Most of the methods applied in the analysis of essential oils rely on chromatographic procedures, which enable component separation and identification. However, additional confirmatory evidence is required for reliable identification, avoiding equivocated characterizations.
In the early stages of research in the essential oil field, attention was devoted to the development of methods in order to acquire deeper knowledge on the profiles of volatiles; however, this analytical task was made troublesome due to the complexity of these real-world samples. Over the last decades, the aforementioned research area has benefited from the improvements in instrumental analytical chemistry, especially in the chromatographic area, and, nowadays, the number of known constituents has drastically increased.
The primary objective in any chromatographic separation is always the complete resolution of the compounds of interest, in the minimum time. To achieve this task the most suitable analytical column (dimension and stationary phase type) has to be used, and adequate chromatographic parameters must be applied to limit peak enlargement phenomena. A good knowledge of chromatographic theory is, indeed, of great support for the method optimization process, as well as for the development of innovative techniques.
In gas chromatographic analysis, the compounds to be analyzed are vaporized and eluted by the mobile gas phase, the carrier gas, through the column. The analytes are separated on the basis of their relative vapor pressures and affinities for the stationary bed. On the other hand, in liquid chro-matographic analysis, the compounds are eluted by a liquid mobile phase consisting of a solvent or a mixture of solvents, the composition of which may vary during the analysis (gradient elution), and are separated according to their affinities for the stationary bed. In general, the volatile fraction of an essential oil is analyzed by GC, while the nonvolatile by liquid chromatography (LC).
At the outlet of the chromatography column, the analytes emerge separated in time. The analytes are then detected and a signal is recorded generating a chromatogram, which is a signal versus time graphic, and ideally with peaks presenting a Gaussian distribution-curve shape. The peak area and height are a function of the amount of solute present and its width is a function of band spreading in the column , while retention time can be related to the solute's identity. Hence, the information contained in the chromatogram can be used for qualitative and quantitative analysis.
6.3.1 Use of GC and Linear Retention Indices in Essential Oils Analysis
The analysis of essential oils by means of GC began in the 1950s, when professor Liberti  started analyzing citrus essential oils only a few years after James and Martin first described gas-liquid chromatography (GLC), commonly referred to as GC , a milestone in the evolution of instrumental chromatographic methods.
After its introduction, GC developed at a phenomenal rate, growing from a simple research novelty to a highly sophisticated instrument. Moreover, the current-day requirements for high resolution and trace analysis are satisfied by modern column technology. In particular, inert, thermostable, and efficient open-tubular columns are available, along with associated selective detectors and injection methods, which allow on-column injection of liquid and thermally labile samples. The development of robust fused-silica columns, characterized by superior performances to that of glass columns, brings open-tubular GC columns within the scope of almost every analytical laboratory.
At present, essential oil GC analyses are more frequently performed on capillary columns, which, after their introduction, rapidly replaced packed GC columns. In general, packed columns support larger sample size ranges, from 10 to 20 |mL, and thus the dynamic range of the analysis can be enhanced. Trace-level components can be easily separated and quantified without preliminary fractionation or concentration. On the other hand, the use of packed columns leads to lower resolution due to the higher pressure drop per unit length. Packed columns need to be operated at higher column flow rates, since their low permeability requires high pressures to significantly improve resolution . It is worthy of note that since the introduction of fused-silica capillary columns considerable progress has been made in column technology, a great number of papers regarding GC applications on essential oils have been published.
The choice of the capillary column in an essential oil GC analysis is of great importance for the overall characterization of the matrix; the stationary phase chemical nature and film thickness, as well as the column length and internal diameter, are to be considered. In general, essential oil GC analyses are carried out on 25-50 m columns, with 0.20-0.32 mm internal diameters, and 0.25 |mm stationary phase film thickness. It must be noted that the degree of separation of two components on two distinct stationary phases can be drastically different. As is well known, nonpolar columns produce boiling-point separations, while on polar stationary phases compounds are resolved according to their polarity. Considering that essential oil components, such as terpenes and their oxygenated derivatives, frequently present similar boiling points, these elute in a narrow retention time range on a nonpolar column. In order to overcome this limit, the analytical method can be modified by applying a slower oven temperature rate to widen the elution range of the oil or by using a polar stationary phase, as oxygenated compounds are more retained than hydrocarbons. However, choosing different stationary phases may provide little improvement as resolution can be improved for a series of compounds but new coelutions can also be generated.
