2.3.3. UV/Vis Spectroscopy

UV/Vis absorption spectra of p-GQDs, ₂₅GQD^IPA-EDA, ₅₀GQD^IPA-EDA, ₂₀₀GQD^IPA-EDA, ₂₀₀GQD-5EDA, and ₂₀₀GQD-10EDA were acquired using a LLG-uniSPEC 2 Spectrophotometer (Lab Logistic Group, Meckenheim, Germany). All spectra were recorded at room temperature in the wavelength range of 200–800 nm. GQDs were dispersed in ultrapure water in the concentration of 0.25 mg·mL⁻¹ for measurements.

To calculate the values of the optical bandgap, Eg, the Tauc equation was used.

where “hυ” is the energy of the photon, B is a proportionality constant, and n is an exponent which is equal to 1/2 for direct transitions [34].

For analysis of the antioxidative properties of GQD samples, 2,2-diphenyl-1-picrylhydrazyl (DPPH), Rhodamine B (RHB), and KMnO₄ tests were used. The DPPH assay was conducted by mixing GQD samples with freshly produced methanol solutions of DPPH at concentrations ranging from 0 to 200 g/mL. After 1 h in the dark, UV/Vis spectra were recorded using an LLG-uniSPEC 2 Spectrophotometer (Lab Logistic Group, Meckenheim, Germany). Ascorbic acid (vitamin C, AA) was used as a standard reference. Using the following equation, the radical-scavenging activities (RSA) of GQD samples were assessed:

where A_C is the intensity of absorption of the control (DPPH in methanol), and A_GQD is the intensity of the absorption band of the mixture of GQDs with DPPH, also in methanol. The method is based on the fact that DPPH is a stable radical with high-intensity absorption at 518 nm. In the presence of an antioxidant, the intensity of the absorption band at 518 nm is lowered proportionally to the antioxidant concentration, whereby DPPH changes color from violet to yellow. Measurements were replicated three times. The RSA of each GQDs sample was determined using the RHB assay. Mixtures of GQDs in different mass concentrations (10–800 μg·mL⁻¹), RHB, and H₂O₂ were subjected to 360 nm UV light for 1 h. After the incubation, UV/Vis spectra were recorded using an LLG-uniSPEC 2 Spectrophotometer (Lab Logistic Group, Meckenheim, Germany). AA was used as a standard. RSA values of GQDs samples were calculated using the following formula:

where A_GQDs is the value of absorption at 554 nm measured from GQDs–RHB–H₂O₂ mixture, and A_c is that of the GQDs–RHB solution. Due to the contribution of GQD absorbance at higher concentrations, this absorption was subtracted. All measurements were replicated three times.

The antioxidative ability of GQDs was also tested using the KMnO₄ reduction assay. We used the previously reported protocol [29,35] and modified the concentration of KMnO₄. We mixed an acidified solution of KMnO₄ at pH 3 in the concentration of 300 mM and added different amounts of GQDs, from 2.5 to 1000 μg·mL⁻¹. Mixtures were held in the dark for 1 h, and the UV/Vis spectra were recorded. When KMnO₄ is reduced, it changes color from purple to colorless.