Halphen Test Research Articles

Methods to differentiate between cotton tract area ghee and cotton seed oil adulterated ghee.

Ghee, the clarified butter fat is one of the principal dairy products in India. In some places of India, cotton seed is fed extensively to dairy animals which changes the physico-chemical constants and fatty acid profile of the milk fat. Ghee is often adulterated with cotton seed oil and is marketed as cotton tract area ghee. Physico-chemical constants like RM value, Polenske value, BR reading, saponification value, iodine value and colorimetric tests i.e., Halphen and DPPH radical test were employed to differentiate ghee adulterated with cotton seed oil and that from cotton tract area. Chromatographic techniques like HPLC and GC-MS were also explored. Physico-chemical constants were not useful to differentiate the two types of ghee. Cyclopropenoic acids were observed only in ghee adulterated with cotton seed oil and not in cotton tract ghee. The RP-HPLC could able to distinguish the cotton tract area ghee from ghee adulterated with cotton seed oil on the basis of presence of β-sitosterol in the latter. Halphen test was positive for cotton tract ghee, but not for the cotton seed oil adulterated ghee. Methylene blue reduction and DPPH radical test were also found to be useful to distinguish both types of ghee.

Effects of Heat and Hydrogenation on Cyclopropenoid Fatty Acid Composition of Baobab (Adansonia Suarezensis) Seed Oil

ABSTRACTThe seeds of the baobab Adansonia suarenzensis (Bombacaceae) contain 46.2% oil used for cooking by the malagasy population. This oil gives a positive Halphen test showing the presence of cyclopropenoic fatty acids (CPEFA). Composition analysis of derivatized fatty acid methyl esters was done by gas‐liquid chromatography (GLC). The A. suarenzensis seed oil contains mainly palmitic (46%) stearic (3.5%) oleic (21%) and linolenic (12%) acids. Effect of heat on CPEFA content of boabab oil was studied at 133°C and 180°C. Major decomposition of CPEFA at 180°C shows that deodorisation would be the main step in the oil refining process. Hydrogenation of the baobab oil using an industrial hydrogenating catalyst results in CPEFA content decreasing rapidly.

Fatty acid composition of seed oils from sixAdansonia species with particular reference to cyclopropane and cyclopropene acids.

The oil content of sixAdansonia species (Bombacaceae family) of Madagascar (Adansonia grandidieri, A. za, A. digitata, A. fony, A. madagascariensis andA. suarenzensis) and Africa (A. digitata) ranges from 8 to 46%. All the oils give a positive response to the Halphen test. Malvalic, sterculic and dihydrosterculic acids were detected using gas liquid chromatography-mass spectrometry (GLC-MS). Epoxy or hydroxy fatty acids were not found in these oils. Fatty acid composition was determined by GLC using glass capillary columns coated with BDS and Carbowax 20 M. Results obtained for cyclopropenic fatty acids (CPEFA) were compared to those given by glass capillary GLC after derivatization with silver nitrate in methanol, by hydrogen bromide titration and by proton magnetic resonance (PMR). Good agreement was observed for the results given by the various methods. Malvalic acid content ranges from 3 to 28%, sterculic acid from 1 to 8% and dihydrosterculic acid from 1.5 to 5.1%. Odd-numbered fatty acids (Pentadecanoic and hepatadecanoic) were also observed in minute amounts (0.1-1.1%). Among the normal fatty acids, we observed mainly palmitic (21-46%), oleic (15-40%) and linoleic (12-32%). The relationship between fatty acid composition andAdansonia species is discussed.

Cyclopropenoid Fatty Acids in Malagasy baobab: Adansonia grandidieri (Bombacaceae) Seed Oil

AbstractAdansonia grandidieri (bombacaceae family) seed oil gives a positive Halphen test. Composition analysis of derivatized fatty acid methyl esters, in presence of silver nitrate in anhydrous methanol, after chromatography fractionation on silicagel column, were made by gas‐liquid chromatography (GLC) using a packed DEGS column. Presence of malvalic and sterculic acids were detected. GLC analysis using glass capillary columns coated with Carbowax 20 M and BDS shows that A. grandidieri seed oil contains mainly palmitic (41%), oleic (22%) and linoleic (12%) acids. Cyclopropenic fatty acid concentration was 14% with 6% for malvalic and 8% for sterculic acids. A slight proportion of dihydrosterculic acid (1.5%) was observed. GLC fatty acid methyl esters analysis, without derivatization, on the two glass capillary columns coated with Carbowax 20 M and BDS gave the same results for cyclopropenic acids content.

