On the kinetics of the autoxidation of fats. II. Monounsaturated substrates

The autoxidation of methyl oleate and oleic acid shows some differences as compared to the autoxidation of linoleate,e.g., the formation of water at an early stage. Linearization of experimental data on the autoxidation to high oxidation degrees of methyl oleate and other monounsaturated substrates shows that the rate equations previously derived for methyl linoleate in the range of 1–25% oxidation are valid, provided the correct expression for the remaining unreacted substrate is used. With monounsaturated substrates, part of the oxygen is consumed by a secondary oxidation reaction almost from the beginning, and only a certain constant fraction α of the total O₂ consumption is consumed in hydroperoxide formation. The fraction α is different for methyl oleate, oleyl alcohol, oleic acid andcis 9-octadecene, but the rate constant for the hydroperoxide formation is the same for all of them when experimental conditions are the same. The main difference between oleate and linoleate autoxidation is the much faster decomposition of the oleate hydroperoxides relative to their slow formation.     

Concurrent oxidation of accumulated hydroperoxides in the autoxidation of methyl linoleate

Summary 1. Kinetic studies showed that concurrent oxidation of preformed hydroperoxides may be expected to take place at all stages of the autoxidation of methyl linoleate. The rate of oxidation relative to the rate of autoxidation of unoxidized ester is determined chiefly by the extent of the accumulation of hydroperoxides. 2. Infrared spectral analysis of hydroperoxides oxidized to various degrees indicated thattrans, trans diene conjugation and isolatedtrans double bonds produced in the autoxidation of methyl linoleate are related to the concurrent oxidation of the accumulated hydroperoxides. 3. The low absorptivity observed for diene conjugation, compared to that which may be expected for the exclusive production ofcis, trans diene conjugated hydroperoxide isomers during the autoxidation of methyl linoleate is attributed to the concurrent oxidation of accumulated hydroperoxides. 4. The effect of antioxidants in giving a well-defined induction period in the oxidation of hydroperoxides isolated from autoxidized methyl linoleate indicated that the oxidation proceeds by a chain reaction. 5. The primary reaction products of the oxidation of hydroperoxides isolated from autoxidized methyl linoleate were found to be polymers formed in a sequence of reaction involving the diene conjugation. 6. Studies on the autoxidation of methylcis-9,trans-11-linoleate showed thatcis, trans isomerization of the conjugated diene took place with the concurrent production of isolatedtrans double bonds and loss of diene conjugation. Hormel Institute publication no. 138. Presented before the American Oil Chemists’ Society, Philadelphia, Pa., Oct. 10–12, 1955. This work was supported by a grant from the Hormel Foundation.     

Effect of α-tocopherol on the volatile thermal decomposition products of methyl linoleate hydroperoxides

α-Tocopherol and 1,4-cyclohexadiene were tested for their effect on the thermal decomposition of methyl linoleate hydroperoxide isomers. The volatiles generated by thermolysis in the injector port of a gas chromatograph at 180°C were analyzed by capillary gas chromatography. In the presence of either α-tocopherol or 1,4-cyclohexadiene, which are effective donors of hydrogen by radical abstraction, volatile formation decreased in all tests, and significant shifts were observed in the relative distribution of products in certain hydroperoxide samples. When an isomeric mixture of methyl linoleate hydroperoxides (cis, trans andtrans, trans 9- and 13-hydroperoxides) was decomposed by heat, the presence of α-tocopherol and 1,4-cyclohexadiene caused the relative amounts of pentane and methyl octanoate to decrease and hexanal and methyl 9-oxononanoate to increase. A similar effect of α-tocopherol was observed on the distribution of volatiles formed from a mixture of thetrans,trans 9- and 13-hydroperoxides. This effect of α-tocopherol was, however, insignificant with purecis,trans 13-hydroperoxide of methyl linoleate. The decrease in total volatiles with the hydrogen donor compounds, α-tocopherol and 1,4-cyclohexadiene, indicates a suppression of homolytic β-scission of the hydroperoxides, resulting in a change in relative distribution of volatiles. The increase in hexanal and methyl 9-oxononanoate at the expense of pentane and methyl octanoate in the presence of hydrogen donor compounds supports the presence of a heat-catalyzed heterolytic cleavage (also known as Hock cleavage), which seems to mainly affect thetrans,trans isomers of linoleate hydroperoxides.     

