Identi cation

Gould, K. J., C. N. Manners, D. W Payling, J. L. Suschitky, and E. VNfells. 1988. Predictive structure-activity relationships in a series of pyranoquinoline derivatives. Anew primate model for the identi cation of antiallergic activity. J. Med. Chem31 1445-1453. 

Lindenbaum, S. and McGraw, S.E. (1985). The identi cation and characterization of polymorphism in drugs by solution calorimetryPharm. Manufact, 2 27-30. 

Comparing GC/FTIR and GC/MS, advantages and limitations of each technique become visible. The strength of IR lies—as discussed before—in distinguishing isomers, whereas identi cation of homologues can only be performed successfully by MS. The logical and most sophisticated way to overcome these limitations has been the development of a combined GC/FTIR/MS instrument, whereby simultaneously IR and mass spectra can be obtained. 

FIGURE 2.10 Identi cation of limonene in celery oil by C-NMR spectroscopy. 

During the last years, a number of articles have been published by Casanova and coworkers (e.g., Bradesi et al. (1996) and references cited therein). In addition, papers dealing with computer-aided identi cation of individual components of essential oils after C-NMR measurements (e.g., Tomi et al., 1995), and investigations of chiral oil constituents by means of a chiral lanthanide shift reagent by C-NMR spectroscopy have been published (Ristorcelli et al., 1997). 

Chamblee, T. S., B. C. Clark, T. Radford, and G. A. lacobucci, 1985. General method for the high-performance liquid chromatographic prefractionation of essential oils and avor mixtures for gas chromatographic-mass spectrometric analysis Identi cation of new constituents in cold pressed lime oil. 

Mondello, L P. Dugo, K. D. Bartle, G. Dugo, and A. Cotroneo. 1995. Automated LC-GC A powerful method for essential oils analysis Part V. Identi cation of terpene hydrocarbons of bergamot, lemon, mandarin, sweet orange, bitter orange, grapefruit, clementine and Mexican lime oils by coupled HPLC-HRGC-MS (ITD). 10 33-42. 

Tomi, R, P. Bradesi, A. Bighelli, and J. Casanova, 1995. Computer-aided identi cation of individual components of essential oils using carbon-13 NMR spectroscopy. J. Magnet Resonance Anal., 1995 25-34. 

As illustrated by the previous paragraph, one of the crucial points of using plants as sources for essential oils is their heterogeneity. A rst prerequisite for reproducible compositions is therefore an unambiguous botanical identi cation and characterization of the starting material. The rst... 

Avoidance of admixtures and adulterations by reliable botanical identi cation. 

Identi cation and authentication of the plant material, especially botanical identity and deposition of specimens. 

In the eld of agriculture, attempts are being made at the identi cation of ecologically more friendly natural biocides, including essential oils, to replace synthetic pesticides and herbicides. Essential oils are also used to improve the appetite of farm animals, leading to more rapid increases in body weight as well as to improved digestion. 

The Rf value is characteristic for any given compound on the same stationary phase using the identical mobile phase. Hence, knownvalues can be compared to those of unknown substances to aid in their identi cation [24]. On the other hand, separations in PC involve the same principles as those in TLC, differing in the use of a high-quality Iter paper as the stationary phase instead of a thin adsorbent layer, by the increased time requirements and poorer resolution. It is worthy to highlight that TLC has largely replaced PC in contemporary laboratory practice [22]. 

Most of the methods applied in the analysis of essential oils rely on chromatographic procedures, which enable component separation and identi cation. However, additional con rmatory evidence is required for reliable identi cation, avoiding equivocated characterizations. 

In addition, the potential of combined GC mass spectrometry (GC-MS) for determining volatile compounds, contained in very complex avor and fragrance samples, is well known. The subsequent introduction of powerful data acquisition and processing systems, including automated library search techniques, ensured that the information content of the large quantities of data generated by GC-MS instruments was fully exploited. The most frequent and simple identi cation method in GC-MS consists of the comparison of the acquired unknown mass spectra with those contained in a reference MS library. 

A mass spectrometer produces an enormous amount of data, especially in combination with chromatographic sample inlets [42]. Over the years, many approaches for analysis of GC-MS data have been proposed using various algorithms, many of which are quite sophisticated, in efforts to detect, identify, and quantify all of the chromatographic peaks. Library search algorithms are com monly provided with mass spectrometer data systems with the purpose to assist in the identi cation of unknown compounds [43]. 

However, as is well known, compounds such as isomers, when analyzed by means of GC-MS, can be incorrectly identi ed, a drawback that is often observed in essential oil analysis. As is widely acknowledged, the composition of essential oils is mainly represented by terpenes, which generate very similar mass spectra hence, a favorable match factor is not suf cient for identi cation, and peak assignment becomes a dif cult, if not impracticable, task (Figure 7.1). In order to increase the reliability of the analytical results and to address the qualitative determination of compositions of complex samples by GC-MS, retention indices can be an effective tool. The use of retention indices in conjunction with the structural information provided by GC-MS is widely accepted and routinely used to con rm the identity of compounds. Besides, retention indices when incorporated to MS libraries can be applied as a Iter, thus shortening the search routine for matching results and enhancing the credibility of MS identi cation [44]. 

FIGURE 7.2 Fast gas chromatography analysis of a lime essential oil on a 5 m x 5 mm (0.05 pm Im thickness) capillary column, applying fast temperature programming. The peak widths of three components are marked to provide an illustration of the high ef ciency of the column, even under extreme operating conditions (for peak identi cation, see Ref. [50]). (From Mondello, L. et al., 7. Sep. Sci., 27, 699, 2004. With permission.)... 

GC O systems are often used in addition to either a FID or a mass spectrometer. With regard to detectors, splitting column ow between the olfactory port and a mass spectral detector provides simultaneous identi cation of odor active compounds. Another variation is to use an in-line, nondestructive detector such as a TCD [64] or a photoionization detector [65]. Especially when working with GC-0 systems equipped with detectors that do not provide structural information, retention indexes are commonly associated to odor description supporting peak assignment. 

FIGURE 7.5 High-performance liquid chromatography of Italian genuine bitter orange oil. For peak identi-cation, refer to the text (I.S.—Internal Standard). (From Dugo, P. et al., J. Agric. Food Chem., 44, 544, 1996. With permission.)... 

The oxygen heterocyclic compounds present in the nonvolatile residue of citrus essential oils have also been extensively investigated by means of HPLC-atmospheric pressure ionization-mass spectrometry (HPLC-API-MS) [99]. The mass spectra obtained at different voltages of the sample cone have been used to build a library. Citrus essential oils have been analyzed with this system, using an optimized NP-HPLC method, and the mass spectra were compared with those of the laboratory-constructed library. This approach allowed the rapid identi cation and characterization of oxygen heterocyclic compounds of citrus oils, the detection of some minor components for the rst time in some oils, and also the detection of authenticity and possible adulteration. 

One of the best examples of the application of comprehensive NPLC x RPLC in essential oil analysis is represented by the analysis of oxygen heterocyclic components in cold-pressed lemon oil, by using a normal phase with a microbore silica column in the D and a monolithic C18 column in the with a 10-port switching valve as interface [133]. In Figure 7.12, an NPLC x RPLC separation of the oxygen heterocyclic fraction of a lemon oil sample is presented. Oxygen heterocyclic components (coumarins, psoralens, and PMFs) represent the main part of the nonvolatile fraction of cold-pressed citrus oils. Their structures and substituents have an important role in the characterization of these oils. Positive peak identi cation of these compounds was obtained both by the relative... 

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