Synthesis and biological characterization of 10, 11‐methylene‐9‐nonadecene; an analog of sterculic acid

AbstractA method is presented for the preparation of 10, 11‐methylene‐9‐nonadecene, a structural analog of the biologically active 9, 10‐methylene‐9‐octadecene (sterculene). When tested for its ability to inhibit stearyl CoA desaturase in vivo, 10, 11‐methylene‐9‐nonadecene failed to alter the stearic‐oleic acid ratio in the yolks of eggs from hens that had been fed 75 mg per day for 8 days. The compound did not give the Halphen test nor react rapidly with silver nitrate or methyl mercaptan in alcohol.

Deposition of cyclopropenoids in the tissue lipids of rainbow trout fed methyl sterculate.

AbstractGroups of young rainbow trout were fed diets containing 0, 100 and 200 ppm methyl sterculate. Their tissue lipids were analyzed for cyclopropene content using the modified Halphen test. Halphenpositive compounds, calculated as sterculic acid, accumulated in all tissues analyzed and reached concentrations approximately equal to those of the dietary lipid. Gas liquid chromatography used in conjuction with mild hydrogenation of the cyclopropene ring revealed that these Halphen‐positive compounds were predominantly, if not entirely, sterculic acid which was esterified in the triglycerides. To facilitate analysis of cyclopropenes in liver tissue, a method was developed whereby interfering pigments were removed prior to the Halphen test.

Use of the Halphen reaction for the determination of the cyclopropenoid content of lipids

An application to cottonseed oils of a quantitative version of the Halphen test for the determination of cyclopropenoid material has been published by other workers, but for other oils containing higher levels of cyclopropenoids, although the absorption at the 495 nm peak is linearly related to the concentration of each oil examined, the relationship differs among the oils. However, transmethylation of oil before applying the Halphen reaction has been found to give results that are in better agreement with titration with hydrogen bromide for oils with widely differing cyclopropenoid content. The use of pressurised capsules for carrying out the reaction with reduced loss of solvent has proved advantageous, as flatter peaks are obtained when optical absorption is plotted against time. The application of the modified technique to oils containing a wide range of concentrations of total cyclopropenoid material in the component fatty acids is described and discussed.

An Evaluation of Five Methods for the Quantitative Determination of Cyclopropenoid Fatty Acids

Abstract An evaluation of 5 methods for determining 5% or less cyclopropenoid fatty acids was made in terms of accuracy, precision, and sensitivity. GLC of the methyl mercaptan derivatives of cyclopropenoid fatty acids and titration with hydrobromic acid in toluene were found to be unsuitable. GLC of the silver nitratemethanol derivatives (quantitation by an internal standard) and titration with hydrobromic acid in acetic acid were equally precise, and the former required less sample; however, both methods gave low recoveries. GLC of silver nitrate-methanol derivatives (quantitation by peak area normalization) and a hydrobromic acid-benzene back-titration method gave good recoveries. The Halphen test was found to be the best general method when calibrated with a cyclopropenoid fatty acid standard analyzed by the silver nitrate-methanol GLC method (quantitation by peak area normalization) or the hydrobromic acid-benzene back-titration method.

Observations on the destruction of cyclopropene fatty acids of cottonseed oil on exposure to visible light

AbstractThe destruction of cyclopropene fatty acids of cottonseed oil on exposure to light from a 500 W tungsten bulb was studied under various conditions in the laboratory. The Halphen test was used to determine the destruction. Visible light exerted a strong catalytic effect and oxygen, at least in traces, was necessary. In 4–12 hr the Halphen response was negative for refined cottonseed oil (0.2–10 ml) sealed in a 50 ml flask at 1 mm Hg without apparent peroxide development. No significant difference was noticed in the peroxide development pattern of the exposed and unexposed samples in the airoven stability test.

Photolytic synthesis of methyl sterculate containing mixture from methyl stearolate and diazomethane

AbstractA method for synthesis of a mixture containing methyl sterculate is described. The presence of the cyclopropene compound was verified by Halphen Test, infrared absorption and the effect on fatty acid distribution in egg yolks produced by hens consuming the synthetic product.