Considering gas chromatographic analyses using flame ionization detector (FID), thermal conductivity detector (TCD), or other detectors which do not provide structural information of the analyzed molecules and retention data, more precisely retention indices, are used as the primary criterion for peak assignment. The retention index system was based on the fact that each analyte is referenced in terms of its position between the two n-paraffins that bracket its retention time. Furthermore, the index calculation is based on a linear interpolation of the carbon chain length of these bracketing paraffins. The most thoroughly studied, diffused, and accepted retention index calculation methods are based on the logarithmic-based equation developed by Kovats in 1958 , for isothermal conditions, and on the equation propounded by van den Dool and Kratz in 1963 , which does not use the logarithmic form and is used in the case of temperature-programming conditions. Values calculated using the latter approach are commonly denominated in literature as retention index (I), linear retention index (LRI), or programmed-temperature retention index (PTRI or IT ), while the ones derived from the former equation are usually referred to as Kovats index (KI).
In general, retention index systems are based on the incremental structure-retention relationship, namely, that any regular increase in a series of chemical structures should provide a regular increase in the corresponding retention times. This means that the retention index concept is not restricted to the use of n-alkanes as standards. In practice, any homologous series presenting a linear relationship between the adjusted retention time, being logarithmic based or not, and the carbon number can be used.
In the characterization of volatiles, the most commonly applied reference series is n-alkanes. However, the latter commonly present fluctuant behavior on polar stationary phases. In consideration of the fact that retention index values are correlated to retention mechanisms, alternative standard series of intermediate polarity have been introduced, such as 2-alkanones, alkyl ethers, alkyl halides, alkyl acetates, and alkanoic acid methyl esters . Shibamoto  suggested the use of polar compounds series, such as ethyl esters, as an alternative. The most feasible choice, when analyzing volatiles, is to apply reference series as n-alkanes, fatty acid ethyl esters (FAEEs), or fatty acid methyl esters (FAMEs), employed according to the stationary phase to be used.
Additionally, it is highly advisable to use two analytical columns coated with stationary phases of distinct polarities to obtain two retention index values and enhance confidence in assignments [37-39]. Identifications made on a single column can only be accepted if used in combination with spectroscopic detection systems. When n-alkanes are used, it is accepted that the reproducibility of retention indices between different laboratories are comprised within an acceptable range of ±5 units for methyl silicone stationary phases, and ±10 units for polyethylene glycol phases. A further aspect of great importance, which is frequently overseen, is the analytical reproducibility of retention indexes. Moreover, it is worthwhile to highlight that in practice it was found that the use of an initial isothermal hold in the GC oven temperature program does not provide additional resolution .
Mass spectrometry (MS) can be defined as the study of systems through the formation of gaseous ions, with or without fragmentation, which are then characterized by their mass-to-charge ratios (m/z)
and relative abundances . The analyte may be ionized thermally, by an electric field or by impacting energetic electrons, ions, or photons.
During the past decade, there has been a tremendous growth in popularity of mass spectrometers as a tool for both, routine analytical experiments and fundamental research. This is due to a number of features including relatively low cost, simplicity of design and extremely fast data acquisition rates. Although the sample is destroyed by the mass spectrometer, the technique is very sensitive and only low amounts of material are used in the analysis.
In addition, the potential of combined gas chromatography-mass spectrometry (GC-MS) for determining volatile compounds, contained in very complex flavor 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 identification 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 . 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 commonly provided with mass spectrometer data systems with the purpose to assist in the identification of unknown compounds .
However, as is well known, compounds such as isomers, when analyzed by means of GC-MS, can be incorrectly identified; a drawback which 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 sufficient for identification and peak assignment becomes a difficult, if not impracticable, task (Figure 6.1). 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 confirm the identity of compounds. Besides, retention indices when incorporated to MS libraries can be applied as a filter, thus shortening the search routine for matching results, and enhancing the credibility of MS identification .
According to D. Joulain and W. A. Konig , provided data contained in mass spectral libraries have been recorded using authentic samples, it can be observed that the mass spectrum of a given sesquiterpene is usually sufficient to ensure its identification when associated with its retention index obtained on methyl silicone stationary phases. Indeed, for the aforecited class of compounds, there would be no need to use a polyethylene glycol phase, which could even lead to misinterpretations caused by possible changes in the retention behavior of sesquiterpene hydrocarbons as a result of column aging or deterioration. Moreover, according to the authors, attention should be paid to the retention index and the mass spectrum registration of each individual sesqui-terpene, since many compounds with rather similar mass spectra elute in a narrow range; more than 160 compounds can elute within 100 retention index units on a methyl silicone-based column, for example, 1400-1500.
Nowadays in daily routine work, apart from increased analytical sensitivity, demands are also made on the efficiency in terms of speed of the laboratory equipment. Regarding the rapidity of analysis, two aspects need to be considered: (i) the costs in terms of time required, for example, as is the case in quality control analysis, and (ii) the efficiency of the utilized analytical equipment.
When compared to conventional GC, the primary objective of fast GC is to maintain sufficient resolving power in a shorter time, by using adequate columns and instrumentation in combination
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