Hydroperoxide formation during autoxidation of conjugated linoleic acid methyl ester

The aim of this study was to investigate whether hydroperoxides are formed in the autoxidation of conjugated linoleic acid (CLA) methyl ester both in the presence and absence of α‐tocopherol. The existence of hydroperoxide protons was confirmed by D₂O exchange and by chemoselective reduction of the hydroperoxide groups into hydroxyl groups using NaBH₄. These experiments were followed by nuclear magnetic resonance (NMR) spectroscopy. The ¹³C and ¹HNMR spectra of a mixture of 9‐hydroper‐oxy‐10‐trans,12‐cis‐octadecadienoic acid methyl ester (9‐OOH) and 13‐hydroperoxy‐9‐cis, 11‐trans‐octadecadienoic acid methyl ester (13‐OOH), which are formed during the autoxidation of methyl linoleate, were studied in detail to allow the comparison between the two linoleate hydroperoxides and the CLA methyl ester hydroperoxides. The ¹³CNMR spectra of samples enriched with one of the two linoleate hydroperoxide isomers were assigned using 2D NMR techniques, namely Correlated Spectroscopy (COSY), gradient Heteronuclear Multiple Bond Correlation (gHMBC), and gradient Heteronuclear Single Quantum Correlation (gHSQC). The ¹³C and ¹H NMR experiments performed in this study show that hydroperoxides are formed during the autoxidation of CLA methyl ester both in the presence and absence of α‐tocopherol and that the major isomers of CLA methyl ester hydroperoxides have a conjugated monohydroperoxydiene structure similar to that in linoleate hydroperoxides.     

Analysis of autoxidized fats by gas chromatography-mass spectrometry: II. Methyl linoleate

The gas chromatography-mass spectrometry (GC-MS) approach developed in the preceding paper was applied for qualitative and quantitative investigations of autoxidation products of methyl linoleate. A GC-MS computer summation method was standardized with synthetic 9- and 13-hydroxyoctadecanoate. Equal amounts of 9- and 13-hydroperoxides were found in all samples of linoleate autoxidized at different temperatures and peroxide levels. The results are consistent with the classical free radical mechanism of autoxidation involving a pentadiene intermediate having equivalent sites for oxygen attack at carbon-9 and carbon-13. Minor oxygenated products of autoxidation indicated by GC-MS include keto dienes, epoxyhydroxy monoenes di- and tri-hydroxy monoenes. These hydroxy compounds are presumed to be present in the form of hydroperoxides. The quantitative GC-MS method was found suitable for the analysis of autoxidized mixtures of oleate and linoleate. By this method, it is possible to determine the origin of the hydroperoxides formed in mixtures of these fatty esters. Presented at the AOCS Meeting, Chicago, September 1976.     

Study on the oxidative rate and prooxidant activity of free fatty acids

Oleic, linoleic and linolenic acids were autoxidized more rapidly than their corresponding methyl esters. Addition of stearic acid accelerated the rate of autoxidation of methyl linoleate and the decomposition of methyl linoleate hydroperoxides. Therefore, the higher oxidative rate of FFA’s than their methyl esters could be due to the catalytic effect of the carboxyl groups on the formation of free radicals by the decomposition of hydroperoxides. Addition of stearic acid also accelerated the oxidative rate of soybean oil. This result suggests that particular attention should be paid to the FFA content that affects the oxidative stability of oils.     

High pressure liquid chromatography of autoxidized lipids: II. Hydroperoxy-cyclic peroxides and other secondary products from methyl linolenate

A previous study of autoxidation products by high pressure liquid chromatography (HPLC) of methyl oleate and linoleate was extended to methyl linolenate. Autoxidized methyl linolenate was fractionated by HPLC either after reduction to allylic alcohols on a reverse phase system, or directly on a micro silica column. Isolated oxidation products were characterized by thin layer and gas liquid chromatography and by ultraviolet, infrared, nuclear magnetic resonance and mass spectrometry. Secondary products from the autoxidation mixtures (containing 3.5–8.5% monohydroperoxides) included epoxy unsaturated compounds (0.2–0.3%), hydroxy or hydroperoxy-cyclic peroxides (3.8–7.7%), epoxy-hydroxy dienes (<0.1%), dihydroxy or dihydroperoxides with conjugated diene-triene and conjugated triene systems (0.9–2.9%). Cyclization of the 12- and 13-hydroperoxides of linolenate would account for their lower relative concentration than the 9- and 16-hydroperoxides. Dihydroperoxides may be derived from the 9- and 16-linolenate hydroperoxides. Cyclic peroxides and dihydroperoxides are suggested as important flavor precursors in oxidized fats.     

