*Article* **Reductive Amination Reaction for the Functionalization of Cellulose Nanocrystals**

**Omar Hassan Omar 1 , Rosa Giannelli 2 , Erica Colaprico 2 , Laura Capodieci 3 , Francesco Babudri 2 and Alessandra Operamolla 4,\***

**Abstract:** Cellulose nanocrystals (CNCs) represent intriguing biopolymeric nanocrystalline materials, that are biocompatible, sustainable and renewable, can be chemically functionalized and are endowed with exceptional mechanical properties. Recently, studies have been performed to prepare CNCs with extraordinary photophysical properties, also by means of their functionalization with organic light-emitting fluorophores. In this paper, we used the reductive amination reaction to chemically bind 4-(1-pyrenyl)butanamine selectively to the reducing termini of sulfated or neutral CNCs (S\_CNC and N\_CNC) obtained from sulfuric acid or hydrochloric acid hydrolysis. The functionalization reaction is simple and straightforward, and it induces the appearance of the typical pyrene emission profile in the functionalized materials. After a characterization of the new materials performed by ATR-FTIR and fluorescence spectroscopies, we demonstrate luminescence quenching of the decorated N\_CNC by copper (II) sulfate, hypothesizing for these new functionalized materials an application in water purification technologies.

**Keywords:** cellulose nanocrystals; nanocellulose; pyrene; organic light-emitting material; reductive amination; nanocellulose functionalization; nanocellulose fluorescence

## **1. Introduction**

The nanostructures isolated from native cellulose, such as cellulose nanofibers (CNFs), cellulose nanocrystals (CNCs) and bacterial cellulose (BC) [1–3], have recently attracted tremendous interest, thanks to their outstanding chemical and mechanical properties [4–8]. These nanomaterials of biologic origin are very appealing for numerous applications and have raised the industrial interest, since cellulose is abundant and can be isolated in crystalline nanostructures with low environmental impact [9]. Besides, nanocelluloses are colorless or even transparent and harmless for human health [10,11]. Their potential in industrial application is mainly related to their sustainable nature, which makes them good candidates as substitutes of oil derivatives for the manufacture of goods, in order to reduce pollution and better commit to the principles of green and blue economies [2]. The current research stream is exploring their use in high-volume industrial applications, such as in the fields of water treatment, in the paint and coating industry, in the building industry, in the hygiene sector and in the paper industry [12].

Siding the industrially oriented research, new emerging fields of application of nanocellulose include hydrogels and aerogels [13,14], emulsion stabilizers [15], biocatalyst

**Citation:** Hassan Omar, O.; Giannelli, R.; Colaprico, E.; Capodieci, L.; Babudri, F.; Operamolla, A. Reductive Amination Reaction for the Functionalization of Cellulose Nanocrystals. *Molecules* **2021**, *26*, 5032. https://doi.org/10.3390/ molecules26165032

Academic Editors: Tersilla Virgili and Mariacecilia Pasini

Received: 29 June 2021 Accepted: 17 August 2021 Published: 19 August 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

immobilizers [16], biosensors [17], drug delivery systems [18], adsorbents for contaminants [19,20], nanocomposites for environmental remediation [21], photonic films and transparent substrates for optoelectronic devices, as well as new nanostructured electroactive materials [22–26].

Among the various typologies of nanocellulose, CNCs are defined, according to the standards of the Technical Association of Pulp and Paper Industry (TAPPI), as crystalline rods featuring a high aspect ratio, a diameter between 5 and 50 nm and a length ranging from 100 to 500 nm. CNCs are the smallest nanostructures which may be isolated from native cellulose and display a high content of crystalline domains. CNCs were discovered in the 1950s by Rånby and Battista [27,28], who independently isolated them by acid hydrolysis. However, only in the last 15 years was the high potentiality of cellulose nanocrystals re-evaluated in light of the new intriguing applications listed above.

CNCs display a high specific surface area (SSA) and expose pending hydroxyl groups on their surface: these chemical functionalities may be exploited to carry out surface functionalization of the nanocrystals. For this reason, the concept of topochemical functionalization of nanocellulose, i.e., a functionalization involving only the surface of the nanocrystals, has become crucial with respect to bulk functionalization, i.e., the complete dissolution of the cellulose crystalline structure due to complete derivatization of the cellulose polymer [29]. In this respect, only mild reaction protocols, with controlled reaction times, can prevent crystalline phase transitions or, even worse, dissolution of the cellulose polymer [30]. This approach sometimes brings some complications, such as the necessity to perform a sequence of chemical reactions on nanocellulose to convert the hydroxyl into an activated group, with the difficulty of obtaining a fine control over the surface functionalities, their number and homogeneity.

An alternative and straightforward strategy for nanocellulose functionalization relies on a reaction used in polysaccharide technology, the reductive amination reaction [31], which modifies only the reducing ends of the cellulose nanocrystals, leaving the remaining nanocrystal surface unfunctionalized or available for further chemical manipulation. According to a model proposed so far [32], CNCs correspond to elementary fibrils and are rigid rods composed of 36 parallel cellulose polymer chains, kept together by intermolecular hydrogen bonds. The number 36 comes from the nature of the enzymatic complex which synthesizes cellulose in plants, the cellulose synthase (CESA) complex, composed by 6 rosettes each made of 6 enzymes which polymerize D-glucopyranose from UDPglucose [33]. The trans-membrane enzymatic complex extrudes the 36 cellulose chains in parallel organization, and the chains associate into a cellulose I crystal structure [34] as soon as they leave the cell wall. Once cellulose nanocrystals have been recovered from a cellulose source by acid hydrolysis, they present the same crystalline association as in their native structure with the reducing termini of the polymer chains on the same side of the cellulose nanorod (as depicted in Figure 1).

The extensive functionalization of CNCs' surface may drastically change their surface properties, for instance modifying their suspension-forming ability in water or the way they interact with oleophilic or oleophobic molecules. Conversely, an incomplete functionalization on the CNCs' surface, for instance with a degree of substitution (DS) ≤0.37 [35], would not allow to understand the exact position of the new functional groups, since most of the reactions which may be performed on the cellulose polymer are only selective, but not specific towards the primary alcohol group [31]. This, for some applications, could be tricky. For this reason, an issue arises in which a mild reaction strategy allows specific functionalization of nanocellulose. In the present work, we use the reducing end functionalization to introduce 1-pyrenyl units as substituents on cellulose nanocrystals: the organic fluorophores are introduced to endow the nanocrystals with an additional property, i.e., pyrene-luminescence, while their hydrophilicity is preserved, being the pyrene units introduced only at the reducing end of the nanocrystal, as depicted in Figure 1. We demonstrate that pyrene-emission (and its excimer) is detectable in the functionalized nanocrystals in spite of the low amount of dye linked to the nanocrystals. Second, we

demonstrate pyrene luminescence quenching in the presence of Cu(II) salts. These experiments demonstrate that, using the reductive amination reaction, it is possible to introduce an additional property on CNCs while leaving the remaining pending hydroxyl groups available for interaction with the solution or for further functionalization. In our view, this strategy is important in order to have the surface of the nanocrystal available for anchoring on filtration devices.

**Figure 1.** A cellulose nanorod, represented according to the model proposed in [32], with 1-pyrenyl units attached to the reducing ends (highlighted in yellow) by reductive amination.

#### **2. Results**

#### *2.1. Synthesis of the Pyrene Dye*

For performing the reductive amination reaction, we needed a pyrene dye functionalized with a pending primary amine functionality. A good precursor of the 1-pyrenyl butanamine **4** was the commercially available 4-(1-pyrenyl)buthanol 1. The reaction sequence leading to compound **4** starting from **1** and the relative yields are reported in the Scheme 1. We first prepared the tosylate derivative **2** via a room temperature tosylation reaction of the alcohol group with a yield of 55%; following this, we converted the tosyl group into an azide by a nucleophilic substitution performed using sodium azide in DMSO at room temperature. The azide is a good precursor of primary amines, and was isolated in 87% yield. Finally, **4** was afforded in satisfactory yield (50%) by submitting the pyrene azide **3** to a hydrogenation reaction in the presence of palladium, supported on charcoal as a catalyst and EtOH as a solvent. The catalytic hydrogenation was much more efficient in furnishing **4** than the Staudinger reaction, which yielded a mixture of the primary amine with phosphine oxide. Our synthetic sequence slightly differs from other synthetic sequences reported by others [36,37], but allowed the isolation of **4** in good overall yield and good purity. The identity of **4** was assessed by 1H- and 13C-NMR spectroscopies and by LCMS-IT-TOF spectrometry (compare Figures S1–S6, Supplementary Materials).

