A Toast to Dynamic NMR Spectroscopy: Towards a Better Comprehension of Palatable Emulsions

VOL. 21 NO. 5 (2009) AARTICLERTICLE A toast to dynamic NMR spectroscopy: towards a better comprehension of palatable emulsions David Carteau, Isabelle Pianet and Dario M. Bassani Institut des Sciences Moléculaires, CNRS UMR 5255, Université Bordeaux 1, 351 Cours de la Libération, 33405 Talence France. E-mail: [email protected] Introduction and aniseed. One of the most intriguing cules that are in different environments.5 Memories of summer vacations on the properties of t-A, however, is its capabil- In many occurrences, however, the NMR Mediterranean would not be complete ity to undergo spontaneous emulsifica- spectra of emulsions are not very inform- without recollections of the refresh- tion upon rapid dilution of a concentrated ative due to inhomogeneous line-broad- ing anise-flavoured alcoholic beverages solution with a miscible but non-solubilis- ening of the signals. Surprisingly, this is served during the hot afternoons. Though ing solvent. This phenomenon, for which not the case for emulsions of t-A in water, varying in name and composition across the term “ouzo effect” has been coined though, as convincingly shown in Figure cultures (Raki in Turkey, Arak in Lebanon, by some authors,2 is responsible for the 1. The sharp and clearly distinguishable Ouzo in Greece, Sambuca in Italy and cloudy aspect of the above-mentioned signals are attributed to t-A located in Pastis in France), extracts from star anis beverages as the alcohol content of the different environments: a moderately (Illicium floridanum) are a common prin- hydro-alcoholic solution is dropped from polar aromatic environment within the cipal ingredient of the plant extracts used above 40% to ca 5% v/v and affects the aggregates, and a hydrophilic aqueous in their production. Besides their pungent photochemistry of t-A.3 Self-emulsification phase. Because the exchange between anis aroma, another well-known trade- phenomena are the subject of continuing these two environments is much slower mark of these drinks is their transition investigation due to their importance in than the frequency difference of the from a clear solution to an opalescent the chemical and pharmaceutical indus- chemical shift of the protons, the result- milky-white substance when they are tries, and yet more applications, such as ant spectrum shows a sharp signal for diluted with water. This sudden change the formation of nanoparticles and nano- each proton in either of the environ- in physical appearance will not go unno- capsules, are emerging. ments. ticed by a scientist, who may summarily In the case of t-A, dynamic light scat- The possibility of following spectro- assign it to the precipitation or separation tering (DLS) and small-angle neutron scopically the occurrence of t-A in the of the hydrophobic organic fragrances scattering (SANS) techniques have been aggregated and aqueous phase simul- due to the addition of water. We know employed to elucidate the origins of the taneously opens exciting possibilities this not to be entirely correct,1 however, self-emulsification process.4 However, to for further elucidating the emulsification as it should lead to a rather un-appetising obtain information on the origin of the process. For example, the quantification of biphasic mixture in which the essential droplets formed at the initial stages of the component in each phase—a some- oils would float on top of a large volume emulsification, it is necessary to employ times inextricable problem—becomes of water and alcohol. The truth behind techniques that are capable of directly trivial and can be performed in real-time, this apparently simple phenomenon is monitoring the exchange between the limited only by the rate of data acquisi- actually much more complex—and very free (dissolved) and the aggregated fraction of the spectrometer. But NMR spec- interesting indeed! tions of the immiscible component. For troscopy can do so much more than The principal component extracted this, nuclear magnetic resonance (NMR) take simple 1H spectra! By using specific from star anis is trans-anethole spectroscopy is particularly well adapted pulse sequences, for example, it is possi- (E-4-methoxyphenylpropene, t-A), as it is capable, in favourable cases when ble to generate spin populations whose which is a naturally-occurring flavour- the individual components possess exchange and decay can be followed. In ing compound present in numerous different spectral signatures, to directly this particular study, the use of exchange essential oils, including fennel, star anis monitor the exchange between mole- spectroscopy (EXSY) and diffusion- www.spectroscopyeurope.com SPECTROSCOPYEUROPE 11 VOL. 21 NO. 5 (2009) AARTICLERTICLE dynamic equilibrium.6 The principle is relatively simple: radiofrequency pulses are applied to create a magnetic non- equilibrium between exchanging species in chemical