Supporting Information. Monitoring Interfacial Lipid Oxidation in Oil-in-Water Emulsions using Spatially-Resolved Optical Techniques

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1 Supporting Information Monitoring Interfacial Lipid Oxidation in Oil-in-Water Emulsions using Spatially-Resolved Optical Techniques Chiranjib Banerjee, Michael Westberg, Thomas Breitenbach, Mikkel Bregnhøj, and Peter R. Ogilby* Department of Chemistry, Aarhus University, DK-8000 Aarhus, Denmark * To whom correspondence should be addressed: progilby@chem.au.dk Contents Materials and Sample Preparation FTIR Spectrometer and Raman Microscope Monitoring Oxygenation with IR Spectroscopy Oxidation Monitored using Microscope-Based Raman Spectroscopy Documenting a Constant Yield of O 2 (a 1 g ) upon Spatially Localized Irradiation References S2 S3 S4 S8 S9 S10 S1

2 Materials and Sample Preparation. 2,3-Dimethyl-2-butene ( 99%), methyl linoleate ( 99%), 2, 2'-Azobis(2- amidinopropane)dihydrochloride (AAPH, 97% ) and C 60 (99.5 %) were obtained from Sigma- Aldrich and used as received. 5,10,15,20-Tetrakis(1-methyl-4-pyridyl)-21H,23H-porphyrin, tetratosylate salt, (TMPyP, 98%, Porphyrin Systems) and azobisisobutyronitrile (AIBN, 98%, A. K. Scientific, Inc.) were also used as received. Cod liver oil, without added stabilizers, was provided by Maritex A/S, a subsidiary of TINE, BA (Sortland, Norway), and stored at 20 ºC until use. Details involving the characterization of these particular samples of cod liver oil are published. 1 Sodium caseinate (CAS, Miprodan 30), with a reported protein content of 93.5%, was donated by Arla Foods Ingredients (Viby J, Denmark). C11-BODIPY 581/591 (Invitrogen, Thermo Fisher Scientific) was used as received. D 2 O (99.9% D) was obtained from EurisoTop and H 2 O was purified by filtration (Milli-Q system by Millipore Corporation). H 2 O- and D 2 O-based phosphate buffer solutions were prepared using commercially available PBS tablets (Sigma Aldrich). Emulsions were prepared with 70% cod liver oil (by weight). CAS was first dispersed in the buffer solution with gentle stirring at room temperature for 2 h. The emulsion was prepared by adding the oil slowly to the buffer solution using a homogenizer operating at 8000 rpm for 3 min. The amount of casein in the emulsions used was 1.4 % (by weight). The O 2 (a 1 g ) photosensitizers, C11-BODIPY 581/591 and radical initiators were always added after emulsion formation. All emulsions were prepared using materials exposed to the ambient atmosphere. For the Raman and SIM experiments, the emulsion was drop cast on a cover slip, and a second cover slip was then put on top of the solution. S2

3 FTIR Spectrometer and Raman Microscope. FTIR spectra were recorded using a Bruker IFS-66 v/s spectrometer with a tungsten lamp and an InSb detector. 2 Data were recorded over the range cm -1 using 1 mm quartz cuvettes. Prior to measurement, samples were diluted with CS 2 to obtain a maximum absorbance less than ~ 1. We used a home-built wide-field microscope for the Raman studies. Briefly, a He-Ne laser with a nm beam was passed through a nm clean-up filter, focused onto the backport of a Nikon TE 2000-S inverted microscope, and directed to the sample via a dichroic mirror designed for Raman measurements (633.0 nm long pass, Semrock). A 40 long-workingdistance objective with NA = 0.6 was used, allowing us to focus this probe light to a spot with an Airy disk diameter of ~ µm in the x-y plane of the sample (i.e., spatial resolution of ~ 700 nm). Raman scattered light was collected through a 635 nm long pass filter and the spectra were recorded using a CCD camera with a grating monochromator (Princeton Instruments Acton POR 2300i; 1200 g/mm BLZ grating). Hardware and acquisition of spectra were controlled with WinSpec/32 software (Roper Scientific, ver ), and the data accumulation time was 20 s. S3

4 Monitoring Oxygenation with IR Spectroscopy. The data shown in Figure S1 illustrate several important points. First, we are indeed able to observe the expected IR spectral changes upon exposure of three different unsaturated compounds to O 2 (a 1 g ). Second, and perhaps more importantly, the IR spectral changes first appear only after a rather aggressive treatment with O 2 (a 1 g ). For example, with methyl linoleate and cod liver oil, appreciable spectral changes become apparent only after a 3 h irradiation period of the photosensitizer C 60 (this is an efficient sensitizer with a O 2 (a 1 g ) quantum yield of unity 3 ). Moreover, these experiments were conducted using neat samples and, as such, we preclude solvent-mediated O 2 (a 1 g ) deactivation channels that compete with the oxidation of the compound. Nevertheless, spectral evidence of the onset of oxygenation is still slow, despite the fact that the rate constants for reaction with O 2 (a 1 g ) are not negligible (e.g., ~ L mol -1 s -1 for 2,3-dimethyl-2-butene and ~ L mol -1 s -1 for methyl linoleate). 4 [These reaction rate constants were recorded for 2,3-dimethyl-2-butene and methyl linoleate dissolved in a solvent and, as such, are not likely to accurately represent what occurs in the neat and more viscous sample. However, we have independently shown that, for reactions involving O 2 (a 1 g ) with rate constants much smaller than those at the diffusion-controlled limit, an increase in sample viscosity results in an increase in the reaction rate constant. 5,6 ] After this initial 3 h irradiation, further changes in the IR spectra appear more rapidly (Figure 1). We infer that secondary reactions (e.g., radical propagation) accelerate the appearance of changes in the IR spectra. S4

