Introduction Status of the molecular-level analysis of complex materials 1 1 4 Fig. 1 de novo structural analysis 5 2  Fig. 2 2 1 6  Fig. 3 7 9 10 12 13 16 17 18 19 22 23 24 25  1 2 26 27 nonrepetitive complex systems. 28 29 9 18 18 30 35 20 36 40 quantitative 3 Consequently, any comparative analysis of nonrepetitive unknowns with reference materials is very unlikely to provide satisfactory molecular resolution, because rather tiny variations in chemical binding may strongly and often unpredictably affect the properties commonly used for detection, such as retention times and spectral signatures. These fundamental restrictions that are intrinsic to comparative and target analysis are not easily circumvented and they necessitate an independent, spectroscopic “bottom-up” approach to the molecular-resolution characterisation of these complex unknowns. Information transfer in organic structural spectroscopy and separation technologies 41 4 Fig. 4 x y n n x y a b c d C D 12 \documentclass[12pt]{minimal}
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\begin{document}$$n = 8 - 14$$\end{document}  42 43 4 5 13 44 45 46 53 Due to the huge peak capacity of FTICR mass spectrometry, FT mass spectra provide the most convincing direct experimental evidence for the extraordinary molecular diversity of complex materials at present. In these, the molecular-level intricacy of the complex unknowns is most adequately converted into very highly resolved and, consequently, extremely information-rich signatures. 54 55 56 Instrumentation and methods 13 7 13 9 8 8 9 5 8 5 8 8 9 36 Fig. 5 57 12  11 58 1 13 1 6 6 60 86 19 6 Fig. 6 top panels molecules ions middle panels bottom panels 5  High-precision frequency-derived organic structural spectroscopy 1 6 7 B 0 59 6 7 7 60 62 63 64 65 1 13 15 31 66 63 67 70 71 7 8 9 10 11 Fig. 7 60 200 72 6 compositional space 13 9 9 3 13 9 9 3 6 12  Fig. 8 9  Fig. 9 8  Fig. 10 73 11  Fig. 11 left n m q 4 4 18 green circumfenced area 9 right n m q 6 8  Table 1 Characteristics and significance of key molecular-level resolution techniques Molecular-level resolution technique Advantages Current weaknesses and future developments NMR spectroscopy 33 74 35 Relative insensitivity compared with other analytical techniques 32 75 81 Intricate physics and chemistry of intra- and intermolecular interactions in complex mixtures may interfere with the direct relationship between chemical shift and molecular structure and, because of relaxation-induced variable line widths, quantification 82 84 85 1 13 15 31 86 87 88 89 92 93 94 95 99 FTICR mass spectrometry 100 Molecular-level structural information is mainly restricted to ionizable compounds 63 69 101 104 71 Fragmentation provides further molecular-level structural information beyond molecular composition Quantification is difficult, even for identical molecules in mixtures, because of the variable ionization efficiencies of individual compounds, which strongly depend on the experimental conditions and mixture composition Column adsorption and fractionation as well as electrochemical and redox reactions associated with the spray conditions may interfere with authentic sample representation 105 110 Mass-selective imaging is feasible with high spatial and mass resolution; qTOF mass spectrometry allows for very fast scan rates, and is perfectly suited for hyphenation with high-performance separation techniques (CE and UPLC) as well as mass-selective imaging 100 High-performance separation techniques (UPLC/HPLC and capillary electrophoresis) Large separation capacity and extensive miniaturisation; is cost-effective; can be highly automated 111 112 Electrophoretic mobility and chromatographic retention time carry structure-specific information, which can be adapted to a wide range of experimental conditions in order to probe size, shape, charge characteristics and reactivity Sensitive and versatile suite of separation methods and of structure-specific (and nondestructive) detection systems, such as (laser-induced) fluorescence, UV/VIS, radioisotope or mass-selective detection CE complements NMR information about primary chemical structures (covalent bonds) by providing data on the corresponding secondary and tertiary structure Feasibility of up-scaling from capillary zone electrophoresis (CZE) to a preparative level by means of free flow electrophoresis (FFE) and from UPLC to any preparative LC method Further miniaturisation offers hyphenation options down to single-cell analysis and compartments within 60 200 72 6 compositional space 8 9 10 11 73 113 114 6 7 115 117 12 4 12 Fig. 12 volumetric pixel (voxel) space 8–14 2–5 4–5 2–4 5 104 115 117 120 121 122  8 8 n m q 4 8 8 + − 8 8 + 8 m 8 8 n m q 8 11 9 n m q 123 n m q 10 11 11 6 7 Key trends relating molecular composition to the number of feasible isomers For any exceedingly complex material, it is logical to postulate that many isomers will contribute to any given molecular formula. Analogously, the intensities of the mass spectral peaks, which superimpose all of the isomers present, will be a function of the abundances of these isomers in these materials and the ionization efficiency of each isomer under the given experimental conditions. n 2n+2 37 11 n m q These considerations imply that the number of feasible C,H,O-isomers for a given mass will reach maximum values at intermediate H/C and O/C ratios, and that these numbers will (sharply) decline at extreme (high and low) H/C and O/C ratios, respectively. 11 n m q 58 11 n m q 4 11 Series 1 represents highly unsaturated molecules in which the number of isomers declines sharply with decreasing H/C ratio. Series 2 presents the maximum number of isomers at intermediate H/C (and O/C) ratios and the decline in the number of isomers at both high and low H/C (and O/C) ratios, as anticipated (see above). 13 22 13 28 13 22 5 14 10 6 14 30 13 22 12 18 7 11 14 2 10 10 3 4 10 10 3 9 6 4 9 6 4 8 2 5 7 14 5 6 10 6 8 2 5 7 14 5 14 10 6 10 10 3 7 6 10 6 4 10 10 3 6 10 6 Mass spectral intensities and number of feasible isomers in marine and terrestrial NOM n m q 18 20 18 n m q 11 9 18 18 124 9 11 18 18 n m n m q 9 11 9 The discrete analytical volumetric pixel space defines our current capacity to depict molecular-level variance in complex systems 8–14 2–5 4–5 2–4 119 125 128 129 130 119 121 129 84 131 134 135 140 141 142 143 144 5 12 145 147 Electronic supplementary material Below is the link to the electronic supplementary material.
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