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DPPH Rf-shift assay to overcome spectral interference: A rapid, portable TLC-based approach for semi-quantitative
Garima Singh1, Perinkulam Ravi Deepa1, Prabhat Nath Jha1
1Department of Biological Sciences, Birla Institute of Technology and Science, Pilani, Pilani Campus - 333031, Rajasthan, India.
Analytica Chimica Acta
|February 16, 2026
Summary
A new DPPH Rf-Shift Assay accurately quantifies antioxidant activity, overcoming limitations of conventional methods. This reliable, color-independent technique enhances compound evaluation in food and pharmaceutical research.
Area of Science:
- Analytical Chemistry
- Food Science
- Pharmaceutical Research
- Biomedical Research
Background:
- Accurate antioxidant activity assessment is crucial for food, pharmaceutical, and biomedical applications.
- Conventional DPPH spectrophotometric assays suffer from spectral interference, high sample usage, and costly instrumentation.
- Existing alternative methods like TLC/HPLC-DPPH assays offer limited improvements in quantification and accessibility.
Purpose of the Study:
- To develop a rapid, reliable, and color-independent method for quantifying antioxidant activity.
- To overcome the limitations of conventional assays, particularly spectral interference from colored compounds.
- To provide accurate antioxidant assessment for both colored and non-colored compounds and complex extracts.
Main Methods:
- Development of the DPPH Rf-Shift Assay, a Thin-Layer Chromatography (TLC)-based method.
- Separation of oxidized DPPH radical (DPPH*) from its reduced form (DPPH-H) based on polarity differences.
- Quantification of antioxidant activity using the ratio of Rf_DPPH-H/Rf_DPPH* via a TLC Documentation System.
Main Results:
- The DPPH Rf-Shift Assay accurately captured concentration-dependent antioxidant responses for reference compounds.
- Conventional spectrophotometric DPPH assays overestimated activity in colored compounds due to spectral interference.
- The novel assay resolved discrepancies, providing clear, reproducible quantification of radical scavenging activity.
Conclusions:
- The DPPH Rf-Shift Assay is a novel, portable, and cost-effective method for antioxidant measurement.
- It effectively overcomes color interference issues inherent in traditional antioxidant assays.
- This method expands analytical capabilities for evaluating antioxidant activity in diverse samples, including colored extracts.
