SOP: Determination of Total Anthocyanins and Degradation Index by the pH Differential Method

GIFSQ — Global Institute for Food Safety & Quality

Principle

A simple direct total-anthocyanin determination cannot be made when chlorophyll or its degradation products, or brown degradation products of anthocyanins, interfere. The pH differential method developed by Fuleki and Francis measures absorbance in samples diluted with pH 1.0 and pH 4.5 buffers: at pH 1.0 the measured absorption covers both the degraded and the intact anthocyanins, while the difference in absorption between the pH 1.0 and pH 4.5 media comes only from the intact (non-degraded) pigments. Because an aqueous medium is used instead of alcoholic HCl, a hypsochromic shift of 19–24 nm occurs at pH 1.0 in the visible region, so the extinction coefficients must be established in the aqueous medium.

Apparatus required

• Spectrophotometer
• Volumetric flasks

Reagents

a. pH 1.0 buffer: 0.2 N KCl–0.2 N HCl (25 : 67).

b. pH 4.5 buffer: 1 N sodium acetate–1 N HCl–water (100 : 60 : 90).

c. 0.1 N HCl.

d. Distilled water.

Procedure

Note the absorption maximum of the juice: dilute 200 ml of cranberry juice to 1 litre with 0.1 N HCl; dilute 25 ml of this to 50 ml with water; allow to equilibrate in the dark for 1 hr; find the absorption maximum (510 nm in the case of cranberry juice) using a spectrophotometer

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The pH differential method for total anthocyanins: dilute 10 ml aliquots of juice to 250 ml and 50 ml with the pH 1.0 and 4.5 buffers respectively

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Allow the diluted samples to equilibrate in the dark at room temperature for 2 hr

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Note the absorbance at 510 nm, using a distilled water blank

The pH values selected for the pH differential method must satisfy the following:

• The difference in absorbance between the two pH values should be the greatest possible at the wavelength of measurement.
• Small variations in pH around the values selected should cause only slight changes in absorbance.
• The anthocyanins should be stable at the pH values at which the measurements are made.

Establishment of the extinction coefficient

Extract the pigments from the plant material, purify, separate into individual pigments chromatographically, and crystallize or precipitate with ether by following any one of the procedures in SOP: Extraction, Purification and Separation of Anthocyanin Pigments (the source’s Method I); establish the extinction coefficient in the aqueous medium similarly for the anthocyanins present in the test material.

Calculation

In juices with no degraded pigment, total anthocyanins may be determined by measuring at pH 1.0 (or by SOP: Estimation of Total Anthocyanins): measure the absorbance at 510 nm on a sample diluted with pH 1.0 buffer, calculate the total absorbance per 100 ml of the juice as in SOP: Estimation of Total Anthocyanins, and calculate the total anthocyanin content as:

Total anthocyanin in mg/100 ml = Total absorbance per 100 ml ÷ 87.3

(where 87.3 is the average absorbance of a solution containing 1 mg/ml at pH 1.0 and 510 nm, from the source’s Table 4-3)

Where degradation products are present, use the pH differential method:

1. Calculate the total absorbance per 100 ml of the juice at pH 1.0 and at pH 4.5.
2. Find the difference in total absorbance (ΔA) between the juice at pH 1.0 and 4.5:

ΔA per 100 ml of juice = Total absorbance at pH 1.0 − Total absorbance at pH 4.5

1. Calculate the total anthocyanin content in mg/100 ml of juice:

Total anthocyanin in mg/100 ml = ΔA per 100 ml of juice ÷ 77.5

1. Calculate the degradation index:

Degradation index = 1 − [Total anthocyanin in mg/100 ml at pH 1.0 ÷ Total anthocyanin by the pH differential method]

Where,

• Total absorbance per 100 ml = absorbance at 510 nm scaled to 100 ml of juice
• 87.3 = average absorbance of 1 mg/ml of cranberry anthocyanins at pH 1.0 and 510 nm
• 77.5 = difference in the average absorbance (1 mg/ml) between pH 1.0 and pH 4.5 at 510 nm

Note: the degradation-index formula is carried as printed in the source; its numerator and denominator are the total anthocyanin at pH 1.0 and the pH-differential result respectively.

Precautions

• Allow all diluted samples to equilibrate in the dark before measuring (1 hr for the absorption maximum, 2 hr for the differential measurements).
• Verify the absorption maximum of the juice before the pH differential measurements.

References

Ranganna, S., Handbook of Analysis and Quality Control for Fruit and Vegetable Products, 2nd edition.

1. Fuleki, T. & F.J. Francis, J. Food Sci., 33, 72 (1968).