\label{HPLC}
A high-performance liquid chromatography Prominence from Shimadzu (solvent delivery module LC-20AT, on-line degasser DGU-20A5, communications bus module CBM-20, auto sampler SIL-20A HT, column oven CTO-10AS VP and photodiode array detector SPD-M20A) was employed for monitoring the reaction. A Synergi Polar-RP column (ether-linked phenyl phase with polar endcapping, 150 A, 4.6 mm, 4 um, Phenomenex) was used for product separation. Solutions containing 4% of methanol and 96% of ammonium acetate (pH=6.5) were used as mobile phase.
In particular, H\(_{2}\)Bip chromatographic peak appears in the same retention time as the corresponding to its oxidized product (Bip). Thereby, integrations of the peaks at different wavelengths were performed such as reported in the literature \cite{Vignoni2009}. Assuming that the peak considered is only due to the reactant and one known product, the concentration of both compounds can be calculated by resolving system of linear equations:
\[\label{eq:area1} Area_{\lambda_{1}}=m_{\lambda_{1}}^{R}\left[R\right]+m_{\lambda_{1}}^{P}\left[P\right]\]
\[\label{eq:area2} Area_{\lambda_{2}}=m_{\lambda_{2}}^{R}\left[R\right]+m_{\lambda_{2}}^{P}\left[P\right]\]
where \(Area_{\lambda_{1}}\) and \(Area_{\lambda_{2}}\) are the values resulting from integration of the chromatogram peaks at analysis wavelengths \(\lambda_{1}\) and \(\lambda_{2}\), \(\left[R\right]\) and \(\left[P\right]\) are the concentrations of the reactant and the product, \(m_{\lambda_{1}}^{R}\), \(m_{\lambda_{1}}^{P}\), \(m_{\lambda_{2}}^{R}\) and \(m_{\lambda_{2}}^{P}\) are the factors obtained from the calibration curves for the reactant and the product at \(\lambda_{1}\) and \(\lambda_{2}\). Another system of equations can be constructed using the above criteria, but at different wavelengths, and thus the results obtained can be corroborated. On the other hand, in the case of Bip, the peak of the reactant could be well separated from those corresponding to its products.