A study of the chemiluminescence behavior of myoglobin with luminol and its analytical applications

Abstract

A chemiluminescence signal at 425 nm was observed when ferric state myoglobin was mixed with luminol in alkaline medium. Because the signal was remarkably enhanced in the presence of Fe(CN)₆ ⁴⁻, analytical applications were investigated in a flow-injection system. The increase in chemiluminescence was linearly dependent on myoglobin concentration in the range 0.1 to 100 nmol L⁻¹, and the limit of detection was 0.04 nmol L⁻¹ with relative standard deviation 3.2% (3σ). It was also found that binding of Mb with the ligands CN⁻, SCN⁻, and F⁻ significantly inhibited the chemiluminescence reaction. The linear dynamic ranges for the ligands were 1.0–300.0, 0.1–3.0, and 0.5–100.0 nmol L⁻¹, and the limits of detection (S/N=3) 0.4, 0.04, and 0.2 nmol L⁻¹, for F⁻, CN⁻, and SCN⁻, respectively. The relative standard deviations were 5.32%, 6.13%, and 3.38% for 0.1 nmol L⁻¹ CN⁻, 0.5 nmol L⁻¹ SCN⁻, and 1.0 nmol L⁻¹ F⁻, respectively. At a flow rate of 2.0 mL min⁻¹ the assay could be accomplished in 1 min, including sampling and washing. The method has been successfully applied to the determination of myoglobin in human urine and F⁻ in water samples. A possible mechanism of chemiluminescence production by myoglobin and luminol is presented.