PHOTOOXIDATION AND PHOTOSENSITIZED OXIDATION OF LINOLEIC ACID, MILK, AND LARD DISSERTATION. By JaeHwan Lee, M.S. * * * * *

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1 PHOTOOXIDATION AND PHOTOSENSITIZED OXIDATION OF LINOLEIC ACID, MILK, AND LARD DISSERTATION Presented in Partial Fulfillment of the Requirement for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By JaeHwan Lee, M.S. * * * * * The Ohio State University 2002 Dissertation Committee: Dr. David B. Min, Adviser Dr. Howard Q. Zhang Dr. Polly D. Courtney Dr. Hua Wang Approved by Adviser Food Science and Nutrition

2 ABSTRACT Photooxidation and photosensitized oxidation on the formation of volatile compounds in linoleic acid, milk, and lard were studied by a combination of solid-phase microextraction (SPME)-gas chromatography (GC)-mass spectrometry (MS) and headspace oxygen content. Photooxidation is the oxidation under light in the absence of photosensitizers such as chlorophyll and riboflavin. Photosensitized oxidation is the oxidation under light in the presence of photosensitizers. Total volatile compounds in linoleic acid without added chlorophyll under light and in the dark did not increase for 48 hr at 4 C. Total volatile compounds in linoleic acid with added 5 ppm chlorophyll under light at 4 C for 0, 6, 12, 24, and 48 hr, increased from 8.9 to 11.6, 21.7, 26.1, 29.3 ( ) in electronic counts, respectively. 2-Pentylfuran, an undesirable reversion flavor compound in soybean oil, 2- octene-1-ol, 2-heptenal, and 1-octene-3-ol were formed by photosensitized oxidation only. Light excited photosensitizers like chlorophyll can generate singlet oxygen from ordinary triplet oxygen. 2-Pentylfuran, 2-heptenal, and 1-octene-3-ol can come from C10, C12, and C10 hydroperoxide of linoleic acid, respectively, which can be formed by singlet oxygen oxidation but not by triplet oxygen oxidation on linoleic acid. The singlet ii

3 oxygen oxidation mechanisms for 2-pentylfuran, 2-heptenal, 1-octene-3-ol, and 2-octene- 1-ol from linoleic acid were proposed. Milk with or without added riboflavin, ascorbic acid, sodium azide, and butylated hydroxyanisol (BHA) was stored at 4 C under light and in the dark. Pentanal, dimethyl disulfides, hexanal, and heptanal were formed only in the light stored milk and increased significantly as the added riboflavin concentration increased from 5 to 10, 50 ppm (P<0.05). As fat content in milk increased from 0.5 to 1.0, 2.0, and 3.4%, pentanal, hexanal, and heptanal increased significantly (P<0.05) but dimethyl disulfide concentration did not change. BHA and ascorbic acid, hydrogen donating free radical scavengers, reduced hexanal and heptanal formation. Sodium azide, a singlet oxygen quencher, prevented dimethyl disulfide formation. Formation of pentanal is different from that of hexanal and heptanal in milk. Singlet oxygen and free radicals play important roles in the formation of volatile compounds in photosensitized milk. Volatile compounds and headspace oxygen content from lard containing 0 and 5 ppm chlorophyll in air-tight 10-mL bottles were studied at 55 C under light and in the dark. Total volatile compounds in lard with 5 ppm chlorophyll under light were 19 times more than that in samples with 0 ppm chlorophyll for 48 hr. Headspace oxygen content in lard with 5 ppm chlorophyll under light changed from 21 to 15% for 48 hr but that in lard with 0 ppm chlorophyll did not change significantly (P>0.05). Photosensitizer-free lipid model system using lard was developed. Photooxidation mechanism seems to be a free radical chain reaction like autoxidation. Photosensitizers like chlorophyll and riboflavin play important roles in the formation of volatile compounds. Singlet oxygen, which can be generated from triplet iii

4 oxygen by light excited photosensitizers, is involved in the formation of volatile compounds in linoleic acid, milk, and lard. Photosensitizer content in food should be minimized and food products should be packed with light impermeable package to prevent the photosensitized oxidation on lipids. iv

5 Dedicated to my mother, my wife, my daughter, and my late father. v

6 ACKNOWLEDGMENT I wish to thank my adviser, Dr. David B. Min for his energetic guidance and enthusiasm which made this dissertation possible. He gave me a chance to study in this charming field and showed me a way to taste the world of knowledge on lipid and flavor. It is my greatest pleasure to be his advisee in my academic study. I also thank my committee members, Drs. Ahmed E. Yousef, Howard Q. Zhang, Polly D. Courtney, and Hua Wang for their valuable suggestions, consistent advisory and concerns throughout my graduate program. I also thank my colleagues and graduate school friends, Jeff Boff, Raymond Diono, Hyun-Jung Chung, and Dr. TaeHyun Ji for their memorable friendship and assistance. Most importantly, I would like to express my greatest appreciation to my mother, my wife, my daughter, and my late father. vi

7 VITA January 26, Born in Seoul, Republic of Korea B.S. Food Science and Technology Seoul National University, Seoul, Korea M.S. Food Science and Technology Seoul National University, Seoul, Korea CheilJedang Food Company, Korea Graduate Research Associate The Ohio State University, Columbus, Ohio, U.S.A. PUBLICATIONS 1. Steenson DF, Lee JH, and Min DB. Solid Phase Microextracion Analyses of Volatile Compounds of Soybean and Corn Oils, J. Food Sci. 2002, 67(1), Chung MS, Lee JH and Min DB. Effects of Pseudomonas putrifaciens and Acinetobacter spp. on the Flavor Quality of Raw Ground Beef, J. Food Sci. 2002, 67(1), Yang WT, Lee JH and Min DB. Quenching Mechanisms and Kinetics of α- Tocopherol and β-carotene on the Photosensitizing Effect of Synthetic Food Colorant FD & C Red No.3, J. Food Sci. 2002, 67 (2), vii