Selective hydrogenation of the cyclopropene acid groups in cottonseed oil

AbstractThe cyclopropene acid groups in cottonseed oil can be modified by a light hydrogenation which will not produce large amounts oftrans isomers or lower the iodine value to a significant extent. Optimum conditions, as indicated by this investigation, are 105–115C, 20 psig hydrogen pressure, 0.1% electrolytic nickel as catalyst, and a low hydrogen‐dispersion rate. Under milder conditions of hydrogenation the elimination of the cyclopropenes was accompanied by a lower formation oftrans isomers and a lower hydrogenation of noncyclopropenes, but the time required increased. In one hydrogenation carried out with commercial nickel catalyst, the 0.4% of malvalic acid groups in the cottonseed oil was hydrogenated completely whereas the iodine value was reduced by only 1.7 units and only 2.1% oftrans isomers was formed. AVinterization of cottonseed oils which had been hydrogenated to the point of eliminating their response to the Halphen test and in which only small amounts of saturated acid groups andtrans isomers had been formed gave yields equal to or better than those of the original oil. Hydrogȳnation actually increased the ease of winterization.

Some Physiological Properties of Halphen-Positive Cottonseed Oils

INTRODUCTIONCOTTONSEED oils bleached with sulfurous acid-treated alumina were reported to differ from conventional cottonseed oils only in the intensity of the red color and in the degree of response to the Halphen test (Pons et al., 1963, 1964). These oils were found to be free of sulfur, to hydrogenate at normal rates and to be indistinguishable organoleptically from normal refined, bleached, and deodorized cottonseed oils (Pons et al., 1964).Physiological differences are reported here between the properties of conventional salad oils of cottonseed origin and oils bleached with sulfurous acid-treated alumina. Hens ingesting cottonseed oils bleached with this reagent produced normal eggs, whereas hens ingesting conventional cottonseed salad oils produced abnormal eggs. The abnormalities appear in the colors and pH values of the yolks and whites and in the fatty acid patterns of the yolk fat (Frampton et al., 1961, 1962; Schaible and Bandemer, 1946; Lorenz et al., 1934; …

A simplified process for the elimination of the halphen‐test response in cottonseed oils

AbstractSince certain biological effects have been ascribed to the cyclopropenoids that give a positive response to the Halphen test, processes were explored to eliminate this response from cottonseed oils. Heating alkali‐refined, Halphen‐positive cottonseed oils in a modified laboratory deodorizer in the presence of cottonseed oil fatty acids, capric acid, citric acid, or phosphoric acid was found to be an effective method of eliminating this response.These treated, Halphen‐negative cottonseed oils, the untreated Halphen‐positive cottonseed oil, and a corn oil control were incorporated into rations fed to different groups of laying hens. Hens that ingested either the Halphen‐negative cottonseed oil or the corn control produced normal eggs, whereas hens fed the Halphen‐positive cottonseed oil produced eggs with pink whites, decreased pH of whites, and increased pH of yolks on storage, and lower ratios of oleic acid to stearic acid in the yolk lipids.The simple processes presented舒particularly the use of cottonseed oil fatty acids舒appear to offer a practical means of inactivating the cyclo‐propenoids in cottonseed oil and thus eliminating the biological effects attributed to them.

A Positive Halphen Test with a Non-Cyclopropenoid Compound ,

THE Halphen color test is a reaction considered characteristic of the naturally occurring cyclopropenoid fatty acids (Carter and Frampton, 1964; Magne, 1965). Halphen reactions with sterculene (1,2-dioctylcyclopropene) and sterculyl alcohol, using the method of Klaus and Deutschman (1960), did not produce deviations from the normal behavior of the Halphen reagents on cyclopropenoid containing oils (Nordby et al., 1962) indicating that the color of the Halphen test is not affected by the carboxyl group of the fatty acid. Apparently, the methylene of the cyclopropene ring must be unsubstituted to give a positive test, as methyl w-(2-n-octyl-3-carbethoxycycloprop-1-enyl) octanoate failed to give the reaction (Brooke and Smith, 1957).This communication reports that the Halphen test carried out in pyridine as the solvent gives an intense red color with the allenic compound ethyl-2,3-decadienoate. The Halphen test with the allenic compound methyl-1,2-nonadiene-3-carboxylate results in a reddish-orange color. The preparation, isolation and structure elucidation of these…

Analysis of cyclopropenoid and cyclopropanoid acids in fats and oils

AbstractThe analysis of cyclopropenoid acids may be considered, from a historical standpoint, to have started with the discovery of the Halphen test. Although this test as orginally conceived was utilized as a means of detecting adulteration of premium edible oils with cottonseed oil, it has since been shown to be a characteristic test for cyclopropenoid fatty acids and has been adapted with various modifications as a quantitative colorimetric test for these substances. More recently, spectrophotometric methods particularly in the IR region have been applied to the analysis of these substances. The 9.8 Ɓ band, characteristic of the cyclopropane, and the 9.91 Ɓ band, characteristic of the cyclopropene group, as well as the 11.0 Ɓ band, characteristic of some of the noncyclic degradation derivatives, have been utilized. Gasliquid chromatography (GLC) has been applied to the methyl esters of cyclopropanoid and hydrogenated cyclopropenoid acids. The reactivity of the cyclopropene ring toward hydrohalogens has been the basis of several analytical methods developed for use with cyclopropene acid‐containing oils. Both aqueous and nonaqueous solutions of hydrohalogens have been employed. The hydrohalogenation methods are the most precise methods currently available for these analyses but only GLC has the inherent potential of identifying the specific cyclopropenoid or cyclopropenoids involved.

