Spectra and Reference Library

It is a well-known fact that the ability to perform a thorough search of the literature is of great importance to the research chemist of similar importance to the spectroscopist is the library of reference spectra that he must acquire and maintain. The ability to locate a spectrum quickly and to identify an unknown by comparison of its spectrum with those on file makes spectroscopy one of the most important analytical and research tools a chemistry laboratory can possess. 

The spectrograms for this library may be obtained by purchase of those commercially available. Of greater importance than these commercially available spectra are the reference spectra run in the user s own laboratory. These latter references are more likely to reflect the specific products and research projects of interest to that particular laboratory. 

This chapter is designed to acquaint the reader with the various spectral reference systems commercially available, and to present some suggestions that may be valuable in maintaining a useful spectrum library. 

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Plenary 15. B Scluader et al, e-mail address beriilrard.scluader uni-essen.de (NIR-FTRS). A review of the use of Raman spectroscopy in medical diagnostics. Its possibilities, limitations and expectations. Emphasizes the need for a library of reference spectra and the applications of advanced analysis (chemometry) for comparing patient/library spectra. 

With the availability of computerized data acquisition and storage it is possible to build database libraries of standard reference spectra. When a spectrum of an unknown compound is obtained, its identity can often be determined by searching through a library of reference spectra. This process is known as spectral searching. Comparisons are made by an algorithm that calculates the cumulative difference between the absorbances of the sample and reference spectra. For example, one simple algorithm uses the following equation... 

When considering libraries of spectra for identification purposes, the effect of sample preparation on spectral characteristics is also important. Two FUR sampling methods have been adopted for IR analysis of TLC eluates in the presence of a stationary phase, namely DRIFTS [741] and PAS [742], of comparable sensitivity. It is to be noted that in situ TLC-PA-FTIR and TLC-DRIFT spectra bear little resemblance to KBr disc or DR spectra [743,744]. This hinders spectral interpretation by fingerprinting. For unambiguous identification, the use of a reference library consisting of TLC-FTIR spectra of adsorbed species is necessary. 

Over a long time period it may well not be possible to duplicate library cell culture conditions. What happens when the lot of media used in the final culture step prior to pyrolysis has been consumed Can culture media suppliers assure nutritional identity between batches Media types for growth of fastidious strains invariably include natural products such as brewer s yeast, tryptic soy, serum, egg, chocolate, and/or sheep blood. Trace components in natural products cannot be controlled to assure an infinite, invariable supply. The microtiter plate wells used here do not hold much media. Even so, the day will come when all media supplies are consumed and a change in batch is unavoidable. When that happens, if there were no effective way to compensate spectra for the resulting distortions, it would be necessary to re-culture and re-analyze replicates for every strain in the reference library. Until recently the potential for obsolescence was a major disincentive for developing PyMS spectral libraries of bacteria. Why this is no longer an insurmountable problem is discussed in the next section. 

The analysis of chemicals by reference to a set of library mass spectra was facilitated by the establishment almost 40 years ago of databases such as the NIST/EPA/NIH reference library of electron impact mass spectra (http //www.nist.gov/srd/nistla.htm). Experimentally derived mass spectra are compared to spectra in the library, and the matches are graded by various algorithms. This comparison is valid because electron impact ionization requires that the sample be vaporized and thus isolated from its sampling history. 

Mass spectra of numerous single compounds are available in reference libraries. However these spectra have not always been obtained in the same conditions as those used in DE or DI EI-MS modes and the spectra of molecules of specific interest in the field of cultural heritage have not been systematically registered. It is thus of importance to achieve mass spectra on a set of standard molecular constituents in order to study their mass spectral fingerprint in detail before investigating the more complex mass spectra of multicomponent materials. 

Today, GC-MS (see Section 4.1.1) is a golden standard for detection and quantification of drugs and poisons volatile under GC conditions, whereas nonvolatile compounds require LC-MS (see Section 4.1.2). The GC-MS technique is much more popular for identification purposes than LC-MS, because of the easy availability of the reference mass spectra for many xenobiotics and their derivatives, either in printed or computer form. The most popular libraries are the NIST library, which contains the mass spectra of 130,000 compounds, the Wiley Registry of Mass Spectral Data, which contains 390,000 reference spectra, and the Pfleger-Maurer-Weber library, with 6,300 mass spectra and other data, such as chromatographic retention indexes. 

The importance of an appropriate transformation of mass spectra has also been shown for relationships between the similarity of spectra and the corresponding chemical structures. If a spectra similarity search in a spectral library is performed with spectral features (instead of the original peak intensities), the first hits (the reference spectra that are most similar to the spectrum of a query compound) have chemical structures that are highly similar to the query structure (Demuth et al. 2004). Thus, spectral library search for query compounds—not present in the database—can produce useful structure information if compounds with similar structures are present. 