8 4. Foley DM, Pickett K, Varon J, Lee JH, Min DB, Caporaso F and Prakash A. Pasteurization of Fresh Orange Juice using Gamma Irradiation: Microbiological, Flavor and Sensory Analyses. J. Food Sci. 2002, 67(4), Lee JH, Kang JH, and Min DB. Optimization of Solid Phase Microextraction on the Headspace Volatile Compounds in Kimchi, a Traditional Korean Fermented Vegetable Product. J. Food Sci (In press). 6. Kang JH, Lee JH, Min S, and Min DB. Study of enzymatic and chemical changes of Kimchi, a Traditional Korean Fermented Vegetable Product. J. Food Sci (In press). 7. Lee JY, Lee JH, Choi YO, and Min DB. Singlet oxygen oxidation and its effects on the soy flour off-flavor. J. AOCS (In press). 8. Lee JH, Ozcelik B, and DB Min. Electron donation mechanisms of β-carotene as a free radical scavenger. J. Food Sci (In press). 9. Ozcelik B, Lee JH, and DB Min. Effects of Light, Oxygen and ph on the 2,2- Diphenyl-1-Picrylhydrazyl (DPPH) method to evaluate antioxidants. J. Food Sci. (In press). 10. Lee JH, Diono R, Kim GY, and DB Min. Optimization of Solid Phase Microextraction on the Headspace Volatile Compounds in Parmesan Cheese. J. Agric. Food Chem. Jf (In press). 11. Kim GY, Lee JH, and DB Min. Study of Light-Induced Volatile Compounds in Goat Cheese. J. Agric. Food Chem jf a (In press). viii

9 PUBLISHED ABSTRACT JH Lee and DB Min. Nuclear magnetic resonance (NMR) study on the electron donating ability of β-carotene by using 2,2-diphenyl-1-picrylhydrazyl (DPPH). Institute of Food Technologist, Anaheim Ca JH Kang, JH Lee, YT Ko and DB Min. Changes of headspace volatile compounds, oxygen, carbon dioxide and microorganisms in Kimchi stored at 5 c for 25 days. Institute of Food Technologist, Anaheim Ca JH Lee, JH Kang, YT Ko and DB Min. Optimization of solid phase microextraction (SPME) on the headspace volatile compounds in Kimchi. Institute of Food Technologist, Anaheim Ca JY Lee, JH Lee, EO Choe and DB Min. Analysis of volatile compounds and headspace oxygen and carbon dioxide in full fat soy flour. Institute of Food Technologist, Anaheim Ca Ozcelik B, Lee JH and Min DB. The effects of chlorophyll, light and oxygen on the stability of 2,2-diphenyl-1-picrylhydrazyl radical in acetone and soybean oil. Institute of Food Technologist, New Orleans LA Lee JH, Ozcelik B and Min DB. Evaluation of electron donating ability of β-carotene by using soybean oil and 2,2-diphenyl-1-picrylhydrazyl (DPPH) in acetone system. Institute of Food Technologist, New Orleans LA Diono R, Lee JH and Min DB. Solid phase microextraction-gas chromatographic analysis of cheese flavor compounds. Institute of Food Technologist, New Orleans LA ix

10 Chung MS, Lee JH and Min DB. Effects of Pseudomonas putrifaciens and Acinetobacter spp. on the Flavor Quality of Raw Ground Beef. Institute of Food Technologist, Dallas, TX FIELD OF STUDY Major Field : Food Science and Nutrition x

11 TABLE OF CONTENTS Page Abstract. ii Dedication... v Acknowledgement vi Vita.. vii List of Tables xiii List of Figures xiv Chapters: 1. Introduction Literature review Lipid Oxidation The chemistry of oxygen Formation of singlet oxygen Triplet oxygen oxidation with fatty acids Singlet oxygen oxidation with fatty acids Comparison of triplet and singlet oxygen oxidation Effects of singlet oxygen oxidation on food quality Photosensitizer Mechanisms of photosensitizer Riboflavin Chlorophyll Photooxidation and Photosensitized Oxidation of Linoleic Acid Abstract Introduction.. 32 xi

12 3.3 Materials and Methods Results and Discussion Conclusion Reference Photosensitized Oxidation of Milk Abstract Introduction Materials and Methods Results and Discussion Conclusion Reference Photooxidation and Photosensitized Oxidation of Lard Abstract Introduction Materials and Methods Results and Discussion Conclusion Reference Conclusion Bibliography APPENDIX A: Figures of sample preparation APPENDIX B: Figures of gas chromatograms xii

13 LIST OF TABLES Table Page 1 Comparison of singlet and triplet oxygen Relative oxidation rates of triplet and singlet oxygen with oleate, linoleate and linolenate Hydroperoxides formed by singlet and triplet oxygen oxidation of oleic, linoleic and linolenic acid Effects of singlet oxygen on the food quality Function of various agents on the mechanism of photosensitization 24 6 Volatile compounds identified from linoleic acid with or without added chlorophyll stored at 4 C for 48 hr in GC peak areas ( ) Effects of ascorbic acid, sodium azide, and BHA on the formation of dimethyl disulfide, hexanal, and heptanal in milk containing 50 ppm added riboflavin Effects of riboflavin, ascorbic acid, sodium azide, and BHA on the formation of pentanal in electronic counts ( ) in milk stored at 4 C under fluorescent light for 4 hr Volatile compounds identified from lard with 0 and 5 ppm chlorophyll stored under light at 55 C for 48 hr in GC peak areas ( ).. 93 xiii

14 LIST OF FIGURES Figure Page 1 Molecular orbital of triplet oxygen Molecular orbital of singlet oxygen Singlet oxygen formation by chemical, photochemical and biological methods The chemical mechanism for the formation of singlet oxygen in the presence of sensitizer, light and triplet oxygen Energy required for hydrogen removal from linoleic acid General mechanisms of triplet oxygen oxidation with linoleic acid 13 7 Reactions of singlet oxygen with olefins by: 1,4-cycloaddition, the ene reaction, and 1,2-cycloaddtion Difference of triplet and singlet oxygen oxidation mechanisms Type I pathway of sensitizer Structure of riboflavin xiv

15 11 Effects of 0, 5 and 15 minutes illumination on electron spin resonance spectrum of 2,2,6,6-4-piperidone-N-oxyl in water solution of riboflavin and 2,2,6,6- tetramethyl-4- piperidone Structure of chlorophyll Gas chromatograms of headspace volatile compounds in linoleic acid containing chlorophyll stored under light for 48 hr The effects of chlorophyll on the volatile compounds in linoleic acid stored at 4 C under light and dark for 48 hr Proposed formation mechanisms of 2-pentylfuran from linoleic acid by singlet oxygen oxidation Proposed formation mechanisms of 2-heptenal from linoleic acid by singlet oxygen oxidation Proposed formation mechanisms of 1-octene-3-ol and 2-octene-1-ol from linoleic acid by singlet oxygen oxidation Headspace volatile compounds in whole milk under the fluorescent light for 0 (a), 2 (b), 4 (c), and 8 hr (d) at 4 C. 1: pentanal, 2: dimethyl disulfide, 3: hexanal, 4: heptanal Headspace volatile compounds in whole milk stored under light or in the dark at 4 C and control for 8 hr. 1: pentanal, 2: dimethyl disulfide, 3: hexanal, 4: heptanal xv

