3. Essential oil characterization

by extraction with hydrocarbons from aromatic plants or, more frequently, flowers —and absolutes, which are separated from concrete or pomade (obtained by

The EO industrial production involves field distillation, in order to avoid the high transportation costs of large vegetal material loads from which only about 1% is going to be obtained as EO. Steam generation is one of the main components of the operation costs. Current trends point toward the use of lignocellulosic waste as biofuel for the furnace. Still capacity is determined by the crop size. The goal is to maintain the still operating for at least 300 days of the year and to schedule the harvests to avoid long storage (more than a week) of the cut vegetal material waiting for its distillation. This is mainly to prevent mold formation. Patchouli and vetiver are two exceptions to this rule, because a curing period of several days or months (vetiver) recommended to enhance oil yield and organoleptic quality.

The reality is that a large part of Colombian small growers have low purchasing power, low economic performance and productivity, and not very sophisticated technology level in rural operations and processes. Traditional agricultural production faces a complex problem that includes low prices, low profitability, and the increasingly acute lack of rural labor, because young people migrate to the cities. The EO industry is a very important rural development alternative in which the harvested vegetal material is no longer the final product, but the start of an addedvalue product chain. Several pilot projects, financed by the Ministry of Agriculture and Rural Development and Colciencias (Colombia's Science Funding Agency), have been carried out in the past 15 years by CENIVAM with the participation of small rural farmers associations. The common goal of these projects has been the development of the EO value chain. The economic, agronomic, and quality viability of EOs obtained in several productive units have been studied. Each unit has characteristics, as follows: 5–8 ha crop extension, 20–22 families of small growers

enfleurage) by alcohol.

Essential Oils - Oils of Nature

Figure 2.

122

Rural production and distillation of essential oils in Colombia (Barbosa, Santander).

#### 3.1 Gas chromatographic analysis

The analytical technique routinely used for the instrumental chemical analysis of EOs is gas chromatography (GC), because the constituents of oils are volatile (monoterpenoids, esters, etc.) or semi-volatile substances (sesquiterpenoids, phenolic derivatives, etc.), whose molecular masses and boiling points do not exceed 300 a.m.u. and 300 °C, respectively. A chromatographic system comprises four fundamental blocks: (1) sample introduction system (injector), (2) separation system (column), (3) detection system for analytes eluted from the column (detector), and (4) data analysis and operation control system.

The GC can have conventional, e.g., flame ionization detector (FID), or thermal conductivity detector (TCD), and spectral detectors can have an external device attached, for example, a headspace sampler, a pyrolyzer, a purge and trap (P&T) system or a thermal desorption setup, among others. Each block of the chromatographic system has its own function and its "responsibility" for the quality of the analysis and the results obtained; for example, the function of the injection system is to transfer the sample to the column quantitatively, without discrimination by molecular weight or by the volatility of the components and without their chemical alteration (decomposition, isomerization, or polymerization) (Figure 3). The "responsibility" of the chromatographic column in the EO analysis is high: the clear, complete (ideally) separation of all the components of the mixture must be accomplished. The separation is based on achieving different distribution constants of the components between the two phases, stationary and mobile. This is obtained by establishing the optimal operational conditions (temperature, type of mobile phase, its velocity, stationary phase polarity, carrier gas pressure, temperature program, etc.) (Figure 4) and by correctly choosing the chromatographic column, i.e., its dimensions (length, internal diameter), chemical composition of stationary phase, its polarity and thickness, and among other factors. For the EO analysis, long columns (50 and 60 m) are used, since the oils are complex multicomponent mixtures and, above all, they have structurally very similar compounds (isomers), which require that the column has a very high resolution, which, among other factors, is achieved by increasing its length. The EOs contain compounds of very different polarities, both nonpolar (terpene hydrocarbons) and polar (alcohols, aldehydes). This implies that for their analysis, columns with different stationary phase polarities will be required.

