Preparation of oxidized nanocellulose by using potassium dichromate

Nanocellulose derivatives are being used in a wide variety of high-quality functional applications. One of them is oxidized nanocellulose, which has been used in biomedical and pharmaceutical applications due to its biodegradable, biocompatible, hemostatic, and antibacterial properties. In this work, oxidized nanocellulose was synthesized using potassium dichromate as an oxidizing agent. The structure of oxidized nanocellulose was investigated by means of ultraviolet spectrophotometry, Fourier transform infrared spectroscopy, X-ray diffraction, atomic force microscopy, and thermogravimetric analysis. The results showed that the primary hydroxyl groups of NC were selectively oxidized to carboxyl groups and their content of 1.36 mmol/g was achieved. The appearance of a new peak (1721 cm−1) in the FTIR- spectra related to the C=O group was observed. The change of oxidized nanocellulose’s crystal index from 88.0% to 82.5% was revealed, and the sizes of the unit cells of both nanocellulose and oxidized nanocellulose were calculated. The thermal stability of oxidized nanocellulose decreased compared to nanocellulose. The oxidation process of nanocellulose leads to a change in the shape and size of particles from acicular to spherical with a narrow particle size distribution. It was shown that oxidized nanocellulose has the ability to accumulate charges on its surface.


Introduction
Nanocellulose (NC) is a new modified form of cellulose; it has a high crystallinity, surface area, degree of dispersion, degradability. Due to their properties such as biocompatibility, biodegradability, and low toxicity, materials based on NC have been widely used in recent years in various fields, including biomedicine (Lin et al. 2014;Reshmy et al. 2021;Tracheet al. 2020;Sezali et al.2021; Thomas et al. 2020). NC can be prepared from various cellulose-containing raw materials (wood, straw, cotton cellulose, etc.) by hydrolysis with aqueous solutions of inorganic acids, ultrasonic dispersion, microwave irradiation, etc. (Zaini et al. 2013;Atakhanov et al. 2020;Moran et al. 2008;Yang et al. 2012;Atakhanov et al. 2019;Yadav et al. 2021).The surface of the NC contains numerous hydroxyl (OH) groups, which provide the main reaction site for modification. By introducing additional functional groups into the structure of NC, it becomes possible to regulate its properties and expand the scope of its application (Braun et al. 2009;Hasani et al. 2008, Morandi et al. 2009). Etherification, oxidation, silylation, and grafting of macromolecules are typical ways (Sun et al. 2015(Sun et al. , 2014Salam et al. 2015;Chen et al. 2015;Kedzior et al. 2016).
The oxidation process of NC is of scientific interest since, depending on the reaction conditions, the final product may contain both carbonyl (aldehyde, ketone) and carboxyl groups (Gensh et al. 2013;Yuldoshev et al. 2016;Huang et al. 2016;Luo et al. 2013). Oxidized nanocellulose (ONC) is a new NC derivative with unique performance properties. Currently, research is being actively carried out on the development of haemostatic drugs, cosmetic products, and medical implants based on ONC (Czaja 2014;Isogai 2018). ONC can be also used as filler in polymer nanocomposites in order to give special properties to materials (Mahendra et al. 2020;Barnes et al. 2019;Gabriel et al. 2022;Voronova et al. 2022). Furthermore, the NC derivatives based materials have become extensively interesting recently in scientific and industrial fields as promising sustainable substrate materials for their versatile usage and also in flexible energy storage devices (Wang et al. 2017;Fang et al. 2014;Zu et al. 2016).
There are several methods of preparing ONC by using different oxidizing reagents (Levanic et al. 2020;Besbes et al. 2011;Isogai et al. 2018). One of the most popular process is oxidation of NC mediated by 2,2,6,6-tetramethylpiperidine-1-oxyl radical (TEMPO) (Isogai et al. 2011). Catalytic oxidation property of TEMPO has opened a new direction in effective and selective chemistry for the conversion of cellulose hydroxyl groups into aldehyde, ketone and carboxyl groups under mild conditions (Isogai 2018;Hassan et al. 2021). To prepare ONC with a high content of carboxyl groups, the TEMPO-NaBr-NaClO system has been widely used with ultrasonic dispersion at various pH values (Moon et al. 2011, Hondo et al. 2019. In recent years, other methods of oxidizing NC have been reported. Given the high cost of TEMPO, ammonium persulfate (APS) is a potential alternative, and it enables synthesis of ONC with higher degree of crystallinity (DC) values (Filipova et al. 2018;Oun et al. 2018). The ONC produced by using the APS-oxidation method has superior mechanical properties and thermal stability compared with the ONC produced by the TEMPO-mediated oxidation method (Oun et al. 2017;Zhang et al. 2016). However, when APS is used for NC oxidation the content of carboxyl groups of ONC remains lower and a large amount of APS is needed for the oxidation process (Yang et al. 2019). Developing new approaches to NC oxidation by using more accessible reagents under mild conditions is of scientific and practical interest. The aim of this work was to prepare oxidized nanocellulose by using potassium dichromate and to study its properties and structure.