16 20 Effects of riboflavin on the formation of pentanal, dimethyl disulfide, hexanal and heptanal in photosensitized milk under light at 4 C for 2hr. * indicates that the compound was not detected Effects of fat content on the formation of pentanal, heptanal, hexanal, and dimethyl disulfide in photosensitized milk under light for 8 hr The suggested formation mechanisms of hexanal in photosensitized milk containing riboflavin Gas chromatograms of headspace volatile compounds in lard with 0 and 5 ppm chlorophyll stored under light for 24 hr at 55 C. Numbered volatiles are shown in Table The effects of chlorophyll on the volatile compounds in lard stored under light and dark for 48 hr at 55 C The effects of chlorophyll on the headspace oxygen content in lard stored under light and dark for 48 hr at 55 C The effects of chlorophyll on 2-hexenal, 2-heptenal, 2-pentylfuran, and 2-octenal in lard stored under light for 48 hr at 55 C Proposed formation mechanisms of 2-hexenal, 2-heptenal, and 1-octene-3-ol from photosensitized lard by singlet oxygen oxidation Solid phase microextraction. 107 xvi

17 29 Fluorescent light box used for the sample preparation of linoleic acid and milk Sample preparation of lard (Chapter 5) Light box used for lard samples (Chapter 5) Gas chromatogram of linoleic acid with added 5 ppm chlorophyll stored under light for 2 day (Chapter 3) Gas chromatograms of milk with added 0 and 5 ppm riboflavin stored under light for 2 hr (Chapter 4) Gas chromatograms of milk with added 10 and 50 ppm riboflavin stored under light for 2 hr (Chapter 4) Gas chromatograms of milk with 0.5 and 1.0% fat content stored under light for 8 hr (Chapter 4) Gas chromatograms of milk with 2.0 and 3.4% fat content stored under light for 8 hr (Chapter 4) Gas chromatograms of milk with added 50 ppm riboflavin with or without 100 ppm ascorbic acid stored under light for 4 hr (Chapter 4) Gas chromatograms of milk with added 50 ppm riboflavin with or without 100 ppm BHA stored under light for 4 hr (Chapter 4) xvii

18 39 Gas chromatograms of milk with added 50 ppm riboflavin with or without 100 ppm sodium azide stored under light for 4 hr (Chapter 4) Gas chromatograms of lard with 5 ppm chlorophyll stored under light for 0 and 6 hr (Chapter 5) Gas chromatograms of lard with 5 ppm chlorophyll stored under light for 12 and 24 hr (Chapter 5) Gas chromatograms of lard with 5 ppm chlorophyll stored under light for 36 and 48 hr (Chapter 5) Gas chromatograms of lard with 0 ppm chlorophyll stored under light for 0 and 6 hr (Chapter 5) Gas chromatograms of lard with 0 ppm chlorophyll stored under light for 12 and 24 hr (Chapter 5) Gas chromatograms of lard with 0 ppm chlorophyll stored under light for 36 and 48 hr (Chapter 5) xviii

19 CHAPTER 1 INTRODUCTION Lipid oxidation of unsaturated fatty acids can produce undesirable off-odor and toxic compounds, and cause loss of important nutrients in foods (Boff and Min 2002). The major mechanisms of lipid oxidation are autoxidation, lipoxygenase catalyzed oxidation, and light-induced oxidation (Chan 1977; Frankel 1985a, 1985b). A chemical reaction of atmospheric triplet oxygen and organic compounds, usually unsaturated compounds, is called as autoxidation (Chan 1987). Autoxidation of lipid is a free radical chain reaction and can be initiated by metal catalysis, high temperature, and UV irradiation (Frankel 1985b). Light-induced oxidation causes the quality loss of foods and beverages, and makes the products less acceptable or unacceptable to consumers (Skibsted 2000). Many studies have been reported on the formation of light-induced off-flavor in foods, including dairy products and oil products (Bradley 1980; Faria 1983; Kim and Morr 1996). Reversion flavor in soybean oil and sunlight flavor in milk are well-known examples of light-induced off-flavor (Min 1998; Jung et al. 1998). 1

20 The mechanisms of light-induced lipid oxidation can be photosensitized oxidation and photooxidation depending on the presence or absence of photosensitizers, respectively. Photosensitizers in foods stored under light can accelerate lipid oxidation either by Type I or Type II mechanisms. Singlet state of photosensitizer can absorb light energy and become excited singlet state photosensitizer. The excited singlet state photosensitizer becomes to the excited triplet state photosensitizer by intersystem crossing mechanisms. The excited triplet state photosensitizer reacts with triplet oxygen to form singlet oxygen (Type II mechanism) or abstracts electron or hydrogen atom from a substrate to generate radicals (Type I mechanism) (Foote 1976; Bradely 1992; Lu et al. 1999). Chlorophyll in plant materials, riboflavin in dairy foods, and myoglobin derivatives in meat products are well-known photosensitizers that can accelerate oxidation reactions (Lee and Min 1988; King 1996; Li and Min 1998). It was reported that photosensitizing function of riboflavin induced the decrease of the important nutrients including amino acids, Vitamin A, C and D (Jung et al. 1995; Li et al. 2000; Whited et al. 2002). The singlet oxygen oxidation (Type II mechanism of photosensitizers) is very important due to the greater reaction rate of singlet oxygen than that of triplet oxygen in foods even at very low temperatures (Raws and VanSanten 1970). Singlet oxygen oxidation can generate new compounds, which are not found in ordinary triplet oxygen oxidation in foods (Boff and Min 2002). The objectives of this dissertation were (1) to study the formation of volatile compounds in photosensitized oxidation of linoleic acid model systems, (2) to study the 2

21 formation of volatile compounds in photosensitized oxidation of milk, (3) to develop a photosensitizer-free model system using lard, and (4) to propose the formation mechanisms of volatile compounds in linoleic acid, milk, and lard by photosensitized oxidation. 3

