Halphen reaction

Halphen Reaction. The halphen test is a very sensitive and reliable method for detecting the presence of cottonseed oil in another oil. A reaction with sulfur in carbon disulfide mixed with equal amounts of amyl alcohol gives a cherry red color when cyclopropenoid fatty acids unique to the Malvacae family, which includes cottonseed and okra, are present. This test is capable of detecting 0.25% or less cottonseed oil in an oil blend. The oil is no longer responsive to the halphen test after hydrogenation, which decreases the iodine value 2-5 units. 

Jones (36) reviewed the natural antinutrients of cottonseed protein products— gossypol and the cyclopropenoic fatty acids (CPFA malvalic and sterculic acids). The CPFAs participate in forming the pink color complex in the Halphen reaction, a test specific for the admixture of cottonseed oil with other oils and fats. They also inhibit A9 desamrase, an enzyme that converts stearic acid into oleic acid, and thus increase hardness of fats from animals (e.g., pig backfat and lard) raised or finished on feedstuffs containing high levels of polyunsamrated oils like corn. Feed industry practice is to limit cottonseed lipids to no more than 0.1-0.2% in the diet of laying hens to avoid pink discoloration of egg whites and alterations of the vitelline membrane that cause pasty yolks. 

Camphor Oil.—Light camphor oil (a by-product in the preparation of safrole), D --= 0-87-0-94, b.pt. about 170-180°, is especially used to adulterate or replace oil of turpentine. It raises the sp. gr. and the b.pt. of the latter. With the Halphen and Grimaldi, and the Herzfeld colour reactions (see preceding section) it behaves much like pinewood oil. It can be identified only when it is contaminated with safrole, which, according to Coen,1 reacts as follows ... 

Some of the tests involved relatively simple colour reactions such as the Baudouin reaction for sesame oil, and the Halphen test for cottonseed oil. In both cases a compound characteristic to an oil is used to determine the presence of the oil. Here again the test detected a component that today would be detected and quantified by gas chromatography (GC) or high performance liquid chromatography (HPLC). It was even possible to determine the presence of cholesterol or phytosterols, although, after separation, the identification as to which type was present depended on microscopic examination and fractional... 

Color reactions. Some oils give specific color reactions caused by particular ingredients. The Halphen test for detecting cottonseed oil is one example. This test estimates the presence of cottonseed oil in vegetable or animal fats or oils as the result of a pink color formed between the reagent (sulfur and carbon disulfide) and cyclopropenoic fatty acids normally present in cottonseed oil. 

Cottonseed oil contains up to 0.5% of a pair of unique fatty acids malvalic (18 1) and sterculic (19 1). These acids are characterized by the presence of a cyclopropene group at or near the center of the fatty acid chain. Under appropriate conditions these give colored compounds and the development of a red color in the Halphen test (reaction with sulfur in carbon disulfide in the presence of amyl alcohol) is due to the cyclopropene acids and therefore characteristic of cottonseed oil (and other minor oils containing cyclopropene acids). This test for cottonseed oil was developed over a century ago in 1897. Inclusion of cyclopropenoid acids in animal diets causes undesirable physiological effects such as reduced egg production, poor hatching, and pink egg whites in chickens, and in rats, decreased growth and sexual development and... 

Traditional procedures for quantification of total cyclopropene acid content have been reviewed by Christie (1970) and include titration with hydro-bromic acid and GC of the methyl mercaptan derivatives, products from reaction with silver nitrate/methanol and from methanethiol addition. However, a colorimetric test involving reaction with sulphur/carbon disulphide (the Halphen test) was found to be one of the most reliable methods for determining the small levels of cyclopropene acids present in oils containing cottonseed oil (Coleman, 1970). Another approach is GC of the relatively stable cyclopropane acids after hydrogenation with hydrazine (Conway, Ratnayake and Ackman, 1985)... 

Some of the frequently used colour reactions of classical fat analysis, like the Halphen-test, can be carried out on the adsorbent layer after thin-layer fractionation this enables us to discover which substances are responsible for these colour reactions [144]. The sterol fraction is especially easily identified with various spray reagents (see Chapter L) and functions thus as a characterising feature ( marker ). 

Halphen s Beaction.—This is a special test to determine the presence or absence of cotton-seed oil fatty acids in mixtures. Equal parts of the fatty acids, amyl alcohol, and a 1 per cent, solution of sulphur in carbon bisulphide, are heated in a test-tube placed in a water-bath until effervescence ceases, then in boiling brine for one hour or longer when only small quantities are present. The presence of cotton-seed oil is denoted by a pink coloration. The reaction is rendered much more rapid, according to Bupp (Z. Untersuch. Nahr. Genussm., 1907, 13, 74), by heating in a stoppered flask. 

Cottonseed Oil. The B.P, modification of Halphen s test, in which the oil (2 5 g) and reagent (2-5 ml of equal volumes of amyl alcohol and 1 per cent precipitated sulphur in carbon disulphide) are heated under slight pressure, will detect less than 5 per cent of cottonseed oil and may be made roughly quantitative up to 20 per cent by the use of comparison mixtures. Kapok oil also gives this reaction, and fats from animals fed on cottonseed cake may show a slight positive test. The reaction is not given by oils which have been previously heated to above 200°. The amyl alcohol may be replaced by a drop of pyridine which is said to make the test more sensitive (Gastaldi24). 

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