Effects of NaOH Addition on Cellulose Nanocrystal Functionalization with 2,4-Dichlorophenoxyacetic Acid

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Clouse, Elise G. Collins, Tanmay Rahman, Mariya V. Khodakovskaya, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3968506/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract This article investigates the necessity of sodium hydroxide (NaOH) addition for the amine functionalization of sulfated cellulose nanocrystals (CNCs) and its effect on nanocrystal reactivity with the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D). The use of CNCs as a nanocarrier of active biomolecules has grown in the past decade. Previously, CNCs were produced by laboratory sulfuric acid hydrolysis protocols that imparted sulfate half-ester groups with hydrogen counterions. Because of this, researchers cited the need to add a deprotonating base such as NaOH before amination, a common precursor reaction for further biomolecule functionalization. However, current commercially produced sulfated CNCs have a sodium counterion instead of hydrogen. This work explores whether the use of commercial sulfated CNCs negates the need for sodium hydroxide addition in amine functionalization. We investigated the effect of 10 wt% sodium hydroxide solution on the amination of 1 wt% and 2 wt% CNC dispersions. Following this step, CNCs were then further modified via EDC/NHS chemistry to attach 2,4-D. Thermogravimetric analysis coupled with infrared spectroscopy was used to qualitatively confirm attachment. Elemental analysis determined that the degree of amine substitution for all dispersions ranged from 5.4–6.7%. 2,4-D attachment to amine groups varied from 3.9–6.5% when NaOH was present to 7.1% when NaOH was not added. These results highlight how the evolution in CNC extraction methods has resulted in NaOH addition no longer being necessary for successful reactions when using commercially sourced sulfated CNCs with a sodium counterion. Cellulose Nanocrystals Surface Functionalization Herbicides Sodium Hydroxide Figures Figure 1 Figure 2 Figure 3 1. Introduction With a growing shift to a green economy and public concern surrounding the use of synthetic materials in medical and food applications, biomass-derived materials, such as cellulose, are a focus of engineering innovation (Kargarzadeh et al., 2018 ). Cellulose is a structural material within the cell walls of all plants on Earth, and acid hydrolysis can yield cellulose nanocrystals (CNCs) from its hierarchical structure (Trache et al., 2020 ). The most common form of CNC production is sulfuric acid hydrolysis of wood pulp and cotton. In brief, the feedstock material is mixed with 64% sulfuric acid and stirred at elevated temperatures, commonly 45°C, for one to two hours. Following this reaction, CNCs are diluted and separated from residual acid. The acid hydrolysis process imparts sulfate half-ester groups bound to the surface of CNCs in their protonated acid form, containing an H + counterion. At the commercial scale, these H + CNCs are then neutralized to sodium counterion form with the addition of NaOH (Reid et al., 2017 ). Na + binds to the negatively charged particle surface and generates a hydration force between particles, which increases stability (Dong & Gray, 1997 ). Almost all industrially produced CNCs are in the neutral Na + form, with a single plant able to produce 300 tons of nanomaterial annually (Hao et al., 2023 ). Cellulose nanocrystals (CNCs) have been proven to be an effective nanocarrier of active biomolecules in agricultural and medical applications in part due to their unique properties such as high aspect ratio and needle-like shape, low toxicity to mammals and plants, and renewability (DeLoid et al., 2019 ; Salari et al., 2019 ; Vinzant et al., 2023 ). However, the commercial success of CNC nanocarriers will be dependent on the efficiency of chemical reactions and conjugation performed on a large scale. An influential paper in the field of functionalized CNCs is the report of FITC-CNC for use in bioimaging applications (Dong & Roman, 2007 ). In this work, the surface hydroxyl groups on H + CNCs are deprotonated using 1 M NaOH based on the work of Porath and Fornstedt (Porath & Fornstedt, 1970 ). Through deprotonation, the alcohol groups become more electron-rich alkoxide anions which alters the reactivity of CNCs (Brewster & McEwen, 1961a). These deprotonated groups then undergo sequential reactions with epichlorohydrin and ammonia to form an amine-functionalized CNC. This report on aminated CNCs was foundational for their use as a functional nanocarrier since amines can donate electrons to other atoms, including protons, metal ions, carbon atoms of low electron density, and oxidizing agents, making them useful in a wide range of reactions (Brewster & McEwen, 1961b). However, since industrially produced CNCs are not in the protonated acid form and contain a Na + counterion, the need for NaOH addition may not be necessary to achieve successful reactions since commercial CNCs are inherently different than those synthesized at the lab scale. By revisiting the seminal work of Dong et al . (Dong et al., 2014 ; Dong & Roman, 2007 ), this work analyzes the necessity of base addition in CNC amination reactions. Commercially sourced Na + form CNCs were functionalized with a primary amine group through a two-step aqueous reaction with epichlorohydrin and ammonia. After confirmation of primary amine functionalization, CNC-NH 2 underwent carbodiimide crosslinking with the model herbicide 2,4-D to produce biomolecule-functionalized CNC (2,4-D-CNC). In addition to understanding the effect of base addition on the degree of surface substitution, this work also describes the quantification of 2,4-D loading to the CNC surface by considering the nanocrystal size, crystallinity, and surface functionality. Accurate quantification and efficient reactions are essential for the use of CNCs in agricultural and medical applications to ensure a therapeutic dose is delivered. 2. Materials and Methods 2.1 Materials Commercial sulfuric acid hydrolyzed CNCs in an aqueous gel (6.71 wt%) were purchased from CelluForce (Montreal, Quebec, CA). Epichlorohydrin, 2,4-dichlorophenoxyacetic acid (2,4-D), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), N-hydroxysulfosuccinimide (NHS), aqueous ammonia (28–30% ACS grade), NaOH, and phosphate-buffered saline (PBS) were purchased from VWR Chemicals (Radnor, PA, USA). All materials were used as received. Full characterization of feedstock CNC is presented in Table S1 and Figure S1 . 2.2 Preparation of Functionalized CNC 2.2.1 Amine Functionalization Amine functionalization of CNC was carried out using a modified method previously described by Dong et al. and outlined in Fig. 1 (Dong et al., 2014 ). After dilution with ultra-pure water to the appropriate initial concentration, NaOH (10 wt% aqueous solution) was added in a mass excess (1.15:1 NaOH:CNC) to the appropriate dispersions in a dropwise manner under ice bath cooling and stirring. Samples are identified by the initial CNC suspension, for example a dispersion with an initial concentration of 1 wt% with NaOH added is labeled CNC-1-NaOH and a dispersion with an initial concentration of 2 wt% and no NaOH added is labeled CNC-2 throughout the entirety of this paper. The conductivity and pH of all systems investigated are shown in Table 1 . Table 1 CNC Conjugate Sample Identification, pH, and Conductivity. Sample ID pH of Initial System* Conductivity of Initial System* (µS/cm) CNC-1-NaOH 13 48,000 CNC-1 7.1 120 CNC-2-NaOH 13 85,000 CNC-2 6.8 220 * pH and conductivity measurements were obtained at 25°C. After bottle rolling overnight, epichlorohydrin (1.5:1 EPI:CNC, mass basis) was added to the dispersions in a round-bottom flask under ice bath cooling and vigorous stirring. The dispersions were then heated to 60°C and allowed to react for six hours. Following completion of the reaction, dispersions were cooled then dialyzed (Spectra/Por 4 membrane, 12 to 14 kDa MWCO) until the pH of the dialysis water reached neutral, indicating the removal of all unreacted species, taking approximately 2–3 days. Following epoxy functionalization of CNC, the suspensions were again added to a round bottom flask, aqueous ammonia was added in excess (1.1:1 NH 4 :CNC, mass basis), and the mixture was heated to 60°C with continuous stirring for six hours. After completion of the reaction, the solution was transferred to dialysis tubing and dialyzed against ultra-pure water until a constant pH was reached. The solution was then sonicated under ice bath cooling (Sonics Vibracell 750 W, 25% amplitude) for 10 minutes to reduce the presence of aggregates. Then the CNC-NH 2 suspension underwent a second dialysis to ensure the removal of unbound molecules. 