Structural elucidation of unknowns

Hyphenation of chromatographic separation techniques (SFC, HPLC, SEC) with NMR spectroscopy as a universal detector is one of the most powerful and time-saving new methods for separation and structural elucidation of unknown compounds and molecular compositions of mixtures [171]. Most of the routinely used NMR flow-cells have detection volumes between 40... 

GC-AAS has found late acceptance because of the relatively low sensitivity of the flame graphite furnaces have also been proposed as detectors. The quartz tube atomiser (QTA) [186], in particular the version heated with a hydrogen-oxygen flame (QF), is particularly effective [187] and is used nowadays almost exclusively for GC-AAS. The major problem associated with coupling of GC with AAS is the limited volume of measurement solution that can be injected on to the column (about 100 xL). Virtually no GC-AAS applications have been reported. As for GC-plasma source techniques for element-selective detection, GC-ICP-MS and GC-MIP-AES dominate for organometallic analysis and are complementary to PDA, FTIR and MS analysis for structural elucidation of unknowns. Only a few industrial laboratories are active in this field for the purpose of polymer/additive analysis. GC-AES is generally the most helpful for the identification of additives on the basis of elemental detection, but applications are limited mainly to tin compounds as PVC stabilisers. 

There are many software tools available to help with the acquisition, processing and interpretation of NMR data. Attempts have been made to automate the verification process and even perform full structural elucidations of unknown compounds. As you might guess from the complexity of the interpretation chapters, these software solutions are not foolproof It remains to be seen whether they ever will be good enough but there have certainly been some major steps forward in all of these areas. 

Of particular importance with the use of LC-NMR as an experimental technique is that it is suited for only a limited number of applications in reference to structure elucidation. As will be discussed in greater detail, the sensitivity issues that arise between the amount of compound one is able to load onto a particular chromatographic stationary phase, and hence elute into the flow-cell of an LC-NMR probe, limit what type of structural analysis that can be performed. It is this author s current opinion that most complete structure elucidations of unknown molecular entities are not amenable to LC-NMR. In these... 

The detection system should assist in the structural elucidation of unknown organic compounds. Moreover, accurate quantitative determinations should be feasible. 

Mass spectrometry is an indispensable analytical tool in chemistry, biochemistry, pharmacy, and medicine. No student, researcher or practitioner in these disciplines can really get along without a substantial knowledge of mass spectrometry. Mass spectrometry is employed to analyze combinatorial libraries [1,2] sequence biomolecules, [3] and help explore single cells [4,5] or other planets. [6] Structure elucidation of unknowns, environmental and forensic analytics, quality control of drugs, flavors and polymers they all rely to a great extent on mass spectrometry. [7-11]... 

Hyphenated analytical techniques such as LC-MS, which combines liquid chromatography and mass spectrometry, are well-developed laboratory tools that are widely used in the pharmaceutical industry. Eor some compounds, mass spectrometry alone is insufficient for complete structural elucidation of unknown compounds nuclear magnetic resonance spectroscopy (NMR) can help elucidate the structure of these compounds (see Chapter 20). Traditionally, NMR experiments are performed on more or less pure samples, in which the signals of a single component dominate. Therefore, the structural analysis of individual components of complex mixtures is normally time-consuming and less cost-effective. The... 

The possibilities for structural elucidation of unknowns are restricted in particular, the distinction between isomers is only possible in exceptional cases. 

The main, and most crucial, part of the experimental set-up is the probe, which has to be pressure-and temperature-stable at the same time and must have a flow cell with an inlet and an outlet to supply and take away the samples. For the development of SFC-NMR probes, one has to keep in mind the aim of the application, i.e. the structure elucidation of unknown compounds. For the investigation of new, unknown substances, the resolution of the probe has to... 

In order to carry out complete structural elucidation of unknown compounds (especially for complex molecules), the RF probe should enable a variety of heteronuclear NMR techniques to be performed. In particular, inverse detection H-15N and 1H-13C experiments such as heteronuclear multiple quantum coherence (HMQC) [29,30] and heteronuclear single quantum coherence (HSQC) [31] find almost ubiquitous application in myriad research environments. Although the microliter-scale probes described above feature both heteronuclear and homonuclear capabilities, no commerical product is... 

Structure elucidation of unknown organic compounds is usually performed by the combined use of H and 13C NMR spectroscopy. The latter spectroscopy provides direct information about the carbon skeleton of the investigated molecule, thus revealing valuable structural features such as carbonyl and carboxyl moieties, which cannot be deduced by H NMR spectroscopy. 

