Unequivocal structural identification

The general procednre is to nse reconstrncted ion chromatograms at appropriate m/z values in an attempt to locate componnds of interest and then look at the mass spectrum of the unknown to determine its molecnlar weight. MS-MS can then be employed to obtain spectra from this and related compounds to find ions that are common to both and which may therefore contain common stmctmal features. Having the same m/z value does not necessarily mean the ions are identical and further MS-MS data or the elemental composition may be required. If these data do not allow unequivocal structure identification, then further MS" information may be required. 

These exceedingly unstable substances, which are invoked as the active intermediates in bioluminescence,20 were first prepared and isolated by Adam and Liu.2 The /-butyl system (2a) was the originally synthesized derivative by dehydrative cyclization of the a-hydroperoxy acid (6) by dicyclohexylcarbodiimide (DCC) [Eq. (5)]. It was not possible to isolate the pure material in view of its great thermal instability but its characteristic carbonyl band at 1875 cm-1 in the infrared (IR) served as unequivocal structure identification of these novel cyclic peroxides. 

Glassy state structural components distinction defines their behavior distinctions in both deformation [46] and relaxation [47] processes. It is known [48, 49], that in its turn polymers glassy state includes a substates number, differing by mechanical properties temperature dependences. A breaking (bend) on the corresponding parameter dependence, for example, of yield stress on temperature, is a typical indication of transition from one substate to another. At present unequivocal structural identification of these states is... 

Once a material is synthesized, it is necessary to establish which mesophases it forms and at what temperatures the transitions occur. Two techniques are used routinely in all laboratories for this purpose, namely polarized optical microscopy and differential scanning calorimetry, and it is important that these are used in conjunction with one another. In addition, the technique of X-ray scattering is often used to give unequivocal phase identification when microscopy cannot do so, and also to provide additional insights into the structures adopted. These three primary techniques will now be described in more detail. 

Although mass spectrometry (LC/MS and LC/MS/MS) is by far the most widely used technique for metabolite profiling and identification, it does not always provide sufficient information for unequivocal structural elucidation of metabolites. NMR spectroscopy is an excellent complementary tool for... 

FTIR and Raman spectroscopy are frequently used to characterize polymorphs. Both the molecular conformation and the crystal packing may lead to differences in the FTIR spectra of polymorphs the differences are more pronounced for compounds capable of hydrogen bonding. Characteristic shifts in C=0, N-H, and O-H stretching frequencies often lead to unequivocal polymorphic identification. Differences in fine structure and in the positions and intensities of IR bands enabled seven crystalline forms of the steroid prasterone 3 to be distinguished."" The presence of characteristic absorption bands due to included solvent molecules allows ready distinction between polymorphs and pseudopolymorphs. 

The mass spectrometer provides the most definitive identification of aU of the HPLC detectors. It allows the molecular weight of the analyte to be determined - this is the single most discriminating piece of information that may be obtained - which, together with the structural information that may be generated, often allows an unequivocal identification to be made. 

Proteolytic cleavage has proven to be an efficient tool for exploring the structure and function of the Na,K-ATPase. Exposure and protection of bonds on the surface of the cytoplasmic protrusion provides unequivocal evidence for structural changes in the a subunit accompanying E1-E2 transition in Na,K-ATPase [52]. Localization of the proteolytic splits provided a shortcut to identification of residues involved in E1-E2 transition [33,53,54] and to detection of structure-function correlations [33]. Further proteolysis identifies segments at the surface of the protein and as the cytoplasmic protrusion is shaved off all ATP-dependent reactions are abolished. 

In chromatography-FTIR applications, in most instances, IR spectroscopy alone cannot provide unequivocal mixture-component identification. For this reason, chromatography-FTIR results are often combined with retention indices or mass-spectral analysis to improve structure assignments. In GC-FTIR instrumentation the capillary column terminates directly at the light-pipe entrance, and the flow is returned to the GC oven to allow in-line detection by FID or MS. Recently, a multihyphenated system consisting of a GC, combined with a cryostatic interfaced FT1R spectrometer and FID detector, and a mass spectrometer, has been described [197]. Obviously, GC-FTIR-MS is a versatile complex mixture analysis technique that can provide unequivocal and unambiguous compound identification [198,199]. Actually, on-line GC-IR, with... 

Capillary HPLC-MS has been reported as a confirmatory tool for the analysis of synthetic dyes [585], but has not been considered as a general means for structural information (degradant identification, structural elucidation or unequivocal confirmation) positive identification of minor components (trace component MW, degradation products and by-products, structural information, thermolabile components) or identification of degradation components (MW even at 0.01 % level, simultaneous mass and retention time data, more specific and much higher resolution than PDA). Successful application of LC-MS for additive verification purposes relies heavily and depends greatly on the quality of a MS library. Meanwhile, MB, DLI, CF-FAB, and TSP interfaces belong to history [440]. 

The Most Intense Peaks. It is not so easy to extract valuable information dealing with the most intense peaks in mass spectra. In contrast to other physicochemical methods (IR, NMR, UV), registration of an ion peak of an integer m/z value does not provide an unequivocal identification of its structure or even composition. Even accurate mass measurements (high resolution mass spectrometry) defining the elemental composition of an ion does not establish its structure, as it could be formed directly from the M+, with minimal distortion of the authentic structure, or as a result of numerous rearrangement processes. 

A second method uses permethylation of the dephosphated (48% aqueous HF, 48 h, 4°C) and 2H-reduced fipid A. This approach allowed the assignment of amide-bound fatty acids linked to GlcN(I) and GlcN(II), as well as the identification of the backbone structure as a HexpN disaccharide (85). Mass-spectrometric analysis of the products was performed by using either a short g.l.c. column (0.3 X 5 cm) or by direct insertion-probe analysis (87). In the case of C. violaceum (85), the mass spectra obtained from the permethyl-ated HexpN disaccharide bearing attached TV-methylacyl residues revealed unequivocally that both amino groups carried 12 0(3-OH). 

Infrared Spectroscopy. The use of IR (9.10.11.12) and FTIR (3.4) for coal mineralogy has been reported. Painter and coworkers (3) demonstrated that FTIR can provide a virtually complete analysis. Painter, Brown and Elliott (4), and others (9.10.11) discuss sample preparation, reference minerals, and data analysis. The advantages of IR are 1) high sensitivity to molecular structure, 2) unequivocal identification of a number of minerals, 3) small sample size (a few milligrams), and 4) rapid analysis time (once LTA is available). Disadvantages include 1) reliance on reference minerals, 2) requires careful attention to sample preparation, and 3) limited selectivity (discrimination among similar minerals). 

Whereas the identification of a [1 + 1] or a [2 + 2] cyclization product was unequivocal by MS, it was more difficult to distinguish between the concave bimacrocycles and the bis-cyclophanes. But a combination of NOE investigations with complexation studies followed by NMR, and finally X-ray analyses proved the bimacrocyclic concave structure of the concave acids and bases 21,29 and 38 [15, 20, 27a]. 

Level II methods are those that are not unequivocal but are used to determine the concentration of an analyte at the level of interest, and to provide some structural information. For example, these methods may employ molecular, functional-group, or immunochemical properties as the basis of the analytical scheme. Hence, these methods are often reliable enough to be used as reference methods. Level II methods commonly separate the determinative from the identification procedures, and may also be used to corroborate the presence of a compound or class of compounds. Tims, a combination of two level II methods may provide attributes suitable for a level I method. The majority of analytical methods presently available and used by regulatory control agencies are level II methods. 

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