Materials
Potassium dichromate (99.5 wt%), and sulfuric acid (98.0 wt%) were purchased from Sigma Aldrich from Sigma-Aldrich Chemical Co. Ltd. (Tianjin, China). Sodium hydroxide (NaOH, 98.0%) was obtained from Daejung (South Korea). All the above reagents were of analytical purity and used as received without further purification. NC (degree of polymerization (DP) 120, DC = 88%) was prepared from cotton cellulose (Atakhanov et al. 2020) and used as raw material for preparing ONC. Deionized water was used in all experiments unless otherwise specified.

Preparation of ONC
ONC was prepared in an aqueous medium by using a calculated amount of K 2 Cr 2 O 7 and H 2 SO 4 , varying the reaction time from 3 h (sample ONC-3) to 9 h (sample ONC-9). In a three-neck flask, 3.0 g NC was dispersed in 50 mL of deionized water for 20 min by using ultrasonic instrument. Next, the pH of the suspension was adjusted to 2-3 by adding of sulphuric acid. 3.63 g potassium dichromate was added into the flask and maintained with constant magnetic stirring at 40 °C in a glycerine bath for 3-9 h, bubbled with nitrogen gas. The product was centrifugated and was repeatedly washed with deionized water under centrifugation at 8000 rpm for 15 min until a final pH of 5 was obtained in the system. Then the ONC suspension was dialyzed using dialysis membranes in deionized water for 3 days. Finally, the ONC was separated from the suspension by centrifuging and dried in a freeze-dryer for later use.

Fourier transform infrared (FTIR) spectroscopy.
FTIR studies were carried out with an Inventio-S IR Fourier (Bruker, Germany). The spectral resolution was 0.085 cm −1 and the range of measurement was 4000-500 cm −1 .

Ultraviolet spectrophotometry (UV)
UV spectra were recorded with a Specord 210 UVspectrophotometer (Analytic Jena, Germany) by using quartz cells 1 cm in diameter and 1 nm slit; the scanning range of measurement was 190-1000 nm, a scanning speed was 5 nm/s.

X-ray diffraction (XRD)
XRD studies were carried out with an XRD Miniflex 600 (Rigaku, Japan) with monochromatic CuKα radiation isolated by a nickel filter with a wavelength of 1.5418 Å at 40 kV and the current strength of 15 mA. The samples were examined in the form of a powder. The spectrum was recorded in the interval 2θ = 2°-70°. The data processing of experimental diffraction patterns, peak deconvolution, describing the peaks used by Miller indices, peak shape, and the basis for the amorphous contribution were conducted using the software "SmartLab Studio II" and data base PDF-2 (2020 Powder diffraction file, ICDD). The Rietveld method was applied using the pseudo-Voigt function. The experiment was conducted in reflection (Bragg-Brentano) mode and fixed slits were used which an angle value is 1.25°. We used the Nishiyama cellulose Iβ file for the neutron diffraction study, which has all of the hydrogen (including deuterium) atoms present.
The crystallite size, τ (Å), was calculated by the Scherrer equation (Nam et al. 2016;Langford et al. 1978) where K is the correction factor (0.9), λ is the wavelength of the X-ray radiation (1.5418 Å), β is the full width at half maximum of the diffraction peak in radians, and θ is the diffraction angle of the peak. The crystal index (CrI) (%) was calculated by the following equation: where I t is the total intensity of the (200) peak for NC at 22.76°, for ONC-3 at 22.66°, and for ONC-9 at 22.67° 2θ, and I a is the amorphous intensity at 18.86° 2θ for NC, at 18.91° 2θ for ONC-3 and ONC-9.

