Glucose Concentrations in Coconut Water via Microplate Spectrometry and Digital Image Colorimetry

Comparison of analytical methods is an essential skill for undergraduate students in chemistry and related sciences. This work describes a colorimetric assay for glucose quantification using the glucose oxidase/peroxidase assay with o-dianisidine. Students also studied the specificity of this assay against fructose and applied it to determine glucose concentrations in an actual coconut water sample. Results were obtained using both standard microplate spectrophotometry (MPS) and digital image colorimetry (DIC) approaches. Both methods are high-throughput, requiring small amounts of reagents and data collection time; therefore, three replicates of experiments were possible in one 4 h laboratory session. The green of the RGB channels yields the highest sensitivity, as expected from a maximum wavelength of absorption of the produced chromophore of 540 nm, corresponding to the green region of light. Analysis of students’ results shows that the linear quantifiable range obtained by most groups is 6–100 μg/mL of glucose for the MPS and 7–100 μg/mL for the DIC. Notably, fructose did not produce a detectable colored product under the same assay conditions, highlighting the selectivity of the assay. The paired samples t-test showed no significant difference (p > 0.05) between glucose concentrations determined by the MPS and DIC methods across all samples, suggesting that the DIC method is a viable and accessible alternative for undergraduate teaching laboratories. This experiment provides an engaging quantitative learning experience of fundamental analytical chemistry concepts, including spectrophotometry, calibration curves, and statistical data analysis with an actual and relevant sample, while exploring practical applications in food and biochemistry.