2.2.2 Covalent Attachment of 2,4-D Molar equivalents (0.001 mols) of 2,4-D, EDC, and NHS were added under stirring to 80 mL of PBS. Aqueous CNC-NH 2 (2:1 CNC:2,4-D, mass basis) was added to the PBS mixture and allowed to proceed for 40 hours at room temperature under stirring in a sealed flask. After the reaction, the same dialysis and sonication protocol used for amination was repeated. 2.3 Rheology of Initial CNC Dispersions Rheological measurements were obtained for the dispersions outlined in Table 1 using an Anton Paar MCR302e rotational rheometer at 25°C. A coaxial cylinder geometry with a 26.65 mm bob diameter, 28.92 mm cup inner diameter, and 39.98 mm gap length was utilized. Flow curves were generated for each sample by subjecting them to a shear rate ranging from 0.1 s − 1 to 100 s − 1 . The time required to achieve steady state for each sample was determined via a transient test at 0.1 s − 1 ; the sampling time required to get the flow curve was reduced logarithmically as the shear rate increased. It should be noted that some low shear rate data points for CNC-1 were below the sensitivity threshold of the torque transducer, which were not displayed. 2.4 Characterization of CNC Conjugates 2.4.1 Elemental Analysis Before elemental analysis, all samples were oven-dried overnight at 80°C under vacuum. CHNS analysis of samples was conducted at Atlantic Microlabs (Norcross, GA) following the outline of ASTM D5373-02. Energy dispersive x-ray spectroscopy (EDX) analysis was conducted on an Oxford INCA (Abingdon, UK) EDX system connected to a Zeiss EVO50 Scanning Electron Microscope with a mean accelerating voltage of 20 kV and a magnification of 1200x. 2.4.2 Coupled Thermogravimetric Analysis – Fourier Transform Infrared Spectroscopy Thermogravimetric analysis (TGA) was conducted using a TA Instruments (New Castle, DE) TGA Q50 interfaced with a ThermoScientific (Waltham, MA) Nicolet FTIR equipped with evolved gas analysis (iZ10) capabilities. Samples were loaded into the TGA under constant tank air flow (90 mL/min), heated at 5°C/min to 120°C, and held isothermal for two hours to remove any absorbed moisture. Dried samples were then ramped at 5°C/min to 700°C and held isothermal for 30 minutes. At the air-flow outlet of the TGA, a quartz-lined stainless steel transfer line enabled the passage of evolved gas from the TGA furnace into a Fourier transform infrared spectroscopy (FTIR) gas cell. The transfer line and the gas cell were kept at 225°C and 250°C, respectively, to prevent gas condensation. Spectral acquisition began at the end of the 120°C isothermal hold. Gas-phase IR spectra were recorded in the spectral range of 600–4000 cm − 1 with a 4 cm − 1 resolution. IR peaks were assigned using a combination of Thermo Scientific OMNIC™ spectroscopy software and literature (Socrates, 2004). 3. Results and Discussion 3.1 Quantification of Amine Attachment Methods to accurately quantify the covalent functionalization of CNCs remain unstandardized and highly debated in cellulose research. A key challenge is that a small number of hydroxyl groups are available for substitution. However, accurate quantification of surface groups is critical for CNC use in sensitive fields such as medicine and agriculture. Analysis techniques for quantifying nitrogen in small molecules include TGA, FTIR, liquid chromatography, and mass spectroscopy. However, these methods become problematic when the nitrogen is bound to the surface of CNCs. Because of their size, CNCs are incompatible and could damage traditional chromatography and mass spectroscopy systems, and schemes to remove the functional groups from CNC can result in inaccuracies due to incomplete removal. Amine peaks traditionally seen using FTIR become more challenging for quantification due to their overlap with CNC peaks, such as O-H and C-O stretching. Similar issues occur with TGA because amine degradation occurs in the same temperature range as CNC itself. Energy dispersive x-ray spectroscopy (EDX) has also been reported for nitrogen quantification, but the position of the nitrogen peak for this method lies between the carbon and oxygen peaks. For functionalized CNCs, where the total concentration of nitrogen is much less than that of carbon and oxygen, it is difficult to peak deconvolute due to low signal and limited resolution. In contrast, ultimate analysis (CHNS) is commonly used to quantify nitrogen. It is an excellent option for cellulose since it analyzes nitrogen separately from carbon and oxygen and breaks down CNC via combustion, enabling whole-sample analysis. The CHNS analysis results in Table 2 show that the amination reaction was successful regardless of the presence of NaOH. The decrease in sulfur content from feedstock CNC to functionalized CNC is because CHNS calculates the weight percent of elements based on total mass. As additional carbon, hydrogen, oxygen, and nitrogen groups are functionalized to the surface, the ratio of sulfur mass to total mass decreases. Table 2 CHNS Quantification and Degree of Surface Substitution Estimation for CNC-NH 2 Under Various Reaction Conditions. C (wt%) H (wt%) N (wt%) S (wt%) Degree of Surface Substitution Feedstock CNC* 41.1 ± 0.2 6.3 ± 0.2 N.D. 0.97 ± 0.02 -- CNC-1 40.6 ± 0.0 6.5 ± 0.0 0.14 ± 0.01 0.48 ± 0.02 6.2 ± 0.4% CNC-1-NaOH 41.2 ± 0.0 6.6 ± 0.0 0.15 ± 0.00 0.59 ± 0.01 6.7 ± 0.0% CNC-2 40.9 ± 0.1 6.4 ± 0.0 0.14 ± 0.00 0.41 ± 0.02 6.2 ± 0.0% CNC-2-NaOH 41.1 ± 0.2 6.5 ± 0.1 0.12 ± 0.02 0.55 ± 0.01 5.4 ± 0.9% Uncertainty is quantified by the standard error for a minimum of three runs. *Feedstock CNC CHNS analysis was conducted on a vario MICRO cube CHNS elemental analyzer for two runs. The error represents the maximum and minimum. N.D. = Not Detected. Correlating values obtained through quantitative characterization to the degree of CNC surface modification is still an underdeveloped area of cellulose research. Few papers consider the specific morphology of the CNC being used as well as any unreactive functional groups that may be present on the surface. We followed the method of Eyley and Thielemans (Eq. 1 ) to determine the number of available surface hydroxyl groups per gram of CNC surface (Eyley & Thielemans, 2014 ). $${N}_{OH}=\frac{{n}_{1}+{n}_{2}}{\rho {N}_{A}{L}_{1}{L}_{2}c}\left(\frac{{L}_{1}+{L}_{2}}{{d}_{\left(110\right)}}+\frac{{L}_{1}+{L}_{2}}{{d}_{\left(1\stackrel{-}{1}0\right)}}\right)+2(\rho {N}_{A}{L}_{3}{d}_{\left(110\right)}{d}_{\left(1\stackrel{-}{1}0\right)}{)}^{-1}$$ 1 Where L 1 , L 2 , and L 3 are the height, width and length of individual CNCs, d (110) and d (1−10) are the horizontal and vertical unit cell of CNC, n 1 and n 2 are the number of primary and secondary -OH groups facing (110) and (1–10) in the unit cell, ρ is the density of crystalline cellulose Iβ, c is the until cell dimension, and N A is Avogadro’s number. Using Eq. 1 , AFM size characterization of feedstock CNC, and average dimensions of cellulose Iβ from literature (Habibi et al., 2006 ; Yoo & Youngblood, 2016 ), it was estimated that the CNC used in this investigation has an average number of surface hydroxyl groups (N OH ) of 1.9 mmol/g. But this equation does not consider the presence of sulfate half-ester groups on the surface of the feedstock CNCs, a byproduct of sulfuric acid hydrolysis. To account for these groups, the results from CHNS analysis in Table 2 were used to obtain a sulfate half-ester content of 0.3 mmol/g. Subtracting this from the 1.9 mmol/g of available surface hydroxyl groups, this leaves 1.6 mmol/g available for reaction. Degree of substitution is defined as the ratio of mmol of modification per gram of CNC conjugate to 1.6 mmol/g, or the maximum possible loading capacity. Results from this calculation are shown in Table 2 . 