Lxv. Note on Knoop's Test for Histidine

IN performing this test Knoop [1908] adds bromine water to the solution to be tested until there is just a very slight excess. On heating, the excess bromine disappears and in the presence of histidine a brownish red colour is developed; if the latter is in sufficient concentration a dark precipitate settles out. The solution to be tested should be slightly acid. The only substance other than histidine found to give this test is iminazolylethylamine or histamine. The test, according to Knoop, is given with histidine at a dilution of 1: 1000. When using this test the writer found that different intensities of colour were produced from solutions of the same concentration of histidine. This peculiar behaviour was accounted for by slight variations in the bromine excess. The colour produced is very soluble in excess of bromine and in dilute solutions of histidine an initial excess may prevent all colour development. The difficulty of adding just sufficient bromine especially to slightly coloured solutions has been overcome by adding a definite excess followed by washing the solution repeatedly with chloroform in a small separating funnel until the chloroform is no longer coloured. By this procedure even strongly coloured meat extracts and other fluids may be successfully tested by Knoop's method. The excess of bromine appears to oxidise the colouring matters and after washing with chloroform such solutions are almost colourless, so that on heating the brown colour due to histidine is readily detected. Most uniform results have been obtained when the washed solution is transferred to a test tube and set in a boiling water-bath. That the chloroform does not extract any of the histidine compound was shown by adding to a series of test tubes equal amounts of 0.1 % histidine monohydrochloride and adding increasing small amounts of bromine water from a pipette until definite excess of bromine was present. By heating all the test tubes together on the water-bath it was found that there was first a rise, then a fall in colour values corresponding with insufficiency or excess of bromine. The maximum thus obtained was less than that obtained by the chloroform method.