that’s the main problem with testing pectin using the DPPH assay. Pectin is highly hydrophilic and tends to precipitate or form gels in ethanol, while DPPH is insoluble in pure water, so directly mixing them causes turbidity and unreliable absorbance readings. This means the “classical” DPPH protocol in pure ethanol or methanol doesn’t work properly for polysaccharides like pectin.

The common solution is to carry out the reaction in a hydroalcoholic system. You dissolve pectin in water or buffer (where it is soluble) and prepare DPPH in ethanol or methanol. When you mix the two, the final solvent system contains both water and alcohol. If you keep the organic phase at about 60–70% and water at 30–40%, the DPPH remains soluble while pectin stays dispersed without heavy precipitation. This balance allows the radical–antioxidant reaction to proceed, and the absorbance at 517 nm can still be measured.

However, even under optimized solvent ratios, some pectins still aggregate or scatter light, which interferes with the spectrophotometric reading. That’s why many researchers instead prefer ABTS•⁺ assay, FRAP, or other aqueous-compatible radical scavenging methods when evaluating polysaccharides. These avoid the solubility conflict altogether and provide more reliable data on antioxidant capacity.

Pectin presents a particular challenge for antioxidant evaluation using the DPPH radical scavenging assay, because while pectin is water-soluble, it readily precipitates or forms gels in ethanol, which is the standard solvent for DPPH. At the same time, DPPH is not soluble in pure water, meaning that a direct aqueous assay is not possible. This incompatibility leads to turbidity, light scattering, and inaccurate absorbance readings at 517 nm when the classical method is applied. Therefore, a modified strategy is required to achieve reliable results without losing solubility of either component.

The most common approach is to conduct the reaction in a hydroalcoholic solvent system. Pectin can first be dissolved in water or phosphate buffer, while DPPH is prepared in ethanol or methanol. When mixed, the final medium should contain around 60–70% alcohol and 30–40% water, a balance that keeps DPPH soluble while minimizing pectin precipitation. For example, in a 200 µL reaction, 70 µL of aqueous pectin solution can be combined with 70 µL of 0.1 mM DPPH in ethanol and 60 µL water, producing a final 65:35 ethanol:water ratio. After incubation in the dark at room temperature for about 25 minutes, the decrease in absorbance at 517 nm is measured. Blanks with pectin alone must be included to correct for background turbidity.

Even with optimized ethanol–water ratios, some pectin samples still form hazes or micro-gels that interfere with the spectrophotometric reading. To minimize this, researchers may adjust the mixing order (adding ethanol first, then aqueous pectin, then DPPH), use methanol instead of ethanol, apply brief sonication, or filter solutions through a 0.45 µm membrane before analysis. Data are then expressed as percentage radical scavenging or as IC₅₀ values, giving a quantifiable measure of antioxidant activity. However, reproducibility may still be a concern with the DPPH method for polysaccharides due to their physical behavior in mixed solvents.

For these reasons, many investigators consider alternative methods such as the ABTS•⁺ radical cation assay, which is fully compatible with aqueous systems and polysaccharides. ABTS radicals are generated chemically and can be diluted in water or buffer, where both the radical and pectin remain stable. This eliminates precipitation issues and provides cleaner, more reproducible results. While a modified DPPH system can still be applied with care, the ABTS assay or other aqueous-based antioxidant evaluations (e.g., FRAP, hydroxyl radical scavenging) are often preferable when studying the antioxidant potential of pectin.

(A) a modified DPPH protocol optimized for pectin using a hydroalcoholic system, and (B) an ABTS protocol as an aqueous-friendly alternative. Both include reagent preparation, step-by-step mixing, controls/blanks, data calculations, and practical notes to avoid precipitation or gelation.

Materials and reagents

All reagents were analytical grade. Pectin (specify source, degree of esterification if known) was dissolved in 10 mM phosphate buffer (pH 6.8). 2,2-Diphenyl-1-picrylhydrazyl (DPPH) and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) were purchased from [supplier]. Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) was used as the antioxidant standard. All solutions were prepared fresh and protected from light during experiments.