3.2 Effect of NaOH Addition on Amine Degree of Substitution To determine if the degree of substitution results in Table 2 are related to transport accessibility within the dispersion, which is a function of viscosity, flow curves were obtained for all dispersions and the data were model fitted using the power law model ( Equation S1, Table S2, and Figure S2 ). From the flow behavior of CNC-1 and CNC-2 shown in Fig. 2 , CNC-2 has higher viscosity compared to CNC-1 for all shear rates due to increased concentration. Both dispersions were nearly Newtonian as indicated by the power law index value being close to unity. When NaOH is added, the viscosity at all shear rates increased compared to their no NaOH counterparts. The behavior of CNC-1-NaOH and CNC-2-NaOH dispersions can be described by three regions. Both dispersions showed shear thinning behavior at lower shear rates until they reached a critical shear rate value of 7.5 s − 1 for CNC-1-NaOH and 13.3 s − 1 for CNC-2-NaOH. Both solutions showed shear thickening after this point due to aggregation before returning to shear thinning at even higher shear rates. When NaOH is added to dispersions, the concentration of Na + increases, which has the same effects on CNC as NaCl, resulting in screened charges and aggregation of the nanoparticles (Huntington et al., 2022 ). Similar behavior was observed by Danesh et al. where they found two shear thinning regions at low and high shear rates with a plateau at intermediate shear rates when NaCl was added in amounts greater than 20 mM in 1 wt% sulfated CNC dispersions with a sodium counterion (Danesh et al., 2020 ). The three dispersions with the lowest viscosities rates resulted in higher degree of surface substitutions. For CNC-2-NaOH, which had the highest viscosity over all shear rates, the lowest degree of substitution was observed likely due to the system having a higher diffusion coefficient and the presence of aggregates, which can lower available surface area, inhibiting the amount of aqueous ammonia that can react. This confirms that amine functionalization is dependent on both transport of the molecule to the surface and the availability of hydroxyl groups. 3.3 Qualitative Confirmation of 2,4-D Functionalization The CNC-NH 2 produced from each method above underwent an EDC/NHS reaction to covalently bind the active biomolecule 2,4-D to the surface of the nanocrystal for use as a nanocarrier of the active agent. 2,4-D was selected for conjugation with CNC based on the extensive use of such herbicide in commercial agriculture (Peterson et al., 2016 ). After conjugation, samples underwent TGA-IR analysis to qualitatively confirm the attachment of the herbicide (Fig. 3 ). Feedstock 2,4-D completely degrades in a narrow temperature window between 170 and 240°C, with a thermal degradation temperature of 178°C, and degrades completely in air, leaving only a residual mass of 0.03 wt%, as shown in Fig. 3 A. Meanwhile, commercial CNC does not begin thermal degradation (the temperature where 5% mass loss occurs) until 261°C and has a residual mass of 1.64 wt%. The 2,4-D-CNC conjugates behave independently from either feedstock material yet similarly to one another. All conjugates have a thermal degradation temperature between 232 and 246°C and a final residual mass of 1.02 to 1.14 wt%. These values lie between thermal degradation and residual mass values of 2,4-D and CNC indicating the presence of both in the final conjugates. Traditional FT-IR spectroscopy is limited for 2,4-D-CNC since the spectral bands of the targeted trace material overlap with the spectra bands of CNC (Chan & Kazarian, 2006 ; Nori et al., 2023 ). A solution to this problem lies in TGA-IR. Since 2,4-D thermally degrades at a lower temperature than feedstock CNC, we can observe higher concentrations of 2,4-D off-gas at temperatures before CNC degradation. The evolution of gas phases of 2,4-D-CNC during degradation was explored by coupling FT-IR spectroscopy with TGA and collecting the spectra of evolved gases over a wide temperature range. The Gram-Schmidt plot in Fig. 3 B shows the spectral intensity as a function of time and temperature. 2,4-D-CNC has a signature peak at approximately 30 min (~ 300°C) that is not present for feedstock CNC. Further investigation into the IR spectra at this peak (Fig. 3 C) shows that the evolved gas of 2,4D-CNC differs from the spectra of unmodified CNC, as shown in Figs. 3 D and 3 E. In Fig. 3 E, the evolution of peaks at 903 cm − 1 , 994 cm − 1 , and 1107 cm − 1 which are due to the evolution of ester containing compounds and C-H deformation. Most importantly, 826 cm − 1 and 1173 cm − 1 can be attributed to C-Cl vibrational coupling and C-O-C asymmetric stretching from a halo containing compound, which appear during the thermal decomposition of the 2,4-D bound to CNC in the conjugates. In Fig. 3 D, notable new peaks appear at 2801 cm − 1 , 2897 cm − 1 , and 2976 cm − 1 . The first two are due to C-H stretching from an alkane. Stretching from an aliphatic amine occurs at 2976 cm − 1 , from the degradation of the secondary amine used to link CNC and 2,4-D. 3.4 Quantitative Confirmation of 2,4-D Functionalization Although surface modification of CNC has gained significant interest in the past decade, there still exists a gap in understanding when it comes to quantifying the degree of surface substitution and recognizing the limitations of current analysis techniques, especially with halides such as chlorine. Traditional methods for chlorine detection such as titration, chromatography, and UV-vis become more complex in the presence of large biomolecules such as CNCs. In addition, careless handling during sample preparation and analysis can cause contamination, and chlorine is well-known to cause contamination in trace element analysis (Jin, 2016 ). Recognizing these limitations and those described in Section 3.1, energy dispersive x-ray spectroscopy (EDX) was chosen to quantify the amount of chlorine present in the 2,4-D-CNC conjugates. EDX is the most common and simple method for chlorine detection in literature, and unlike nitrogen, the K α peak for Cl occurs around 2.6 compared to 0.3 and 0.5 for carbon and oxygen, respectively (Larnøy et al., 2011 ; Singh & Khatri, 2012 ; Tanaka et al., 2015 ). Table 3 EDX Quantification and Degree of Surface Substitution Estimation for 2,4-D-CNC Under Various Reaction Conditions. Cl (wt%) Degree of Hydroxyl Substitution Degree of Primary Amine Substitution Feedstock CNC N.D. -- -- 2,4-D-CNC-1 0.05 ± 0.01 0.4 ± 0.1% 7.1% 2,4-D-CNC-1-NaOH 0.03 ± 0.01 0.3 ± 0.1% 3.9% 2,4-D-CNC-2 0.05 ± 0.01 0.4 ± 0.1% 7.1% 2,4-D-CNC-2-NaOH 0.04 ± 0.01 0.4 ± 0.1% 6.5% Uncertainty is quantified by the standard error for a minimum of six scans. Using N OH calculated previously and the same method for determining degree of surface substitution, the amount of covalently bound 2,4-D can be estimated from the weight percentage of chlorine present via EDX analysis (Fig. 4 and Table 3 ). The degree of surface substitution was calculated based on the average chlorine content detected for each sample. The degree of hydroxyl substitution ranged from 0.3–0.4% for all samples. This correlates to 3.9 to 7.1% of amine groups covalently bound to a 2,4-D molecule. Interestingly, the addition of NaOH to initial dispersions did not increase the final degree of 2,4-D substitution. The investigation supports the hypothesis that NaOH is not a necessary additive in the functionalization of commercially sourced CNCs. In future work, by removing NaOH, the viscosity of the CNC suspensions will remain lower and more workable, allowing for higher initial concentrations of CNCs to be functionalized, and potentially increasing the final degree of biomolecule substitution. 4. Conclusions This investigation built upon previous work on amine attachment to CNCs and investigated if NaOH is a necessary additive for functionalization when commercial sulfated CNCs are used. CNCs at 1 wt% and 2 wt% were aminated with and without NaOH. The reaction succeeded under all conditions, demonstrating that deprotonation with NaOH is unnecessary when using commercial CNCs with a sodium counterion. Rheological studies and elemental analysis found that for dispersions containing 2 wt% CNC with base added, the degree of substitution was limited, and NaOH increased the presence of aggregates and transport limitation within the dispersion. Amine functionalized CNCs were then reacted with 2,4-D to determine if adjustment to amine functionalization affects the nanocrystals resulting reactivity with biomolecules. FTIR and EDX showed that 2,4-D attachment was not hindered by the removal of NaOH in the first step and the highest degree of surface substitution was seen for both cases without base addition. These results highlight the changing field of cellulose functionalization and show that deprotonation of CNCs with NaOH is not required for amination when commercial sulfated CNCs are used. Established results create a base for a simpler and cost-effective methodology for the conjugation of CNC with variable chemicals used in medical and agricultural applications. Declarations Author Contributions Delaney E. Clouse, Mariya V. Khodakovskaya, and Virginia A. Davis contributed to the studies conception and design. Material preparation, data collection, and analysis were performed by Delaney E. Clouse, Elise G. Collins, and Tanmay Rahman. The first draft of the manuscript was written by Delaney E. Clouse and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. Ethical Approval Not applicable. Funding This work was supported by USDA-NIFA 2021-67022-33995. The authors have no relevant financial or non-financial interests to disclose. Availability of Data and Materials Data is presented within the manuscript and supplementary information. Requests for additional data should be made to the corresponding author. References Brewster R, McEwen W (1962) Organic chemistry, 3rd edn. 