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Left) array of 1H PFGSTE spectra measured with linearly increasing field gradient, for a mixture containing TSP, choline and acetone in D 2O, and (right) the resultant DOSY spectrum, showing TSP signals in blue, choline in green and acetone in red. I. Timari, C. Wang, A. L. Hansen, G. Costa Dos Santos, S. O. Yoon, L. Bruschweiler-Li and R. Bruschweiler, Anal. Chem., 2019, 91, 2304 CrossRef CAS PubMed . J. R. N. Haler, D. Morsa, P. Lecomte, C. Jérôme, J. Far and E. De Pauw, Methods, 2018, 144, 125–133 CrossRef CAS PubMed. T. Castaing-Cordier, D. Bouillaud, J. Farjon and P. Giraudeau, in Annual Reports on NMR Spectroscopy, ed. G. A. Webb, Academic Press, 2021, pp. 191–258 Search PubMed . J. Mauhart, S. Glanzer, P. Sakhaii, W. Bermel and K. Zangger, J. Magn. Reson., 2015, 259, 207 CrossRef CAS PubMed .

J. V. Duynhoven, E. V. Velzen and D. M. Jacobs, Annu. Rep. NMR Spectrosc., 2013, 80, 181 CrossRef . Interestingly, the relevance of UF 2D NMR for mixture analysis is not limited to single-scan implementations. In fact, UF 2D NMR spectra acquired in a single scan have limitations in terms of spectral width, resolution and sensitivity. These limitations can be alleviated through the acquisition of a few (typically 2 to 8) scans, that makes it possible to increase the accessible spectral width and/or the sensitivity. 150 Such hybrid UF 2D NMR experiments have proven particularly useful for quantitative analyses of complex mixtures for, e.g., metabolomics applications. 151–155 UF 2D NMR spectra are virtually free of t 1 noise, and this was found to improve the limit of quantification of signal-averaged UF 2D spectra compared to conventional 2D spectra. 151 After discussing the most relevant techniques for the structural characterisation of supramolecular molecules, we move to present three examples where supramolecular systems were successfully characterised by multi-technique approaches. In particular, we highlight the powerful interplay of the already discussed techniques while also mentioning the impact of other methods. 7 Host–guest complex formation of pyridine[4]arenes Molecular recognition describes the specific binding between two molecules via non-covalent interactions, for example between a host and a guest molecule. 80,81 This concept represents a major challenge in supramolecular chemistry because it is particularly important for catalytic processes and molecular sensors. In this context, binding preferences of macrocycles towards cationic, neutral or anionic guests are a critical aspect of fundamental supramolecular research. F. Zhang, S. L. Robinette, L. Bruschweiler-Li and R. Bruschweiler, Magn. Reson. Chem., 2009, 47(suppl 1), S118 CrossRef CAS PubMed . NUS has proven useful for the 2D experiments that are typically used for mixture analysis. 153,174–176 The number of points used to obtain a 2D spectrum with NUS cannot be taken arbitrarily low. Empirically, it was found that at least 64 points were needed in the case of HSQC and DQF-COSY experiments applied on an artificial mixture of 30 metabolites, designed to mimic the composition of human serum. 153 In this example, the resulting durations were of about 30 min for each experiment. This corresponds to an acceleration by a factor of 2 to 3 compared to the conditions that would typically have been used for such experiments. The quantitative information required for the subsequent statistical analysis was found to be preserved at such NUS levels, which makes them a relevant option for metabolomics applications. 153,176,177Nuclear magnetic resonance (NMR) spectroscopy is a powerful approach for the analysis of mixtures. 1,2 The most common NMR experiment has several essential features that are highly favourable for mixture analysis. It relies on a “pulse-acquire” pulse sequence, carried out at thermal spin equilibrium, to yield 1D spectra, most commonly for 1H spins. This experiment is simple to implement, it can be fast, and it already yields a wealth of structural information, which is useful to identify components. It is also non-invasive and non-destructive, meaning that the sample can be retrieved after analysis for storage or other analyses, and also, in the case of monitoring applications, that experiments can be repeated multiple times on the same sample as a function of time. 1D 1H NMR also provides straightforward access to quantitative information on concentrations.

Additional proof came from ion mobility mass spectrometry, which showed smaller CCS values for the species [ 17 2 − H] − and [ 17 2 + S − H] − compared to [ 17 2 + PF 6] − and [ 17 2 + PF 6 + S] − (with S = acetone, ΔCCS = 8 Å, Fig. 12A on the left side). This again underlines the exo complexation of PF 6 −, since the biggest change in CCS values should be caused by the species that is attached to the cavity rather than encapsulated in its interior. Moreover, the authors assumed a spherical shape for the molecules and calculated their diameter based on CCS values. For the dimeric species in general, the comparison of those values (between 2.2 and 2.3 nm) with the hydrodynamic radius obtained from DOSY NMR (2.0 nm) and with the X-ray crystal structure (1.9 nm) proved similar structures of the host–guest complex in solution, gas phase and in the solid state. Complexity The most commonly used NMR experiment for mixture analysis is the 1D 1H pulse-acquire experiment. Protons have a spin I = 1/2, over 99% natural abundance, and the largest gyromagnetic ratio among stable nuclei, resulting in favourable sensitivity from an NMR point of view. However, the chemical-shift dispersion in 1H spectra is limited to 10–15 ppm. In addition, the large abundance of 1H nuclei result in the ubiquitous presence of 1H– 1H couplings, that yield diverse multiplet structures and broaden NMR signals. Overall, the spectra of all but the simplest mixtures are usually complex. Y. Ben-Tal, P. J. Boaler, H. J. A. Dale, R. E. Dooley, N. A. Fohn, Y. Gao, A. Garcia-Dominguez, K. M. Grant, A. M. R. Hall, H. L. D. Hayes, M. M. Kucharski, R. Wei and G. C. Lloyd-Jones, Prog. Nucl. Magn. Reson. Spectrosc., 2022, 129, 28 CrossRef CAS PubMed . C. Lhoste, B. Lorandel, C. Praud, A. Marchand, R. Mishra, A. Dey, A. Bernard, J.-N. Dumez and P. Giraudeau, Prog. Nucl. Magn. Reson. Spectrosc., 2022, 130–131, 1 CrossRef CAS PubMed .L. Castanar, M. Perez-Trujillo, P. Nolis, E. Monteagudo, A. Virgili and T. Parella, Chem. Phys. Chem., 2014, 15, 854 CrossRef CAS PubMed . B. Gouilleux, B. Charrier, S. Akoka, F.-X. Felpin, M. Rodriguez-Zubiri and P. Giraudeau, TrAC, Trends Anal. Chem., 2016, 83, 65 CrossRef CAS .