Reactions of fatty materials with oxygen. XX. Recent developments in the autoxidation of methyl oleate and other monounsaturated fatty materials

Summary Some significant developments since 1947 in the autoxidation of methyl oleate and other monounsaturated fatty materials have been reviewed and critically evaluated. Subjects discussed are preparation and characterization of hydroperoxides, and mechanism, kinetics, and secondary products of autoxidation. Major developments in the field have resulted largely from the use of newer instruments (polarograph, infrared spectrophotometer) and separation techniques (urea complexes, molecular distillation, countercurrent distribution). Direct experimental evidence is now available which demonstrates that a) hydroperoxides are the predominating, but not the exclusive, primary products of autoxidation; b) the hydroperoxides obtained from methyl oleate are mostly, if not entirely,trans; c) substantially all the methyl oleate undergoes single attack in the chain before any significant amount of multiple attack occurs, and d) α,β-unsaturated carbonyl compounds are among the most important secondary products of autoxidation. Paper XIX is reference 66a. Presented at the Fall Meeting of the American Oil Chemists' Society, Philadelphia, Pa., October 10–12, 1955. A laboratory of the Eastern Utilization Research Branch, Agricultural Research Service, U. S. Department of Agriculture.     

Role of the bases and phosphoryl bases of phospholipids in the autoxidation of methyl linoleate emulsions

Methyl linoleate, emulsified in borate buffer with sodium lauryl sulfate, was used to study the pro- or antioxidant effect of 0-phosphocholine, 0-phosphoethanolamine, 0-phosphoserine, as well as the corresponding nonphosphoryl compounds. Oxygen uptake was calculated from rate data obtained at 37 C with an oxygen electrode. The results were similar for the corresponding phosphoryl and nonphosphoryl bases. 0-Phosphocholine and choline had little effect at either pH 7.9 or 10.2. 0-Phosphoethanolamine and ethanolamine significantly increased oxygen uptake at pH 7.9, but significantly decreased uptake at pH 10.2. 0-Phosphoserine and serine decreased oxygen uptake at both pH values. The catalytic activities of the bases investigated may be attributed to their functional groups. The phosphoryl and β-hydroxy groups exhibited no catalytic activity in the autoxidation of methyl linoleate emulsions at either pH 7.9 or 10.2. The α-carboxyl amino group of 0-phosphoserine and serine decelerated autoxidation at both pH values. The amino group H₃N⁺ of the primary amine accelerated autoxidation, but the H₂N: group and the reverse effect. Since the quaternary amino group (CH₃)₃N⁺ did not affect autoxidation at either pH 7.9 or 10.2, we concluded that the presence of the N−H bond may be necessary for the prooxidant activity of an amine, and that the presence of a pair of free electrons on the nitrogen of an amine is necessary for its antioxidant activity. Kinetically, the autoxidation of methyl linoleate emulsion without added base was in agreement with Farmer’s proposed mechanism involving a bimolecular dissociation of hydroperoxides. However, methyl linoleate emulsion at pH 7.9 and 37 C in the presence of ethanolamine or 0-phosphoethanolamine was autoxidized by a mechanism involving a combined mono- and bimolecular dissociation of hydroperoxides. Submitted as partial requirement for a Ph.D. degree in Agricultural Chemistry. Presented in part at the AOCS Meeting, San Francisco, April 1969.     

