Rapid Determination of Glucose in Tobacco Products by Coulometric Titration with Enzymatic Catalysis

The treated tobacco product sample （1.000 0 g） was taken, and 20 mL of water was added. The mixture was shaken and extracted at 30 ℃ for 1 h, then filtered through qualitative filter paper. The pH of the filtrate was adjusted to 5.8 with 0.1 mol·L⁻¹ acetic acid solution. 20 mL of 0.05 mol·L⁻¹ acetate buffer solution （pH 5.8） was added, and the solution was preheated at 37 ℃ for 10 min. Then 1 mL of 2.0 g·L⁻¹ glucose oxidase solution was added, and the solution was reacted at 37 ℃ for 20 min. 2 mL of 0.10 mol·L⁻¹ sodium hydroxide solution was added, the mixture was mixed thoroughly, and the volume was made up to 50 mL with water. Based on the reaction in which glucose is catalytically oxidized by glucose oxidase to produce gluconic acid-δ-lactone and hydrogen peroxide, two coulometric titration methods were proposed. For the acid-base coulometric titration, 0.6 mol·L⁻¹ potassium chloride solution was used as the electrolyte solution. H⁺ was electrolytically generated under a current of 20.0−50.0 mA for the titration of excess OH⁻ in the sample solution. For the redox coulometric titration, 0.8 mol·L⁻¹ cerous sulfate solution was used as the electrolyte solution. Ce⁴⁺ was electrolytically generated under a current of 1.0−5.0 mA for the titration of hydrogen peroxide in the sample solution. The glucose in tobacco product samples was determined in both methods by measuring the electrolysis time required to reach the titration endpoint （i.e., the titration time） according to Faraday's law of electrolysis. As shown by the results, linear relationship between the corresponding titration time and mass concentrations of glucose was kept in the range of 50.0‒800.0 mg·L⁻¹. The detection limit （3s） of the acid-base coulometric titration method was 2.04 mg·L⁻¹, and detection limit （3s） of the redox coulometric titration method was 3.13 mg·L⁻¹. Test for recovery was made by the standard addition method, giving results in the range of 98.1%−103%, with RSDs （n=5） of the determined values less than 2.0%. This method was applied to the analysis of actual samples, and there was no significant difference in the determination results of glucose between this method and high performance liquid chromatography-evaporative light scattering detector method in YC/T 447—2012.