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Front Chem 8:392. https://doi.org/10.3389/fchem.2020.00392 Vinzant K, Clouse D, Amit S, Ivanov I, Davis V, Khodakovskaya M (2023) Cellulose nanocrystals are a renewable and biocompatible nanocarrier of agrochemicals directly to plant cells. Adv Sustain Syst 2300511. https://doi.org/10.1002/adsu.202300511 Yoo Y, Youngblood J (2016) Green one-pot synthesis of surface hydrophobized cellulose nanocrystals in aqueous medium. ACS Sustain Chem Eng 4:3927–3938. https://doi.org/10.1021/acssuschemeng.6b00781 Additional Declarations No competing interests reported. Supplementary Files CelluloseSupplementalforSubmission021824.pdf Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3968506","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":274129876,"identity":"8a2732c1-bbb0-4953-8dcc-188ef5ab6563","order_by":0,"name":"Delaney E. Clouse","email":"","orcid":"","institution":"Auburn University","correspondingAuthor":false,"prefix":"","firstName":"Delaney","middleName":"E.","lastName":"Clouse","suffix":""},{"id":274129877,"identity":"b6452aba-bcca-490c-afa2-24abb1c9775f","order_by":1,"name":"Elise G. Collins","email":"","orcid":"","institution":"Auburn University","correspondingAuthor":false,"prefix":"","firstName":"Elise","middleName":"G.","lastName":"Collins","suffix":""},{"id":274129878,"identity":"32ee2d22-4470-409b-8f7f-971bd4448a29","order_by":2,"name":"Tanmay Rahman","email":"","orcid":"","institution":"Auburn University","correspondingAuthor":false,"prefix":"","firstName":"Tanmay","middleName":"","lastName":"Rahman","suffix":""},{"id":274129879,"identity":"7ed79dc2-4cb1-4781-97cc-a110823fd13b","order_by":3,"name":"Mariya V. Khodakovskaya","email":"","orcid":"","institution":"The University of Arkansas at Little Rock","correspondingAuthor":false,"prefix":"","firstName":"Mariya","middleName":"V.","lastName":"Khodakovskaya","suffix":""},{"id":274129880,"identity":"68d70122-5314-470e-ba80-c21a8b062bd6","order_by":4,"name":"Virginia A. Davis","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAxUlEQVRIiWNgGAWjYDCCA2DEIMfAwIMQIUqLMWlaQCCxgWgtfMfPGB782WaX3j8j95h05Q4GOb4bCfi1SJ7JMTjM25acO+NGXprk2TMMxpKEtBgcSEs4zHCGObfhRo6ZZGMbQ+IGglrOP0s4+ONMfbo8VEs9YS03kg8c4Kk4nGAA1QJkEPLLjccHDvNUHDfceOZdsmVjm4ThzDMP8GvhO5/Y/PGHQbW83PHcgzcb22zk+Y4TsAUdSJCmfBSMglEwCkYBdgAA6bpMw6FuGfwAAAAASUVORK5CYII=","orcid":"","institution":"Auburn University","correspondingAuthor":true,"prefix":"","firstName":"Virginia","middleName":"A.","lastName":"Davis","suffix":""}],"badges":[],"createdAt":"2024-02-19 01:24:31","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3968506/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3968506/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":51523818,"identity":"a968308c-147f-432e-ade8-1c59c6f05bbc","added_by":"auto","created_at":"2024-02-23 04:50:30","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":86134,"visible":true,"origin":"","legend":"\u003cp\u003eFunctionalization of 2,4-D to CNC. Image not drawn to scale.\u003c/p\u003e","description":"","filename":"CelluloseFigure1020924.png","url":"https://assets-eu.researchsquare.com/files/rs-3968506/v1/f56df9694cdef8f6c2b0b876.png"},{"id":51523814,"identity":"137b1540-686c-4bdb-a3df-049547f18b94","added_by":"auto","created_at":"2024-02-23 04:50:30","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":71357,"visible":true,"origin":"","legend":"\u003cp\u003eSteady shear rheology of initial CNC dispersions. Solid lines represent viscosity calculated using the power-law model. Error bars represent the standard error of three runs.\u003c/p\u003e","description":"","filename":"CelluloseFigure2020924.png","url":"https://assets-eu.researchsquare.com/files/rs-3968506/v1/e983c69d49fd377f071ffe56.png"},{"id":51523815,"identity":"fb3a2bc9-eaa7-47b6-b20d-50ba64d492f9","added_by":"auto","created_at":"2024-02-23 04:50:30","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":519152,"visible":true,"origin":"","legend":"\u003cp\u003e(A) TGA degradation curves of 2,4-D, CNC, and 2,4-D-CNC conjugate in an air atmosphere, (B) Gram-Schmidt plot of 2,4-D-CNC conjugates in air, (C) gas phase IR spectra of 2,4-D-CNC conjugates at 300 °C in air, (D) notable gas phase IR spectra between 800-1200 cm\u003csup\u003e-1\u003c/sup\u003e at 300 °C in air, and (E) notable gas phase IR spectra between 2600-3050 cm\u003csup\u003e-1\u003c/sup\u003e at 300 °C in air.\u003c/p\u003e","description":"","filename":"CelluloseFigure3020924.png","url":"https://assets-eu.researchsquare.com/files/rs-3968506/v1/6bd05b6fe72585e5be575bcb.png"},{"id":53975740,"identity":"698c1741-f4bf-447b-931b-e79d8bedb7c3","added_by":"auto","created_at":"2024-04-03 00:29:44","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":862531,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3968506/v1/510e5e0a-3b19-423b-b2c8-467f0ec3eb04.pdf"},{"id":51523819,"identity":"9730fb0a-6897-4c58-a3a7-5c4db095fd0f","added_by":"auto","created_at":"2024-02-23 04:50:30","extension":"pdf","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":403013,"visible":true,"origin":"","legend":"","description":"","filename":"CelluloseSupplementalforSubmission021824.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3968506/v1/4382e3f19a9b14aedaf60895.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Effects of NaOH Addition on Cellulose Nanocrystal Functionalization with 2,4-Dichlorophenoxyacetic Acid","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eWith a growing shift to a green economy and public concern surrounding the use of synthetic materials in medical and food applications, biomass-derived materials, such as cellulose, are a focus of engineering innovation (Kargarzadeh et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Cellulose is a structural material within the cell walls of all plants on Earth, and acid hydrolysis can yield cellulose nanocrystals (CNCs) from its hierarchical structure (Trache et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The most common form of CNC production is sulfuric acid hydrolysis of wood pulp and cotton. In brief, the feedstock material is mixed with 64% sulfuric acid and stirred at elevated temperatures, commonly 45\u0026deg;C, for one to two hours. Following this reaction, CNCs are diluted and separated from residual acid. The acid hydrolysis process imparts sulfate half-ester groups bound to the surface of CNCs in their protonated acid form, containing an H\u003csup\u003e+\u003c/sup\u003e counterion. At the commercial scale, these H\u003csup\u003e+\u003c/sup\u003e CNCs are then neutralized to sodium counterion form with the addition of NaOH (Reid et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Na\u003csup\u003e+\u003c/sup\u003e binds to the negatively charged particle surface and generates a hydration force between particles, which increases stability (Dong \u0026amp; Gray, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e1997\u003c/span\u003e). Almost all industrially produced CNCs are in the neutral Na\u003csup\u003e+\u003c/sup\u003e form, with a single plant able to produce 300 tons of nanomaterial annually (Hao et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eCellulose nanocrystals (CNCs) have been proven to be an effective nanocarrier of active biomolecules in agricultural and medical applications in part due to their unique properties such as high aspect ratio and needle-like shape, low toxicity to mammals and plants, and renewability (DeLoid et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Salari et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Vinzant et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). However, the commercial success of CNC nanocarriers will be dependent on the efficiency of chemical reactions and conjugation performed on a large scale. An influential paper in the field of functionalized CNCs is the report of FITC-CNC for use in bioimaging applications (Dong \u0026amp; Roman, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). In this work, the surface hydroxyl groups on H\u003csup\u003e+\u003c/sup\u003e CNCs are deprotonated using 1 M NaOH based on the work of Porath and Fornstedt (Porath \u0026amp; Fornstedt, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e1970\u003c/span\u003e). Through deprotonation, the alcohol groups become more electron-rich alkoxide anions which alters the reactivity of CNCs (Brewster \u0026amp; McEwen, 1961a). These deprotonated groups then undergo sequential reactions with epichlorohydrin and ammonia to form an amine-functionalized CNC. This report on aminated CNCs was foundational for their use as a functional nanocarrier since amines can donate electrons to other atoms, including protons, metal ions, carbon atoms of low electron density, and oxidizing agents, making them useful in a wide range of reactions (Brewster \u0026amp; McEwen, 1961b).