Deae cellulose

Lyophilized melanin was applied to silica gel TLC (Thin layer chromatography) plates and a chromatogram was developed using the solvent system of n-butanol: acetic acid: water (70:20:10). The developed plates were dried in an oven and the spots were visualized with ninhydrin. The TLC purified melanin was applied to a DEAE-Cellulose (Bio-Rad, 1 × 30 cm) (Diethylaminoethyl cellulose) column which had been equilibrated with 25 mM Tris–HCl buffer (pH 8.6) containing 50 mM sodium chloride. Elution was performed at a flow rate of 100 ml/h with 1:1 volume gradient from 0.1 M to 2 M NaCl in the same buffer^{33 (link)}. The purified melanin was dissolved in deionised water and absorbance was recorded in the UV/VIS range at wavelengths between 200–450 nm in a spectrophotometer. Fourier transform infrared (FT-IR) spectral analysis was carried out in KBr disks on a Thermo-Nicolet Nexus 6700 FTIR spectrophotometer (Thermo Fisher Scientific Inc., Madison, WI, USA), and spectra were recorded in the wave number region of 400–4000 cm⁻¹. The heat stability of the melanin was determined by increasing the temperature in a range from 40–100 °C and incubating for 10 min at the respective temperatures. The solubility of the melanin pigment was determined in deionized water, and in organic solvents such as ethanol, methanol, acetone, chloroform, hexane, ethyl acetate and benzene.