Atomic force microscopy (AFM)
Morphological studies were performed by using AFM Agilent 5500 (Agilent, USA). The silicon cantilevers with a stiffness of 9.5 N/m 2 were used and frequency was 262 kHz. The AFM scan area (x-y-z) was 4.5-4.5-1 µm.

Thermogravimetric analysis (TGA)
Thermal analysis of the samples was carried out with TG-DSC/DTA synchronous thermal analyzer STA PT1600 (Linseis, Germany) by heating ~ 20 mg of the sample in an air atmosphere at a heating rate of 10 °C/min from 25 °C to 900 °C.

Dynamic light scattering (DLS)
The particle size ONC of was estimated by using DLS Photocor compact (Photocor, Russia). The measurements were carried out at 298 K using a thermally stabilized semiconductor laser with a wavelength of 635.6 nm and a power of 25 mW.

Determination of carboxyl group content
The content of carboxyl groups (mmol/g) was determined by conductometric titration on a Mettler Toledo conductometer (Switzerland). 50 mg ONC was dispersed in 20 mL of 0.01 M HCl solution for 15 min by using magnetic stirring. Then, the suspension was titrated with a 0.01 M NaOH solution.
The number of carboxyl groups was calculated using an equation (Habibi et al. 2006) and expressed in mmol/g. Each batch was titrated three times.

Impedance spectroscopy
The measurements were carried out on thin films deposited on ITO substrates using Metrohm Autolab (Autolab PGSTAT302N, Netherlands), sample area (5.5 mm 2 ), on which silver paste was applied to ensure the necessary electrical contact between the test sample and the meter. The impedance spectra were recorded in the frequency range of 1 Hzto 106 Hz with scan rates of 10 mV/s. The results were analyzed using the NOVA 2.0-Advanced electrochemistry software.