Fig. 6 Structural elucidation of unknown metabolites. The combination of accurate mass (a), which provides molecular formulas, and tandem MS (b) enables the development of hypotheses of metabolite structures, which can subsequently be validated through chemical synthesis...

At sufficient concentration and in the absence of disturbing background resonances, NMR is the superior method both for the identification of known chemicals and for the structural elucidation of unknown chemicals. Its usefulness in identification is attributable to the fingerprint nature of spectra, while the usefulness in structural elucidation rests on the structural specificity of the spectra. The wide variety of routine 1-D and 2-D experiments available is of assistance in both identification of chemicals and structure elucidation. 

In the field of CWC-related analysis, the chemicals should be identified by comparing their spectra to spectral libraries or to spectra measured from authentic chemicals. In infrared spectroscopy of CWC-related chemicals, the spectral interpretation is not enough for unambiguous identification, but it is an important tool in the structural elucidation of unknown chemicals. 

Levsen K, Preiss A, Spraul M. 2003. Structure elucidation of unknown pollutants of environmental samples by coupling HPLC to NMR and MS. In Namiesnik J, Chrzanowski W, Zmijewska P, editors, New horizons and challenges in environmental analysis and monitoring, Workshop, Gdansk (PL), August 18-29. p 150-180. 

Although low in identification value compared to other spectroscopic methods, UV spectra may be used to assist in structural elucidation of unknown peaks. 

Ultimately, the definitive structure elucidation of unknown molecules is most often accomplished via NMR. NMR has traditionally been performed on the purified natural product isolated using bioassay-guided fractionation. With the advent of hyphenated techniques such as LC/NMR, these data can now be obtained prior to purification [130,131]. LC/NMR can prove useful even in the dereplication phase, particularly when LC/UV/MS data are insufficient for unambiguous peak identification. LC/NMR has played an important role in natural products structure elucidation, where several related compounds (factors) are often encountered in a single sample. For example, isobaric or isomeric mixtures that may prove difficult or impossible to differentiate by MS, can often be readily distinguished by NMR. Several thorough reviews of LC/NMR in natural products discovery and phytochemical analysis have recently appeared [132,133]. 

In conclusion, parallel synthesis and more generally, combinatorial chemistry, seems to be an attractive approach in aroma research to help identifying new odorants with interesting sensory properties. It allows rapid synthesis of a large number of components in a reasonable time. The reference compoimds can be very usefril for structure elucidation of unknown odorants. 

Mass spectrometry is an excellent technique for both qualitative and quantitative analysis. On the basis of molecular weight alone, many compounds can be identified (with the exception of isomers). Using a combination of molecular weight and fragmentation patterns, unambiguous identification is possible as well as structural elucidation of unknown compounds. MS can also be used for quantitative work using either internal or external standards for reference. Limits of detection are very low so sensitivity is normally excellent. 

There are two limitations of electron impact ionization, which in other ways is nearly an ideal ion source for identification of organic compounds. The sample must be stable as neutral molecules in the gas phase prior to ionization. There are many compounds, however, that are simply not stable at the temperatures required for vaporization. Radical molecular ions are formed with excess energy that may be sufficient for complete elimination of molecular mass information from the mass spectrum, even at low electron energies (e.g. 15-20 eV). Molecular mass information is almost always required for structure elucidation of unknown compounds. A softer ionization technique, such as chemical ionization, may be used in these circumstances to establish the compound molecular mass. 

Tandem mass spectrometry or ms/ms was first introduced in the 1970s and gained rapid acceptance in the analytical community. The technique has been used for structure elucidation of unknowns (26) and has the abflity to provide sensitive and selective analysis of complex mixtures with minimal sample clean-up (27). Developments in the mid-1980s advancing the popularity of ms/ms included the availabiUty of powerfiil data systems capable of controlling the ms/ms experiment and the viabiUty of soft ioni2ation techniques which essentially yield only molecular ion species. 

Mass spectrometry is today the method used most frequently for identification and structure elucidation of unknown compounds. GC/MS investigations have been used in the combination with thermal desorption as extraction procedure, for the analysis of the dichloromethane extract and for the derivatization products (Thurow et al., 1997). Investigations have been carried out with an HP 5973 MSD (Hewlett Packard Inc.) in combination with an cold injection system CIS 3 (Gerstel GmbH, Germany). 

---