Unknown compounds, identification spectra

The library search is a mathematical comparison of the unknown compound s spectrum with that of all reference compounds in the database. The aim of the comparison is to find the spectrum that most resembles that of the unknown compound. At the end of the search, the computer software makes a list of all spectra that resemble the unknown spectrum. The software lists the spectra relative to the unknown, along with a reliability or correlation index, irrespective of the library used. Because the object of the library search is to help the analyst and not act as a substitute for him/her, the analyst must manually examine the results. The best approaches to identification are interactive approaches in which the analyst can define filters to reduce the field of investigation. Several different algorithms are used for comparison and can lead to different spectra listings. 

PMR spectrometry is an extremely useful technique for the identification and structural analysis of organic compounds in solution, especially when used in conjunction with infrared, ultraviolet, visible and mass spectrometry. Interpretation of PMR spectra is accomplished by comparison with reference spectra and reference to chemical shift tables. In contrast to infrared spectra, it is usually possible to identify all the peaks in a PMR spectrum, although the complete identification of an unknown compound is often not possible without other data. Some examples of PMR spectra are discussed below. 

The fingerprint region lies between 1300-400 cm-1 which is considered to be the most valuable component of the spectra and mainly comprises of a specifically large number of unassigned vibrations. Therefore, IR-spectroscopy aids in the identification of unknown compound by comparing its spectrum with a standard spectra recorded under exactly similar experimental parameters. Thus, pharmaceutical substances that exhibit the same infrared spectra may be inferred as identical. 

Unknown compounds are detected frequently, and laboratories eventually develop some level of comfort in recognizing them as artifacts that are not significant clinically, at least for the purpose of ruling out a possible inborn error of metabolism [21]. However, there are instances when an unknown compound is found in multiple specimens from the same patient and cannot be associated with ongoing drug and known dietary intake. This was the set of circumstances that led to the identification of 2-octenylsuccinic acid as the compound referred to by some laboratories as pseudo-orotic acid [11, 18]. On the other hand, the spectrum shown in Fig. 3.1.10, tentatively identified as 4-hydroxy 2-hexenoic acid by GC-MS/MS, belongs to a compound that appears in the urine of patients with disorders of propionate me-... 

The capability to identify compounds outside the target analyte list is one of the greatest advantages offered by GC/MS. Searching the EPA/NIST spectral library, the computer finds the best matching library spectra for an unknown compound and tentatively identifies it using the best fitting spectrum. The retention time information is not available for this compound as it is not part of the calibration standard, hence only tentative identification. These tentatively identified compounds (TICs) can be also quantified to provide an estimated concentration value. 

The third of the three adjacent peaks (29.207 minutes) is readily identified as 2-hydroxybenzaldehyde (salicylaldehyde) by comparison of the MS or IR spectrum with library data. It should be noted that the automated search routine in the MS software picked 3-and 4-hydroxybenzaldehyde as better matches than the 2-hydroxy compound, even though the 76 ion in the spectrum of the unknown is present only in the mass spectrum of the 2-hydroxy compound. However, the IR search routine correctly identified the 2-isomer. This illustrates that casual operators who rely on automated search routines for compound identification are much less likely to make errors when they have access to both IR and MS searching. 

Frequently industrial hygiene analyses require the identification of unknown sample components. One of the most widely employed methods for this purpose is coupled gas chromatography/ mass spectrometry (GC/MS). With respect to interface with mass spectrometry, HPLC presently suffers a disadvantage in comparison to GC because instrumentation for routine application of HPLC/MS techniques is not available in many analytical chemistry laboratories (3). It is, however, anticipated that HPLC/MS systems will be more readily available in the future ( 5, 6, 1, 8). HPLC will then become an even more powerful analytical tool for use in occupational health chemistry. It is also important to note that conventional HPLC is presently adaptable to effective compound identification procedures other than direct mass spectrometry interface. These include relatively simple procedures for the recovery of sample components from column eluate as well as stop-flow techniques. Following recovery, a separated sample component may be subjected to, for example, direct probe mass spectrometry infra-red (IR), ultraviolet (UV), and visible spectrophotometry and fluorescence spectroscopy. The stopped flow technique may be used to obtain a fluorescence or a UV absorbance spectrum of a particular component as it elutes from the column. Such spectra can frequently be used to determine specific properties of the component for assistance in compound identification (9). 

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 ... 

The spectrum is examined first. Before starting any interpretation, it is strongly recommended that a computer or a manual library search is performed to check whether this spectrum belongs to an existing collection. Identification of an unknown compound in this way depends directly on the quality and comprehensiveness of the collection used. However, only libraries of electron ionization spectra are efficient. Other ionization techniques yield spectra that are much too dependent on the instruments and experimental conditions. 

A cortunon use of computed vibrational frequencies is to ascertain the identity of an unknown structure by comparison with experimental IR spectra. A few recent examples of the positive identification intermediates will sirflice here. In the attempt to prepare benzocyclobutenylidene (1), an unknown compound was detected. By comparing the experimental IR spectrum with the computed IR... 

COMPOUND IDENTIFICATION The identification of a compoimd from its mass spectrum may be achieved by matching the spectrum against libraries of spectra. However, if the compound is not already known, e.g. a drug intermediate or an unknown metabolite which is not in the library collections, the fragmentation patten must be interpreted from first principles using as much other analytical information (UV, IR, NMR, etc.) as may be available. 

Applications of IR spectroscopy to qualitative analysis are mainly for the identification of unknown compounds. For a pure substance, an exact match of the IR spectrum of the compound with that of the... 

The mode of peak identification that the diode array detector provides allows identification of unknown peaks, not only by retention time but also by their UV spectra. The UV spectmm is very reproducible and its full shape is much more compound-specific than is generally assumed. It is a powerful tool that, beyond other applications, it can also be used as a scouting technique to find out the possible identity of an unknown sample. However, spectral identity is a necessary but not a sufficient precondition for compound identification. The information that is acquired by the comparison of an unknown and a known spectrum can be used as strong indication of compound similarity or confirmation of identity. Combinatorial estimation of retention time and spectral comparison results will give us a more definite idea. 

Once the electrical signal leaves the photomultiplier tube, it is fed to a recorder if a printout is required, or, more usually, to a screen where the absorption spectrum can be displayed. Most modern spectrophotometers are now interfaced to a personal computer to allow storage of large amounts of data, or to allow access to a library of stored spectra on the hard drive of the machine. This allows comparison of stored spectra with the experimentally derived results from the laboratory and aids in the identification of unknown compounds. 

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