Unknown, identification with proton

As noted above, whole-cell MALDI-TOF MS was intended for rapid taxonomic identification of bacteria. Neither the analysis of specific targeted bacterial proteins, nor the discovery of new proteins, was envisioned as a routine application for which whole cells would be used. An unknown or target protein might not have the abundance or proton affinity to facilitate its detection from such a complex mixture containing literally thousands of other proteins. Thus, for many applications, the analysis of proteins from chromatographically separated fractions remains a more productive approach. From a historical perspective, whole-cell MALDI is a logical extension of MALDI analysis of isolated cellular proteins. After all, purified proteins can be obtained from bacteria after different levels of purification. Differences in method often reflect how much purification is done prior to analysis. With whole-cell MALDI the answer is literally none. Some methods attempt to combine the benefits of the rapid whole cell approach with a minimal level of sample preparation, often based on the analysis of crude fractions rather... 

In the case of an unknown chemical, or where resonance overlap occurs, it may be necessary to call upon the full arsenal of NMR methods. To confirm a heteronuclear coupling, the normal H NMR spectrum is compared with 1H 19F and/or XH 31 P NMR spectra. After this, and, in particular, where a strong background is present, the various 2-D NMR spectra are recorded. Homonuclear chemical shift correlation experiments such as COSY and TOCSY (or some of their variants) provide information on coupled protons, even networks of protons (1), while the inverse detected heteronuclear correlation experiments such as HMQC and HMQC/TOCSY provide similar information but only for protons coupling to heteronuclei, for example, the pairs 1H-31P and - C. Although interpretation of these data provides abundant information on the molecular structure, the results obtained with other analytical or spectrometric techniques must be taken into account as well. The various methods of MS and gas chromatography/Fourier transform infrared (GC/FTIR) spectroscopy supply complementary information to fully resolve or confirm the structure. Unambiguous identification of an unknown chemical requires consistent results from all spectrometric techniques employed. 

We have only begun to explore the intricate world of identification of structure by spectroscopy. It is important that you recognize that structures are assigned, not because of some theoretical reason or because a reaction ought to give a certain product, but because of so mid evidence from spectra. You have seen three powerful methods—mass spectra, l3C NMR, and IR spectroscopy in this chapter. In Chapter 11 we introduce the most important of all—proton H) NMR and, finally, in Chapter 14 we shall take each of these a little further and show how the structures of more complex unknown compounds are really deduced. The last problem we have discussed here is not really solvable without proton NMR and in reality no-one would tackle any structure problem without this most powerful of all techniques. From now on spectroscopic evidence will appear in virtually every chapter. Even if we do not say so explicitly every time a new compound appears, the structure of this compound will in fact have been determined spectroscopically. Chemists make new compounds, and every time they do they characterize the compound with a full set of spectra. No scientific journal will accept that a new compound has been made unless a full description of all of these spectra are submitted with the report. Spectroscopy lets the science of organic chemistry advance. 

Unnatural Products Chemistry. The complete identification of unknown compounds that we have successfully resolved using PB/LC/MS will clearly require additional analytical information, such as provided via liquid chromatography ICP/MS (detecting nonmetals such as chlorine and sulfur), FT-IR, UV or proton and heteroatom NMR. This situation is analogous to that of a natural products chemist faced with making a complete structural assignment of an unknown compound isolated from some matrix such as seaweed instead of a leachate from a hazardous waste site. The natural products chemist would exploit the complete array of analytical instrumentation and not attempt identification based solely upon low resolution (quadrupole) mass spectrometry. 

Identification of unknown compounds NMR spectroscopy provides the forensic analyst with one of the most powerful techniques for identification of unknown compounds. The full range of structural elucidation techniques of modern spectrometers is available. First, the analyst obtains a high-resolution proton (NMR) spectrum in an appropriate deuterated solvent. The chemical shifts and integration in the spectrum give an indication of the types (aliphatic, olefinic, aromatic, etc.) and relative numbers of protons present in the molecule. The appearance of the coupling patterns in the molecule often provides very useful structural information. If the identity of the unknown cannot be determined from the results of the NMR study alone, the analyst next obtains information. The NMR spectrum gives a count of the number of nonequivalent carbon atoms, as well as the types of carbon (aliphatic, aromatic, carbonyl, etc.) present in the unknown. The number of protons attached to each carbon may... 

Finally, one of the most difficult and challenging aspects in this field is to address the analysis of nontarget pesticide residues [30]. Eor this kind of analysis HRMS combined with databases and accurate mass measurements to achieve confirmation and identification are necessary. This approach works quite well for nontarget and known pesticide residues, thus, a standard is available for identification. However, for unknown substances, confirmation and identification are more difficult and will usually require HRMS and accurate mass measurements of both the protonated molecule and its fragment ions. 

NMR spectroscopy is very useful for identifying organic compounds, provided that they can be obtained in a reasonably pure state. Kdnig has published a table of chemical shifts of functional groups found in common surfactants (39). This allows use of proton magnetic resonance to identify components of commercial products, where the range of possible structures is limited. Carminati and coworkers recommend the use of C NMR for the identification of unknown surfactants, both alone and in formulated products. With experience, not only the surfactants, but other components of products can be identified (40). 

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