The detection system differentiates the analyte molecules from those of the mobile phase (carrier gas), to which the detector is transparent. The response of the

of specialized software (data system), its accessories, interfaces, and analog-digital converters, the work of the chromatographic system and all its operational parts (hardware) is harmonized. For the EO analysis, which are very complex mixtures, two GC detectors are mainly used, namely, the flame ionization detector and the mass selective detector (MSD) or the mass spectrometry (MS) detector. The GC-

Ylang-ylang essential oil obtained from flowers by hydro-distillation and analyzed by GC-MS on a polar column (DB-WAX, 60 m), using different temperature programs: A. 12 °C/min and B. 8 °C/min (chromatogram fragment). With higher temperature rate, poorer separation of germacrene D and benzyl

Study of Essential Oils Obtained from Tropical Plants Grown in Colombia

DOI: http://dx.doi.org/10.5772/intechopen.87199

3.2 Tentative and confirmatory identification of essential oil components

The preliminary or presumptive (tentative) identification of the EO components may be obtained once the retention indices are determined. The analysis in modern equipment uses a program for the column temperature; in these cases, linear retention indexes are calculated, which are part of many databases and bibliographic references [5, 6]. The confirmatory identification of a compound in a complex mixture analyzed by GC needs to obtain its "fingerprint," which is the mass spectrum (MS) represented by a unique combination of charged fragments (ions) generated during the breakup of the previously ionized molecule. The complementarity of the chromatographic analysis (screening) with confirmatory spectral data (mass spectra) is achieved using the combination of two techniques, GC and MS. The GC-MS coupling complements the quantitative analysis carried out by GC-FID and provides important additional information, i.e., the mass spectra of all components,

EOs contain both nonpolar (monoterpene and sesquiterpene hydrocarbons) and polar compounds (their oxygenated derivatives, aliphatic alcohols, ketones, oxides, phenolic compounds and their derivatives, phenylpropanoids, and rarely acids, among others). Their analysis is performed by GC-FID (quantitative analysis) and

FID is used to quantify the oil components.

Figure 4.

125

acetate is observed.

through which their identity can be established.

Figure 3.

Ylang-ylang essential oil obtained by hydro-distillation from flowers. GC-MS analysis. DB-WAX column (60 m). Injection modes: A. Split 1, 100; B. Splitless. Injection volume—1 μL (250°C). Many "new" compounds appear in the chromatogram obtained in splitless mode.

detector is based on the measurement of one of the physical properties of the system, e.g., ion current, thermal conductivity, photon emission, etc. The analog signal becomes digital, graphic, i.e., a chromatographic peak, which is characterized by its area (A), which is proportional to the analyte quantity or concentration (C). This permits to establish an interdependent relationship, A = f (C), and to carry out a quantitative analysis, to determine not only how many components there are in a mixture but in what proportion (quantity) they are present. Through a combination Study of Essential Oils Obtained from Tropical Plants Grown in Colombia DOI: http://dx.doi.org/10.5772/intechopen.87199

Figure 4.

Ylang-ylang essential oil obtained from flowers by hydro-distillation and analyzed by GC-MS on a polar column (DB-WAX, 60 m), using different temperature programs: A. 12 °C/min and B. 8 °C/min (chromatogram fragment). With higher temperature rate, poorer separation of germacrene D and benzyl acetate is observed.

of specialized software (data system), its accessories, interfaces, and analog-digital converters, the work of the chromatographic system and all its operational parts (hardware) is harmonized. For the EO analysis, which are very complex mixtures, two GC detectors are mainly used, namely, the flame ionization detector and the mass selective detector (MSD) or the mass spectrometry (MS) detector. The GC-FID is used to quantify the oil components.

#### 3.2 Tentative and confirmatory identification of essential oil components

The preliminary or presumptive (tentative) identification of the EO components may be obtained once the retention indices are determined. The analysis in modern equipment uses a program for the column temperature; in these cases, linear retention indexes are calculated, which are part of many databases and bibliographic references [5, 6]. The confirmatory identification of a compound in a complex mixture analyzed by GC needs to obtain its "fingerprint," which is the mass spectrum (MS) represented by a unique combination of charged fragments (ions) generated during the breakup of the previously ionized molecule. The complementarity of the chromatographic analysis (screening) with confirmatory spectral data (mass spectra) is achieved using the combination of two techniques, GC and MS. The GC-MS coupling complements the quantitative analysis carried out by GC-FID and provides important additional information, i.e., the mass spectra of all components, through which their identity can be established.

