3.2.1 Discovery of singlet oxygen
Joseph Priestley discovered oxygen in 1775, but it took several decades before the scientific world found out about its diamagnetic properties. It was Faraday who took the credit for discovering this major difference between oxygen and the other permanent gasses such as nitrogen. In the twentieth century the oxygen molecule was completely exposed. The paramagnetic properties of oxygen (triplet oxygen) could finally be explained at a molecular level and in 1934 a higher energy state of oxygen was also discovered by spectroscopy and reported as singlet oxygen. The importance of singlet oxygen in chemical reactions was not recognized for almost 30 years until the 1960s. Singlet oxygen was rediscovered by a group of chemists interested in photooxidation experiments in organic chemistry (Corey and Taylor, 1964). The insight into singlet oxygen chemistry has progressed from that point in time.
3.2.2 Properties and reactivity of singlet oxygen
It is important to specify which of the specific forms of the oxygen molecule is present under certain conditions in order to understand its diverse reactive properties towards food components. Therefore, it is necessary to take a look at the molecular structure in more detail and more specifically the distribution of the outer shell electrons. The distribution of the electrons of the individual oxygen atoms in the molecular orbital diagram reveals many chemical properties of both singlet and triplet oxygen. Filling the molecular orbitals with electrons in the diagram for the oxygen molecule specifically results in a 58 Oxidation in foods and beverages and antioxidant applications
situation where the last two electrons need to be distributed over the outer two orbitals (-orbitals). The most stable form of oxygen (triplet oxygen) is obtained when the electrons are placed in different orbitals according to the Hund's rule, while the exited and less common form (singlet oxygen) is obtained when the two electrons are present in one orbital (Table 3.1). These names for the different forms of molecular oxygen are derived from the nomenclature of the spin multiplicity of the molecule which is defined a 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 for two electrons with the same spin in two individual orbitals. Therefore the molecular spin is Ý + Ý = 1. The spin multiplicity of the grounds state of the oxygen molecule then becomes 3 ( 2 1 1), which is referred to as triplet oxygen. For the excited form of the oxygen molecule there are two electrons with an opposite spin present in one orbital which leads to a molecular spin of ÿÝ + Ý = 0. The spin multiplicity of the excited state of the oxygen molecule then becomes 1 ( 2 0 1), which is referred to as singlet oxygen.
In singlet oxygen the electronic repulsion between the two negatively charged electrons that are forced in one orbital brings about a highly energetic and reactive molecule. The energy difference of 22.5 Kcal/mole is the main driver for the higher reactivity of singlet oxygen (Korycka-Dahl and Richardson, 1978). However, the electron distribution also generates a very different chemical reactivity for both compounds. Triplet oxygen is a biradical because it contains two free electrons and therefore it will only react with other radicals. Singlet oxygen, on the other hand, has an electron pair instead of two free radicals. As a consequence singlet oxygen will be looking for a `tenant' to fill its empty molecular orbital in order to reach an energetically more favorable state. Because an electron pair would fit nicely into the highest vacant molecular orbital, singlet oxygen will react rapidly with all electron rich compounds.
Table 3.1 Properties of triplet and singlet oxygen
Property Type of oxygen
3O2(triplet) 1O2(singlet)
-orbitals " # "#
Energy level 0 22.5 Kcal/mole
Nature Diradical Non-radical
Reacts with Radicals Electron-rich
compounds
The impact of singlet oxygen on lipid oxidation in foods 59
3.2.3 Reaction of singlet oxygen with lipids
Oxidation plays a fundamental role in the reduction of the quality of fats and oils and therefore this matrix is also very useful to further discuss oxidation by singlet oxygen versus oxidation by triplet oxygen. A significant amount of research data is available on the impact of the so-called autoxidation in which triplet oxygen is involved. Initially a lipid radical is formed in this oxidation process, which requires a significant amount of energy. Therefore, autoxidation is strongly accelerated by increased temperatures. Once the lipid radical is formed it will react with other radicals such as triplet oxygen. In polyunsaturated lipids with several double bonds present, the radical can be stabilized via different resonance forms (Frankel, 2005). The observation that polyunsaturated lipids have a higher relative oxidation rate confirms the role of radical formation in the reaction mechanism of autoxidation (Table 3.2).