\u003c/p\u003e \u003cp\u003eHowever, since industrially produced CNCs are not in the protonated acid form and contain a Na\u003csup\u003e+\u003c/sup\u003e counterion, the need for NaOH addition may not be necessary to achieve successful reactions since commercial CNCs are inherently different than those synthesized at the lab scale. By revisiting the seminal work of Dong \u003cem\u003eet al\u003c/em\u003e. (Dong et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Dong \u0026amp; Roman, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2007\u003c/span\u003e), this work analyzes the necessity of base addition in CNC amination reactions. Commercially sourced Na\u003csup\u003e+\u003c/sup\u003e form CNCs were functionalized with a primary amine group through a two-step aqueous reaction with epichlorohydrin and ammonia. After confirmation of primary amine functionalization, CNC-NH\u003csub\u003e2\u003c/sub\u003e underwent carbodiimide crosslinking with the model herbicide 2,4-D to produce biomolecule-functionalized CNC (2,4-D-CNC). In addition to understanding the effect of base addition on the degree of surface substitution, this work also describes the quantification of 2,4-D loading to the CNC surface by considering the nanocrystal size, crystallinity, and surface functionality. Accurate quantification and efficient reactions are essential for the use of CNCs in agricultural and medical applications to ensure a therapeutic dose is delivered.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Materials\u003c/h2\u003e \u003cp\u003eCommercial sulfuric acid hydrolyzed CNCs in an aqueous gel (6.71 wt%) were purchased from CelluForce (Montreal, Quebec, CA). Epichlorohydrin, 2,4-dichlorophenoxyacetic acid (2,4-D), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), N-hydroxysulfosuccinimide (NHS), aqueous ammonia (28\u0026ndash;30% ACS grade), NaOH, and phosphate-buffered saline (PBS) were purchased from VWR Chemicals (Radnor, PA, USA). All materials were used as received. Full characterization of feedstock CNC is presented in \u003cb\u003eTable \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e and \u003cb\u003eFigure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Preparation of Functionalized CNC\u003c/h2\u003e \u003cdiv id=\"Sec5\" class=\"Section3\"\u003e \u003ch2\u003e2.2.1 Amine Functionalization\u003c/h2\u003e \u003cp\u003eAmine functionalization of CNC was carried out using a modified method previously described by Dong \u003cem\u003eet al.\u003c/em\u003e and outlined in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e (Dong et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). After dilution with ultra-pure water to the appropriate initial concentration, NaOH (10 wt% aqueous solution) was added in a mass excess (1.15:1 NaOH:CNC) to the appropriate dispersions in a dropwise manner under ice bath cooling and stirring. Samples are identified by the initial CNC suspension, for example a dispersion with an initial concentration of 1 wt% with NaOH added is labeled CNC-1-NaOH and a dispersion with an initial concentration of 2 wt% and no NaOH added is labeled CNC-2 throughout the entirety of this paper. The conductivity and pH of all systems investigated are shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eCNC Conjugate Sample Identification, pH, and Conductivity.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSample ID\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003epH of Initial System*\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eConductivity of Initial System* (\u0026micro;S/cm)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCNC-1-NaOH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e48,000\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCNC-1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e120\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCNC-2-NaOH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e85,000\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCNC-2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e220\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e* pH and conductivity measurements were obtained at 25\u0026deg;C.\u003c/p\u003e \u003cp\u003eAfter bottle rolling overnight, epichlorohydrin (1.5:1 EPI:CNC, mass basis) was added to the dispersions in a round-bottom flask under ice bath cooling and vigorous stirring. The dispersions were then heated to 60\u0026deg;C and allowed to react for six hours. Following completion of the reaction, dispersions were cooled then dialyzed (Spectra/Por 4 membrane, 12 to 14 kDa MWCO) until the pH of the dialysis water reached neutral, indicating the removal of all unreacted species, taking approximately 2\u0026ndash;3 days. Following epoxy functionalization of CNC, the suspensions were again added to a round bottom flask, aqueous ammonia was added in excess (1.1:1 NH\u003csub\u003e4\u003c/sub\u003e:CNC, mass basis), and the mixture was heated to 60\u0026deg;C with continuous stirring for six hours. After completion of the reaction, the solution was transferred to dialysis tubing and dialyzed against ultra-pure water until a constant pH was reached. The solution was then sonicated under ice bath cooling (Sonics Vibracell 750 W, 25% amplitude) for 10 minutes to reduce the presence of aggregates. Then the CNC-NH\u003csub\u003e2\u003c/sub\u003e suspension underwent a second dialysis to ensure the removal of unbound molecules.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e \u003ch2\u003e2.2.2 Covalent Attachment of 2,4-D\u003c/h2\u003e \u003cp\u003eMolar equivalents (0.001 mols) of 2,4-D, EDC, and NHS were added under stirring to 80 mL of PBS. Aqueous CNC-NH\u003csub\u003e2\u003c/sub\u003e (2:1 CNC:2,4-D, mass basis) was added to the PBS mixture and allowed to proceed for 40 hours at room temperature under stirring in a sealed flask. After the reaction, the same dialysis and sonication protocol used for amination was repeated.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Rheology of Initial CNC Dispersions\u003c/h2\u003e \u003cp\u003eRheological measurements were obtained for the dispersions outlined in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e using an Anton Paar MCR302e rotational rheometer at 25\u0026deg;C. A coaxial cylinder geometry with a 26.65 mm bob diameter, 28.92 mm cup inner diameter, and 39.98 mm gap length was utilized. Flow curves were generated for each sample by subjecting them to a shear rate ranging from 0.1 s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e to 100 s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. The time required to achieve steady state for each sample was determined via a transient test at 0.1 s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e; the sampling time required to get the flow curve was reduced logarithmically as the shear rate increased. It should be noted that some low shear rate data points for CNC-1 were below the sensitivity threshold of the torque transducer, which were not displayed.