX, elemental analysis, and the mass yield of the carbamation reaction). The results showed that DSNCO is always lower than DSTDI, which confirms that the o-NCO groups of TDI are not unreactive during CNC carbamation (Table1) and can still react with neighboring hydroxyl groups or undergo some side reactions [30]. This may explain the inability of some reports to graft certain functional polymers on a CNC surface in amounts equivalent to the amount of TDI placed earlier on that surface as not all of those TDIs had free isocyanate groups [19]. This also confirms that elemental analysis is not an accurate method for estimating the amount of NCO groups on a carbamated CNC surface as a perfect p-NCO/o-NCO selectivity could be impossible to achieve. For instance, the CNCs carbamated here at 75 ◦C had a DSTDI of 14.2%, which is close to the maximum possible DS on the surface of these 7.0 nm-thick CNCs (14.3 % assuming the C3 hydroxyl groups are also reactive). However, DSNCO is significantly lower (4.9%), which means that only one third (4.9%/14.2%) of o-NCO stayed unreacted after carbamation. The results also showed that a lower temperature of 55 ◦C leads to a higher amount of free isocyanates than the traditionally used 75 ◦C (Table1) although 75 ◦C led to a higher amount of TDI reacting with the CNCs. A better selectivity can be achieved by further lowering the temperature to 35 ◦C. It is reported that the activation energy of the reaction of o-NCO with a hydroxyl group is significantly higher than that of p-NCO. The difference, however, can vanish by increasing the reaction temperature making both isocyanates react at comparable rates [22]. Therefore, a higher p-NCO/o-NCO selectivity can be achieved using low carbamation temperatures. These interesting outcomes triggered us to explore in a future publication the nature of TDI-carbamation of CNCs and optimize it towards the highest DSNCO/DSTDI.

Table 1. The eﬀect of carbamation temperature on the amount of free isocyanates on the CNC surface.

T(◦C) Final pH mmol NCO DSNCO (%) N/C Ratio DSTDI (%) DSNCO/DSTDI (%) 35 2.51 0.00 0.59 0.00 3.2 0.0 0.045 0.001 4.5 0.1 72 1.6 ± ± ± ± ± ± 55 2.55 0.01 1.07 0.11 5.8 0.6 0.077 0.001 9.6 0.2 61 6.4 ± ± ± ± ± ± 75 2.54 0.00 0.91 0.06 4.9 0.3 0.096 0.001 14.2 0.3 34 2.2 ± ± ± ± ± ±

4. Conclusions A simple and accurate method was developed for the determination of the free isocyanates which are available on the surface of CNCs upon carbamation with 2,4-toluene diisocyanate. It relies on the increase in pH upon the hydrolysis of the free isocyanates to amine groups using HCl-acidified DMSO as a hydrolysis medium. Acidified DMSO was shown to be efficient to hydrolyze isocyanates to amine groups in a short time. The method showed that elemental analysis significantly overestimates the Surfaces 2019, 2 453 amount of free isocyanates on the surface of CNCs upon TDI-based carbamation, as it relies on the invalid assumption that only one of the two isocyanates of TDI is reactive during CNC carbamation. This method also showed that the carbamation temperature has a negative impact on the amount of free isocyanates and recommends, therefore, the use of a lower temperature (55 0C for example) to increase the efficiency of such carbamation reaction.

Supplementary Materials: The following are available online at http://www.mdpi.com/2571-9637/2/2/32/s1, Figure S1: The estimation of DSTDI of carbamated CNCs using the N/C molar ratio obtained from EDX, Figure S2: An example of an EDX measurement of a carbamated CNC sample, Table S1: The estimation of DSTDI using EDX results based on N/C, N/O, and C/O Ratios, Table S2: The estimation of DSTDI using EDX, Elemental Analysis, and Mass Yield. Funding: The author would like to thank the Fraunhofer Institute for Wood Research (WKI) for funding this research work through the Wilhelm-Klauditz Fellowship. Acknowledgments: Many thanks to Kirsten Schiffmann and Nadine Nöcker from the Fraunhofer Institute for Surface Engineering and Thin Films (IST) for the EDX measurements, and to Janin Schloesser from the Technical University of Braunschweig for the elemental analysis measurements. Conflicts of Interest: The author declares no conflict of interest.

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