**Scheme 1.** Synthetic sequence leading to 4-(1-pyrenyl)butanamine **4**.

## *2.2. Synthesis of Sulfated and Neutral CNCs*

Cellulose nanocrystals were prepared by acid hydrolysis with either sulfuric acid or hydrochloric acid at controlled reaction conditions. The starting material was Avicel PH-10.1, a cotton linter-based commercial stationary phase for column chromatography. As extensively reported in the literature, sulfuric acid hydrolysis yields surface negatively charged S\_CNC, due to a parallel process of surface sulfatation involving the primary hydroxyl groups on the surface glucopiranose units of the nanocrystals [38]. As explained in the next sections, the surface sulfatation determined aggregation of the cellulose nanocrystals in the presence of the salts used in the following experiments. For this reason, we also prepared neutral cellulose nanocrystals (neutral = no bonded surface charge) by hydrochloric acid hydrolysis from the same starting material. The uncharged nanocrystals were named N\_CNC. N\_CNC displayed a higher aggregation tendency in distilled water, as an effect of the absence of any surface charge. However, they could be characterized by FE-SEM microscopy by deposition from DMSO suspensions, in which they showed better stability. Figure 2 shows the FE-SEM micrographs recorded on (a) S\_CNC deposited from water and (b) N\_CNC deposited from DMSO on silicon substrates. Thanks to these microscopic observations, we could measure an average length of 149 ± 32 nm and 151 ± 17 nm for S\_CNC and N\_CNC respectively, while in both cases. the thickness was detected by AFM to be 10 ± 2 nm.

**Figure 2.** FE-SEM micrographs of (**a**) S\_CNC deposited from 10 mg L−1 water suspension, and (**b**) N\_CNC deposited from 1 mg L−1 DMSO suspension. Scale bar: 200 nm.

## *2.3. Synthesis of Pyrene Derivatives of CNCs*

The functionalization of cellulose nanocrystals with the dye **4** was conveniently performed in water solvent, dispersing the cellulose nanocrystals in a pH 6.0 and 50 mM phosphate buffer. A 1% molar ratio between the dye and the nanocellulose (mmol of **4** vs. mmol of glucopyranose) was used to perform the reaction. The functionalization was carried out in the presence of a molar excess of sodium cyanoborohydride. The synthesis of the pyrene-functionalized cellulose nanocrystals is depicted in Scheme 2. At the end of the reaction time, the nanocrystals were purified by washing with methanol, followed by dialysis against mQ water to eliminate traces of unreacted dye and excess salts. Finally, the samples were freeze-dried and used in the next experiments. An AFM check of the preserved nanocrystals' morphology was performed on their thin films. Figure S7, reported in the Supplementary Materials, demonstrates the preserved nanorod aspect of the S\_CNC sample after the reductive amination.

**Scheme 2.** Reductive amination between **4** and sulfated or neutral cellulose nanocrystals.

ATR-FTIR spectroscopy performed on the freeze-dried samples was very useful to have an indication of the success of the functionalization. Figure 3 shows the ATR-FTIR spectra of the 4-(1-pyrenyl)butanamine **4** (blue trace), of S\_CNC (black trace) and of the modified S\_CNC sample **7a** (red trace). The spectrum of the cellulose nanocrystals displayed the common features of cellulose I, with a strong absorption band at 3340 cm−1 attributed to -OH groups' stretching, forming intramolecular hydrogen bonds. Absorption bands attributed to C-H stretching modes below 3000 cm−1 were also clearly visible and centered in the nanocelluloses at 2900 cm−1 . The signal centered at 1644 cm−1 in CNCs could be attributed to residual water contained in the cellulose nanocrystals. The intense band between 920 and 1220 cm−1 in the nanocrystals was attributed to stretching modes of single C-O-C bonds of the acetal units.

**Figure 3.** ATR-FTIR spectra of (**a**) 4-(1-pyrenyl)butanamine **4**, (**b**) pyrene-modified S\_CNC **7a** and (**c**) unmodified S\_CNC.

Butanamine **4** presented distinctive signals, such as a broad absorption at ~3350 cm−1 (stretching of -NH2 group), characteristics of absorption of aromatic C-H stretching at 3044 cm−1 , -NH2 group bending modes at ~1574 and in the region below 900 cm−1 , besides aromatic stretching modes in the region between 1600 and 1400 cm−1 and intense aromatic C-H bending modes below 900 cm−1 . In particular, the intense absorption band centered at 841 cm−1 could be attributed to aromatic C-H bending and produced a small overtone at ~1670 cm−1 .

The modified S\_CNC **7a** showed typical features of the cellulose nanocrystals in their spectrum, such as the broad absorption band attributed to the -OH stretching, but with the appearance of features representative of the butanamine **4**. First, the increase in intensity of the -OH stretching band with respect to the acetal stretching band pointed at an increase in the sample of hydrogen bond-donating groups (hence the presence of secondary amine functionalities and additional hydroxyl groups in the reducing termini introduced by the reaction), with the contemporary loss in intensity of acetal absorption due to the reducing ends turned from hemiacetal into secondary amines. The broad band of -OH stretching also presented a small shoulder corresponding to the resonance frequency of aromatic C-H stretching at 3044 cm−1 . Presence of the aromatic units was also evident from the increase in intensity of the band attributed to crystallized water, overlapped to the overtone of the bending absorption of aromatic C-H. Finally, the strong absorption at 841 cm−1 , attributed to aromatic C-H bending, appeared as a distinctive absorption in the spectrum of modified S\_CNC **7a**.

## *2.4. Photophysical Characterization*

Emission spectra of the pristine S\_CNC and N\_CNC and of the pyrene-modified nanocrystals **7a** and **7b** are shown in Figure 4, while the positions of the emission maxima are listed in Table 1. Emission spectra were collected in DMSO to evidence the presence or not of the excimer of pyrene, whose emission is centered at ~470 nm. The pyrene butanamine **4** showed a distinctive emission profile, with characteristic emission maxima at 378, 399, 420 and 475 nm (Figure 4e). The last emission peak was absent in water solution and detectable in DMSO (compare Supplementary Figure S8 for the relevant emission spectrum of **4** in water). As expected, modified S\_CNC and N\_CNC **7a** and **7b** showed the distinctive emission peaks of butanamine **4** at the following wavelengths: 378, 398, 419 and 470 nm. Surprisingly, both pristine S\_CNC and N\_CNC showed an emission in the UV region, upon excitation at 345 nm, with considerably lower intensity than the pyrene-modified nanocrystals **7a** and **7b**. This emission was not observed in commercial sulfated cellulose nanocrystals (panel f, Figure 4), which were investigated as a control sample. As it will be explained in the Discussion Section, this phenomenon was attributed to the cellulose source used for the preparation of cellulose nanocrystals by acid hydrolysis.

#### *2.5. Luminescence Quenching Experiments*

Luminescence quenching experiments were investigated on both pyrene-modified S\_CNC and N\_CNC **7a** and **7b** in the presence of increasing concentration of diverse salts in water suspensions. The scope of the experiments was to have a preliminary information on whether the pyrene-modified cellulose nanocrystals interacted preferentially with some salts and were therefore suitable to develop sensing systems in water or to being immobilized in membranes for water purification. Several salts were tested: NaCl, FeCl3, CuSO4 · 5H2O, Hg(OAc)2. The Figure 5 shows the result of CuSO4 slow addition to a water suspension of N\_CNC (left panel) and pyrene-modified N\_CNC 7b (right panel). The same experiments were performed on S\_CNC, but the pyrene-modified S\_CNC 7a yielded inconsistent response to the addition of each salt (data not shown in this paper), due to aggregation and precipitation of the nanocrystals. Pyrene-modified N\_CNC 7b, instead, yielded a rational response only in the presence of copper sulfate, while were apparently unperturbed by the other salts. These results will be better discussed in the next paragraph.