22 CHAPTER 2 LITERATURE REVIEW 2.1. Lipid oxidation The chemistry of oxygen There are two types of oxygen in nature: one is triplet state and the other is singlet state under magnetic field. Joseph Prestley discovered triplet oxygen, which is most abundant and in normal air we are breathing, in Avogadro reported in 1811 that oxygen is a diatomic molecule and Faraday reported paramagnetic characteristics of oxygen in The paramagnetic property of oxygen was due to the parallel spins of two outer electrons in the degenerate orbitals (Boff and Min 2002). The molecular orbital of triplet oxygen is shown in Figure 1. The spin multiplicity of molecule is defined as 2S+1, where S is the total spin quantum number. One spin is designated as (+1/2). In case of ground state of oxygen, S is 1 (=1/2+1/2). The spin multiplicity of oxygen is 3 (=2*1+1), which can be shown under magnetic field as 3 distinctive energy states. Therefore, ground state of oxygen is called triplet state and is a di-radical compound. Radical compounds can readily react with radical compounds and non- 4

23 radical compounds react with non-radical compounds. Therefore, triplet oxygen can react with radical compounds. Molecular Atomic σ* π* π* Atomic 2Px 2Py 2Pz π σ π 2Pz 2Py 2Px Energy σ* 2S σ 2S 1S σ* σ 1S Figure 1. Molecular orbital of triplet oxygen (Boff and Min 2002) 5

24 Herzberg discovered singlet oxygen, which has a higher energy state using spectroscopy in The importance of singlet oxygen was rediscovered by Foote and Wexler (1964) and Corey and Taylor (1964). Singlet oxygen has been studied for the last 30 years in food chemistry, biochemistry, medicine, and organic chemistry. The molecular orbital of singlet oxygen is shown in Figure 2. Molecular orbital of singlet oxygen is different from that of triplet oxygen, where the electrons are paired in the π antibonding orbital. Singlet oxygen is non-radical state. The total spin quantum number (S) of singlet oxygen is 0 (=+1/2-1/2, where each spin is in opposite position) and the multiplicity of state is 1 under magnetic field. Singlet oxygen is 1 state and this type of singlet oxygen plays an important role in the oxidations in foods due to the relatively long lifetime. The energy state of 1 singlet oxygen is 22.5 kcal/mole and the lifetime is ranging 50 to 700 microseconds depending on the solvent types, which is long enough to react with other food components with electron rich moiety (Korycka-Dahl and Richradson 1978; Girotti 1998). The activation energy of singlet oxygen is 0 to 6 kcal/mole, which is low enough for the oxidation reactions irrespective of temperature effects (Yang and Min 1994). The comparisons of singlet and triplet oxygen characteristics are shown in Table 1. Singlet oxygen has been studied in lipid and vitamin areas, which are most sensitive to the oxidative damage by singlet oxygen. 6

25 Atomic Molecular σ* π* π* Atomic 2Px 2Py 2Pz π σ π 2Pz 2Py 2Px Energy σ* 2S σ 2S 1S σ* σ 1S Figure 2. Molecular orbital of singlet oxygen (Boff and Min 2002) 7

26 3 O 2 1 O 2 Energy Level Nature Reaction 0 Diradical Radical compound 22.4 kcal/mole Electron rich compounds Non-radical, electrophilic (Boff and Min 2002) Table 1. Comparison of singlet and triplet oxygen Formation of singlet oxygen Singlet oxygen can be formed by the reactions of enzyme, chemical compounds, and sensitizers exposed under light (Krinsky 1977). The formation of singlet oxygen is shown in Figure 3. Some formation mechanisms are not scientifically proven. 8

27 R CO O + R C O O ENZYMES 3 O SENSITIZER ENDOPEROXIDES PRODUCTS R C O + R C O H SENSITIZER H 2 O 2 + O C l - PRODUCTS H 2 O + C l - OZONIDES 1 O 2 H 2 O 2 + O 2 - H 2 O + O H - O H - + O H - H 2 O 2 + H O 2 2H + H 2O 2 Y e - O H - O H + O - 2 O 2 - O O Y + O 2 - Figure 3. Singlet oxygen formation by chemical, photochemical and biological methods (Krinsky 1977) The most well documented formation mechanism of singlet oxygen is the photochemistry using sensitizers. Chlorophyll, myoglobin, riboflavin, porphyrins, and synthetic colorants are well-known photosensitizers, which can change triplet oxygen to singlet oxygen (Foote and Denny 1968; Lledias and Hansberg 2000). The chemical mechanisms of formation of singlet oxygen by photosensitizers are shown in Figure 4. 9

28 Excited state k = /sec Ground state Fluorescence hν 1 Sen* Intersystem Crossing k = /sec Phosphorescence 1 Sen k = /sec + 3 O 2 k = /sec 1 O 2 3 Sen* Figure 4. The chemical mechanism for the formation of singlet oxygen in the presence of sensitizer, light and triplet oxygen. Ground state of singlet sensitizer ( 1 Sen) can become an excited singlet sensitizer ( 1 Sen*). 1 Sen* can return to 1 Sen state by emitting fluorescence or change to excited triplet sensitizer ( 3 Sen*) by intersystem crossing mechanisms. 3 Sen* loses the higher energy either by emitting phosphorescence or by reacting with triplet oxygen. Singlet oxygen can be formed through the energy transferring reaction from the 3 Sen* to triplet oxygen by triplet-triplet annihilation mechanisms. The lifetime of 3 Sen* is longer than 1 Sen*. The returned 1 Sen can begin another cycle of generation of singlet oxygen. Kochevar 10

29 and Redmond (2000) reported that a sensitizer molecule may generate 10 3 to 10 5 molecules of singlet oxygen before becoming inactive Triplet oxygen oxidation with fatty acids Triplet oxygen is a diradical compound, which can react with radical compounds. Food components are not radicals. In order to react with triplet oxygen, one hydrogen atom should be removed from food components to become radicals. Energy required for hydrogen removal from linoleic acid is shown in Figure 5. The removal of hydrogen from saturated fatty acid requires approximately 100 kcal/mol but that of hydrogen at position 11 of linoleic acid is only about 50 kcal/mol (Boff and Min 2002). Therefore, hydrogen between double bonds is most easily removed due to low energy requirement. 100 kcal/mol 50 kcal/mol 75 kcal/mol C H 3 ( C H 2 ) 2 C H 2 C H 2 C H C H C H 2 C H C H C H 2 ( C H 2 ) 6 C O O H Figure 5. Energy required for hydrogen removal from linoleic acid. (Boff and Min 2002) 11

