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J. Farjon, C. Milande, E. Martineau, S. Akoka and P. Giraudeau, Anal. Chem., 2018, 90, 1845 CrossRef CAS PubMed . B. Shapira, A. Karton, D. Aronzon and L. Frydman, J. Am. Chem. Soc., 2004, 126, 1262 CrossRef CAS PubMed . Introduction Starting with the discovery of the inclusion complexes in the late 19th century, supramolecular chemistry developed into an independent field of research until the mid-1960s. 1 From then on, the discipline received considerably more attention culminating in the 2016 chemistry Nobel Prize for molecular machines. Supramolecular chemists initially were driven by the synthesis of ‘chemically beautiful’ molecules, but more recently there has been a shift in focus to synthesise compounds with potential application in a range of disciplines including medicine, 2,3 catalysis 4,5 and materials science. 6 A possible approach to improve the separation of overlapping spectra is to use multivariate processing methods. They consist of algorithmically solving the problem:

A. M. R. Hall, P. Dong, A. Codina, J. P. Lowe and U. Hintermair, ACS Catal., 2019, 9, 2079 CrossRef CAS . L. Rouger, B. Gouilleux, M. Pourchet-Gellez, J. N. Dumez and P. Giraudeau, Analyst, 2016, 141, 1686 RSC .

S. J. Elliott, Q. Stern, M. Ceillier, T. El Daraï, S. F. Cousin, O. Cala and S. Jannin, Prog. Nucl. Magn. Reson. Spectrosc., 2021, 126–127, 59 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. 153,176,177 Another interesting analysis, arguably also related to the structural characterisation of 18 and 19 in their aggregated forms, is the determination of the average number of monomers in a polymer (polymerisation degree). The authors showed that this is possible with fluorescence spectroscopy, because they were able to assign one peak/shoulder (≈620 nm) to the monomers and a second band (≈700 nm) to the assemblies. Due to broad overlapping signals, reliable deconvolution was not possible for the assembly of 18. However, the authors obtained average polymerisation degrees between 10 and 140 for 19 in solution, with the value depending on the concentration and the temperature of the system. These studies shed light on thermodynamic properties of polymeric 19, but further details are beyond the scope of this review. Cryo-TEM have had a major impact on the structural characterisation of biological compounds, however its use for supramolecular molecules has not received as much attention. 71 Compared to traditional TEM, cryo-TEM facilitates the resolution of structures without staining, which can be necessary to enhance the contrast between sample and substrate. Staining is preferentially avoided as it can cause structural changes, but even without it, for TEM each sample has to be dried onto a substrate, which can lead to artefact inclusion. 72 Avoiding both staining and drying is therefore a useful improvement for an unambiguous characterisation of supramolecular molecules, which can readily be achieved by cryo-TEM. 71,72

L. L. Fillbrook, M. D. Nothling, M. H. Stenzel, W. S. Price and J. E. Beves, ACS Macro Lett., 2022, 11, 166 CrossRef CAS PubMed .I. Reile, R. Aspers, J. M. Tyburn, J. G. Kempf, M. C. Feiters, F. Rutjes and M. Tessari, Angew. Chem., Int. Ed., 2017, 56, 9174 CrossRef CAS PubMed .