**Figure 4.** Emission spectra of cellulose nanocrystals recorded in 0.2 mg mL−1 DMSO suspensions: (**a**) S\_CNC, (**b**) pyrene-modified S\_CNC **7a**, (**c**) N\_CNC, (**d**) pyrene-modified N\_CNC **7b**, (**e**) spectrum of 4-(1-pyrenyl)butanamine **4** in the same solvent and (**f**) commercial sulfated CNCs. Excitation wavelength = 345 nm. Emission spectra of S\_CNC and N\_CNC were recorded using a 1 cm cuvette. Emission spectra of pyrene-modified S\_CNC and N\_CNC **7a** and **7b** were recorded using a 0.1 cm cuvette.

**Table 1.** Emission maxima of all samples measured in DMSO.

1 Emission peaks of 4-(1-pyrenyl)butanamine **4** in water: 378, 399, 420 nm (Supplementary, Figure S8). Relevant absorption spectra of **4** in DMSO, CHCl3 and water are reported in the Supplementary, Figures S9 and S10. UV-Vis Absorption spectra of S\_CNC and pyrene-modified S\_CNC 7a are reported in the Supplementary, Figures S11 and S12.

**Figure 5.** Emission spectra of N\_CNC recorded in 0.2 mg mL−1 water solutions in the presence of Cu2+ salts: (**a**) pristine N\_CNC and (**b**) pyrene-modified N\_CNC **7b**. The insets show the molar concentration of salts used.

#### **3. Discussion**

The reductive amination reaction allowed us to carry out a covalent functionalization of the cellulose nanocrystals with 4-(1-pyrenylbutanamine) **4**, availing of a very mild reaction protocol performed in water. Purification of the sample was straightforward, as it was performed by dialysis in water. To assess the effectiveness of the functionalization, we acquired the ATR-FTIR spectra of S\_CNC and their pyrene-modified derivative **7a**, identifying the signals of the pyrene aromatic system in the spectrum of the hybrid system. In order to confirm the effectiveness of the reaction, we also coupled an aliphatic amine with D-glucopyranose in the same conditions, successfully isolating the corresponding amination product (data not shown in this paper). This allowed us to understand that the proposed synthetic protocol is reliable for the effective conversion of the imine intermediate (the Schiff's base generated as a result of the nucleophile attack of the primary amine to the aldose unit) into a secondary amine by sodium cyanoborohydride. The amount of dye bonded to the nanocrystals was very low, as the reductive amination reaction was performed with a molar ratio of **4**:glucopiranose units of 1:100, and the linked pyrene dyes were not detectable by UV-Vis spectroscopy, as they were covered by the scattering produced by the nanocrystals (see Supplementary Figures S10 and S11 acquired for the S\_CNC samples).

Then, we started the luminescence characterization. Unexpectedly, recording the reference spectra for S\_CNC and N\_CNC, we found an emission from both samples, upon excitation of the relevant suspensions at the wavelength of 345 nm, that peaked at 378 and 400 nm for S\_CNC and 383 nm for N\_CNC (compare Table 1). The relevant spectra for DMSO suspensions are the ones reported in Figure 4a,c. This emission was found on spectra recorded in DMSO and in water (for the relevant emission spectrum recorded from N\_CNC water suspensions, please refer to Figure 5a, black trace). Suspecting a possible contamination of the products during their preparation in our laboratories, we repeated the preparation of both typologies of cellulose nanocrystals, by hydrolysis with sulfuric acid or hydrochloric acid, several times in the same and also in different laboratories, each time recording the same result. To finally understand the possible origin of this emission from our cellulose nanocrystals, in the same conditions, we analyzed commercial CNCs purchased from CelluForce. The relevant emission spectrum is reported in Figure 4f. The commercial cellulose nanocrystals did not show any emission feature. Commercial CNCs from cellulose are produced, as declared by the company, by sulfuric acid hydrolysis from wood pulp. Hence, we started to hypothesize whether the emission from the CNCs produced in our laboratories was dependent on the nature of the starting cellulose material, the microcrystalline cellulose Avicel PH-10.1. This microcrystalline cellulose derives from cotton by hydrolysis with diluted mineral acids [39], has a high cellulose I content with very

high crystallinity index and it is often used as a reference for XRD or Raman assessment of the crystallinity index of cellulose [40,41].

The luminescent properties of cellulose are the subject of controversial literature reports. Actually, our CNCs displayed a similar emission to the one observed from Avicel by Castellan et al. [42], attributed to heteroaromatics of the furan and pyron type, possibly formed in traces by degradation of the polysaccharide chain. A similar hypothesis was formed by Kalita et al. [43], who attributed the autofluorescence at ~400 nm found in their synthetic CNCs to the presence of fluorescent subunits identified in the FTIR spectra as lignin fragments, such as *p*-coumeric and ferulic acid or phenolic structures. Ding et al. [44] attributed the autofluorescence of cellulose to a partial double-bond character displayed by the glycosidic bond, whose presence was also hypothesized in disaccharides such as cellobiose and maltose. However, it was demonstrated by Arndt and Stevens [45], using UV-circular dichroism, that mono- and di-saccharides absorb only between 150 and 190 nm. A final interpretation was that by Gan et al. [46], who hypothesized that the emission recorded from cellulose nanocrystals was the consequence of a Stokes scattering [47,48] enhanced by CNC-oriented assembly [49]. In our case, it is very difficult to understand which can be the origin of the luminescence observed in CNCs, as the FTIR spectra of our pristine CNCs also did not reveal the presence of aromatic structures. However, the native emission intensity was very low if compared to the luminescence of pyrene-modified nanocrystals, and it was not responsive to the titration experiments that we performed with salts (compare Figure 5a).

Pyrene-modified S\_CNC and N\_CNC **7a–b** displayed the distinctive emission features of 4-(1-pyrenyl)butanamine **4**, with emission peaks at 378, 398, 419 and 470 nm. This result was remarkable considering the very low amount of dye used during the functionalization reaction (1 molar % with respect to glucopyranose units). The emission wavelength of 470 nm observed in DMSO was attributed to pyrene excimer formation. At this point, we decided to investigate how interaction with cations influenced the emission intensity from the cellulose nanocrystals, with the aim to understand whether a luminescence quenching induced by any of the salts used could suggest a possible application of the systems that we had prepared in water purification technology. During this investigation, we had to discard experiments conducted on pyrene-modified S\_CNC **7a**, because interaction with cations of these surface-negatively charged nanorods induced their aggregation, preventing any reasonable conclusion on their luminescence behavior. We took this behavior as a demonstration that the terminal functionalization had left the surface of the S\_CNC unperturbed, exposed to the external solution and therefore free to interact with the other solutes. Conversely, Zhang et al. [50] reported an extensive surface functionalization of S\_CNC with pyrene units, and no aggregation was observed in the presence of a wide number of cations. Pyrene-modified N\_CNC **7b** yielded a luminescence quenching response only in the presence of increasing concentrations of copper sulfate, while it was apparently unperturbed by NaCl and Hg(OAc)2. FeCl3 was discarded as a quencher, since it displayed an absorption in the relevant region for the experiments (overlapping absorption of the pyrene dye). Thus, among the salts investigated, the pyrene-modified N\_CNC **7b** selectively interacted only with copper sulfate.

The presence of two vicinal -OH and secondary amine groups, formed on the C1 and C2 positions of the terminal polysaccharide unit of the nanocrystal in the pyrene-modified N\_CNC **7b**, could potentially act as a bidentate ligand for metal cations, with potential assistance from other neighboring -OH groups, while the free -OH pending groups on the CNC surface remain available for establishing H-bonds, which could be, for instance, used to anchor the nanocrystals on a surface or on paper, to obtain a sustainable filtration device. A similar principle was exploited by Lu et al. [51] to prepare a hydrophilic fluorescent paper sensor for nitroaromatic compounds (NACs) exploiting pyrene sensing ability, towards whom our pyrene-cellulose derivatives could be exploited as well.