A. Modified DPPH radical scavenging assay (hydroalcoholic system) — optimized for pectin

Reagent preparation. Prepare pectin stock at 5 mg·mL⁻¹ in 10 mM phosphate buffer, pH 6.8. Prepare serial dilutions of pectin in the same buffer to obtain final assay concentrations (for example: 5.0, 2.5, 1.25, 0.625, 0.3125 mg·mL⁻¹). Prepare DPPH 0.1 mM in absolute ethanol (freshly) and store in amber container. Prepare blank solvent (ethanol and buffer) to match final solvent composition. Prepare Trolox standards (1000 → 15.6 µM, two-fold dilutions) in buffer/ethanol mixture matching final solvent composition for calibration if expressing Trolox equivalents.

Assay setup (recommended 96-well microplate protocol; total volume 200 µL). The aim is a final solvent composition of ~65:35 (v/v) ethanol:water. Per well (example):

Add 70 µL of pectin solution (at the appropriate concentration) or buffer for control wells.

Add 60 µL of buffer or water (use to back-fill so solvent ratio is constant across wells).

Add 70 µL of DPPH 0.1 mM in ethanol to start the reaction (final DPPH = 0.035 mM in the well; final ethanol ≈ 65% v/v).

Mix gently by pipetting up/down once. Incubate the plate in the dark at room temperature for 25 minutes.

Controls and blanks. Include the following in each plate:

DPPH control (A₀): all reagents except sample (replace sample with buffer) — measures initial radical absorbance.

Sample blank (A_bg): sample + solvent (no DPPH) — corrects for turbidity/background absorbance from pectin.

Solvent blank: buffer + ethanol (no sample, no DPPH) — checks baseline.

Trolox standard series (for Trolox equivalents) and/or positive control antioxidant (e.g., ascorbic acid).

Measurement and calculation. Measure absorbance at 517 nm using a microplate reader. Calculate percent scavenging for each sample concentration using:

\%\ \text{inhibition} = \frac{A_0 - (A_{\text{sample}} - A_{\text{bg}})}{A_0}\times 100

Practical notes/precautions.

If visible turbidity, reduce ethanol fraction toward 60:40 (v/v) or decrease pectin concentration; re-equilibrate all wells so solvent composition is identical across the plate.

If gelation or haze persists, try: (i) swapping ethanol for methanol and testing 70:30 MeOH:water; (ii) pre-sonication (≤40 °C, short pulse) and filtration through 0.45 µm before assay; (iii) changing the order of addition (add ethanol to well first, then aqueous pectin while mixing, then DPPH). Always include the sample blank (no DPPH) to correct for scattering/absorbance by pectin.

B. ABTS•⁺ decolorization assay (aqueous-compatible alternative; recommended for polysaccharides)

Reagent preparation. Prepare 7 mM ABTS stock in water and 2.45 mM K₂S₂O₈ in water. Mix equal volumes of ABTS and K₂S₂O₈ and allow the mixture to react in the dark at room temperature for 12–16 h to generate ABTS•⁺. Before assay, dilute the ABTS•⁺ solution with phosphate buffer or water to obtain an absorbance of 0.70 ± 0.02 at 734 nm.

Assay procedure (200 µL total volume, 96-well plate). Add 10–20 µL of pectin sample (prepared in buffer at appropriate concentrations) to each well, then add 180–190 µL ABTS•⁺ working solution (A₇₃₄ ≈ 0.70). Incubate 6 minutes at room temperature in the dark. Measure absorbance at 734 nm.

Controls and calculation. Include ABTS•⁺ control (A₀), sample blank (sample + buffer, no ABTS•⁺), and Trolox standards (e.g., 1000 → 15.6 µM) run in the same plate. Calculate % inhibition:

\%\ \text{inhibition} = \frac{A_0 - (A_{\text{sample}} - A_{\text{bg}})}{A_0}\times 100.

Rationale and recommendation. The ABTS assay is fully aqueous and typically avoids precipitation or gelation problems seen with hydroalcoholic DPPH assays when testing polysaccharides. Use ABTS as the primary method for reporting antioxidant capacity of pectin; include modified DPPH data only if you have validated that no significant scattering or precipitation influenced absorbance (show representative spectra and note solvent composition in methods). When both assays are reported, state solvent ratios, incubation times, and the method used for background correction explicitly.

thank you for the reply H.A Adewuyi