3.2.2. FT-IR Analysis of Biosynthesized AgNP

FT-IR analyses were conducted to identify the biomolecules that might be responsible for reducing the Ag+ ions in the beech bark extract, and also those that may be involved in the stabilization of silver nanoparticle synthesis. Figure 3 shows the spectrum of aqueous beech bark extract, where the bands corresponding to the –O–H bonds at 3414 and 1608 cm−<sup>1</sup> specific to the carbonyl group can be observed.

The recording of the FT-IR spectra in the case of AgNPs obtained by reducing AgNO3 (TS1–TS2) with the polyphenolic compounds in the aqueous beech extract shows the appearance of a band at the value of 1512 cm−<sup>1</sup> and intensifies the band corresponding to the wavenumber 1384 cm−<sup>1</sup> (Figure 4). These show that certain compounds in the aqueous extract modify their structure, thus, generating AgNPs. For TS3–TS4 solutions, there were no different results compared to TS1–TS2.

**Figure 3.** Fourier transform infrared spectra of aqueous bark extract.

**Figure 4.** Fourier transform infrared spectra of gold nanoparticles (AgNPs): A—TS2 (AgNPs obtained with AgNO3 at pH = 9), B—aqueous bark extract, C—TS1 (AgNPs obtained with AgNO3 at pH = 4).

3.2.3. The Analysis by Transmission Electron Microscopy (TEM) of Biosynthesized AgNP

Transmission electron microscopy was used in order to characterize the obtained nanoparticles in terms of their morphology and size. Thus, the TEM photomicrographs obtained were analyzed using ImageJ program, obtaining information about the surface and diameter of the biosynthesized nanoparticles.

In Figure 5, it is observed that the AgNPs synthesized are nanometric and uniformly distributed. Regarding the morphology of the nanoparticles, it can be observed that it varies, encountering polygonal, spherical, and even triangular shapes at TS1 (Figure 5a). In contrast, at basic pH (TS2) only spherical shapes are observed (Figure 5c). The particles synthesized in TS1 have sizes between 10 and 420 nm (Figure 5b), with an average of 118.75 nm, and about 44% of these have a diameter below 100 nm. Regarding the size of the nanoparticles obtained at pH 9 (TS2) they fall in the range of 2–80 nm, with an average of 44.02 nm and 100% of the measured nanoparticles have dimensions smaller than 100 nm (Figure 5d).

Analyzing the synthesized AgNPs in the presence of the extract obtained from the beech bark and AgNO3 (Figure 6), the results are similar to those presented previously. The morphology of the obtained AgNPs at pH = 4 (TS3) is varied, in this case observing polygonal, triangular, and spherical shapes (Figure 6a). In contrast, at basic pH (TS4) it can be observed that the AgNP morphology is constant, with only the spherical shape being met (Figure 6c). AgNPs obtained in the basic medium have dimensions in the range of 2–80 nm, with an average of 32.43 and 100% being smaller than 100 nm in diameter (Figure 6d).

The results obtained with the help of TEM confirm the results presented above, regarding the basic pH which favors the formation of a larger number of smaller nanoparticles. Thus, comparing the tested solution, it is found that at pH = 9 all AgNPs have dimensions below 100 nm, and at pH = 4, about half of AgNPs have dimensions below 100 nm. These findings, related to shape and size, are also confirmed by other researchers who analyzed synthesized nanoparticles in the presence of extracts obtained from the bark of woody plants [26,27]. The particles synthesized in TS3 have sizes between 10 and 200 nm (Figure 6b), with an average of 62.2 nm.

Thus, from literature data, AgNPs with a larger surface area, provide better contact with microorganisms [28]. These particles are able to penetrate into the cell membrane or attach to the bacterial surface based on their size. In addition, they have been reported to be highly toxic to bacterial strains and their antibacterial efficacy is increased by decreasing the particle size [29].

**Figure 5.** Graphical representation of AgNPs synthesized in the presence of the extract obtained from the beech bark and AgC2H3O2: (**a**) TS3— TEM photomicrograph; (**b**) histogram of the distribution of AgNP size distribution in TS3; (**c**) TS4—TEM image; (**d**) histogram of the distribution of AgNP size distribution in TS4. (TS3—beech bark extract, pH = 4, AgC2H3O2; TS4—beech bark extract, pH = 9, AgC2H3O2).

**Figure 6.** Graphical representation of AgNP synthesized in the presence of the extract obtained from the beech bark and AgNO3: (**a**) TS1— TEM photomicrograph; (**b**) histogram of the distribution of the AgNP size distribution in the TS1; (**c**) TS2—TEM image; (**d**) histogram of the distribution of AgNP size distribution in TS2 (TS1—beech bark extract, pH = 4, AgNO3; TS2—beech bark extract, pH = 9, AgNO3).