#### **4. Materials and Methods**

#### *4.1. Synthesis of 4-(1-pyrenyl)Butanamine* **4**

**General:** All reagents were purchased at the highest commercial quality and used without further purification. Reactions were carried out under a nitrogen atmosphere in oven-dried glassware, using dry solvents unless otherwise stated. Dichloromethane was distilled immediately prior to use on phosphorus pentoxide. Anhydrous grade dimethylsulfoxide was used and dispensed under nitrogen. Absolute ethanol was used without further purification. Preparative column chromatography was performed using silica gel 60, particle size 40 ÷ 63 µm from Merck. Merck silica gel 60 F254 aluminium sheets were used for TLC analyses. All new compounds were characterized by 1H-NMR, 13C-NMR, FTIR spectroscopy and HMRS spectrometry. GC-MS analyses were performed on a gas chromatograph equipped with an SE-30 (methyl silicone, 30 m × 0.25 mm id) capillary column and an ion trap selective mass detector. 1H-NMR and 13C-NMR spectra were recorded at 500 MHz and 125 MHz, respectively, on a Bruker Avance AM 500, using the residual proton peak of CDCl3 at 7.26 ppm as reference for 1H spectra and the signals of CDCl3 at 77 ppm for 13C spectra. Coupling constants values are given in hertz. High-resolution mass spectra were acquired on an Agilent high performance liquid chromatography-QTOF spectrometer via direct infusion of the samples using methanol or water as the elution solvent. Melting points (uncorrected) were determined on a Stuart Scientific Melting point apparatus SMP3.

**4-(1-pyrenyl)butyl tosylate 2:** A 2 neck round bottom flask was charged under nitrogen with 0.5 g of 4-(1-pyrenyl)buthanol (1.8 mmol), 0.22 g of dimethylaminopyridine (1.8 mmol), 5 mL of dry dichloromethane and 0.5 mL of triethylamine (3.6 mmol). The system was cooled to 0 ◦C and a solution of tosyl chloride (0.7 g, 3.6 mmol in 5 mL) was added dropwise. The system was kept at 0 ◦C for 2 h, then it was allowed to warm to room temperature and stirred until TLC analysis revealed complete disappearance of the starting material. The reaction was quenched with 30 mL of HCl 1.5 N. The mixture was extracted with dichloromethane (3 × 30 mL). The organic extracts were collected and dried over anhydrous Na2SO4, filtered and the solvent was distilled under reduced pressure. The crude material was purified by column chromatography on silica gel using hexane and ethyl acetate in volumetric ratio 8:2 as the eluent. 0.423 g of a yellow wax were isolated (yield 55%). 1H-NMR (CDCl3, 500 MHz): δ8.23-8.15 (m, 3H), 8.10 (d, 2H, *J* = 8.1 Hz), 8.03 (s, 2H), 8.01 (t, 1H, *J* = 8.1 Hz), 7.80 (d, 2H, *J* = 7.8 Hz), 7.76 (d, 2H, *J* = 7.8 Hz), 7.25 (d, 2H, *J* = 7.3 Hz), 4.09 (t, 2H, *J* = 6.2 Hz), 3.31 (t, 2H, *J* = 7.5 Hz), 2.36 (s, 3H), 1.98–1.72 (m, 2H) ppm. 13C-NMR (CDCl3, 126 MHz): δ144.6, 135.8, 133.10, 131.4, 130.8, 129.9, 129.8, 128.6, 127.8, 127.5, 127.3, 127.15, 126.7, 125.9, 125.10, 125.0, 124.9, 124.8, 123.14, 70.3, 32.7, 28.7, 27.5, 21.5, 14.19 ppm.

**1-(4-azidobutyl)pyrene 3:** A 100 mL round bottom flask was charged with 0.4 g of 4-(1 pyrenyl)butyl tosylate (0.93 mmol), 0.091 g of sodium azide and 5 mL of dry DMSO under nitrogen atmosphere. The mixture was stirred at room temperature for one night. After this time TLC analysis revealed the complete disappearance of the starting material. The mixture was diluited with 30 mL of brine and extracted with ethyl acetate (3 × 30 mL). The organic extracts were collected and washed with brine (2 × 30 mL), dried over anhydrous Na2SO4, filtered and the solvent was distilled under reduced pressure. 0.241 g of a white solid were isolated (yield 87%). Mp = 78–79 ◦C. 1H-NMR (CDCl3, 500 MHz): δ8.26 (d, 1H, *J* = 9.5 Hz), 8.17 (dd, 2H, *J*1 = 7.5, *J*2 = 4.0 Hz), 8.12 (d, 2H, *J* = 8.5 Hz), 8.04 (d, 1H, *J* = 9.5 Hz), 8.02 (d, 1H, *J* = 9.5 Hz), 7.99 (d, 1H, *J* = 7.5 Hz), 7.86 (d, 1H, *J* = 8.0 Hz), 3.38 (t, 2H, *J* = 7.5), 3.34 (t, 2H, *J* = 7.5 Hz), 1.96 (quintuplet, 2H, *J* = 7.5 Hz), 1.78 (quintuplet, 2H, *J* = 7.5 Hz). ppm. 13C-NMR (CDCl3, 126 MHz): δ136.10, 131.4, 130.9, 129.9, 128.6, 127.5, 127.3, 127.18, 126.7, 125.8, 125.10, 125.0, 124.9, 124.8, 124.7, 123.18, 51.4, 33.0, 28.9, 28.8 ppm.

**4-(1-pyrenyl)butanamine 4:** A 250 mL round bottom flask was charged with 0.22 g of 1-(4-azidobutyl)pyrene (0.74 mmol), 0.079 g of Pd(C) (10%) (0.074 mmol) and 15 mL of absolute ethanol. The mixture was degassed by nitrogen bubbling for 10 min. Afterwards hydrogen was blown into the mixture using a syringe and a balloon. The mixture was stirred at the room temperature for 48 h. After this time, TLC analysis revealed complete

disappearance of the starting material. The solvent was removed under reduced pressure and the crude material was purified by column chromatography on silica gel using dichloromethane, methanol and triethylamine in volumetric ratio 90:10:1 as the eluent. 100 mg of a yellow wax was isolated (yield 50%). 1H-NMR (CDCl3, 500 MHz): δ8.26 (d, 1H, *J* = 8.3 Hz), 8.16 (dd, 2H, *J*1 = 8.16 Hz, *J*2 = 4.0 Hz), 8.10 (dd, 2H, *J*1 = 8.10 Hz, *J*2 = 3.0 Hz), 8.06–7.93 (m, 3H), 7.86 (d, 1H, *J* = 7.86 Hz), 3.36 (t, 2H, *J* = 8.0 Hz), 2.76 (t, 2H, *J* = 7.0 Hz), 1.89 (quintuplet, 2H, *J* = 8.0 Hz), 1.58 (m, 2H) ppm. 13C-NMR (CDCl3, 126 MHz): δ136.8, 131.4, 130.9, 129.8, 128.6, 127.5, 127.2, 127.17, 126.5, 125.8, 125.10, 125.0, 124.8 (two signals), 124.6, 123.4, 42.10, 33.7, 33.3, 29.13 ppm. LCMS-IT-TOF calculated for C20H19N: 273.1517, found (M + H)+ : m/z 274.1588.

## *4.2. Hydrolysis of Cellulose, Nanocrystals Isolation and Functionalization*

**General** Avicel PH-101 was used as the starting material for nanocrystalline cellulose isolation. Sonication of nanocellulose suspensions was carried out with a Branson Sonifier 250 (Danbury, CT, USA). Dialysis was carried out at room temperature against mQ water in nitrocellulose tubes with a cut off of 12,600 Da. Commercial nanocrystals used for acquisition of the emission spectrum in the Figure 4f were a donation from the Pharmacy Department of the University of Pisa, and had been purchased from CelluForce.

**Synthesis of sulfated CNCs 5a:** This procedure was adapted from Operamolla et al. [52] 47 mL of deionized water were introduced in a 250 mL three necked roundbottom flask equipped with a water condenser and a mechanical stirrer. Then, the flask was cooled in an ice bath and 47 mL of concentrated H2SO4 were added. After that, 5 g of Avicel PH-101 were added and the suspension was warmed to 50 ◦C for 80 min. The system was cooled to room temperature and the mixture was transferred to polypropylene centrifugation tubes. Centrifugation at 4000 rpm was repeated replacing the supernatant solution with fresh deionized water until the pH was approximately 1. Then, the precipitate was suspended in deionized water with the aid of a Branson sonifier 250 (Danbury, CT, USA) equipped with an ultrasonic horn with 3.5 mm diameter (micro tip) operated in pulsed mode, with a power of 40 W, 0.6 s pulses for 10 min and dialyzed against distilled water until neutrality using a cellulose nitrate membrane with a molecular weight cut-off of 12,400 Da. The resulting suspension was transferred to polypropylene centrifugation tubes and centrifuged at 4000 rpm for 20 min. The supernatant solution was kept and water was removed under reduced pressure, yielding 925 mg of cellulose nanocrystals with an average length of 280 ± 70 nm.