For singlet oxidation the reaction does not proceed via a radical type reaction.
The singlet state oxygen reacts directly with the unsaturated fatty acid via a concerted `ene' addition mechanism (Fig. 3.1). For this reaction mechanism the effect of the level of unsaturation of the lipids on the oxidation rate is only limited because oxidation by singlet oxygen will not proceed via a radical mechanism. The influence of temperature on the reaction rate is also negligible
Table 3.2 Relative oxidation rates of singlet and triplet oxygen with unsaturated lipids
Type of oxygen Degree of unsaturation
C18:1 C18:2 C18:3
Triplet 1 27 77
Singlet 3 104 4 104 7 104
Fig. 3.1 Difference between lipid oxidation mechanism singlet and triplet oxygen.
60 Oxidation in foods and beverages and antioxidant applications
since much lower activation energies are required for oxidation reactions that involve singlet oxygen.
The main importance of the difference in reaction mechanism is the generation of different oxidation breakdown products. In lipid oxidation the two types of oxygen will react differently with the lipid matrix generating distinctive hydroperoxides which decompose further in specific aldehydes and other breakdown products.
In the case of linoleic acid oxidation with triplet oxygen, two hydroperoxides will be generated (Frankel, 2005). Triplet oxygen does not react directly with the double bonds. Due to its molecular structure (biradical) the reaction needs to proceed via an initial formation of a lipid radical. Therefore, the first step in the triplet oxidation of linoleic acid is hydrogen abstraction at carbon 11, which is the most easily removed as it is a bis-allylic methylene group. The radical produced is resonance stabilized and two resonance forms are particularly stable because they contain conjugated double bonds. Therefore the radical will be present preferentially on carbon 9 and 13, and triplet oxygen will react with these alkyls radicals to form a hydroperoxide (Fig. 3.2).
Because singlet oxygen does react via a concerted mechanism there will be a direct reaction with the double bond without any preference for a specific carbon. Consequently hydroperoxides can be formed on carbon 9, 10, 11 and 12 in equal quantities.
A similar reasoning can be applied on other fatty acids (Table 3.3). There is always a preferential formation of conjugated hydroperoxides for poly-unsaturated fatty acids that are oxidized with triplet oxygen. For singlet oxidation the peroxides will be introduced at either end carbon of all double bonds. Owing to the difference in the oxidation mechanism between singlet and triplet oxygen the hydroperoxide distribution will be different. Consequently also the breakdown product of the hydroperoxides will be different. Chemical
Fig. 3.2 Reaction products formed by autoxidation of linoleic acid.
The impact of singlet oxygen on lipid oxidation in foods 61
analysis of the oxidation breakdown products therefore allows differentiating between the types of oxygen that may cause the oxidation.
3.2.4 Methods to study singlet oxygen in foods Detection and evaluation of singlet oxygen in foods
Singlet oxygen detection in food oxidation of foods is difficult due to the short lifetime of the excited molecule. Several analytical techniques have been developed for the detection of singlet oxygen.
Spectrophotometric methods can be used to measure singlet oxygen indirectly. These methods use a compound of which absorption at a suitable wavelength decreases after reaction with singlet oxygen. In organic solvents the molecule 1,3-diphenylisobenzofuran can be used since it reacts readily with singlet oxygen. This reaction results in decreased absorbance at 410 nm (Kochevar and Redmond, 2000). In aqueous systems para-nitrosodimethyl-alanine can be monitored at 440 nm absorbance as the molecule reacts with an imidazole intermediate.