Catalytic elimination of cyclopropene‐containing acids

AbstractA procedure is described for decreasing the cyclopropene ring acids in cottonseed oil triglycerides as indicated by the Halphen test. Continuous reaction of the oil in the presence of aluminum silicate catalyst at elevated temp effectively decreases its cyclopropene acid content. The effect of temp and space velocity are described from temp of 170‐225C. The treated oil produces no observable effect on egg quality or production.

Bleaching of refined cottonseed oil with modified alumina adsorbents

AbstractActivated aluminas, modified by treatment with inorganic and organic acids were tested for their efficiency as bleaching agents and for their ability to eliminate the response of refined cottonseed oil to the Halphen test. The best of these adsorbents, an alumina containing catalytic amounts of adsorbed sulfurous acid, is a bleaching agent that is effective in removing problem pigments from offcolored cottonseed oils, and is superior to activated aluminas in removing green chlorophyll‐like pigments. Oils bleached with the catalytic adsorbent are negative to the Halphen test, and are free of sulfur.Study of the rate of reduction in the Halphen test response as a function of bleaching time and temp indicated that sulfurous acid treated alumina has a pronounced catalytic effect. The Halphen test response is eliminated during a 30 min bleaching period at 225C. Deodorization conditions employed had little effect on Halphen test reduction.

Characterization of oils from low‐gland and glandless cottonseed

AbstractNew varieties of cotton which contain few or no gossypol pigment glands are being developed. Commercial production of such varieties should contribute to improving meal quality and reducing the color problems encountered with cottonseed oil. Samples of gland‐containing, low‐gland, and glandless seed grown in California were obtained, and the oils were extracted and characterized.With regard to the over‐all characteristics the oils from the low‐gland and glandless seed were indistinguishable from regular cottonseed oil. Iodine values, contents of unsaponifiables, cloud‐ and pour‐points, response to the Halphen test, and similar characteristics resembled those of commercial cottonseed oil. No differences were found in behavior during winterization.Determination of the component fatty acids by gas chromatography showed the fatty acid composition of the oils to be typical. Determination of the positions of the double bonds in the unsaturated acyl groups showed no differences between the oils from gland‐containing, low‐gland, and glandless seed. Ultraviolet, visible, and infrared spectra of the oils revealed no differences other than the presence of gossypol in the crude oil from the gland‐containing seed. The infrared spectra of the unsaponifiable fractions obtained from the oils showed some differences, which were not believed to be important.

Heat Inactivation of Substances in Crude Cottonseed Oil Causing Pink Whites and Large Discolored Yolks in Stored Eggs

HENS fed rations containing crude cottonseed oil produce eggs that develop viscous pink whites and very large, fluid salmon colored or large thick brown yolks after six months or more of cold storage (Sherwood, 1928). Lorenz (1939) believed that the substance in crude cottonseed oil that causes “pink-white” egg discoloration is the same as the one that gives the Halphen reaction or something very closely related to it. The material giving the Halphen reaction is heat-labile (Shenstone and Vickery, 1956). The substance in crude cottonseed oil that causes “pink-white” egg discoloration is also heat-labile (Evans et al., 1957). Sterculic acid from Sterculia foetida oil and malvalic acid from certain species of Malva give the Halphen test and cause “pink-white” egg discoloration (Masson et al., 1957; Shenstone and Vickery, 1959).The purpose of the present experiment was to determine the amount of heat necessary to inactivate the “pink-white” causative substance in …

Changes in composition of cottonseed during development and ripening

AbstractAnalyses have been made of cottonseed (Gossypium hirsutum) at different stages of maturity after flowering, and the results have been recorded in tables and graphs. It is seen that no appreciable oil develops in the seed until about the 35th day, when it increases rapidly; the carbohydrates decrease over this period. The oil as first formed is highly acidic, and contains much unsaponifiable matter, although the composition of the fatty acids remains practically constant over this period. The protein and ash contents increase steadily more or less in proportion to the weight of seed, but the crude fibre shows a sharp increase shortly before full maturity is reached. The immature seeds yield oil which does not respond to the Halphen test.