**Synthesis of neutral CNCs 5b:** 50 mL of deionized water were introduced in a 250 mL three necked round-bottom flask equipped with a water condenser and a mechanical stirrer. Then, the flask was cooled in an ice bath and 50 mL of concentrated HCl were added. After that, 5 g of Avicel PH-101 were added and the suspension was warmed to 105 ◦C for 6 h. The system was cooled to room temperature, diluted with 50 mL of distilled water and the mixture was transferred to polypropylene centrifugation tubes. Centrifugation at 1000 rpm for 10 min was repeated 4 times replacing the supernatant solution with fresh deionized water until the pH was approximately 3. Then the precipitate was dialyzed against distilled water until neutrality using a cellulose nitrate membrane with a molecular weight cut-off of 12,400 Da. The resulting suspension was recovered and water was removed by distillation under reduced pressure, yielding 4.713 g of cellulose nanocrystals.

**General Procedure for the reductive amination of cellulose nanocrystals (7a,b):** In a 100 mL round bottom flask 500 mg of cellulose nanocrystals (3.1 mmol) were suspended in a 50mM phosphate buffer (pH = 6, 50 mL) using Branson sonifier 250 (Danbury, CT, USA) equipped with an ultrasonic horn with 3.5 mm diameter (micro tip) operated in pulsed mode, with a power of 40 W, 0.6 s pulses for 10 min in the case of sulfated CNCs and operated in constant mode in the case of neutral CNCs. A solution of 4-(1-pyrenyl)butanamine (8 mg, 0.03 mmol) in phosphate buffer (5 mL) was added, followed by 100 mg of NaBH3CN (14.6 mmol). The mixture was stirred at room temperature for 72 h. After this time, the mixture was diluted with methanol (50 mL) and centrifuged at 4000 rpm for 10 min. The liquid

phase was removed and replaced with fresh methanol, the systems were mixed and centrifuged again at 4000 rpm for 10 min. The washing operation was repeated two more times. Then, the precipitate was resuspended in water (30 mL) and dialyzed against distilled water using a cellulose nitrate membrane with a molecular weight cut-off of 12,400 Da. Water was distilled under reduced pressure and a white solid was isolated (0.286 g, 57% yield in the case of sulfated CNCs; quantitative yield in the case of neutral CNCs).

#### *4.3. Characterization*

**ATR-FTIR Analyses** FTIR spectra were recorded on a Spectrum, Perkin-Elmer, Waltham, MA, USA. Two spectrophotometer equipped with an UATR Accessory. The nanocellulose powders were placed in direct contact with the diamond/ZnSe crystal without the aid of any solvent. For the same analysis on 4-(1-pyrenyl)butanamine, a drop of chloroform solution with the concentration of 1 mg/mL was deposited on the crystal and the solvent was allowed to evaporate at room temperature before the measurement was acquired.

**FE-SEM images** The morphology of the surface of nanocrystalline cellulose was analyzed by FE-SEM, on a Field Emission Scanning Electron Microscope, ZEISS Merlin, Jena, Germany, equipped with a GEMINI IIs column and Beam-Booster, with acceleration voltages between 0.05 and 30 kV and 0.8 nm as the best resolution, four optional detectors for SE and BSE, charge compensation and an in situ sample cleaning system. Silicon slabs were used as substrates for FE-SEM measurements. For S\_CNC investigation, a solution 0.01% by weight of nanocrystals in water was drop cast on a silicon slab and allowed to dry overnight. For N\_CNC investigation, a solution 0.001% by weight of nanocrystals in DMSO was drop cast on a silicon slab and allowed to dry overnight.

**AFM Microscopy** Atomic force microscopy topographies (Figure S6) were taken using a XE-100 SPM, Park, Suwon, Korea, system microscope. Images were acquired in the tapping mode using tips (Type PPP-NCHR) on a cantilever of 125 µm length, about 330 kHz resonance frequency, 42 N m−1 nominal force constant and 10 nm guaranteed tip curvature radius. Surface areas were sampled with a scan rate of 1 Hz. The topographies were analysed using the software XEI (Park System Corporation, version 1.8.0).

**UV-VIS Spectroscopy** Analyses were recorded on a Spectrophotometer, Shimadzu, Kyoto, Japan, using the software Spectrum, with 1 cm cuvettes in distilled water or spectrophotometric grade DMSO. Cellulose nanocrystals were analyzed at concentration of 0.2 mg/mL.

**Emission Spectroscopy** Fluorescence spectra were recorded on Cary Eclipse Instrument, Agilent, Santa Clara, CA, USA, with the software Scan. Cellulose nanocrystals were analysed at a concentration of 0.2 mg/mL, in 0.1 cm cuvettes. Titration experiments with heavy metal salts were performed adding increasing volumes of a mother solution of the salt at a concentration of 100 mg/mL in distilled water.

#### **5. Conclusions**

In this paper, we have shown how the reductive amination reaction can be used as an effective synthetic protocol to covalently bind luminescent dyes to cellulose nanocrystals. Pyrene-modified S\_CNC and N\_CNC **7a,b** displayed the emission profile of 4-(1-pyrenyl) butanamine **4**, with emission peaks at 378, 398, 419 and 470 nm, though a very low amount of dye was used during the functionalization reaction (1 molar % with respect to glucopyranose units). The luminescence behavior of the modified nanocrystals, under excitation at the wavelength of 345 nm, was compared with the behavior of the pristine cellulose nanocrystals, evidencing a low emission from nanocellulose, centered at around 400 nm, whose origin is controversial and under literature debate. However, the emission properties of the pyrene-modified samples were clearly identified, and we could test the effect of interactions with different salts on the luminescence properties. In our experiments, N\_CNC were revealed to be best-suited to investigate interaction with salts, as they lack any surface-negatively charged group which could induce nanocrystals' aggregation in the presence of positive cations. Pyrene-modified N\_CNC **7b** showed luminescence quenching

in the presence of copper sulfate, and this suggests a possible application of these systems in water purification technologies, especially after their immobilization on sustainable devices composed of biodegradable cellulose paper.

**Supplementary Materials:** The following are available online, Figures S1–S12: 1H-NMR and 13C-NMR spectra of compounds **2**, **3** and **4**, AFM topography of a pyrene-modified S\_CNC **7a** film, emission of **4** in water, UV-Vis absorption profiles of 4-(1-pyrenylbutanamine) **4** in chloroform, DMSO and water solution, UV-Vis absorption profile of S\_CNC and pyrene-modified S\_CNC **7a** in DMSO suspension.

**Author Contributions:** Conceptualization, O.H.O., F.B. and A.O.; methodology, R.G., E.C. and L.C.; validation, O.H.O. and L.C.; formal analysis, A.O.; investigation, O.H.O., R.G., E.C., L.C. and A.O.; resources, A.O.; data curation, R.G. and E.C.; writing—original draft preparation, O.H.O. and A.O.; writing—review and editing, O.H.O., F.B. and A.O.; supervision, A.O.; project administration, F.B. and A.O.; funding acquisition, F.B. and A.O. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received financial support from the University of Pisa through the projects PRA\_2020\_21 "SUNRISE: Concentratori solari luminescenti NIR riflettenti" and "BIHO 2021—Bando Incentivi di Ateneo Horizon e Oltre" (D.d. 408, Prot. n. 0030596/2021).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Data are contained in the article and Supplementary Materials.

**Acknowledgments:** The authors are grateful to Lorenzo Veronico for performing preliminary experiments and to Angela Punzi for acquiring high-resolution mass spectra on compound **4**. Andrea Mezzetta is acknowledged for furnishing to the authors a sample of commercial sulfated nanocrystals as a kind contribution.

**Conflicts of Interest:** The authors declare no conflict of interest.

**Sample Availability:** Samples of the compounds shown in this paper are available upon request from the corresponding author.

#### **References**


**2. Differential scanning calorimetry for studying mass transfer within emulsions**

The test consists in submitting an emulsion sample, the volume of which being a few mm3, to a regular cooling and heating performed in a Differential Scanning Calorimeter, the volume of the cell being a few mm3. The emulsion samples are taken time to time from the mother emulsion wherein mass transfers are expected to occur at ambient temperature.