Photo-initiated peroxidation of lipids in micelles by azaaromatics

The monoazaaromatics, pyridine (1), hexyl nicotinate (2), and quinoline (3) and diazaaromatics pyrimidine (4) and purine (5), readily act as photo-initiators for the peroxidation of methyl linoleate in 0.50 M SDS at 37°C giving free radical chain oxidations of linoleate. Quantitative kinetic runs on the order in substrate, RH, and in the rate of chain initiation, R_i, showed that the classical rate law for autoxidation,-d[O₂]/dt=(k _p/(2 k _t ^1/2))[RH]xR _i ^1/2, is applicable to these photo-initiated oxidations. The oxidizability of methyl linoleate under these conditions is 2.92×10⁻² M^−1/2 s^−1/2. These peroxidations were inhibited by chromanol phenolic antioxidants of the vitamin E class, such as lipid-soluble 2,2,5,7,8-pentamethyl-6-hydroxychroman (PMHC) and water-soluble 2-carboxy-2,5,7,8-tetramethyl-6-hydroxychroman (Trolox) and derived rate constants for inhibition of peroxidation were k _inh (PMHC)=4.35×10⁴ M⁻¹ s⁻¹ and k _inh (Trolox)=2.81×10⁴ M⁻¹ s⁻¹ during inhibited oxidation of methyl linoleate photo-initiated by 4. The products from photo-initiated peroxidation of methyl linoleate by 1 through 5 were determined by reduction and high-performance liquid chromatography analyses to be the 9-and 13-positional hydroperoxides of the four geometrical isomers: cis-9, trans-11 (6), trans-10, cis-12 (7), trans-9, trans-11 (8), and trans-10, trans-12 (9)-octadecadienoates typical of the free radical chain mechanism of lipid peroxidation. Products from dye-sensitized oxidation by Methylene Blue or Rose Bengal of methyl linoleate gave a product distribution of six hydroperoxides typical of oxidation by singlet oxygen. Thermal or photo-initiated peroxidation of methyl linoleate in SDS gave some selectivity of oxidation at the 13-position of the linoleate chain. The ratio of 13-to 9-oxidation varied in the range 1.23 to 1.14 as the cis/trans to trans/trans ratio of geometric isomers varied from 0.44 to 1.25 during photooxidation of increased amounts of linoleate in SDS. This selectivity is attributed to loss of the pseudo symmetry around the pentadienyl system in the lipid chain in the SDS system during the peroxidation.     

Effects of α- and γ-tocopherols on formation of hydroperoxides and two decomposition products from methyl linoleate

The antioxidant effects of α-and γ-tocopherols (at 0, 10, 100, 500, and 1000 ppm) were evaluated in a model system based on the autoxidation of methyl linoleate in bulk for 4 d at 40°C. Samples were collected every 24 h and analyzed for the 9 cis,trans, 9 trans,trans, 13 cis,trans, and 13 trans,trans isomers of hydroperoxide, hydroxy, and ketodiene oxidation products by high-performance liquid chromatography. Results showed that both α- and γ-tocopherols are effective hydrogen donors as evidenced by their abilities to inhibit the formation of hydroperoxides, hydroxy compounds, and ketodienes and the cis,trans to trans,trans isomerization of hydroperoxides. Compared with γ-tocopherol, α-tocopherol was a more efficient antioxidant at very low concentrations (10 ppm) but a less efficient antioxidant at the high concentrations (100–1000 ppm). This paradoxical behavior is explained on the basis of differences in ease of hydrogen donation between the two tocopherol homologs. Although α-tocopherol shows some loss of efficiency with increasing concentration, it is not a prooxidant when compared to the control void of antioxidants.     

The chemistry of polymerized oils. V. The autoxidation of methyl linoleate

1. The geometrical forms obtained during autoxidation of methyl linoleate at ordinary temperatures are largely conjugatedcis-trans andtrans-trans as shown by previous workers; there is a possibility that conjugatedcis-cis forms are also produced. 2. Thetrans-trans molecules arise partly, at least, by thermal rearrangement of already formedcis-trans peroxide. 3. Some proportion, at least, of thecis-trans molecules have theirtrans double bonds nearest to the hydroperoxide group. 4. A partial separation of the geometrical forms can be accomplished by reversed phase partition chromatography both on methyl linoleate hydroperoxides and on the corresponding mixed hydroxy compounds; isolation of thetrans-trans forms can be accomplished in the latter case by urea complex fractionation. 5. No position isomers except the known 9- and 13-isomers have been positively identified; there is a possibility that very minor amounts of the 2-isomer are formed; the 9- and 13-isomers are present in about equal amounts; the 11-isomer was not detected by the methods applied. 6. Various ways in which the linoleate autoxidation problem might be advanced further are suggested.     