Results and discussions
Dichromate solutions have long-term stability in low pH, light, and the presence of many organic substances and chlorine ions. In the course of NC oxidation, dichromate ions (Cr 6+ ) are reduced to Cr 3+ , which changes the colour of the solution from orange to green; first, the hydroxyl groups are oxidised to aldehyde groups, and then to carboxyl groups (Scheme 1). A similar carboxylation mechanism occurs during the potassium permanganate oxidation of cellulose nanofibrils (Liu et al. 2021). NC oxidation proceeds heterogeneously. The course of such a process is significantly influenced by the supramolecular structure of NC. The amount of carboxyl group in the ONC samples was determined by conductometric titration (Fig. 1).
The titration curves have a parabolic shape; an initial decrease in conductivity is related to the neutralization of HCl with NaOH solution due to the accumulation of ions with low mobility (cations and anions). After complete neutralization of free HCl, the conductivity plateaued out where NaOH consumption was associated with the weak carboxylic groups on the ONC (Jiang et al. 2013). After reaching the second point of equivalence, the electrical conductivity of the solution increases due to an increase in the concentration of -OH ions with high mobility. Calculations of the results of conductometric titration showed that with an increase in the duration of Scheme 1 Reaction of NC oxidation the reaction, the amount of carboxyl groups increases from 1.21 mmol/g (for ONC-3) to 1.36 mmol/g (for ONC-9).
The formation of carboxyl groups was also confirmed by FTIR studies (Fig. 2). Comparative studies of NC and ONC showed that absorption bands at around 3400 cm -1 are observed, which are related to the stretching vibrations of O-H. In the case of ONC, this absorption band narrows and appears more intensely due to a decrease in the number of hydroxyl groups involved in hydrogen bonds. The stretching vibrations of the CH bonds of methylene and methine groups are manifested near 2800-2950 cm -1 . The presence of adsorbed water was observed at around 1635 cm -1 , the water molecules cannot be total because of cellulose-water interaction (Moran et al. 2008), the absorption bands at 1420 cm -1 , 1335-1375 cm -1 , 1202 cm -1 , 1075-1060 cm -1 correspond to the bending vibrations of -CH-, -CH 2 -, -OH, -CO, the stretching vibrations of C-O and the pyranose rings. In contrast to the spectrum of the NC, a new absorption band appears in the FTIR spectra of ONCs at 1721 cm -1 , which is related to the stretching vibration C=O of the carboxyl group. This suggests the primary hydroxyl groups (on C6) of the anhydroglucose unit were converted into carboxyl groups successfully (Tang et al. 2017;Lin et al. 2018). The intensity of peaks of FTIR spectra of ONC-9 is stronger than ONC-3, indicating a greater amount of carboxyl groups and a higher degree of oxidation. At the same time, in the region of 1425 cm -1 related to bending vibrations of -CH 2 -groups, the signal intensity decreases, and the intensity of the absorption band at 1315 cm -1 related to fan-shaped vibrations of -CH 2 -groups, which are associated with out-ofplane vibrations, increases. This gives information about decreasing DC of the ONC, which was also confirmed by X-ray diffraction studies (below in the text). At the same time, the pyranose ring of the elementary cellulose unit is preserved, that confirms the oxidation of the C6 hydroxyl group.
The results of UV spectroscopic studies show (Fig. 3) that there are three absorbance peaks at roughly 196, 240, and 290 nm peaks associated with C=O of the aldehyde groups (at 240, 290 nm) and the carboxyl groups (at 190-210 nm). It is observed an increase in the number of conjugated bonds leads to a bathochromic shift, which is described by the Woodward rule (Ager et al. 1996).
The crystalline structure of cellulose is one of the important determining factors for its mechanical properties and thermal stability. The crystal structure of ONC was evaluated by XRD analysis (Segal 1959). The results of the X-ray study showed there are four crystal reflections in the region 2θ = 14°, 16°, 22° and 34°, corresponding to the planes 1 1 0, 110, 200, and 004 (Fig. 4.). The CrI of ONC decreased from 88 to 79% as the oxidation time increased, indicating that the crystal structure of NC was partially destroyed. The interplanar distances (d) of ONC crystals have also increased. It is interesting to note that during the TEMPO-oxidation of the cellulose to prepare ONC, an increase in the degree of crystallinity is observed, that is explained by the removal of the amorphous parts of cellulose during the oxidation process using strong oxidants (Qin et al. 2011).
The results of X-ray diffraction analysis showed that the oxidation process of NC affects the crystallite size of NC anisotropically, i.e., the crystallite sizes increase in the two directions (a, c). The analysis results are shown in Figs. S1, S2 and S3 and Table 1.
We assume the oxidation process begins at the surface of the crystallites and then gradually moves into the deeper layers. Theoretical calculations were carried out, and a model was created (Fig. 5) about the available hydroxyl groups for oxidation at carbon C6, which was also shown in the work (Habibi et al. 2006).
According to the sizes of the unit cell and crystallites calculated from the X-ray diffraction analysis data (Table 1) that are more available for external modification, and if all these hydroxyl groups will be oxidized, the degree of oxidation will be 100%. This is approximately 30% of the total number of hydroxyl groups in the elementary units in the crystallite. Theoretical calculations showed that 20-25% of the available hydroxyl groups on the C6 carbon were oxidized to carboxyl groups.