During the cooling of a pure material, the droplets dispersed in the emulsions are expected to freeze. Due to the need to the formation of a solid germ that requires a certain amount of energy, the freezing temperatures are lower than the melting ones and the theory shows they are scattered around a mean temperature T\* referred as the most probable freezing temperature of the droplets. The energy released during the droplets freezing is evidenced on the obtained freezing curve as a peak showing a bell shape if the polydispersity is low. Should the emulsion contains a rather large range of droplet sizes, the freezing curve will show more than one signal and the shape could be asymmetrical. The apex temperature of the signal that gives T\* can be correlated to the mean droplet size. On the contrary, as there is no delay in the melting phenomenon, all the droplets (whatever their size) will melt at the same temperature, which is the one observed for a bulk material. More theoretical developments of these phenomena can be found in literature (Clausse et al., 2005). Typical experimental results are given thereafter.

Figures 1 shows typical cooling curves obtained by DSC of pure water (Figure 1a) dispersed in W/O emulsions. For comparison, DSC of bulk water is given on Figure 1b. These curves represent the basic data needed to interpret the results obtained by DSC on emulsions. For a bulk material, its solidification will give a signal at a temperature higher than the one obtained for dispersed material (-18ºC for bulk water and -39 ºC for dispersed water). Between the melting temperature Tm and the freezing temperature Tc, the material still

chemically with the water of the dispersed droplets. Following this process, an example of

In mixed emulsions the transfer is due to the difference of composition between the two populations of droplets. The phase wherein they are dispersed plays the role of a liquid membrane and should this membrane be permeable to one of the material present in the

In multiple emulsions, the situation is very similar of the one observed in mixed emulsions, except that the phases involved in the mass transfer are the dispersed droplets in the globules (primary emulsion) and the continuous phase wherein the globules are dispersed. A difference in the composition between these two kinds of phases lead to a mass transfer,

Different techniques have been used to detect this kind of mass transfer in emulsions. They are based on the phenomena linked with the mass transfer, mainly: solidification and changes of the composition and the sizes of the phases involved in the mass transfer. Therefore classical techniques as spectroturbidimetry, light scattering, conductivimetry and rheology have been used. In this chapter the results obtained by using a technique that has been developed for charactering emulsions and their evolution due to mass transfer will be thoroughly described. The referred technique called DSC for Differential Scanning Calorimetry is described in the next section. Afterwards the results dealing with simple,

droplets a mass transfer occurs as it can be observed in a direct osmosis device.

the material of the globules playing in that case the role of a liquid membrane.

**2. Differential scanning calorimetry for studying mass transfer within**

The test consists in submitting an emulsion sample, the volume of which being a few mm3, to a regular cooling and heating performed in a Differential Scanning Calorimeter, the volume of the cell being a few mm3. The emulsion samples are taken time to time from the mother emulsion wherein mass transfers are expected to occur at ambient temperature. During the cooling of a pure material, the droplets dispersed in the emulsions are expected to freeze. Due to the need to the formation of a solid germ that requires a certain amount of energy, the freezing temperatures are lower than the melting ones and the theory shows they are scattered around a mean temperature T\* referred as the most probable freezing temperature of the droplets. The energy released during the droplets freezing is evidenced on the obtained freezing curve as a peak showing a bell shape if the polydispersity is low. Should the emulsion contains a rather large range of droplet sizes, the freezing curve will show more than one signal and the shape could be asymmetrical. The apex temperature of the signal that gives T\* can be correlated to the mean droplet size. On the contrary, as there is no delay in the melting phenomenon, all the droplets (whatever their size) will melt at the same temperature, which is the one observed for a bulk material. More theoretical developments of these phenomena can be found in literature (Clausse et al., 2005). Typical

Figures 1 shows typical cooling curves obtained by DSC of pure water (Figure 1a) dispersed in W/O emulsions. For comparison, DSC of bulk water is given on Figure 1b. These curves represent the basic data needed to interpret the results obtained by DSC on emulsions. For a bulk material, its solidification will give a signal at a temperature higher than the one obtained for dispersed material (-18ºC for bulk water and -39 ºC for dispersed water). Between the melting temperature Tm and the freezing temperature Tc, the material still

hydrate formation will be described.

mixed and multiple emulsions will be described.

experimental results are given thereafter.

**emulsions**

liquid is said to be under cooled. The degree of undercooling is defined by ΔT = Tm –Tc. For water, this degree is around 20°C for bulk water of a few mm3 and around 40°C for a population of microsized droplets. Another point to stress is that the shape of the signals is also different. This can be attributed to the way the material solidifies, very rapidly in a bulk sample or progressively in a dispersed phase as it has been indicated previously.

Fig. 1. Cooling curves for water dispersed in an emulsion (a) and for bulk water (b)

From the electrical power dq/dt registers by the calorimeter it is possible to deduce the enthalpy power dh/dt involved by the freezing or melting of the sample. On using rather low scanning rate temperature less than 2K/min, dh/dt can be approximated by dq/dt. Therefore by a previous calibration done with pure materials, the area of the signal correctly delimited permits to determine ΔH, the total heat involved in the liquid-solid transition. This quantity divided by the energy involved per mass unit Δh permits to know the mass m involved in the transition. Δh for the freezing is different for the melting one due to the net influence of the temperature specially for water due to a rather high difference between the values of the heat capacities of ice and liquid water. Nevertheless this quantity can be estimated from data on heat capacities values and as far comparison of areas of signals are done, this point is not a problem by itself. Furthermore when it is possible the amount of material involved in the transition can be determined by the area of the melting signal at a known temperature at which Δh is found in the literature. Should a mass transfer induces a change of the initial mass m(t=0) of a material, the following of the area A(t) of either the

Mass Transfers Within Emulsions Studied by

Differential Scanning Calorimetry (DSC) - Application to Composition Ripening and Solid Ripening 747

material balance, the amount of material transferred can be determined. When it is possible another way to follow the transfer is to determine the areas of the freezing or better the melting signals for the droplets for which the composition does not vary, the transfer being from these droplets to the other droplets of different composition. The way these determinations are done will be more thoroughly described in the sections dealing with.

Fig. 2. Cooling and heating curves for a solution A+B dispersed within an emulsion

composition of the droplets made of A+B due to the transfer.

To summarize two ways for quantifying the mass transfer will be used. One from the determination of y (equation 2) and the other one from the calibration curve T\* versus the

**3. Mass transfer within simple emulsions. Solid ripening in W/O emulsions**  An example is given by the mass transfer between yet solid droplets and still liquid ones due to under cooling phenomena. This transfer not very well known but that has to be taken into account when during the storage of the emulsion, the temperature reaches values below the melting ones, is referred as solid ripening as far the equilibrium state in these conditions is all the dispersed material solid (Clausse et al. 1999b). The other type is encountered when a material is added in the continuous phase of an emulsion. It is expected to diffuse and to react chemically with the material of the dispersed droplets. At the end a stable solid material is also obtained. Formation of solid hydrates in petroleum industry is a typical

example of such a situation. It is this kind of transfer that is described thereafter.

An example of such solid ripening giving rise to the formation of a hydrate is illustrated by the study of trichlorofluoromethane (CCl3F) hydrate formation in W/O emulsions. CCl3F is a volatile liquid poorly soluble in water and forms a hydrate under mild conditions at 8.5°C and 1 bar. Therefore, CCl3F appeared to be a good candidate in order to mimic the conditions of gas hydrate formation in W/O emulsions as a model system (Jakobsen et al. 1996; Fouconnier et al. 1999, 2006). The solid hydrate phase is formed inside the dispersed droplets as the result of a chemical reaction between CCl3F molecules and water molecules present in the droplets. Actually, CCl3F molecules are initially dissolved in the oily

freezing signal or the melting signal allows to evaluate the transferred material z or the ratio of non-transferred material y. They will be given by the following equations:

$$z = m(t=0) - m(t) = \frac{\left[\Delta H(t=0) - \Delta H(t)\right]}{\Delta h} = A(t=0) - A(t) \tag{1}$$

$$y = \frac{m(t)}{m(t=0)} = \frac{\Delta H(t)}{\Delta H(t=0)} = \frac{A(t)}{A(t=0)}\tag{2}$$