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Characterization of CNC Conjugates\u003c/h2\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003e2.4.1 Elemental Analysis\u003c/h2\u003e \u003cp\u003eBefore elemental analysis, all samples were oven-dried overnight at 80\u0026deg;C under vacuum. CHNS analysis of samples was conducted at Atlantic Microlabs (Norcross, GA) following the outline of ASTM D5373-02. Energy dispersive x-ray spectroscopy (EDX) analysis was conducted on an Oxford INCA (Abingdon, UK) EDX system connected to a Zeiss EVO50 Scanning Electron Microscope with a mean accelerating voltage of 20 kV and a magnification of 1200x.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003ch2\u003e2.4.2 Coupled Thermogravimetric Analysis \u0026ndash; Fourier Transform Infrared Spectroscopy\u003c/h2\u003e \u003cp\u003eThermogravimetric analysis (TGA) was conducted using a TA Instruments (New Castle, DE) TGA Q50 interfaced with a ThermoScientific (Waltham, MA) Nicolet FTIR equipped with evolved gas analysis (iZ10) capabilities. Samples were loaded into the TGA under constant tank air flow (90 mL/min), heated at 5\u0026deg;C/min to 120\u0026deg;C, and held isothermal for two hours to remove any absorbed moisture. Dried samples were then ramped at 5\u0026deg;C/min to 700\u0026deg;C and held isothermal for 30 minutes.\u003c/p\u003e \u003cp\u003eAt the air-flow outlet of the TGA, a quartz-lined stainless steel transfer line enabled the passage of evolved gas from the TGA furnace into a Fourier transform infrared spectroscopy (FTIR) gas cell. The transfer line and the gas cell were kept at 225\u0026deg;C and 250\u0026deg;C, respectively, to prevent gas condensation. Spectral acquisition began at the end of the 120\u0026deg;C isothermal hold. Gas-phase IR spectra were recorded in the spectral range of 600\u0026ndash;4000 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e with a 4 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e resolution. IR peaks were assigned using a combination of Thermo Scientific OMNIC\u0026trade; spectroscopy software and literature (Socrates, 2004).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"3. Results and Discussion","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Quantification of Amine Attachment\u003c/h2\u003e \u003cp\u003eMethods to accurately quantify the covalent functionalization of CNCs remain unstandardized and highly debated in cellulose research. A key challenge is that a small number of hydroxyl groups are available for substitution. However, accurate quantification of surface groups is critical for CNC use in sensitive fields such as medicine and agriculture. Analysis techniques for quantifying nitrogen in small molecules include TGA, FTIR, liquid chromatography, and mass spectroscopy. However, these methods become problematic when the nitrogen is bound to the surface of CNCs. Because of their size, CNCs are incompatible and could damage traditional chromatography and mass spectroscopy systems, and schemes to remove the functional groups from CNC can result in inaccuracies due to incomplete removal. Amine peaks traditionally seen using FTIR become more challenging for quantification due to their overlap with CNC peaks, such as O-H and C-O stretching. Similar issues occur with TGA because amine degradation occurs in the same temperature range as CNC itself. Energy dispersive x-ray spectroscopy (EDX) has also been reported for nitrogen quantification, but the position of the nitrogen peak for this method lies between the carbon and oxygen peaks. For functionalized CNCs, where the total concentration of nitrogen is much less than that of carbon and oxygen, it is difficult to peak deconvolute due to low signal and limited resolution. In contrast, ultimate analysis (CHNS) is commonly used to quantify nitrogen. It is an excellent option for cellulose since it analyzes nitrogen separately from carbon and oxygen and breaks down CNC via combustion, enabling whole-sample analysis.\u003c/p\u003e \u003cp\u003eThe CHNS analysis results in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e show that the amination reaction was successful regardless of the presence of NaOH. The decrease in sulfur content from feedstock CNC to functionalized CNC is because CHNS calculates the weight percent of elements based on total mass. As additional carbon, hydrogen, oxygen, and nitrogen groups are functionalized to the surface, the ratio of sulfur mass to total mass decreases.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eCHNS Quantification and Degree of Surface Substitution Estimation for CNC-NH\u003csub\u003e2\u003c/sub\u003e Under Various Reaction Conditions.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eC (wt%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eH (wt%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eN (wt%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eS (wt%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eDegree of Surface Substitution\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eFeedstock CNC*\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e41.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e6.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eN.D.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e0.97\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e--\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eCNC-1\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e40.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e6.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.14\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e0.48\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e6.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eCNC-1-NaOH\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e41.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e6.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.15\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e0.59\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e6.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eCNC-2\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e40.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e6.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.14\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e0.41\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e6.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eCNC-2-NaOH\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e41.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e6.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.12\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e0.55\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e5.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eUncertainty is quantified by the standard error for a minimum of three runs. *Feedstock CNC CHNS analysis was conducted on a vario MICRO cube CHNS elemental analyzer for two runs. The error represents the maximum and minimum. N.D. = Not Detected.\u003c/em\u003e \u003c/p\u003e \u003cp\u003eCorrelating values obtained through quantitative characterization to the degree of CNC surface modification is still an underdeveloped area of cellulose research. Few papers consider the specific morphology of the CNC being used as well as any unreactive functional groups that may be present on the surface. We followed the method of Eyley and Thielemans (Eq.\u0026nbsp;\u003cspan refid=\"Equ1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) to determine the number of available surface hydroxyl groups per gram of CNC surface (Eyley \u0026amp; Thielemans, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2014\u003c/span\u003e).\u003cdiv id=\"Equ1\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ1\" name=\"EquationSource\"\u003e\n$${N}_{OH}=\\frac{{n}_{1}+{n}_{2}}{\\rho {N}_{A}{L}_{1}{L}_{2}c}\\left(\\frac{{L}_{1}+{L}_{2}}{{d}_{\\left(110\\right)}}+\\frac{{L}_{1}+{L}_{2}}{{d}_{\\left(1\\stackrel{-}{1}0\\right)}}\\right)+2(\\rho {N}_{A}{L}_{3}{d}_{\\left(110\\right)}{d}_{\\left(1\\stackrel{-}{1}0\\right)}{)}^{-1}$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e1\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003eWhere L\u003csub\u003e1\u003c/sub\u003e, L\u003csub\u003e2\u003c/sub\u003e, and L\u003csub\u003e3\u003c/sub\u003e are the height, width and length of individual CNCs, d\u003csub\u003e(110)\u003c/sub\u003e and d\u003csub\u003e(1\u0026minus;10)\u003c/sub\u003e are the horizontal and vertical unit cell of CNC, n\u003csub\u003e1\u003c/sub\u003e and n\u003csub\u003e2\u003c/sub\u003e are the number of primary and secondary -OH groups facing (110) and (1\u0026ndash;10) in the unit cell, ρ is the density of crystalline cellulose Iβ, c is the until cell dimension, and N\u003csub\u003eA\u003c/sub\u003e is Avogadro\u0026rsquo;s number.