equilibrium. It is the subse- quent return to the magnetic equilibrium, which is under the influence of the chemical exchange, that 2D EXSY NMR permits one to observe in a particularly vivid representation. Let us take the case of a system in which two compounds are in exchange: free t-A resonating at ω1 and aggregated t-A resonating at ω2. The NMR sequence (Figure 2A) is composed of a first 90° pulse that creates the transverse spin magnetisation. In the course of the following evolution period t1, the transverse magnetisation vectors evolve at their characteristic resonance frequen- 1 cies (in our case ω1 for free t-A and ω2 for Figure 1. H NMR (400.13 MHz) of a 5 mM t-A solution in 5% EtOD / 95% D20 10 min after the mixture preparation. The signals assigned to the free (cyan) and aggregated (magenta) t-A are aggregated t-A) and become “frequency sharp and well separated. Residual solvent peaks are marked by an asterisk (*). labelled”. The exchange process predom- inantly takes place after the second 90° ordered spectroscopy (DOSY) proved Methods pulse, which replaces the magnetisation particularly useful to characterise the The EXSY (Exchange SpectroscopY) along the z axis (longitudinal magnetisa- kinetics of the exchange of t-A between NMR experiment is a powerful meth- tion) during the mixing time period tm. each sub-phase, and the growth of the odology to investigate exchange proc- The last 90° pulse is named the “read- aggregates, over time. esses in molecular systems under a ing pulse”, and rotates the magnetisation Figure 2. (A) EXSY sequence and spectrum of a hydroalcoholic solution of t-A 5 mM recorded with a 60 ms mixing time showing the interconversion between the free (cyan) and aggregated (magenta) forms of t-A. (B) DOSY sequence and spectra at different applied gradients (G), showing the decay of the free and aggregated forms. 12 SPECTROSCOPYEUROPE www.spectroscopyeurope.com VOL. 21 NO. 5 (2009) AARTICLERTICLE acteristic phase. Then two scenarios are possible. If no diffusion occurs, the 180° pulse cancels the effect of the two gradient pulses and all the spins will refo- cus leading to a maximum signal at the echo (i.e. at the end of the second τ period, see Figure 2B). But if the spins have moved during the Δ delay, the degree of dephasing will be proportional to the displacement of the spin during this delay, inducing an attenuation of the signal. The greater the diffusion, the larger the attenuation of the echo signal. This is illustrated in Figure 2B (right) in which the signal of free t-A is more atten- uated than the aggregated t-A.8 Results Figure 1 shows a proton spectrum of t-A obtained approximately 10 min after emulsification. According to expected pattern for the proton spectrum, two distinct sets of readily assigned reso- nances can be distinguished, suggest- ing that t-A can exists in at least two forms. Three types of NMR experiments confirm that these two forms correspond to free and aggregated t-A. First, EXSY experiments acquired on the mixture clearly demonstrate a slow exchange between the two species, confirming Figure 3. (A) 2-dimensional DOSY spectrum of a hydroalcoholic solution of t-A 5 mM, showing that the same compound is undergoing two distinct diffusion coefficient values allowing the assignment of the free (cyan) and aggre- exchange between two states. Second, gated (magenta) forms. (B) Evolution of the molar fraction of free (magenta) and aggregated DOSY experiments performed on the 1 (cyan) t-A over time as determined from solution H NMR. The proportion of t-A that in not mixture show that the two species detected (calculated from the conservation of mass) is shown in yellow and is attributed to t-A have very different diffusion coeffi- located in small droplets. cient values: about 6 × 10–10 m 2 s –1 and 1 × 10–10 m 2 s –1 allowing the unambig- uous assignment of the free and the in the x–y detection plane. The signal is ment of a translational diffusion coeffi- aggregated forms, respectively, as shown recorded all along the t2 period during cient (D), and thus allows differentiation in the DOSY spectrum displayed in Figure which the magnetisation components of species based on their diffusion in 2. Third, proton spectra recorded at differ- precess at their new resonance frequen- solution. The NMR pulse sequence, that ent ethanol concentrations (up to 60% cies owing to the exchange process. was proposed by Stejskal and Tanner in ethanol) show that only the free form The experiment is then repeated for a 1965,7 is based on a spin echo sequence of t-A is present in solutions containing number of equally spaced values of t1, in which two magnetic gradient pulses G, >40% ethanol. and the result is a data matrix s(t1, t2) for of δ duration and separated by a delay When water is added to an alcoholic which a double Fourier transform gives Δ, are inserted during the echo peri- solution of t-A, the mixture instantane- a 2D spectrum S(ω1, ω2).

Load more