Another interesting molecule for indirect detection of singlet oxygen is cholesterol as it reacts with singlet oxygen to form specific oxidation products, more specifically hydroperoxides. The specificity even allows differentiation of Table 3.3 Hydroperoxides formed by oxidation of fatty acids with singlet and triplet oxygen
Fatty acid
Oleate Linoleate Linolenate Singlet oxygen
Saturated hydroperoxides 9-OOH 10-OOH
Conjugated hydroperoxides 9-OOH 9-OOH
13-OOH 12-OOH
13-OOH 16-OOH
Non-conjugated hydroperoxides 10-OOH 10-OOH
12-OOH 15-OOH
Triplet oxygen
Saturate hydroperoxides 8-OOH
9-OOH 10-OOH 11-OOH
Conjugated hydroperoxides 9-OOH 9-OOH
13-OOH 12-OOH
13-OOH 16-OOH From Frankel et al. (1979).
62 Oxidation in foods and beverages and antioxidant applications
oxidation originating from triplet oxygen, as this results in hydroperoxides on position 7a and 7b, while singlet oxygen introduces the hydroperoxide on 6a and 6b (Girotti and Korytowski, 2000).
The more advanced technique of electron spin resonance spectroscopy (ESR) can also be used to detect singlet oxygen. This technique specifically detects free radicals, often by reaction of a radical with a spin-trapping agent, thus forming a stable radical adduct. Consequently ESR seems more suitable to study the reactions in which free radicals play a role such as the oxidation of food products with triplet oxygen. However, one of the spin-trapping agents called TMPD (2,2,6,6-tetramethly-4-piperidone) reacts very specifically with singlet oxygen to form a stable nitroxide radical adduct TAN (2,2,6,6-tetramethyl-4-piperidone-N-oxyl). No other reactive oxygen species have been found to convert TMPD to TAN (Ando et al., 1997). ESR detected the formation of singlet oxygen in meat (Whang and Peng, 1988) and milk (Bradley, 1991).
Another suitable analytical technique to detect singlet oxygen is detection of its chemiluminescence at 1270 nm. A photon can be released at 1270 nm which corresponds to the specific energy differential between excited singlet oxygen and ground-state triplet oxygen. Detection of singlet oxygen by measuring the emission at 1270 nm has been successful in biological systems (Kanofsky, 2000).
Methods of studying oxidation by singlet oxygen
Because of the different chemical nature of 1O2 and 3O2 their reaction with lipids proceeds via very dissimilar oxidation pathways. Both types of oxygen will attack the lipid skeleton at different locations. When these oxidized lipids then decompose further, distinct breakdown products are formed specific for the type of reactive oxygen that lies at the basis of the oxidation process. The occurrence of these products can therefore be used to study oxidation pathways.
Most of the oxidation breakdown products are very volatile and also cause the rancid flavor of products that contain oxidized fats or oils. It is possible to sample these volatiles in the headspace above an oxidized lipid. All components in the headspace can then be separated (via gas chromatography, GC) and these individual constituents of the total flavor can be identified (via mass spectrometry, MS).
If one wants to find out whether the rancidity was caused by singlet or triplet oxygen, the profile of the flavor molecules can reveal some valuable informa-tion. In order to be able to pinpoint which flavor molecules are produced from singlet oxidation and which from the more common triplet oxidation, it is helpful to design an experiment that looks at only one of the two possible oxidation pathways. Triplet oxygen-borne lipid oxidation can be promoted by working at high temperatures combined with the use of a singlet oxygen quencher. Alternatively singlet oxygen-borne lipid oxidation is promoted by the addition of a photosensitizer that is able to transfer energy from light to the oxygen molecule, which leads to the formation of high levels of singlet oxygen.
By working simultaneously at low temperatures the reactivity of remaining triplet oxygen can be slowed down.
The impact of singlet oxygen on lipid oxidation in foods 63
In rapeseed oil, the light-induced promotion of singlet oxidation leads to a strong increase in the concentration of volatile components such as butenal, heptenal and several other specific lipid oxidation products (Fig. 3.3). The headspace analyses of samples subjected to conditions that promote lipid oxidation by triplet oxygen reveal the presence of a distinctly different set of volatile oxidation products. Compounds such as hexanal and nonenal appear which therefore can be considered as markers for triplet oxidation (Van Dyck, 2007).