30 General mechanism of triplet oxygen oxidation is shown in Figure 6. Triplet oxygen oxidation or autoxidation has 3 steps : Initiation, Propagation, and Termination (Min 1998). Initiation is a step for the formation of free alkyl radicals. The pentadienyl radical is formed from linoleic acid by hydrogen abstraction at C11 position and rearranged to the C9 or 13 position of conjugated radicals at equal concentration. During rearrangement, cis double bond changes to trans double bond. Triplet oxygen can react with conjugated double bond radicals of linoleic acid and produce peroxyl radical at C9 and 13 positions. The peroxyl radicals abstract hydrogen from other fatty acids and become hydroperoxide and generate free alkyl radicals, which is called Propagation step. The chain reactions of free alkyl radicals and peroxyl radicals accelerate the autoxidation. Radicals react each other to form nonradical products, which is a Termination step. Triplet oxygen oxidation produces only the conjugated diene hydroperoxides in linoleic and linolenic acids. The relative reaction ratio of triplet oxygen with oleic, linoleic, and linolenic acid are 1 : 12: 25 (Min et al. 1989). Linolenic acid reacts twice faster than linoleic acid due to the 2 pentadienyl groups. The number of double bonds does not change in triplet oxygen oxidation even though cis conformation of double bond changes to trans conformation. The changes during autoxidation are used for the measuring the degree of oxidation in oil and fat systems. Measuring weight gain, Peroxide value, and conjugate diene by spectroscopy are common methods to determine the degree of oxidation before the decomposition of hydroperoxides steps, or primary changes. 12

31 C H 3 ( C H 2 ) 3 C H 2 C H C H C H 2 C H C H INITIATION - H C H 2 R (METAL) C H 3 C H C H C H C H C H C H 2 R ( C H 2 ) O 2 PROPAGATION C H 3 ( C H 2 ) 4 C H C H C H C H C H C H 2 R O O + H C H 3 ( C H 2 ) 4 HYDROPEROXIDE DECOMPOSITION C H C H C H C H C H O O H - OH C H 2 R C H 3 ( C H 2 ) 4 C H C H C H C H C H C H 2 R O C H 3 ( C H 2 ) 3 C H 2 TERMINATION + O H C C H C H C H C H C H 2 R + H C H 3 ( C H 2 ) 3 C H 3 (PENTANE) Figure 6. General mechanisms of triplet oxygen oxidation with linoleic acid (Min 1998) 13

32 2-Thiobarbituric acid value, Oxirane value, p-anisidien value, and measuring carbonyl groups, hydrocarbons, and fluorescent products are used for determining the degree of autoxidation after the decomposition of hydroperoxide, or secondary changes (Shahidi and Wanasundara 1998) Singlet oxygen oxidation with fatty acids Singlet oxygen is non-radical compounds and can directly react with non-radical compounds without radical intermediates like triplet oxygen oxidation. One of the highest degenerate π antibonding molecular orbitals in singlet oxygen is vacant and singlet oxygen tries to fill the empty molecular orbital with electrons. Singlet oxygen can directly react with electron rich compounds such as unsaturated fatty acids with double bonds (Beutner et al. 2000). The reaction mechanisms of singlet oxygen with electron rich compounds are shown in Figure 7. Singlet oxygen can react with any compound with double bonds through the mechanisms of cycloaddition and ene reaction. 14

33 O O + O O Endoperoxide 1,4- Cycloaddition O O + O O H Allyl Hydroperoxide ene Reaction O O + CH 2 CH 2 O O Dioxetane 1,2 Cycloaddition Figure 7. Reactions of singlet oxygen with olefins by: 1,4-cycloaddition, the ene reaction, and 1,2-cycloaddtion (King 1996; Boff and Min 2002) One thing should be noticed is singlet oxygen oxidation can break double bonds and reduce the number of double bond in cycloaddition mechanisms while triplet oxygen does not reduce the number of double bonds. The reaction rates of singlet oxygen with oleic, linoleic, linolenic, and arachidonic acids are 0.7, 1.3, 1.9, and M -1 Sec -1, respectively (Doleiden et al. 1974). Singlet oxygen oxidation depends on the number of 15

34 double bonds instead of types of double bonds, such as conjugated or nonconjugated double bonds Comparison of triplet and singlet oxygen oxidation Triplet and singlet oxygen differ not only the reaction mechanisms but also reaction rates. Triplet oxygen can not directly react with double bonds but singlet oxygen can (Figure 8). 1 O 2 R R ' R O R ' H O Hydroperoxide at 9, 10, 12, and 13 3 O 2 R R ' - H R R ' 9 3 O 2 Hydroperoxide at 9 and 13 Figure 8. Difference of triplet and singlet oxygen oxidation mechanisms 16

35 The reaction rates of triplet and singlet oxygen with linoleic acid are and M -1 Sec -1, respectively (Rawls and VanSanten 1970). Singlet oxygen can react 1450 times faster than triplet oxygen with linoleic acid. Relative reaction rates of singlet and triplet oxygen are shown in Table 2. C18:1 C18:2 C18:3 Triplet Oxygen Singlet Oxygen 3 x x x 10 4 (Min 1998) Table 2. Relative oxidation rates of triplet and singlet oxygen with oleate, linoleate, and linolenate Singlet oxygen can react readily with double bonds in unsaturated fatty acids due to the low activation energy (0 to 6 kcal/mol). Temperature does not affect the reaction rate of singlet oxygen but has a significant effect on the triple oxygen oxidation, which requires high activation energy. Hydroperoxide position formed from oleic, linoleic and linolenic acid by triplet and singlet oxygen are shown in Table 3. (Frankel 1985). Singlet oxygen can form C9 17