\u003c/p\u003e \u003cp\u003eUsing Eq.\u0026nbsp;\u003cspan refid=\"Equ1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, AFM size characterization of feedstock CNC, and average dimensions of cellulose Iβ from literature (Habibi et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Yoo \u0026amp; Youngblood, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), it was estimated that the CNC used in this investigation has an average number of surface hydroxyl groups (N\u003csub\u003eOH\u003c/sub\u003e) of 1.9 mmol/g. But this equation does not consider the presence of sulfate half-ester groups on the surface of the feedstock CNCs, a byproduct of sulfuric acid hydrolysis. To account for these groups, the results from CHNS analysis in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e were used to obtain a sulfate half-ester content of 0.3 mmol/g. Subtracting this from the 1.9 mmol/g of available surface hydroxyl groups, this leaves 1.6 mmol/g available for reaction. Degree of substitution is defined as the ratio of mmol of modification per gram of CNC conjugate to 1.6 mmol/g, or the maximum possible loading capacity. Results from this calculation are shown in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Effect of NaOH Addition on Amine Degree of Substitution\u003c/h2\u003e \u003cp\u003eTo determine if the degree of substitution results in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e are related to transport accessibility within the dispersion, which is a function of viscosity, flow curves were obtained for all dispersions and the data were model fitted using the power law model (\u003cb\u003eEquation S1, Table S2, and Figure S2\u003c/b\u003e). From the flow behavior of CNC-1 and CNC-2 shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, CNC-2 has higher viscosity compared to CNC-1 for all shear rates due to increased concentration. Both dispersions were nearly Newtonian as indicated by the power law index value being close to unity. When NaOH is added, the viscosity at all shear rates increased compared to their no NaOH counterparts. The behavior of CNC-1-NaOH and CNC-2-NaOH dispersions can be described by three regions. Both dispersions showed shear thinning behavior at lower shear rates until they reached a critical shear rate value of 7.5 s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e for CNC-1-NaOH and 13.3 s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e for CNC-2-NaOH. Both solutions showed shear thickening after this point due to aggregation before returning to shear thinning at even higher shear rates. When NaOH is added to dispersions, the concentration of Na\u003csup\u003e+\u003c/sup\u003e increases, which has the same effects on CNC as NaCl, resulting in screened charges and aggregation of the nanoparticles (Huntington et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Similar behavior was observed by Danesh \u003cem\u003eet al.\u003c/em\u003e where they found two shear thinning regions at low and high shear rates with a plateau at intermediate shear rates when NaCl was added in amounts greater than 20 mM in 1 wt% sulfated CNC dispersions with a sodium counterion (Danesh et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe three dispersions with the lowest viscosities rates resulted in higher degree of surface substitutions. For CNC-2-NaOH, which had the highest viscosity over all shear rates, the lowest degree of substitution was observed likely due to the system having a higher diffusion coefficient and the presence of aggregates, which can lower available surface area, inhibiting the amount of aqueous ammonia that can react. This confirms that amine functionalization is dependent on both transport of the molecule to the surface and the availability of hydroxyl groups.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Qualitative Confirmation of 2,4-D Functionalization\u003c/h2\u003e \u003cp\u003eThe CNC-NH\u003csub\u003e2\u003c/sub\u003e produced from each method above underwent an EDC/NHS reaction to covalently bind the active biomolecule 2,4-D to the surface of the nanocrystal for use as a nanocarrier of the active agent. 2,4-D was selected for conjugation with CNC based on the extensive use of such herbicide in commercial agriculture (Peterson et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). After conjugation, samples underwent TGA-IR analysis to qualitatively confirm the attachment of the herbicide (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFeedstock 2,4-D completely degrades in a narrow temperature window between 170 and 240\u0026deg;C, with a thermal degradation temperature of 178\u0026deg;C, and degrades completely in air, leaving only a residual mass of 0.03 wt%, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA. Meanwhile, commercial CNC does not begin thermal degradation (the temperature where 5% mass loss occurs) until 261\u0026deg;C and has a residual mass of 1.64 wt%. The 2,4-D-CNC conjugates behave independently from either feedstock material yet similarly to one another. All conjugates have a thermal degradation temperature between 232 and 246\u0026deg;C and a final residual mass of 1.02 to 1.14 wt%. These values lie between thermal degradation and residual mass values of 2,4-D and CNC indicating the presence of both in the final conjugates.\u003c/p\u003e \u003cp\u003eTraditional FT-IR spectroscopy is limited for 2,4-D-CNC since the spectral bands of the targeted trace material overlap with the spectra bands of CNC (Chan \u0026amp; Kazarian, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Nori et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). A solution to this problem lies in TGA-IR. Since 2,4-D thermally degrades at a lower temperature than feedstock CNC, we can observe higher concentrations of 2,4-D off-gas at temperatures before CNC degradation. The evolution of gas phases of 2,4-D-CNC during degradation was explored by coupling FT-IR spectroscopy with TGA and collecting the spectra of evolved gases over a wide temperature range. The Gram-Schmidt plot in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB shows the spectral intensity as a function of time and temperature. 2,4-D-CNC has a signature peak at approximately 30 min (~\u0026thinsp;300\u0026deg;C) that is not present for feedstock CNC. Further investigation into the IR spectra at this peak (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC) shows that the evolved gas of 2,4D-CNC differs from the spectra of unmodified CNC, as shown in Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE, the evolution of peaks at 903 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, 994 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, and 1107 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e which are due to the evolution of ester containing compounds and C-H deformation. Most importantly, 826 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 1173 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e can be attributed to C-Cl vibrational coupling and C-O-C asymmetric stretching from a halo containing compound, which appear during the thermal decomposition of the 2,4-D bound to CNC in the conjugates. In Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD, notable new peaks appear at 2801 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, 2897 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, and 2976 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. The first two are due to C-H stretching from an alkane. Stretching from an aliphatic amine occurs at 2976 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, from the degradation of the secondary amine used to link CNC and 2,4-D.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Quantitative Confirmation of 2,4-D Functionalization\u003c/h2\u003e \u003cp\u003eAlthough surface modification of CNC has gained significant interest in the past decade, there still exists a gap in understanding when it comes to quantifying the degree of surface substitution and recognizing the limitations of current analysis techniques, especially with halides such as chlorine. Traditional methods for chlorine detection such as titration, chromatography, and UV-vis become more complex in the presence of large biomolecules such as CNCs. In addition, careless handling during sample preparation and analysis can cause contamination, and chlorine is well-known to cause contamination in trace element analysis (Jin, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Recognizing these limitations and those described in Section 3.1, energy dispersive x-ray spectroscopy (EDX) was chosen to quantify the amount of chlorine present in the 2,4-D-CNC conjugates. EDX is the most common and simple method for chlorine detection in literature, and unlike nitrogen, the K\u003csub\u003eα\u003c/sub\u003e peak for Cl occurs around 2.6 compared to 0.3 and 0.5 for carbon and oxygen, respectively (Larn\u0026oslash;y et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Singh \u0026amp; Khatri, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Tanaka et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eEDX Quantification and Degree of Surface Substitution Estimation for 2,4-D-CNC Under Various Reaction Conditions.