Innovative 96-Well Plate Imaging for Quantifying Hydrogen Peroxide in Cow’s Milk: A Practical Teaching Tool for Analytical Chemistry

Initially, students performed a qualitative
analysis to detect
the presence of oxidizing compounds in cow’s milk samples.
This was achieved by observing the oxidation of potassium iodide (KI)
by hydrogen peroxide (H2O2). Subsequently, the
concentrations of H2O2 in the milk samples were
quantified using images of 96-well plates captured with a flatbed
scanner. This method is straightforward, efficient, and ideal for
high-throughput analysis. The RGB values from the 96-well plates were
automatically extracted using the ImageJ plugin, ReadPlate. For the
quantitative analysis, students explored various figures of merit,
including the limit of detection (LOD), limit of quantification (LOQ),
linearity, sensitivity, and recovery. Milk samples were spiked with
H2O2 at three concentrations (0.03%, 0.06%,
and 0.09%), and the measured concentrations in these spiked samples
were compared to evaluate interclass repeatability using one-way ANOVA.
Post hoc tests, including Games-Howell and Tukey, were used to identify
significant differences between concentrations across the classes.
Before conducting the one-way ANOVA, students assessed data normality
using Q-Q plots, the Shapiro–Wilk test, and the Anderson–Darling
test. They also evaluated the homogeneity of variance with Levene’s
and Bartlett’s tests. Additionally, a two-way ANOVA was employed
to analyze the effects of spiking concentrations and laboratory classes
on the recovery. This analysis revealed a significant interaction
between spiking concentrations and laboratory classes on recovery.
All statistical tests were conducted using accessible and user-friendly
freeware, providing students with practical experience in both data
analysis and statistical interpretation.