The thermal stability of the samples was studied with TGA curves (Fig. 6). All thermograms display the characteristic behaviour of endothermic polymeric degradation. TGA analysis revealed that the decomposition temperatures of the ONC samples are lower than those of the neat NC, and that the higher the carboxyl group content, the lower the decomposition temperature. The weight loss for all samples proceeds in three stages. An initial weight loss of approximately 5-9% was observed for samples upon heating to 100 °C. These findings correspond to the vaporization of dampness in the sample (Kargarzadeh et al. 2012).
In this case, the greater the content of the carboxyl groups in the ONC samples, the greater the weight loss. This is probably due to the decomposition process of cellulose, which is catalysed by carboxyl groups; therefore, it proceeds at 200 °C, whereas in the case of the NC sample, the temperature of the decomposition process is approximately 250 °C. These data are consistent with the results reported by other authors ; Sharma et al. 2014), where this phenomenon is also explained by a decrease in the degree of crystallinity and the content of the carboxyl groups.
There are several methods, such as AFM (Beck-Candanedo et al. 2005), transmission electron microscopy (Bercea et al. 2000), dynamic depolarized light scattering (Lima et al. 2002), field emission scanning electron microscopy (Hassan et al. 2021) for determining NC dimensions and morphology. In this work, the sizes and distribution of particles were estimated by the DLS method (Boluk et al. 2014), which showed that the sizes of particles ranged from nanoto micrometers, and that a polymodal distribution of particles was also observed (Fig. 7, Table 2).
NC and ONC are surface-active, they are easily agglomerated and form micron-sized clusters. The size of the ONC particles decreases with an increase in the duration of the oxidation process, which is in good agreement with the results of AFM studies (Fig. 8). It was also revealed that the oxidation process leads to a narrowing of the distribution of particles, which can be described by the Lorenz distribution.
The AFM study showed that NC particles have an acicular shape with a width of 20-80 nm and a length of 180-600 nm (Fig. 8). The oxidation process leads to a decrease in the size of particles with a width of 50-120 nm and a length of 150-400 nm and partial destruction of the acicular shape of NC with a transition to a spherical shape. The long duration of the oxidation process leads to the formation of agglomerates of spherical particles with a size of 20-60 nm.
The oxidation process reduces the amount of larger particles while increasing the amount of small particles, resulting in a monodisperse particle size distribution.
The study of macromolecular substances by electrochemical impedance spectroscopy is possible due to the correlation between the electrophysical properties observed in the experiment and the molecular structure of the substance. By means of impedance spectroscopic measurements, the dipole moment, polarizability, and rotational velocity of a particular group, or of a macromolecule as a whole, i.e., quantities that determine the structure, conformational features, and molecular mobility of a macrochain both in an isolated state and in a condensed state, can be determined.
The electrophysical properties of NC and ONC-9 were studied using impedance spectroscopy. The Nyquist diagrams for NC and ONC-9 are shown in Fig. 9. As seen from the experimental data, in the region of high-frequency values, a capacitive semicircle is partially observed. The semicircle is the result of a combination of polarization resistances, i.e., the sum of the charge transient resistance at the NC/electrode interface and the ONC/electrode in parallel to the total capacitance. At low frequencies, a different behaviour was observed, indicating ONC-9's ability to accumulate charge on their surfaces (Natalia et al. 2013;Hernández-Flores et al. 2020).
It can be seen from the phase diagram ( Fig. 10) that in case of both samples, the intensity of the lowfrequency region is greater than the high-frequency region, since the number of segments is less than the number of elementary links. Increasing the amount of the functional group of ONC-9 leads to an increase in  intensity and frequency. The response of a polymer to an electric field is the stronger, the better the dipoles are oriented in it, and the larger the dipole moment, (Chan et al. 2018) and the dipole moment increases during the transition from NC to ONC.

Conclusions
In summary, the possibility of NC oxidation in a more accessible way under mild conditions using potassium dichromate was shown. The oxidation of NC proceeds at C6 carbon without the destruction of the pyranose ring of cellulose, and the number of carboxyl groups in the ONC is 1.21-1.36 mmol/g. It is discovered that increasing the oxidation process duration decreases the CrI and thermal stability. Based on the results of the X-ray structural analysis, the sizes of both the crystallites and the unit cells of NC and ONC are calculated, where a decrease in size in one direction and an increase in size in the other two directions of measurement are observed. Theoretical calculations were carried out, and a model was created for the hydroxyl groups available for oxidation at carbon (C6), which amounted to approximately 5% of the total number of hydroxyl groups in the elementary units in the crystallite. It was calculated that 60% of the available hydroxyl groups at carbon (C6) were oxidized to carboxyl groups. By using AFM and DLS, it was shown that the oxidation process leads to a decrease in the particle size and a change in the shape of the particles from acicular to spherical, while the particle size distribution becomes monodisperse. The ONC samples are able to accumulate charges on their surfaces. The ONC formed by potassium dichromate  oxidation has potential applications as an environmentally friendly and cost-effective nanomaterial in energy-related fields.