Visionlite software 2

The assessment of the stoichiometry followed the previously established protocol [29 (link)]. Shortly, all experiments were performed in ultraviolet-transparent cuvettes (BrandTech Scientific Inc., The United Kingdom) and absorbance was measured by the use of spectrophotometer Helios Gamma equipped with VISIONlite software 2.2 (ThermoFisher Scientific Inc., USA). Standard Job's method [30 ] and the same transition metal solutions were used as mentioned above. In all measurement of cuprous ions at all pH and ferrous ions at pH 7.5, hydroxylamine was added to buffer to retain metal in its reduced state.
A methanolic solution of a tested substance was mixed with a solution of metal ions for 1 min at different molar concentration ratios ranging generally from 1 : 4 to 4 : 1 (substance : metal) at all tested pH conditions. Afterwards absorption spectra were immediately measured. The blank was composed of a buffer and a solvent at the ratio 2 : 1, respectively.

The stoichiometry was assessed according to the previously published methodology [37 (link)]. The methanolic solutions of alkaloids were mixed with iron ions at different molar ratios, from 1:4 to 6:1, at all pH conditions. The absorbance was measured immediately after 1 min of shaking by Helios Gamma spectrophotometer equipped with VISIONlite software 2.2 (ThermoFisher Scientific Inc., Waltham, MA, USA). The blank sample consisted of buffer pH 7.5 and methanol in a ratio of 2:1. The measurements were performed in UV-transparent cuvettes (BrandTech Scientific Inc., Saint Neots, UK). Two methods were used to find and confirm the stoichiometry: Job’s method [38 ] and the complementary approach [37 (link)].