Fluorescence Quenching of Pheophytin-a by Copper(II) Ions

A method was developed for determination of Cu(II) ions quantitatively by measuring fluorescent intensity of pheophytin-a (Pheoa) solution. The Pheoa was obtained by de-intercalation of magnesium from the porphyrin ring of chlorophyll-a (Chla) extracted from fresh spinach leaves. Its two UV-Vis absorption peaks at 505 and 535 nm in acetone solution have been observed but disappeared when the acetone solution of Pheoa was mixed with a Cu(II) ion aqueous solution. A fluorescence quenching phenomenon was thus observed when the acetone solution of Pheoa was mixed with an aqueous solution of Cu(II) ions. However, other physiologically relevant cations rarely caused any quenching fluorescence of Pheoa under the same experimental conditions. Kinetics of the fluorescence fading process was investigated by measuring the effects of Cu(II) ion concentration, reaction time and reaction temperature on the fluorescence intensity of the Pheoa acetone solution. An activation energy of (10±1) kJ·mol⁻¹ was estimated from Arrhenius empirical relation assuming that the interaction between the Pheoa and the Cu(II) ions was the first order reaction. The calibration graph obtained with the fluorescence was linear over the Cu(II) concentration range of 8.0×10⁻⁵−8.0×10⁻⁷ mol·dm⁻³ with a detection limit of 8.0×10⁻⁷ mol·dm⁻³ for Cu(II) ion.