Good morning Ferdinand, and welcome to the scientific world! 😊 It’s great to see your curiosity and commitment.

To calculate anthocyanin content in mg/g dry basis (b.s. = “base seca” or dry weight basis) using the differential pH method, you’ll need to follow a few steps. I’ll break it down clearly and provide an example at the end.

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What is the Differential pH Method?

It’s a reliable method for quantifying monomeric anthocyanins, like cyanidin-3-glucoside (C-3-G), based on their structural changes at different pH levels. The absorbance is measured at pH 1.0 and 4.5, and the difference reflects the anthocyanin content.

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Steps for Calculation

1.

Prepare Buffers

• pH 1.0 buffer: Usually KCl buffer
• pH 4.5 buffer: Usually sodium acetate buffer

2.

Prepare Sample Dilutions

Dilute your sample in both buffers (same dilution ratio).

3.

Measure Absorbance

Using a UV-Vis spectrophotometer:

• Measure absorbance at λ = 520 nm and 700 nm for each buffer.
• Calculate corrected absorbance (A):

A = (A_{520} - A_{700}){pH1.0} - (A{520} - A_{700})_{pH4.5}

4.

Use the Anthocyanin Equation

\text{Anthocyanin content (mg/L)} = \frac{A \times MW \times DF \times 1000}{\varepsilon \times l}

Where:

• A = corrected absorbance
• MW = Molecular weight of C-3-G = 449.2 g/mol
• DF = Dilution factor
• ε = molar extinction coefficient of C-3-G = 26,900 L·mol⁻¹·cm⁻¹
• l = path length of cuvette (usually 1 cm)

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Convert to mg/g Dry Weight

Once you get mg/L anthocyanins, use:

\text{mg anthocyanin/g dry sample} = \frac{\text{(mg/L) × V}}{\text{W}}

Where:

• V = Volume of extract used (in L)
• W = Weight of dry sample extracted (in g)

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Example

Let’s say:

• Corrected A = 0.75
• DF = 10
• V = 0.01 L (10 mL extract used)
• W = 0.2 g (dry sample)

\text{mg/L} = \frac{0.75 \times 449.2 \times 10 \times 1000}{26900 \times 1} = \approx 125.3 \text{ mg/L}

\text{mg/g dry weight} = \frac{125.3 \times 0.01}{0.2} = \approx 6.27 \text{ mg/g}

📝 Summary:

• Perform absorbance measurements at pH 1.0 and 4.5
• Apply the formula with known constants
• Convert final result to mg per g dry sample

Follow this article;

Fuleki, T. and Francis, F.J., 1968. Quantitative methods for anthocyanins. 1. Extraction and determination of total anthocyanin in cranberries. Journal of food science, 33(1), pp.72-77.

https://doi.org/10.1111/j.1365-2621.1968.tb01365.x