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  Section: Plant Lab Protocols
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Methodology for Anti-nutritional Factor

Cyanogens are cyanogenic glucosides and are widespread in the plant kingdom in trace amounts. These glucosides release hydrogen cyanide by an endogenous enzymic reaction. Relatively high concentrations are found in certain grasses, root crops and fruit kernels. Young sorghum plants particularly ratoon crop, contain high amounts of cyanogens and bring deleterious effects on cattle.


Hydrocyanic acid which is evolved from the sample, forms a red colored compound with sodium picrate and the intensity is measured at 625nm.
Whatman No.1 Filter Paper: Cut filter paper into strips 10-12cm long and 0.5cm wide, and saturate them with alkaline picrate solution.
Alkaline picrate solution: Dissolve 25g sodium carbonate and 5g picric acid in one liter of water.
Standard Hydrogen Cyanide Solution:   Dissolve 0.241g of KCN/liter of water. This gives a solution containing 100mg hydrogen cyanide/mL.

Homogenize 1g of the sample in 25mL water with 3 - 4 drops of chloroform.
Place the homogenate in 500mL conical flask.
Place the saturated filter paper in the hanging position with the help of a cork stopper inside the conical flask.
Incubate the mixture at room temperature (20°C) for 20 - 24h.
The sodium picrate present in the filter paper is reduced to reddish compound in proportion to the amount of hydrocyanic acid evolved.
Elute the color by placing the paper in a clean test tube containing 10mL distilled water and compare it with standards at 625nm.
Preparation of the Standard Curve: (KCN is poisonous. Use propipettes, automatic pipettes or burettes for transfer of cyanide solution.) Place 5mL of the alkaline picrate solution and 5mL of the potassium cyanide solution in a test tube. Heat for 5min in boiling water. Deliver the following volumes from the above KCN alkaline picrate solution to six test tubes: 0.1, 0.2, 0.4, 0.6, 0.8 and 1mL. Bring the volume of each test tube to 10mL by adding distilled water. Press rubber stopper to the tubes and keep them in a cool place. Measure the absorbance at 625nm. The amount of hydrogen cyanide in the above tubes is equal to 5, 10, 20, 30, 40 and 50mg respectively. Prepare a blank with 10mL distilled water.
Calculate the hydrogen cyanide content of the sample from the standard graph.


1. Hogg, P G and Ahlgren, H L (1942) J Ame Soc Agron 34 199.
2. Indira, P and Sinha, S K (1969) Indian J agric Sci 39 1021.

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