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Determination of allyl isothiocyanate in mustard oil


The oil obtained from mustard seeds and rape seeds contains sinigrin and myrosin (glucosinolates), which are sources of goitrogens.

As such they are relatively nontoxic but their hydrolysis products include allyl isothiocyanates and oxazolidinethiones, which are powerful goitrogens, producing varying manifestations of toxicity in nonruminants


The allyl isothiocyanate in the oil is steam distilled into a known excess of silver nitrate solution, and the excess of silver nitrate solution is determined by titration with standard ammonium thiocyanate solution.


Distillation Flask – 500 ml round-bottomed flask.
Any Efficient Reflux Condenser (~ 90 cm long).
Measuring Flask – 200 ml capacity.

  • Reagents
  • Ethyl Alcohol -95%, v/v neutral to phenolphthalein.
  • Silver Nitrate Solution – 0.1 N.
  • Ammonium Hydroxide Solution – 10 % (w/v) Conc. HNO3
  • Ferric ammonium sulfate Indicator – 0.1 % solution in water.
  • Standard Ammonium Thiocyanate (NH4SCN) Solution – 0.1 N


Weigh accurately about 5 g of the material into a 500 ml distillation flask and add to it 25 ml of ethyl alcohol, 250 ml of water, and a few pieces of pumice stone.

Distill the mixture in steam and collect the distillate in a 200 ml measuring flask containing exactly 25 ml of silver nitrate solution and 10 ml of ammonium hydroxide solution.

Collect as rapidly as possible about 150 ml of the distillate.

Attach the reflux air condenser to the measuring flask and heat the mixture for about one hour on a boiling water-bath.

Cool to room temperature, add water to make up to 200 ml, and filter the contents after shaking.

Take 100 ml of the filtrate, add 6 ml of HNO3 and a few drops of ferric ammonium sulfate indicator, and titrate with standard ammonium thiocyanate solution until a permanent red color is obtained.

Carry out a blank test simultaneously along with the sample.


B = Volume, in ml, of NH4SCN solution required for blank,
S = Volume, in ml, of NH4SCN solution required for the sample,
N = Normality of NH4SCN solution, and
W = Weight, in g, of the sample taken for the test.


The difference between the results of two concurrent determinations carried out simultaneously or in rapid succession by the same analyst (repeatability) shall not exceed 0.02% by mass.

The allyl isothiocyanate content in different varieties if mustard varies from 0.1 to 0.7% by mass.


• Sample and blank test should be conducted simultaneously under similar conditions.
• Standardized ammonium thiocyanate should be used.

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