5 Figure S1. FTIR absorption spectra of three separate samples over the range cm -1 (i.e., O-H stretch) as a function of the exposure time to O 2 (a 1 g ) generated upon irradiation of the S5

6 photosensitizer C 60 (0.02 mm of C 60 irradiated at 355 nm with ~30 mw average power). (a) 2,3- dimethyl-2-butene, (b) methyl linoleate, (c) cod liver oil. All oxidations were performed using the neat compound saturated with oxygen gas. For analysis, we diluted the samples with CS 2 to facilitate acquisition of the IR spectrum. The point about initial reactivity has important consequences with respect to monitoring autooxidation under non-accelerated conditions. We show a pertinent example in Figure S2 using cod liver oil. Only small changes are observed in the IR spectrum of an air-saturated solution of the oil after an incubation period of 20 days at 25 C. However, upon adding the radical initiator AIBN, pronounced changes in the IR spectrum are observed. These data clearly indicate the advantage of performing experiments under accelerated oxidation conditions and/or using an analysis technique that is more sensitive (e.g., fluorescence). Admittedly, the processes involved in an accelerated oxidation reaction may differ from those in an autooxidation reaction and, as such, results must be judiciously interpreted as not to infer an incorrect conclusion about the mechanism of autooxidation. S6

7 Figure S2. FTIR spectra of cod liver oil over the range cm -1 (i.e., O-H stretch). Data were recorded from (a) a fresh sample of the oil (black line), (b) a sample of the airsaturated oil that had been stored in the dark for 20 days at 25 C (red line), and (c) a sample into which AIBN had been added, that was maintained at 73 C for 24 h, followed by storage for 20 days in the dark at 25 C (green line). All experiments were performed using neat cod liver oil, which was then diluted with CS 2 to record the IR spectra. S7

8 Oxidation Monitored using Microscope-Based Raman Spectroscopy. Figure S3. Raman spectra of neat cod liver oil recorded over the range cm -1. The spectrum of a fresh, unoxidized sample is shown in black. Accelerated oxidation was initiated by adding AIBN to the oil (initial concentration of 1.2 mm) and then heating at 73 C for 1 day. The sample was then stored in the dark at room temperature for 20 days prior to, once again, recording the Raman spectrum (red trace). The I 1300 /I 1264 intensity ratio changes from 0.71 ± 0.07 to 0.91 ± 0.07 upon oxidation. S8

9 Documenting a Constant Yield of O 2 (a 1 g ) upon Spatially Localized Irradiation Figure S4. Fluorescence intensity of TMPyP recorded upon spatially localized irradiation of this O 2 (a 1 g ) sensitizer plotted against the elapsed irradiation time. The data were recorded from an oil-in-water emulsion, and the focused laser was positioned to irradiate the water soluble TMPyP at a spot immediately adjacent to the oil interface. The insets show images of the fluorescent spot observed at selected times of elapsed irradiation. In our system of spatially localized irradiation, even if sensitizer bleaching occurs, we presume that rapid exchange with the large volume of surrounding solvent will ensure that fresh sensitizer will always be irradiated. We tested this hypothesis using sensitizer fluorescence as a probe (Figure S4). Admittedly, the data in Figure S4 show a very slight systematic increase in the sensitizer fluorescence intensity as the elapsed irradiation time is increased. As also illustrated in Figure S4, the volume from which this fluorescence originates also systematically increases with an S9

10 increase in the elapsed irradiation time. Control experiments performed on simple aqueous solutions of this sensitizer do not show these same changes; it is unique to our emulsion system in which we irradiate a localized spatial domain close to the oil-water interface. These data could reflect a number of phenomena. For example, photooxidative degradation reactions that occur at the protein-covered oil surface may result in the ejection of debris into the surrounding water. Such debris might influence the extent to which light is scattered, and/or it might promote aggregation of the TMPyP leading to a change in its photophysical properties. In any event, this phenomenon only appears to be relevant at the latter period of prolonged irradiation and, as such, experiments can be adjusted accordingly. References (1) Garcia-Moreno, P. J.; Horn, A. F.; Jacobsen, C. J. Agric. Food Chem. 2014, 62, (2) Andersen, L. K.; Ogilby, P. R. Rev. Sci. Instrum. 2002, 73, (3) Scurlock, R. D.; Nonell, S.; Braslavsky, S. E.; Ogilby, P. R. J. Phys. Chem. 1995, 99, (4) Wilkinson, F.; Helman, W. P.; Ross, A. B. J. Phys. Chem. Ref. Data 1995, 24, (5) Ogilby, P. R.; Dillon, M. P.; Kristiansen, M.; Clough, R. L. Macromolecules 1992, 25, (6) Scurlock, R. D.; Kristiansen, M.; Ogilby, P. R.; Taylor, V. L.; Clough, R. L. Polym. Degrad. Stab. 1998, 60, S10