A kinetic-spectrophotometric method for the determination of glucose in solutions

A kinetic-spectrophotometric method is proposed to determine glucose in solutions. Measurements were performed at 400 nm; the negative peak was obtained by subtracting the absorption spectra of myoglobin (Mb) before and after oxidation. In this method, glucose is added to a mixture of Mb and glucose oxidase. Glucose is oxidized by glucose oxidase and oxygen to gluconate and hydrogen peroxide is generated. The liberated hydrogen peroxide oxidizes the Mb heme (Fe2+) into Fe3+. The higher the glucose concentration added, the more the H2O2 generation, and the more the Mb oxidation (Fe2+ to Fe3+) and, as a result, the higher the absorbance at 400 nm (negative peak, lower absorbance value). The increments of added glucose are monitored by measuring the absorbance decay versus time (0–250 s) at 400 nm. Each glucose concentration has an accompanying unique absorbance value at 250 s. The higher the glucose concentrations, the lower the absorbance at 250 s (measured at 400 nm). The calibration curve for glucose was linear from 0.1 to 3.0 mM; the detection limit was found to be 0.025 mM. There was no interference from major substances present; the only interference was from species that react with H2O2 (ascorbic acid, uric acid, and urea) or that react with glucose (Cu2+ and Fe3+). Standard deviation in the determination was ±0.01 mM for a 1.3 mM glucose solution (n = 10).