*3.4. Validation of the Molecular Interaction*

The proposed hypothesis, specifically the molecular interaction, was validated by analyzing the FTIR spectrum obtained from a HAMA hydrogel sample soaked in a Pb(II) solution, as shown in Figure 6a. A comparison of all of the prominent peak positions in terms of wavenumber (cm−1) between the pure HAMA hydrogel and the Pb(II)-soaked HAMA hydrogel is listed in Table 1. It was found that peaks 1, 3, 4, 5, and 6 in both spectra were situated at similar frequency bands. A new peak 7 emerged at 1237.61 cm−<sup>1</sup> for the Pb(II)-soaked HAMA hydrogel, which could have been due to either C-O or C-N stretching. In addition, the original peak 2 (corresponding to the carbonyl stretching of the amide group) for the pure HAMA hydrogel separated into two peaks, i.e., peak 2 situated at 1640.03 cm−<sup>1</sup> and peak 2 situated at 1565.72 cm−<sup>1</sup> for the Pb(II)-soaked HAMA hydrogel, as depicted in Figure 6b. The significant shift of peak 2 implies that the Pb(II) ions mainly interact with the amide group of the HAMA hydrogel. We attribute such a molecular interaction to the effect of the Pb(II)–amide complexation.

**Figure 6.** (**a**) FTIR spectrum of the HAMA hydrogel soaked in a solution of 100 μg/L Pb(II), in which the numbers 2 , 2, 7 (in blue) denote the new peaks. (**b**) Comparison of FTIR spectra in a frequency band from 2050 to 1450 cm−<sup>1</sup> between the pure HAMA hydrogel and the one soaked in Pb(II) solution.

**Table 1.** Comparison of peak positions in terms of wavenumber (cm−1) between the pure HAMA hydrogel and the Pb(II)-soaked HAMA hydrogel.


The resonance structure of an amide group has two possible configurations [41], which are named type I and type II. For a type I configuration (refer to Figure 7a), the lone pair of electrons on the N atom is not involved in the conjugation with the carbonyl group, making the N atom relatively electronegative. Hence, a positively charged metal ion (here is Pb2+) can coordinate with the N atom of the amide group. Such coordination would result in a positive shift of the carbonyl infrared adsorption to a higher frequency [42]. For a type II configuration (refer to Figure 7b), the lone pair of electrons on the N atom is delocalized into the carbonyl group, making the O atom of the carbonyl group more electronegative. Therefore, a positively charged metal ion can also coordinate with the O atom of the amide group, which would bring about a negative shift of the carbonyl infrared adsorption to a lower frequency [42]. As illustrated in Figure 6b, peak 2 with a positive shift of ~12 cm−<sup>1</sup> with respect to peak 2 could be due to the vibration of the Pb(II)-N ligand, which corresponds to the type I complexation. On the other hand, peak 2 with a negative shift of ~62 cm−<sup>1</sup> with respect to peak 2 could be due to the vibration of the Pb(II)-O ligand, corresponding to the type II complexation. Based on the experimental observations, it is highly possible that both nitrogen and the carbonyl oxygen of the amide group of the HAMA hydrogel may be simultaneously involved in the formation of the Pb(II)–amide complex.

**Figure 7.** Schematic representation of (**a**) type I and (**b**) type II Pb(II)–amide complexations.

Despite the contribution from nitrogen, as well as carbonyl oxygen, in the process of the complex formation, the tendency to form a Pb(II)-O ligand seems much higher than that of a Pb(II)-N ligand if one compares the negative shift with the positive one. The negative shift (~62 cm<sup>−</sup>1) is 5 times larger than the positive shift (~12 cm−1), suggesting that the carbonyl oxygen of the amide group could play the dominant role in the construction of the Pb(II)– amide complex. This could be explained by the fact that the type II configuration of an amide group is more stable than the type I configuration [41]. Owing to the delocalization, the electron density distributed on the carbonyl oxygen is much greater than that on the nitrogen, making the carbonyl oxygen behave as though it is being completely negatively charged, thereby attracting more Pb(II) ions.
