SOP: Detection of Leucoanthocyanins
GIFSQ — Global Institute for Food Safety & Quality
Principle
Leucoanthocyanins are colourless precursors that are converted by hot acid into the deeply coloured anthocyanidins. Three tests exploit this chemistry: direct heating with 2 N hydrochloric acid gives a red extract from which the coloured product partitions into iso-amyl alcohol; heating with a butanol–hydrochloric acid reagent develops the red colour directly in an aqueous extract; and the vanillin test in methanolic hydrochloric acid gives a cherry-red condensation product. Leucoanthocyanins are responsible for the development of pink to pinkish-brown colour in fruit and vegetable products made from pear, guava, apple, banana, litchi, broad beans, field beans, black-eyed peas, cabbage, cauliflower and water chestnut.
Apparatus required
- Test tubes
- Boiling water bath
- Separatory funnel
Reagents
a. 2 N HCl.
b. Iso-amyl alcohol.
c. n-Butanol–HCl reagent (95:5).
d. Methanol.
e. Petroleum ether (for removal of chlorophyll, if present).
f. A saturated solution of vanillin in ethanol.
g. Conc HCl.
Procedure
Acid conversion and iso-amyl alcohol extraction
Cover the pieces (fruit, vegetable or seed coat) in a test tube with sufficient 2 N HCl
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Heat in a boiling water bath for 20 min
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Decant the aqueous solution into a small test tube (filter, if necessary) and cool
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Add a few drops of iso-amyl alcohol, shake and allow the layers to separate
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If leucoanthocyanins are present, the HCl extract becomes coloured (red) and on shaking the colour comes into the alcoholic layer, which may be drawn off and used for spotting the chromatograms
Note: the source’s Table 3-5 (Rf values of anthocyanidins) is illegible in the extracted source.
Butanol–HCl test
To 1 ml of the aqueous extract of the plant material, add 10 ml of n-butanol–HCl (95:5)
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Heat in a boiling water bath for 30 min, stirring occasionally in the initial stages
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A red colour develops in the presence of leucoanthocyanins
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For plant material in which leucoanthocyanins are not easily extracted in the aqueous medium, blend the material and heat 1 g of it with 10 ml of the butanol–HCl reagent
Vanillin test
Macerate the plant material with methanol and transfer the supernatant liquid
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If chlorophyll is present, extract with petroleum ether in a separatory funnel until the methanolic layer is no more than faintly green
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Concentrate the methanolic extract, if necessary
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Add 3–5 drops of conc HCl and 3–5 drops of a saturated solution of vanillin in ethanol
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A positive reaction for leucoanthocyanins ranges from a deep cherry-red colouration produced almost immediately to colour formed only on warming; when the test is negative, the colour usually becomes bluer in tint because of the reaction between carotenoid compounds and HCl
References
Ranganna, S., Handbook of Analysis and Quality Control for Fruit and Vegetable Products, 2nd edition.