2.3. Melanin Characterization

The samples were confirmed as melanin via UV-Vis absorbance spectroscopy, electron paramagnetic resonance (EPR), and X-ray photoelectron spectroscopy (XPS).

For the UV-Vis absorbance spectroscopy, a small amount of melanin powder was dissolved in a pH 8 deionized water solution (adjusted with NH₃). The spectra were then obtained utilizing a Perkin Elmer PDA UV/Vis Lambda 265 (Perkin Elmer, Waltham, MA, USA), using the wavelength monitoring functionality, obtaining a wavelength range from 350 nm to 900 nm at 1 nm wavelength intervals. Example data can be seen in Figure 4A, and the featureless, exponential decay spectra were as expected for melanin [69].

(A) An example UV-Vis absorbance spectrum obtained for the melanin sample. The curve shows a simple decaying exponential as expected for the material. (B) An example CW-EPR X-band spectrum obtained for the sample.

For the EPR measurement, a powder sample of the melanin was measured using a Bruker EMX Micro X CW-EPR spectrometer (Bruker, Billerica, MA, USA) with an E4104 X-band cavity at a microwave power of 0.87 mW and at room temperature. The spectra were taken at a modulation frequency of 100 kHz and modulation amplitude of 1 Gauss with a scan width 60 Gauss. The apparent isotropic g factor was calibrated against a DPPH standard and found to be 2.0036, which is as reported elsewhere for solid melanin synthesized in the same manner [43]. An example spectrum can be seen in Figure 4B.

An elemental analysis for melanin can be reliably probed via XPS. For the XPS, a wide-scan survey spectrum was performed on pressed pellets of the powder utilizing a Kratos Axis Supra (Kratos Analytical Ltd, Manchester, UK) using 225 W AlKα X-rays with an emission current of 15 mA and equipped with a quartz crystal monochromator with a 500 mm Rowland circle. Spectra were collected with a pass energy of 40 eV, with the hybrid lens setting, a 0.1 eV step size, and a 1 s dwell time for electron counting at each step. To eliminate differential charging [70], the charge neutralizer integral to the Kratos spectrometer was used as an electron source.