Autoxidation ofAcholeplasma laidlawii membranes

Autoxidation ofAcholeplasma laidlawii membranes (with equimolar ratio of palmitic and linoleic acid) lacks an obvious induction period, and the overall rate of disappearance of substrate does not follow closely that of typical autocatalytic kinetics. Throughout the course of autoxidation, the major oxygenated products isolated were hydroperoxides (as hydroxy esters) and compounds that gave rise to trihydroxy esters. The yield of trihydroxy esters was appreciable even at the early stage of the oxidation and eventually grew to surpass that of hydroperoxides. The positions of the three hydroxyl groups in the trihydroxy esters were determined to be mostly of the 1,2,5-type rather than 1,2,3-type arrangement. To a lesser extent, some degraded products, including dimethyl nonanedioate, methyl myristate, methyl pentadecanoate, methyl hexadecadienoate and methyl heptadecadienoate also were obtained. Dimethyl nonanedioate was a previously known degradation product from 9-hydroperoxide. The shorter chain esters presumably arise from the cleavage of α-hydroperoxides of palmitate and linoleate moieties.     

The peroxidizing effect of α-tocopherol on autoxidation of methyl linoleate in bulk phase

In order to understand the effect of α-tocopherol on the autoxidation mechanism of edible oil under storage conditions, methyl linoleate was allowed to autoxidize at 50 C in bulk phase without any radical initiator. The reaction was monitored by determining the production of four isomeric hydroperoxides (13-cis,trans; 13-trans,trans; 9-cis,trans; 9-trans,trans) by high performance liquid chromatographic analysis after reduction. In the absence of α-tocopherol, the rate of autoxidation depended on the sample size, and the duration of the induction period was affected by the initial level of hydroperoxides. However, the distribution of c-t and t-t hydroperoxide isomers remained constant during the propagation period regardless of the sample size. The addition of α-tocopherol at 0.1 and 1.0% caused a linear increase in the amount of hydroperoxides and elevated the distribution of the c-t isomers. The rate of hydroperoxidation appeared to be governed by the initial concentration of α-tocopherol rather than the sample size or the initial hydroperoxide level. This peroxidizing effect of α-tocopherol was suppressed by the presence of ascorbyl palmitate. A mechanism in which chromanoxy radical participates is proposed for the effect of α-tocopherol on lipid autoxidation in bulk phase. It is therefore suggested that α-tocopherol at high concentrations influences the mechanism of autoxidation of edible oil.     

Effect of α-tocopherol and Trolox on the decomposition of methyl linoleate hydroperoxides

To clarify the mechanisms of antioxidant action, the effect of α-tocopherol and its water-soluble carboxylic acid derivative, Trolox, was studied on the decomposition of methyl linoleate hydroperoxides (MeLoOOH). Decomposition rate and the distribution of autoxidation products formed from MeLoOOH were followed by analyzing the volatile and nonvolatile products by static headspace gas chromatography and normal-phase high-performance liquid chromatography, respectively. Both α-tocopherol and Trolox markedly inhibited the decomposition of MeLoOOH in a concentration-dependent way. In the absence of antioxidants, MeLoOOH was completely decomposed after incubation for 48 h at 60°C, and in the presence of equal molar concentration of antioxidants only 6–7% of initial MeLoOOH was decomposed even after 280 h of incubation. MeLoOOH produced 1.2% methyl linoleate hydroxy compounds (MeLoOH) in the presence of α-tocopherol and 3.8% in the presence of Trolox. Both antioxidants inhibited the formation of volatile decomposition products and the formation of ketodiene compounds. The hydroxy compounds may be formed by the reaction of alkoxy radical and hydrogen donating antioxidants. Conversion of MeLoOOH into stable MeLoOH demonstrated that the antioxidants α-tocopherol and Trolox trap alkoxyl radicals by H-donation.     

Analysis of autoxidized fats by gas chromatography-mass spectrometry: III. Methyl linolenate

The gas chromatography-mass spectrometry (GC-MS) method developed in the preceding papers was extended to the analysis of autoxidation products of methyl linolenate. Four isomeric hydroxy allylic trienes with a conjugated diene system were identified after reduction of the linolenate hydroperoxides. All eight geometrictrans,cis- andtrans, trans-conjugated diene isomers of these hydroxy allylic compounds were identified and partially separated by GC of the trimethylsilyl (TMS) ether derivatives. The proportion found of 9- and 16-hydroperoxides was significantly higher (75–81%) than the 12- and 13-hydroperoxides (18–25%). The tendency of the 12- and 13-hydroperoxides to form cyclic peroxides, cyclic peroxidehydroperoxides, and prostaglandin-like endoperoxides was supported by indirect evidence for the presence of 9,10,12- and 13,15,16-trihydroxyoctadecanoate in hydrogenated derivatives of the highly oxygenated products. The quantitative GC-MS method was used to determine the relative contribution of linolenate, linoleate, and oleate in mixtures to the formation of hydroperoxides. Presented at the AOCS Meeting, New York, May 1977.     