From the daughter spectra of di-n-octylphthalate, we were able to determine two spectrum/substructure correlations the 149+ daughter spectrum to structure I in Figure 3 and the 105+ daughter spectrum to structure III in Figure 3. In order to obtain spectrum substructure relationships for the alkyl portions of the reference molecule di-n-octylphthalate, we would then match other portions of the complete MS/MS spectrum against those of compounds containing alkyl substructures. However, this portion of the reference library has not yet been developed. Thus, to complete the structure elucidation we have used standard methods of spectral interpretation (11). As will be shown, these methods can also lead to useful spectrum/substructure relationships. 

The bottleneck in utilizing Raman shifted rapidly from data acquisition to data interpretation. Visual differentiation works well when polymorph spectra are dramatically different or when reference samples are available for comparison, but is poorly suited for automation, for spectrally similar polymorphs, or when the form was previously unknown [231]. Spectral match techniques, such as are used in spectral libraries, help with automation, but can have trouble when the reference library is too small. Easily automated clustering techniques, such as hierarchical cluster analysis (HCA) or PCA, group similar spectra and provide information on the degree of similarity within each group [223,230]. The techniques operate best on large data sets. As an alternative, researchers at Pfizer tested several different analysis of variance (ANOVA) techniques, along with descriptive statistics, to identify different polymorphs from measurements of Raman... 

An excellent collection of NMR and IR spectra for reference purposes is found in The Aldrich Library of 13 C and 1H FT NMR Spectra and The Aldrich Library of Infrared Spectra. Both are available in most libraries and the former can be purchased from Aldrich Chemical Co. A variety of compilations of mass spectral data are available seeF. S. McLafferty and D. B. Stauffer, The Wiley/NBS Registry of Mass Spectral Data, 5th ed., Wiley Interscience, New York, 1988. 

These same analysis techniques can be applied to chemical imaging data. Additionally, because of the huge number of spectra contained within a chemical imaging data set, and the power of statistical sampling, the PLS algorithm can also be applied in what is called classification mode. In this case, the reference library used to establish the PLS model is... 

The compounds were identified by comparison of the GC retention indices and mass spectra with reference data from authentic components and with data of our own MS library. Detailed spectral data will be published separately. ... 

They generally lack sensitivity and a direct relation to molecular structure. GC-MS is fast, direct, and very sensitive, and the spectrum provides a result which puts identification beyond dispute. Computer-assisted systems are now available which embody extensive drug reference libraries and can be automatically searched to identify unknown spectra. The further development of chemical ionization and mass fragmentography methods using stable isotopes now permits very accurate quantitative work. 

All major mass spectral data collections consist of El mass spectra, mostly recorded under accepted standardized conditions such as an ionization voltage of 70 eV, an emission current of 100-200 xA, and an ion source temperature of 150-200°C. Several types of GC/MS systems may be applied, for instance, magnetic sector, quadrupole, or ion trap analyzers. Ion trap systems are considered less applicable, when data comparison is required with spectra from a reference library. In particular, basic compounds related to VX or the three nitrogen mustards tend to produce protonated molecular ions by self-protonation. Magnetic sector and quadrupole mass spectrometers suffer less from interference of self-protonation, and spectra produced with these types of instruments are generally reproducible. 

Search algorithms have advanced over the years to the point that most of the spectral data are used in the search. The methods are referred to as full-spectra searches because the entire spectral pattern is used in the matching procedure. Again, a number of similarity metrics are used, but most produce similar results. Typically, the spectral range for the search is selectable, and the library and target spectra are all normalized so that the total spectral area is 1.0. Either the Euclidean distance or the dot product between the target and library entries is calculated. The Euclidean distance is defined as... 

Without doubt, however, the greatest difference between the two is that the sophisticated electronics of the newer instrument enable it to be directly connected to an Infrared Data Station , a microcomputer system which can smooth spectra, produce an average spectrum from a number of scans, subtract one spectrum from another, or reformat a spectrum. In addition, it is possible to identify possible structural features of an unknown compound and to match the spectrum against a reference library stored on magnetic disks, to facilitate a rapid identification of unknowns3). It is interesting to note that the basic optical specifications of the two instruments are not all that dissimilar ... 

Spectral methods can provide the deepest insight into the constitution of GC eluates. However, the spectral laboratory has to cooperate closely with a library providing stored reference spectra, and efficient communication between these two facilities is hardly practicable without the use of a computer. 

Although individual laboratories find it useful to compile their own reference library files, access to very large collections of mass spectra and to published data [55] is essential. A compilation of many thousands of spectra by the Aldermaston Mass Spectrometry Data Centre and the Division of Computer Research and Technology at the National Institutes of Health [56-58] has been made available commercially. The file can be searched in a number of ways using an interactive conversational mass spectral search system via a teletype and acoustic link over telephone lines. 

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