36 and 10 trans hydroperoxide from oleic acid by ene reaction. Triplet oxygen can form C8, 9, 10 and 11 position hydroperoxides from oleic acid. Frankel (1985) reported that C8- and 11- hydroperoxides are formed in larger portion than inner hydroperoxide like C9- and 10- hydroperoxides. In case of linoleic acid, singlet oxygen can form hydroperoxides at C9, 10, 12, and 13 positions while triplet oxygen can form C9 and 13 hydroperoxides. Hydroperoxides from triplet oxygen oxidations are all conjugated forms while singlet oxygen can produce both conjugated and nonconjugated hydroperoxides. Singlet oxygen can form C9, 10, 12, 13, 15, and 16 positions of hydroperoxides from linolenic acid. Triplet oxygen can produce C9, 12, 13, and 16 conjugated hydroperoxides from linolenic acid. The concentration of hydroperoxides at C12 and 13 positions from linolenic acid is lower than C9 and 16 hydroperoxides due to the inner cyclization (Frankel 1985). Hydrogen donor can prevent inner cyclization and produce equal amounts of hydroperoxides at C9, 12, 13, and 16 positions by triplet oxygen oxidation Effects of singlet oxygen oxidation on food quality Singlet oxygen can readily react with electron rich moiety in food compounds including fatty acids, amino acids, and vitamins due to the low activation energy. The development of reversion flavor, known as beany or grassy, is a unique characteristic in soybean oil, which prevents the wide consumption of soybean oil (Ho et al. 1978). 2- Pentylfuran and 2-pentenyl furan, which are reported as reversion flavor compounds, can be formed in soybean oil containing chlorophyll stored under light only (Callison 2001). 18

37 Chlorophyll is a well-known photosensitizer in vegetable oils. Boff and Min (2002) reported the formation mechanisms of 2-pentylfuran and 2-pentenyl furan from linoleic and linolenic acids, respectively using singlet oxygen oxidation. 1 O 2 3 O 2 Oleic acid 9-OOH 10-OOH 8-OOH 9-OOH 10-OOH 11-OOH Linoleic acid 9-OOH 10-OOH 12-OOH 13-OOH 9-OOH 13-OOH Linolenic acid 9-OOH 10-OOH 12-OOH 13-OOH 15-OOH 16-OOH 9-OOH 12-OOH 13-OOH 16-OOH Table 3. Hydroperoxides formed by singlet and triplet oxygen oxidation of oleic, linoleic and linolenic acid (Frankel 1985) 19

38 Singlet oxygen can react with tryptophan, histidine, tyrosine, methionine, and cysteine (Foote 1976; Michaeli and Feitelson 1997). The reaction of singlet oxygen and amino acids depends on ph, solvent types, dielectric constants of medium, and the electron rich moiety in amino acids, including the presence of double bonds and sulfur atom (Miskoski and Garcia 1993; Michaeli and Feitelson 1994). Sunlight flavor in milk is one of important reactions between singlet oxygen and amino acid. Dimethyl disulfide, described as sunlight flavor in milk, can be formed in milk stored under light only (Jung et al. 1998). Jung et al. (1998) reported the formation mechanisms of dimethyl disulfide from singlet oxygen oxidation of methionine. Vitamin D, which is an important nutrient for normal mineralization and growth of bones, can be oxidized by singlet oxygen oxidation at the rate of M -1 s -1 (King and Min 1998; Li et al. 2000). Oxidation of vitamin D in model system did not occur in the absence of either photosensitizer or light. Ascorbic acid, which is an important nutrient in foods, can be destroyed easily by oxidation. Jung et al. (1995) reported ascorbic acid was oxidized by singlet oxygen at the rate of M -1 s -1 in ph 6 potassium buffer at 20 C. Yang (1994) reported the reaction rate for ascorbic acid with singlet oxygen as M -1 s -1 in an aqueous solution of ph 7 at 25 C. The summary of effects of singlet oxygen on food quality is shown in Table 4. 20

39 Food types Quality changes Involved chemical compounds Soybean oil Reversion flavor 2-pentylfuran 2-(2-pentenly) furan Milk Sunlight flavor Methionine Dimethyl disulfie Proteins Oxidation of nutrients Tryptophan, Histidine, Tyrosine, Methionine, Cysteins Vitamins Oxidation of nutrients Vitamin C Vitamin D Meat Flavor and color change Myoglobin Hemoproteins (Boff and Min 2002) Table 4. Effects of singlet oxygen on food quality 2.2 Photosensitizer Mechanisms of photosensitizer The reaction mechanisms of photosensitizer with triplet oxygen or other substrates are shown in Figure 9. The excited triplet sensitizer can react directly with compounds by donating and abstracting hydrogen or electrons to produce free radicals or free radical ion, which is known as Type I pathway. The hydrogen abstraction of sensitizer produces free radicals and transferring electron from sensitizer to triplet oxygen 21

40 produces radical anion like superoxide anion. The rate of the Type I pathway depends on the type and concentration of sensitizers and substrates. Phenols and reducible compounds favor Type I pathway (Korycka-Dahl and Richardson 1978). Type I mechanism can be explain using one-electron reduction potentials. In case of riboflavin, low (-0.3 V) potential of ground riboflavin is excited to high (1.7 V) triplet flavin, which is higher than that of target compounds like Try (1.5 V), Tyr (0.93 V) or polyunsaturated fatty acids (0.6V) (Buettner 1993; Decker 1998; Lu et al. 1999). Excited triplet riboflavin can take electron, or hydrogen atom from substrates easily making cation radical, or neutral radical, respectively, due to its relatively high oneelectron reduction potential. These radical compounds can initiate radical reactions with each other or triplet oxygen, which is di-radical. Type II mechanism happens when triplet oxygen exist abundantly. Energy transfer from excited triplet sensitizer to triplet oxygen makes singlet oxygen (Foote 1976). Triplet photosensitizer transfer energy to dissolved oxygen using a dye-oxygen complex or energized singlet oxygen. More than 99% of the reaction between triplet oxygen and excited triplet sensitizer produces singlet oxygen. The rate of Type II pathway depends on the solubility and concentration of oxygen in systems. Oil system with chlorophyll, where triplet oxygen is more soluble, favors Type II pathway while aqueous system like milk favors Type I pathway due to the limitation of triplet oxygen. The result compounds from Type I and Type II pathway accelerate the oxidation reactions by providing reactive free radical species and singlet oxygen. 22

41 H transfer (Hydrogen atom) 3 Sen* + RH Sens H + R back reaction Sens + RH 3 Sen* + R electron transfer Sens - + R + back reaction Sens + R Sens - + R + O 2 - or HO 2 or RO 2 + H 2 O 2 + O 2 Sen H or R + or Sen O - 2 or HO 2 H 2 O 2 Figure 9. Type I pathway of sensitizer Addition of different scavengers and singlet oxygen quenchers (Table 5) can help to understand the mechanisms of kinetics and types of photosensitizers (Edwards and Silva 2001(43)). Nitrogen or oxygen gas bubbling can be another technique to understand the radical process in photosensitized system. 23

