DPPH R_f-shift assay to overcome spectral interference: A rapid, portable TLC-based approach for semi-quantitative...

Accurate assessment of antioxidant activity is essential for evaluating compounds in food, pharmaceutical, and biomedical research. Conventional methods, such as the DPPH spectrophotometric assay, are widely used due to their simplicity but are limited by false-negative responses, spectral interference from coloured compounds, relatively higher sample usage, and dependence on costly instrumentation. Alternative approaches, including TLC/HPTLC-DPPH and HPLC-based assays, only partially overcome these limitations and often face challenges in quantification or accessibility. Therefore, there is a need for a rapid, reliable, and colour-independent method to quantify antioxidant activity in both coloured and non-coloured compounds. We developed the DPPH R_f-Shift Assay, a TLC-based method that quantifies antioxidant activity by separating the oxidized DPPH radical (DPPH∗) from its reduced form (DPPH-H) based on polarity differences, producing a measurable shift in R_f values. Antioxidant activity was quantified using a TLC Documentation System as the ratio of R_f_DPPH-H/R_f_DPPH∗, with values ≥ 1 indicating significant activity. Validation with five reference antioxidants (BHT, α-tocopherol, ascorbic acid, pyrogallol red, β-carotene) and a coloured non-antioxidant control (synthetic azo dye, amaranth) revealed that the DPPH R_f-Shift Assay accurately captured concentration-dependent responses. In contrast, conventional spectrophotometric based DPPH assay overestimated activity for coloured compounds due to spectral interference, producing misleading >100 % scavenging values. The DPPH R_f-Shift Assay resolved these discrepancies, providing clear and reproducible quantification of radical scavenging. The DPPH R_f-Shift Assay is a novel, portable, and cost-effective method that overcomes colour interference limitations in antioxidant measurement. By directly resolving DPPH redox states on TLC and enabling image-based quantification, it allows accurate assessment of antioxidant activity in coloured compounds and complex pigment extracts, expanding analytical capabilities for food, pharmaceutical, and natural product research.