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCl (wt%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDegree of Hydroxyl Substitution\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eDegree of Primary Amine Substitution\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eFeedstock CNC\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eN.D.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e--\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e--\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e2,4-D-CNC-1\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.05\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e7.1%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e2,4-D-CNC-1-NaOH\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.03\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.9%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e2,4-D-CNC-2\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.05\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e7.1%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e2,4-D-CNC-2-NaOH\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.04\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6.5%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eUncertainty is quantified by the standard error for a minimum of six scans.\u003c/em\u003e \u003c/p\u003e \u003cp\u003eUsing N\u003csub\u003eOH\u003c/sub\u003e calculated previously and the same method for determining degree of surface substitution, the amount of covalently bound 2,4-D can be estimated from the weight percentage of chlorine present via EDX analysis (Fig.\u0026nbsp;4 \u003cb\u003eand\u003c/b\u003e Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The degree of surface substitution was calculated based on the average chlorine content detected for each sample. The degree of hydroxyl substitution ranged from 0.3\u0026ndash;0.4% for all samples. This correlates to 3.9 to 7.1% of amine groups covalently bound to a 2,4-D molecule.\u003c/p\u003e \u003cp\u003eInterestingly, the addition of NaOH to initial dispersions did not increase the final degree of 2,4-D substitution. The investigation supports the hypothesis that NaOH is not a necessary additive in the functionalization of commercially sourced CNCs. In future work, by removing NaOH, the viscosity of the CNC suspensions will remain lower and more workable, allowing for higher initial concentrations of CNCs to be functionalized, and potentially increasing the final degree of biomolecule substitution.\u003c/p\u003e \u003c/div\u003e"},{"header":"4. Conclusions","content":"\u003cp\u003eThis investigation built upon previous work on amine attachment to CNCs and investigated if NaOH is a necessary additive for functionalization when commercial sulfated CNCs are used. CNCs at 1 wt% and 2 wt% were aminated with and without NaOH. The reaction succeeded under all conditions, demonstrating that deprotonation with NaOH is unnecessary when using commercial CNCs with a sodium counterion. Rheological studies and elemental analysis found that for dispersions containing 2 wt% CNC with base added, the degree of substitution was limited, and NaOH increased the presence of aggregates and transport limitation within the dispersion. Amine functionalized CNCs were then reacted with 2,4-D to determine if adjustment to amine functionalization affects the nanocrystals resulting reactivity with biomolecules. FTIR and EDX showed that 2,4-D attachment was not hindered by the removal of NaOH in the first step and the highest degree of surface substitution was seen for both cases without base addition. These results highlight the changing field of cellulose functionalization and show that deprotonation of CNCs with NaOH is not required for amination when commercial sulfated CNCs are used. Established results create a base for a simpler and cost-effective methodology for the conjugation of CNC with variable chemicals used in medical and agricultural applications.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDelaney E. Clouse, Mariya V. Khodakovskaya, and Virginia A. Davis contributed to the studies conception and design. Material preparation, data collection, and analysis were performed by Delaney E. Clouse, Elise G. Collins, and Tanmay Rahman. The first draft of the manuscript was written by Delaney E. Clouse and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eEthical Approval\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eFunding\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by USDA-NIFA 2021-67022-33995. The authors have no relevant financial or non-financial interests to disclose.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAvailability of Data and Materials\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData is presented within the manuscript and supplementary information. 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ACS Sustain Chem Eng 4:3927\u0026ndash;3938. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1021/acssuschemeng.6b00781\u003c/span\u003e\u003cspan address=\"10.1021/acssuschemeng.6b00781\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Cellulose Nanocrystals, Surface Functionalization, Herbicides, Sodium Hydroxide","lastPublishedDoi":"10.21203/rs.3.rs-3968506/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3968506/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThis article investigates the necessity of sodium hydroxide (NaOH) addition for the amine functionalization of sulfated cellulose nanocrystals (CNCs) and its effect on nanocrystal reactivity with the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D). The use of CNCs as a nanocarrier of active biomolecules has grown in the past decade. Previously, CNCs were produced by laboratory sulfuric acid hydrolysis protocols that imparted sulfate half-ester groups with hydrogen counterions. Because of this, researchers cited the need to add a deprotonating base such as NaOH before amination, a common precursor reaction for further biomolecule functionalization. However, current commercially produced sulfated CNCs have a sodium counterion instead of hydrogen. This work explores whether the use of commercial sulfated CNCs negates the need for sodium hydroxide addition in amine functionalization. We investigated the effect of 10 wt% sodium hydroxide solution on the amination of 1 wt% and 2 wt% CNC dispersions. Following this step, CNCs were then further modified via EDC/NHS chemistry to attach 2,4-D. Thermogravimetric analysis coupled with infrared spectroscopy was used to qualitatively confirm attachment. Elemental analysis determined that the degree of amine substitution for all dispersions ranged from 5.4\u0026ndash;6.7%. 2,4-D attachment to amine groups varied from 3.9\u0026ndash;6.5% when NaOH was present to 7.1% when NaOH was not added. These results highlight how the evolution in CNC extraction methods has resulted in NaOH addition no longer being necessary for successful reactions when using commercially sourced sulfated CNCs with a sodium counterion.\u003c/p\u003e","manuscriptTitle":"Effects of NaOH Addition on Cellulose Nanocrystal Functionalization with 2,4-Dichlorophenoxyacetic Acid","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-02-23 04:50:25","doi":"10.21203/rs.3.rs-3968506/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"938d06cb-b3d2-4be4-8275-487768f97c91","owner":[],"postedDate":"February 23rd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-04-03T00:29:27+00:00","versionOfRecord":[],"versionCreatedAt":"2024-02-23 04:50:25","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3968506","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3968506","identity":"rs-3968506","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}