The reference signal increased with copper concentration (Fig. 2a) due to the excess of BCA in the medium.
Copper concentra-tion affects sensitivity (hindered with a large excess of the metalion) and the response range (limited for low copper amounts).
A7.9 mol L−1 concentration was selected as a compromise condi-tion and to avoid absorbance values higher than 1.0.
The reference signal was constant from 25 mol L−1 BCA (Fig. 2b), which corre-sponds to a concentration 3-fold higher than Cu(I) in agreement with results reported in previous applications [35].
Excess of ascor-bic acid was employed to reduce Cu(II) and to avoid oxidation of Cu(I) by dissolved oxygen, thus increasing the reagent lifetime.