42 Agents Chemical compounds Effects Singlet oxygen quencher NaN 3, sodium azide, (0.1-5 mm), Histidine (55.0 mm) Type II confirm. If effected, 1 O 2 is involved. Singlet oxygen enhancer D 2 O If effected, 1 O 2 is involved. Electron acceptor K 3 Fe(CN) 6 (0.1 mm) If effected, electron is involved. Type I H 2 O 2 trap Catalase (10.0 U Sigam/ml) Type I Superoxide trap O 2 - Superoxide dismutase (SOD) M 2 O H +! H 2 O 2 + O 2, Type I H O O H +! H 2 O 2 + O 2 H 2 O 2 + UV! 2 OH HO 2 O H + (ph dependent) OH trap Sodium benzoate (1.0 mm) Mannitol, Salicylic acid Type I Cu(II) chelator Neocuproine (100µM) Copper chelating and can be used for antioxidant. (Edwards and Silva 2001) Table 5. Function of various agents on the mechanism of photosensitization 24

43 2.2.2 Riboflavin Riboflavin is a 7,8-dimethyl-10-(1 -D-ribityl) isoalloxazine (Figure 10). Riboflavin is present as free form or flavin mononucleotide (FMN) or flavin adenine dinucleotide (FAD) in all aerobic cells. FAD and FMN can act as oxidases or dehydrogenases to transfer electrons in oxidation-reduction reactions in biological systems. A deficiency in riboflavin may affect the metabolisms of glucose, fatty acids, and amino acids. The excess of riboflavin did not produce toxic effects (King 1996). C H 2 ( C H O H ) 3 C H 2 O H C H 3 N N O C H 3 N O N H Figure 10. Structure of riboflavin The recommended daily allowance (RDA) for riboflavin is 0.6 mg (Cooperman and Lopez 1984). The concentration of riboflavin is reported 0.17 mg/100 g in milk and 3.5 mg/ 100g in liver. The stability of riboflavin is influenced by ph, heat, and light. Riboflavin is stable at acidic and neutral conditions but less stable in basic environment. 25

44 Riboflavin can undergo redox cycling easily and one hydrogen reduced form, flavosemiquinone radical and two hydrogen reduced compound, flavohydoquinone can be formed. Riboflavin is very sensitive to light exposure, especially 450 nm wavelength. The loss of riboflavin was as high as 30% during a 30 min fluorescence light exposure at room temperature, while only a 12% loss occurred in boiling processing. The reduction of riboflavin during light exposure is due to the abstraction of hydrogen atom from coplane of rings. Riboflavin has been known as a photosensitizer which can be excited by light and react with triplet oxygen (Type II) or by substrates (Type I). Type I reaction of riboflavin is favored due to the low concentration of oxygen in water matrix and easy oxidationreduction property of riboflavin (McGinnis et al. 1999; Gutierrez et al 2001). However, the generation of singlet oxygen from riboflavin has been confirmed using Electron Spin Resonance (ESR) and singlet oxygen trapping compound like 2,2,6,6-tetramethyl-4- piperidone (Bradley 1991) (Figure 11). The photosensitizing effect of riboflavin on the oxidation of many compounds, including vitamin D 2, hydroxypyridine, has been reported (Li et al. 2000; Pajares 2001). The destruction of riboflavin under light in nitrogen gas is less than in oxygen gas, which suggested that not only light energy but also singlet oxygen produced by riboflavin itself can be related with the loss of riboflavin. 26

45 Figure 11. Effects of 0, 5 and 15 min illumination on electron spin resonance spectrum of 2,2,6,6-4-piperidone-N-oxyl in water solution of riboflavin and 2,2,6,6- tetramethyl-4- piperidone (Bradley 1991) 27

46 Riboflavin and FMN are photosensitizers and induce the modification of other compounds by transferring energy of visible light. Inactivation effects of riboflavin photosensitization (446~ nm) were studied on many enzymes including catalase, horse-radish peroxidase, lysozme, and aromatic amino acids like Trp and Tyr (Edward and Silva 2001). Ascorbic acid protects riboflavin and other target amino acids by being oxidized first in the presence of oxygen (Lee at al. 1998). The oxidation of ascorbic acid was accelerated by higher light intensity and more riboflavin content. Cysteine showed strong antioxidant activity on the riboflavin-sensitized oxidation on ascorbic acid (Jung et al. 1995). Riboflavin can be found in dairy products without exceptions and induce the changes of food quality. Riboflavin oxidized various substrates such as malondialdehyde, tryptophan, ethylene glycol, and ascorbic acid (King 1996; Jung et al. 1995; Edwards and Silva 2001). Riboflavin can induce sunlight flavor from light exposed milk by singlet oxygen oxidation (Jung et al. 1998; Skibsted 2000). Riboflavin can photooxidize ascorbic acid 1.4 times and 21 times faster than methylene blue and protoporphyrin, respectively (Jung et al. 1995). 28

47 2.2.3 Chlorophyll Chlorophylls are magnesium complexes derived from porphins with tetrapyrrole rings (Figure 12). Chlorophyll a and b, which have different substituents, are found in green plants in an approximate ratio of 3:1 (Elbe and Schwartz 1996). Many chlorophyll derivatives have been identified from plants, marine algae, and photosynthetic bacteria. H C H H 3 C C H H R C H 3 C H 2 N N chl a R : C H 3 H M g H chl b R : C H O H 3 C N N H C H 3 C H 2 H 2 C H C O 2 C H 3 O C O 2 C H 3 C H 3 Figure 12. Structure of chlorophyll 29

48 One of the most important characteristics of chlorophyll and its derivatives is the absorption of visible light between nm and nm. Chlorophyll a and b in ethyl ether showed the maximum absorption at and 642 nm, respectively, in the red region and and nm in the blue region, respectively. Chlorophyll is light sensitive and easily degraded into colorless photodegradation compounds including methyl ethyl maleimide and glycerol (Jen and Mackinney 1970; Schwartz and Lorenzo 1990). Photodegradation of chlorophyll results in the opening of tetrapyrrole ring. Singlet oxygen and hydroxyl radicals, which can be formed from chlorophyll exposed to light in the presence of oxygen, can react with tetrapyrroles of chlorophyll and destroy the porphyrins of chlorophyll and become colorless compounds (Elbe and Schwartz 1996). Chlorophyll has been used to accelerate the lipid oxidation in olive oil (Kiritsakis and Dugan 1985), soybean oil (Jung and Min 1991; Lee et al.1997), and fatty acid model systems (Rawls and VanSanten 1970; Jung et al. 1999). Rawls and VanSanten (1970) reported that chlorophyll works as a photosensitizer through mostly Type II mechanism (singlet oxygen generation) while Type I mechanism of chlorophyll is involved in some degree. 30