Should the material contain additives, the cooling curves obtained will be different and dependent on the amount of solute present (Figure 2). For two compounds A+B forming solutions in all proportions and showing an eutectic point, the freezing curves show either one or two signals as it is shown on figure 2 for 6 compositions shown by dotted lines in the phases diagram T versus composition x given as molar fraction of component B in the solutions. Line starting from 0 *e A* represents the A solidification points versus composition and line starting from 0 *e B* , the B solidification points versus composition, E being the eutectic point of the binary. When a solution is dispersed within an emulsion the solidification of the droplets shows different results depending on the composition. A composition different from the eutectic point E, separates the sequence of the events. This point indicated as ε on the graph is found at the intersection of two lines that give the most probable solidification temperatures of A for x<xε and the most probable solidification temperatures of B for x>xε. The circles mimic the contents of the droplets regards to the solid materials present. They are drawn in front of the respective signals shown by DSC. For example for droplets the composition of which being given by dotted line 2, the cooling curve shows two signals, one showing the partial solidification of A and at a lower temperature the total solidification of the droplet, namely the solidification of B and the solidification of the remaining A still liquid. As it can be seen on the diagram, the signals shift with the composition. The temperature of the apex of the first signal observed gives the most probable solidification temperature as it was yet described for dispersed pure material. The heating curve represented on the figure 2 by the line 1\* for the composition represented by the dotted line 1, shows the eutectic melting followed by the progressive melting of component A until its complete dissolution when the equilibrium line is reached. This diagram is a schematic one for the case considered, solutions that can be mixed in all proportions and that show an eutectic point. In this chapter mixtures of hexadecane and tetradecane that enter in this case will be considered. Generally, especially for water + salt, the diagram is limited as far very concentrated solution in B being impossible to reach. That is the case for water + NaCl, solutions or water +urea solutions also treated in this chapter. For the purpose of studying mass transfer that induces changes in the composition of the

droplets, only the lines that give the most probable solidification temperature of either A or B will be considered. This curve will be referred as the calibration curve that gives the most probable freezing temperature T\* versus the composition of the droplets. The undercooling given by the interval of temperatures between the equilibrium lines, and the lines that give the most probable solidification temperatures is not a constant as the figure lets assuming, but in fact it is changing with the composition and the drawing of the calibration curve needs to study samples of various compositions as it will be described later on. Determining the temperature from the freezing signal allows determining the composition of the droplets and therefore from the knowledge of the formulation of the emulsions and by doing a

freezing signal or the melting signal allows to evaluate the transferred material z or the ratio

[ ( 0) ( )] ( 0) ( ) ( 0) ( ) *Ht Ht z mt mt At At h*

> () () () ( 0) ( 0) ( 0) *mt Ht At y mt Ht At*

Should the material contain additives, the cooling curves obtained will be different and dependent on the amount of solute present (Figure 2). For two compounds A+B forming solutions in all proportions and showing an eutectic point, the freezing curves show either one or two signals as it is shown on figure 2 for 6 compositions shown by dotted lines in the phases diagram T versus composition x given as molar fraction of component B in the

eutectic point of the binary. When a solution is dispersed within an emulsion the solidification of the droplets shows different results depending on the composition. A composition different from the eutectic point E, separates the sequence of the events. This point indicated as ε on the graph is found at the intersection of two lines that give the most probable solidification temperatures of A for x<xε and the most probable solidification temperatures of B for x>xε. The circles mimic the contents of the droplets regards to the solid materials present. They are drawn in front of the respective signals shown by DSC. For example for droplets the composition of which being given by dotted line 2, the cooling curve shows two signals, one showing the partial solidification of A and at a lower temperature the total solidification of the droplet, namely the solidification of B and the solidification of the remaining A still liquid. As it can be seen on the diagram, the signals shift with the composition. The temperature of the apex of the first signal observed gives the most probable solidification temperature as it was yet described for dispersed pure material. The heating curve represented on the figure 2 by the line 1\* for the composition represented by the dotted line 1, shows the eutectic melting followed by the progressive melting of component A until its complete dissolution when the equilibrium line is reached. This diagram is a schematic one for the case considered, solutions that can be mixed in all proportions and that show an eutectic point. In this chapter mixtures of hexadecane and tetradecane that enter in this case will be considered. Generally, especially for water + salt, the diagram is limited as far very concentrated solution in B being impossible to reach. That is the case for water + NaCl, solutions or water +urea solutions also treated in this chapter. For the purpose of studying mass transfer that induces changes in the composition of the droplets, only the lines that give the most probable solidification temperature of either A or B will be considered. This curve will be referred as the calibration curve that gives the most probable freezing temperature T\* versus the composition of the droplets. The undercooling given by the interval of temperatures between the equilibrium lines, and the lines that give the most probable solidification temperatures is not a constant as the figure lets assuming, but in fact it is changing with the composition and the drawing of the calibration curve needs to study samples of various compositions as it will be described later on. Determining the temperature from the freezing signal allows determining the composition of the droplets and therefore from the knowledge of the formulation of the emulsions and by doing a

Δ = −Δ = =− = = = − Δ (1)

Δ = = = = Δ= = (2)

*e A* represents the A solidification points versus composition

*e B* , the B solidification points versus composition, E being the

of non-transferred material y. They will be given by the following equations:

solutions. Line starting from 0

and line starting from 0

material balance, the amount of material transferred can be determined. When it is possible another way to follow the transfer is to determine the areas of the freezing or better the melting signals for the droplets for which the composition does not vary, the transfer being from these droplets to the other droplets of different composition. The way these determinations are done will be more thoroughly described in the sections dealing with.

Fig. 2. Cooling and heating curves for a solution A+B dispersed within an emulsion

To summarize two ways for quantifying the mass transfer will be used. One from the determination of y (equation 2) and the other one from the calibration curve T\* versus the composition of the droplets made of A+B due to the transfer.


Acknowledgements

The author would like to thank the Ministry of Economy, Trade, and Industry (METI), and the Japan Oil, Gas, and Metals National Corporation (JOGMEC) for permission to publish this paper.


**Author details**

*Phytochemicals in Human Health*

stress and free radicals in diabetic cases [143].

**7. Future direction and conclusion**

better application in human pharmacology.

such as retinopathy, nephropathy, and cardiovascular disease are well set up so that dietary antioxidant compounds was fixed to protect from the damages of oxidative

In animal experimental designs of alloxan-induced diabetes, both antioxidant and hypoglycemic effects of *O. basilicum* L., *O. tenuiflorum* L., *O. canum* Sims. and *O. gratissimum* L. have been reported. By treating alloxan-diabetic rabbits [141, 142, 144], made a significant decrease in blood glucose levels as corresponded with diabetic control rabbits. Such a hypoglycemic work was related to its powerful antioxidant potentiality: in evidence, in interact, the rabbits studied with *Ocimum* oil showed further a renewal of the levels of malondialdehyde and most of the enzymatic and nonenzymatic endogenous antioxidants [141, 142, 144]. Similar results were achieved in alloxan-diabetic rats: the control, of *Ocimum tenuiflorum*rich extracts showed significant hypoglycemic, hypolipidemic, and antioxidant effects in all the investigated diabetic rats [145, 146]. In an identical empirical design, *O. gratissimum* L. led to a decrease in the sugar level in plasma and a rise in superoxide dismutase, catalase, and glutathione peroxidase activities in liver and kidney. Furthermore, an opposed reaction against hepatic and renal toxicity in diabetic rats was also observed [147, 148]. Furthermore, the effects of *Ocimum sanctum* leaf polyphenols have been investigated also in insulin-secreting pancreatic β-cells, whose OS-induced alterations contribute to the pathogenesis of diabetes [149].

Despite the many appreciations of science and industry, present practice is filled

The biodiversity of essential oils containing the small molecular terpenoids remains an enchanted field of investigation, and the continuous usage of this reward in a broad field of studies suggested these demands in the consequent [151]. Screening, identifying, and dealing with this vast biodiversity will require a progressing progress of precise, rich-throughput experimental methods including new driving procedures. The beneficial health effects of *Ocimum* oil compounds have also been proven by many randomized, crossover, controlled, human studies on biomarkers of health performed in the last decades. Several preclinical studies suggest that such beneficial effects may be mainly ascribed to the phenolic compounds. Further development of biotechnology with the genomic and metabolomic analyses and genetic engineering will advance a variety of fields involving bioactive compounds ranging from food and animal nutrition to plant protection. Although many biological activities as antimicrobial or antioxidative and other effects have been intensively studied and well documented. However, well studies are needed to further characterize the *in vivo* effects of individual *Ocimum* derivatives applied as specific agents or in a mixture, consisting of their safety analysis on mortals. Moreover, a better evidence of their molecular procedure of activity may appropriate the system to a

with stress. Mobile devices and the web have vastly enhanced the pace of life so that many people feel that they are now going down in an endless-increasing ocean of data, while technical culture has overwhelmed us with growing vulnerability to unhealthy prepared and packed food and a profusion of pesticides, food container components, and many toxic modern chemicals. Urban citizens are nevertheless dealt with growing prosperity disparity, social segregation, excessive turbulence, air, water and soil pollution and disconnection from nature. Therefore, while industrialization experiences served to stronger lifespans and impressive expansions in human populations, it is now agreed that the extremest causes of death and disease

on the globe are preventable lifestyle-related chronic diseases [150].