Ion exchange resins and ethyleneimine polymer as antioxidants II: Autoxidation products

The antioxidant effects of ion exchange resins and ethyleneimine polymer on the autoxidation products of methyl linoleate in a heterogeneous reaction system are discussed. Results from analyses of the various autoxidation products from linoleate samples with and without the antioxidants showed that the addi-tion of the antioxidants did not change the original autoxidation mechanism of methyl linoleate. How-ever, the antioxidants did retard the autoxidation in response to their antioxidant activity and, compared with a linoleate control, changed the yields of some autoxidized products such as an increased amount of conjugated diene hydroperoxides in linoleate samples with added ion exchange resins.     

Antioxidant effects of chlorophyll and pheophytin on the autoxidation of oils in the dark. II. The mechanism of antioxidative action of chlorophyll

To understand the mechanism of the antioxidant effect of chlorophyll on the autoxidation of oils in the dark, antioxidant activities of several derivatives of chlorophyll were compared. Antioxidant activities were observed in chlorophyll derivatives such as protopor-phyrin methyl ester and its magnesium chelated compound. Porphyrin seems to be an essential chemical structure for the antioxidant activity of chlorophyll. Chlorophyll did not decompose the hydroperoxides, but reduced free radicals such as 1,1-diphenyl-2-picrylhydrazyl. Electron spin resonance spectrum of the π-cation radical was recorded during the oxidation of chlorophyll in methyl linoleate solution. These observations suggest that chlorophyll may act as a hydrogen donor to break the chain reaction.     

Distribution of14C after oral administration of (U-14C)labeled methyl linoleate hydroperoxides and their secondary oxidation products in rats

To study the toxicity of low molecular weight (LMW) compounds formed during the autoxidation of oils,¹⁴C-labeled primary monomeric compounds (methyl linoleate hydroperoxides) and secondary oxidation products, i.e., polymer and LMW compounds prepared from autoxidized methyl [U-¹⁴C]linoleate hydroperoxides (MLHPO) were orally administered to rats, and their radioactive distributions in tissues and organs were compared. The polymeric fraction consisted mainly of dimers of MLHPO. For the LMW fraction, 4-hydroxy-2-nonenal, 8-hydroxy methyl octanoate and 10-formyl methyl-9-decenoate were identified as major constituents by gas chromatography-mass spectrometry (GC-MS) after chemical reduction and derivatization. When LMW compounds were administered to rats,¹⁴CO₂ expiration and the excreted radioactivity in urine in 12 hr were significantly higher than those from polymer or MLHPO administration. Maximum¹⁴CO₂ expiration appeared 2–4 hr after the dose of LMW compounds. Radioactivity of the upper part of small intestines six hr after the dose of LMW compounds was higher than the values from administered polymer or MLHPO. The remaining radioactivity in the digestive contents and feces 12 hr after administration of LMW compounds was much lower than the values observed from administered polymer or MLHPO. Among internal organs, the liver contained the highest concentration of radioactivities from polymer, MLHPO and LMW fractions, and an especially higher level of radioactivity was found in liver six hr after the administration of LMW compounds. Six hours after the dose of LMW compounds, a relatively higher level of radioactivity also was detected in kidney, brain, heart and lung. These results show that the LMW compounds from MLHPO autoxidation are more easily absorbed in rat tissues than polymer and MLHPO.     

Autoxidation of methyl linoleate. Separation and analysis of isomeric mixtures of methyl linoleate hydroperoxides and methyl hydroxylinoleates

The mixture of conjugated diene hydroperoxide isomers obtained from autoxidation of methyl linoleate was separated by high performance liquid chromatography (HPLC). Four major isomers were obtained from adsorption chromatography and identified as the 9 and 13 positional isomers having thetrans-trans andcis-trans configurations. The latter geometrical isomers have thetrans double bond adjacent to the hydroperoxide group. The hydroxy compounds (methyl hydroxylinoleates) obtained from the hydroperoxides by NaBH₄ reduction were similarly separated but with improved resolution. This is the first instance of the complete separation of these compounds and provides a rapid method for their analysis. Unlike adsorption chromatography, reversed-phase chromatography separates the mixtures only according to the geometrical isomerism of the double bonds and not according to the position of the hydroxy or hydroperoxide function.