49 CHAPTER 3 Photooxidation and Photosensitized Oxidation of Linoleic Acid 3.1 Abstract Volatile compounds of linoleic acid with or without added 5 ppm chlorophyll under light and dark were studied by solid phase microextraction (SPME)-gas chromatography (GC)-mass spectrometry (MS). Total volatile compounds in linoleic acid without added chlorophyll under light and in the dark did not increase and the same for 48 hr at 4 C. Total volatile compounds in linoleic acid with added 5 ppm chlorophyll under light at 4 C for 0, 6, 12, 24, and 48 hr, increased from 8.9 to 11.6, 21.7, 26.1, 29.3 ( ) in electronic counts, respectively. 2-Pentylfuran, an undesirable reversion flavor compound in soybean oil, 2-octene-1-ol, 2-heptenal and 1-octene-3-ol were formed by photosensitized oxidation only. Light excited photosensitizers like chlorophyll can generate singlet oxygen from ordinary triplet oxygen. 2-Pentylfuran, 2- heptenal, and 1-octene-3-ol can come from C10, C12, and C10 hydroperoxide of linoleic acid, respectively, which can be formed by singlet oxygen oxidation but not by triplet 31

50 oxygen oxidation on linoleic acid. The singlet oxygen oxidation mechanisms for 2- pentylfuran 2-heptenal, 1-octene-3-ol, and 2-octene-1-ol from linoleic acid were proposed. 3.2 Introduction The oxidation of lipid containing foods causes off-flavor, loss of nutrients and production of undesirable compounds during manufacturing and storage, which decreases food quality and makes the foods less acceptable or unacceptable to consumers. Lipid can be oxidized by free radicals, lipoxygenase, and singlet oxygen (Chan 1977; Frankel 1985; Zhuang et al. 1998). Autoxidation or triplet oxygen oxidation of lipids is a free radical chain reaction on unsaturated fatty acids. Free radical chain reaction can be initiated by metal catalysis heat, or ultraviolet irradiation, and produce hydroperoxides of unsaturated fatty acids (Frankel 1985). The decomposition of lipid hydroperoxides produces volatile compounds, including aldehydes, alcohols, ketones, and hydrocarbons, which are responsible for the increase of off-odor and the decrease of food qualities (Min 1998). Light-induced lipid oxidation has been studied on unsaturated fatty acids and vegetable oils with or without addition of photosensitizers (Frankel et al. 1976; Frankel et al. 1981; Faria 1983). Even though the energy of visible light is not absorbed by unsaturated fatty acids, photooxidation can induce oxidation in linoleic acid and safflower oil exposed to ultraviolet (UV), incandescent, and fluorescent light, which may 32

51 be due to the presence of impurities including photosensitizers (Faria 1983). UV light photooxidation may accelerate lipid oxidation by generating the alkoxyl radicals and hydroxyl radicals from hydroperoxides (Faria 1983). Photosensitizers, including chlorophyll, riboflavin, and methylene blue, can accelerate lipid oxidation (Yang and Min 1994). Singlet state of photosensitizer can absorb light energy and become excited singlet state photosensitizer. The excited singlet state photosensitizer becomes to the excited triplet state photosensitizer by intersystem crossing mechanisms. The excited triplet state photosensitizer reacts with triplet oxygen to form singlet oxygen (Type II mechanism) or abstracts electron or hydrogen atom from a substrate to generate radicals (Type I mechanism) (Foote 1976; Bradely 1992). Singlet oxygen can play important roles in the photosensitized oxidations in foods due to the relatively long lifetime. The energy state of singlet oxygen is 22.4 kcal/mole and the lifetime is ranging 50 to 700 microseconds depending on the solvent types, which is long enough to react with other food components with electron rich moiety (Korycka- Dahl and Richradson 1978; Girotti 1998). The activation energy of singlet oxygen is 0 to 6 kcal/mole, which is low enough for the oxidation reactions (Yang and Min 1994). The reaction rates of triplet and singlet oxygen with linoleic acid are and M -1 s -1, respectively (Rawls and VanSanten 1970). Linoleic acid is one of the major unsaturated fatty acids in vegetable oils including soybean oil (51%), corn oil (61%), and sunflower oil (67%) (Chu and Kung 1998). The isomeric structures of hydroperoxides from fatty acids by autoxidation and photosensitized oxidation have been studied extensively (Frankel et al. 1981; Frankel 1985). However, the effects of photooxidation and chlorophyll photosensitized oxidation 33

52 on the formation of volatile compounds from linoleic acid and the formation mechanisms of light-induced volatiles were not fully understood. The objectives of this work were (1) to study the effects of light and chlorophyll on the formation of volatile compounds from linoleic acid model system and (2) to propose the formation of volatile compounds from linoleic acid by photosensitized oxidation. 3.3 Materials and Method Materials Chlorophyll b, linoleic acid (99%), 1-pentanol, hexanal, 2-hexenal, 2-heptenal, 1- octene-3-ol, 2,4 heptadienal, 2,4 octadienal, 2-nonenal, octanoic acid, and 2,4 decadienal were purchased from Sigma Chemical Co. (St. Louis, MO., U.S.A.). Teflon-coated rubber septa, aluminum caps, serum bottles, glass liners, the fiber assembly holder, and 65 µm Polydimethylsiloxane/Divinylbenzene (PDMS/DVB) were purchased from Supelco, Inc. (Bellefonte, PA., U.S.A.). 2-Pentylfuran was purchased from Karl Industries Inc. (Aurora, OH., U.S.A.). Sample preparation for the light and dark storage of linoleic acid model system Chlorophyll was dissolved in linoleic acid to obtain 5 ppm (weight/volume) while magnetic stirring. Samples of 0.10 g linoleic acid with or without added 5 ppm chlorophyll were put in 10-mL serum vials (25 40 mm, 20 mm diameter from Supelco, 34