Halphen test

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... 

Halphen Test for Detecting Cottonseed Oil (Cb 1-25) estimates the presence of cottonseed oil in vegetable or animal fats or oils as the result of a pink color formed between the reagent and cyclopropenoic fatty acids (sterculic and malvalic) normally present in cottonseed oil. 

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. 

Measurement of safflower oil s various chemical and physical characteristics is quite straightforward and only minor changes have occurred over 50 years in the rules governing the safflower trade. In 1990, the requirement for certification that safflower oil demonstrate a negative halphen test was dropped. The emergence of better and better GLC technology eliminated the need for a color test of cottonseed oil adulteration. 

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 ). 

The chemistry of cyclopropane and cyclopropane ring-containing fatty acids has been reviewed eisewhere [159], The presence of such functional groups can be detected by a number of spectroscopic procedures and also by various chemical techniques. For example, cyclopropane rings give a pink coloration with carbon disulfide (the Halphen test) and a brown coloration with silver nitrate. The ring is disrupted by ozonolysis or... 

Both are responsible for the characteristic color shown by cottonseed oil in the so-called Halphen test. A probable intermediate in the biosynthesis of malvalic from sterculic acid, D-2-hydroxysterculic acid, has been isolated from seed oils (Earle et al., 1964 Morris and Hall, 1967). 

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. 

Theroux P, Waters DD, Halphen C, Oebaisieux JC, Mizgala HE Prognostic value of exercise testing soon after myocardial infarction. N Engl J Med 1979 301 341. 

One feature distinguishing cottonseed oil from other commercial oils is its content of C20, C22 and C24 saturated fatty acids. The presence of cyclo-propenoid acids (0.1-1.3%) (Bailey etal., 1966 Pan-dey and Suri, 1982) is of even greater importance. These acids are responsible for giving a positive Hal-phen test (Halphen, 1897) which has been used to characterize cottonseed oil for almost 100 years. The major cyclopropenoid acids are sterculic (C19) and malvalic (Cis) (Shenstone and Vickery, 1959, 1961) (Sections 1.8 and 3.2.14). 

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. 

Rosin Test (Qualitative) n See Halphen-Hicks Test and Lieber-Man-Storch Method. 

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). 

---