*3.1. Characterization of NOCNF*

The characterization on the surface functionalization of NOCNF extracted from untreated jute fibers was first carried out by FTIR and conductometric titration. Figure 1i demonstrates the FTIR spectra of jute fibers and prepared NOCNF. The characteristic peaks of cellulose are ascribed at (i) 3327 cm−<sup>1</sup> to O–H stretching vibrations, and at (ii) 2904 cm−<sup>1</sup> to CH and CH2 stretching. The prominent peak in NOCNF at 1591 cm−<sup>1</sup> presented the carboxylate groups (–COONa) appeared in NOCNF, which represents the oxidation of anhydroglucose unit at C6 position. Additional peaks at 1372, 1150, 1100, and 1030 cm−<sup>1</sup> were due to stretching and bending vibrations in glycosidic bonds in cellulose. Other peaks in the FTIR of jute fibers such as 1512; 1732, 1456, 1235 and 808 cm−<sup>1</sup> are because of aromatic symmetrical streching of C=C bonds in the lignin and in hemicellulose units respectively. Interestingly, the peaks belonging to lignin and hemicellulose completely disappear or significantly reduce in NOCNF, indicating that the nitro-oxidation was resonably effective in removing the lignin and hemicellulsoe impurities from raw jute fibers. The quantitative determination of lignin and hemicelulose in NOCNF was also perfomed to find the exact amount of lignin and hemicellulsoe, which is explained in the next section.

**Figure 1.** (**i**) Fourier transform infrared spectrometry (FTIR) of carboxycellulsoe nanofibers (NOCNF) and jute fibers, and (**ii**) conductometric titration graph to determine the carboxylate group on NOCNF (volume of NaOH consumed = 0.705 mL), inset the photograph of NOCNF suspension.

The quantitative determination of carboxylate groups in NOCNF was performed by conductometric titration method. The following equation (Equation (1)) was used to determine the degree of oxidation (*DO*):

$$DO = \frac{MX\left(V\_2 - V\_1\right)}{w} \tag{1}$$

where *M* is the molarity of NaOH in mol/L, *V2* and *V1* is the final and initial volume of NaOH in mL, *w* is the weight of the NOCNF dried fibers added in grams.

The conductometric titration plot of NOCNF shown in Figure 1ii indicates its calculated DO value which is 0.94 mmol/g. This DO value shows that NOCNF contains a moderate degree of oxidation, which is further confirmed by zeta potential measurement. The zeta potential measurement demonstrates the presence of −115 ± 4 mV charge on the NOCNF surface. NOCNF showed good dispersion in water (inset photograph in Figure 1ii) because of the repulsion caused in between the fibers due to similar charges.

The lignin and hemicellulose analysis of raw jute fibers is presented in Table 1. It shows that the total hemicellulose (sugar content) and total lignin content (klason lignin (KL) + acid soluble lignin (ASL)) in the raw jute fibers was 68.9% and 17.55%, respectively. However, the total hemicellulose and lignin content in NOCNF was found as 65% and 1.94% respectively. The results indicate that the reasonable hemicellulose and residual lignin are still present in the NOCNF after the nitro-oxidation.


**Table 1.** Characteristic of carboxycellulsoe nanofibers (NOCNF) obtained from jute.

<sup>a</sup> KL = klason lignin, ASL = acid soluble lignin.

The TEM image of NOCNF is shown in Figure 2i. The average fiber length observed for NOCNF was 524 ± 203 nm and width were in the range of 7 ± 2 nm. However, the AFM of the NOCNF indicated the average fibers thickness was 2.9 nm. In this study, the NOCNF obtained has greater width and thickness as compared to the cross section of most of the cellulose fibers where width is in the range of 4–5 nm and thickness ~1.5 nm [37]. This is probably due to the chosen nitro-oxidation conditions are relatively mild, where the presence of residual hemicellulose and lignin contents were still high.

**Figure 2.** (**i**) Transmission electron microscopy (TEM) and (**ii**) atomic force microscopy (AFM) of NOCNF extracted from raw jute fibers.

The dynamic light scattering (DLS) data measurement of NRL is presented in Figure 3. The data shows that the effective diameter of NRL molecule is 637 nm with polydispersity value of 0.005. This indicates that the NRL is composed of polyisoprene molecules with almost the same size. The TEM measurement of NOCNF indicated its fibers length in the range of 524 ± 203 nm, which is almost like the NRL particles' size. We have assumed that similar sizes of two interacted molecules NOCNF and NRL will provide the better chances of their physical interaction.

**Figure 3.** Dynamic light scattering (DLS) data of natural rubber latex (NRL) (average diameter = 637 nm with size polydispersity (PDI) = 0.005.