**194**

Debjoy Bhattacharjya1 , Sinchan Adhikari2 , Arijit Biswas2 , Anil Bhuimali1 , Parthadeb Ghosh2 and Soumen Saha1,2\*

1 Cytogenetics and Plant Breeding Section, Department of Sericulture, Raiganj University, Raiganj, West Bengal, India

2 Cytogenetics and Plant Biotechnology Research Unit, Department of Botany, University of Kalyani, Kalyani, West Bengal, India

\*Address all correspondence to<EMAIL_ADDRESS>
© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


*2.4. Thermogravimetric Analysis*

Figure 5 shows the thermal stability of activated carbon (a), as well as the catalysts, Fe-C (b) and Fe-C-K (e). At a temperature of 110–250 ◦C, the water molecules are evaporated [17]. In Figure 5, the evaporation of water molecules occurred between 110–250 ◦C, as shown by Equations (1)–(4). At roughly 450 ◦C, partial thermal degradation of the carbonsupported iron-based catalyst with and without potassium promoters occurs at low and high temperatures, as illustrated in Figure 5b–d,f,g, and shown by Equations (5) and (6). Above 550 ◦C, however, full thermal damage occurs, as shown by Equations (7)–(10). The following steps describe the overall process of thermal degradation of the Fe-C catalyst with and without potassium promoter (Equations (1)–(10)). First, goethite decomposes (Equation (1)) between 200 and 250 degrees Celsius [27,28]. The shape of Fe2O3 (hematite) is then reduced to FeO. In addition, the reaction of iron oxide with CO at 250–300 ◦C produces Fe2O3(magnetite) (Equations (2)–(6)). In the XRD peaks, the production of magnetite was also plainly visible. The creation of CO2 is linked to the reduction of iron oxides to generate the iron carbide carbonaceous material at temperatures above 350 ◦C, as shown by Equations (7)–(10) [29].

$$2\text{FeO(OH)} \rightarrow \text{Fe}\_3\text{O}\_3 + \text{H}\_2\text{O} \tag{1}$$

$$\text{C3FeCO}\_3 \rightarrow \text{Fe}\_3\text{O}\_4 + 2\text{CO}\_2 + \text{CO} \tag{2}$$

$$\text{FeCO}\_3 \rightarrow \text{FeO} + \text{CO}\_2 \tag{3}$$

$$\text{FeC}\_2\text{O}\_4\cdot2\text{H}\_2\text{O} \rightarrow \text{FeC}\_2\text{O}\_4 + 2\text{H}\_2\text{O} \tag{4}$$

$$3\text{FeC}\_2\text{O}\_4 \rightarrow \text{Fe}\_3\text{O}\_4 + 4\text{CO} + 2\text{CO}\_2\tag{5}$$

$$2\text{ 3Fe2O3} + \text{CO} \rightarrow 2\text{Fe3O4} + \text{CO2} \tag{6}$$

$$6\text{FeO}(\text{OH}) + \text{CO} \rightarrow 2\text{Fe}\_3\text{O}\_4 + \text{CO}\_2 + 3\text{H}\_2\text{O} \tag{7}$$

$$\text{FeC}0\_3 \rightarrow \text{FeO} + \text{CO}\_2 \tag{8}$$

$$\text{xFeyO}\_4 + \text{xCO} \rightarrow \text{3Fe}\_2\text{C} + \text{xCO}\_2 \tag{9}$$

xFeO + xCO → FexC + xCO2 (10)

**Figure 5.** TGA analysis of activated carbon (**a**), Fe-C catalyst (**b**), Fe-C LT-FTS (**c**), Fe-C HT-FTS (**d**), Fe-C-K catalyst (**e**), Fe-C-K LT-FTS (**f**), and Fe-C-K HT-FTS (**g**).


**Author details**

Eugenio Hernan Otal1†, Manuela Leticia Kim1† and Mutsumi Kimura1,2,3\*

1 Faculty of Textile Science and Technology, Department of Chemistry and Materials, Shinshu University, Ueda, Nagano, Japan

2 COI Aqua-Innovation Center, Shinshu University, Ueda, Nagano, Japan

3 Research Initiative for Supra-Materials, Shinshu University, Ueda, Nagano, Japan

\*Address all correspondence to<EMAIL_ADDRESS>
† These authors contributed equally.

© 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*Fluoride Detection and Quantification, an Overview from Traditional to Innovative… DOI: http://dx.doi.org/10.5772/intechopen.102879*


**12. Conclusion**

The information on energy recovery helps to establish an overall energy balance. The improvement of energy recovery through optimizing configuration, operation, microbiology and materials will make AFCs more attractive. On the other hand, adopting proper strategies to reduce the energy requirement of operation may compensate for low energy recovery. Incorporating other energy producing processes such as biogas production, algal biomass harvesting, biohydrogen etc., will increase the energy independency. Further, modifying the process for desalination, nutrient recovery and production of valuable chemicals will also

AFC has attractive properties that ensure further development and applications of this technology. It can be easily combined with green roofs to create electricity where photosynthetic and electrochemical reactions are carried out by a continuously growing population of microorganisms in living solar cells. This makes the system capable of self-repair, giving long lifetime and low maintenance. Moreover, using these reproducing organisms living in solar cells does not require any special catalysts that in solar cells are costly and toxic. Therefore, it can be used in natural surroundings with no risk of pollution. AFC also has organic material as intermediate energy carriers between the photosynthetic and the electrochemical portions of the cell which help them in generating electricity at night [48]. Closed loop AFC systems can preserve nutrients for the organisms which enable long-term, low-maintenance power production. Integrated AFC will add value to other applications such as food, agriculture, biomass for bio-energy production etc. [49, 50]. Similarly, it can be coupled with wastewater and surface water treatment to supply extra organic matter for energy production and in turns providing treated water [51].

Algae fuel cells are not without limitations. They need high cost infrastructure and energy for harvesting and growth. Another problem associated with microbial fuel cells is the pH membrane gradient which reduces cell voltage and power output. This problem is caused by acid production at the anode, alkaline production at the cathode and the nonspecific proton exchange through the membrane. The high cost of membrane commonly used in laboratories as a proton-permeable membrane would also limit the applications [52]. In addition, the slow rate of oxygen reduction at cathode electrodes is also a major limiting factor for power generation. Need for improved engineering on downstream algae biofuel processing from AFC for sustainable energy production is another challenge. It includes effective strategies for nutrient circulation and light exposure in designing photo-bioreactors that are reasonably cheap for large-scale deployment in low-cost systems. The secondary challenge related to this is the extraction of crude algae oil which is mostly addressed from the engineering side. The extraction technologies which are successfully demonstrated are relatively expensive. On the other hand, challenges associated with the management of algae bio-oil conversion to usable liquid

fuels need improved catalysts similar to petroleum crude.

maximize the benefits of AFC.

98 Microalgal Biotechnology

**11. Challenges**

**10. Application and adaptability**

AFC is a developing technology with a huge potential to capture solar energy and convert it to electricity. Similarly, the regenerated biomass during the process can be converted into secondary biofuels like solid biomass, bioethanol, biogas, etc. which is an added advantage. This technology also remediates wastewater, removes heavy metals, dye decolorizes, etc. Even though various studies have focused on increasing the performance parameters, physical and catalytic parameter variations, improvement of power generation, cost effective electrode materials, selection of bioactive organisms and finding out an alternative membrane to give cost effective solution need to be addressed. In near future, algae will become a sustainable technology and development in this research area. The possibility of using bioengineering, molecular biology, biotechnology and electrical engineering together to improve the efficiency of AFC is not a farfetched idea. Some studies like life cycle analysis based on commercial-scale, increasing power density, optimization technological methods on AFC configuration need special attention and investigation.