EOs contain both nonpolar (monoterpene and sesquiterpene hydrocarbons) and polar compounds (their oxygenated derivatives, aliphatic alcohols, ketones, oxides, phenolic compounds and their derivatives, phenylpropanoids, and rarely acids, among others). Their analysis is performed by GC-FID (quantitative analysis) and

detector is based on the measurement of one of the physical properties of the system, e.g., ion current, thermal conductivity, photon emission, etc. The analog signal becomes digital, graphic, i.e., a chromatographic peak, which is characterized by its area (A), which is proportional to the analyte quantity or concentration (C). This permits to establish an interdependent relationship, A = f (C), and to carry out a quantitative analysis, to determine not only how many components there are in a mixture but in what proportion (quantity) they are present. Through a combination

compounds appear in the chromatogram obtained in splitless mode.

Ylang-ylang essential oil obtained by hydro-distillation from flowers. GC-MS analysis. DB-WAX column (60 m). Injection modes: A. Split 1, 100; B. Splitless. Injection volume—1 μL (250°C). Many "new"

Figure 3.

Essential Oils - Oils of Nature

124

by GC-MS (qualitative analysis), in two columns, with polar and nonpolar stationary phases. In columns with the nonpolar stationary phase, poly(dimethylsiloxane), PDMS, or 5% phenyl-PDMS, the elution of components happens depending on their boiling temperatures (or volatilities), that is, the retention times, tR, increase with the decrease of the volatility and with the increase of the molecular masses and boiling points of the components (Figure 5). The compounds reach the end of the column in the increasing order of their boiling points. In the polar column, poly (ethylene glycol), the elution order of the components is more difficult to predict, because it is related to the intermolecular forces between the analyte and the stationary phase and depends both on the dipole moment of the molecule (the polarity) and on the possibility of hydrogen bond formation between the substance and the stationary phase.

The elution order of some compounds in columns with different stationary phases may be reversed. This often helps, together with the mass spectra and the fragmentation pattern study, to differentiate, for example, terpene alcohols from their acetates, since the latter sometimes do not exhibit molecular ions, M+• in their mass spectra. When the chromatographic parameters (tR, tRR, or retention indices) and spectroscopic parameters, i.e., mass spectra (characteristic fragmentation pattern; see Figure 6) of the analyte and reference substance (certified standard) coincide, a complete or confirmatory compound identification is achieved. However, when retention indexes and mass spectra are used, extracted from the specialized literature [6, 7] or from the databases (e.g., spectral libraries, NIST, WILEY, Adams [7], others), and compared with the spectroscopic and chromatographic parameters of the EO component, their coincidence leads only to a recognition of the chemical structure, but not to its unambiguous, absolute identification, which requires the use of a standard compound, a pure substance with certified chemical structure. Frequently, it is necessary to isolate the compound from the mixture and purify it for further characterization through the UV, IR, MS, X-ray diffraction, NMR, elemental analysis, or high-resolution mass spectrometry (HRMS). Each one of the mentioned spectroscopic techniques contributes with some structural information, but the combined results allow to assemble the puzzle and elucidate the chemical structure unequivocally.

The biggest challenge in EO analysis is the complete separation of its components (Figure 7) because their frequent coelution occurs due to their very close or equal distribution constants. Some conventional strategies, e.g., change of the column (polarity), temperature program, use of selective detectors, etc., can often fail or be insufficient to determine all the compounds present in the oil. Multidimensional chromatography makes it possible to separate the peaks of partially or totally co-eluted substances. For this, it uses a second column, usually orthogonal, through the "heart-cutting" operation by means of pneumatic switching valves—today with the micro-fluidic technology, between the two columns and diverting part of the eluent from the first to the second column. This method has played an important role in the development of separation techniques for complex mixtures, including EOs [8, 9]. Multidimensional chromatography requires at least two detectors and can have configurations of up to three columns in the same oven or in separate chromatographic ovens. Along with this, today one of the most modern, complete solutions for the separation of multicomponent mixtures—although not very affordable for most laboratories because of its high price—is comprehensive or total gas chromatography (Comprehensive GC GC), whose applications and developments have grown day after day for more than two decades [10, 11].

In comprehensive gas chromatography (GC x GC), two columns are used, linked together by means of a modulator. In contrast to conventional multidimensional gas chromatography, the GC x GC requires a single detector with high processing frequency; both columns can be in the same oven or in two separate ovens. There are different types of modulators, e.g., rotary thermal modulator ("sweeper"), cryogenic "jet" system, modulators of valves, or longitudinal cryogenic modulator,

nonpolar [poly(dimethylsiloxane)] and polar [poly(ethylenglycol), PEG] columns.

Study of Essential Oils Obtained from Tropical Plants Grown in Colombia

DOI: http://dx.doi.org/10.5772/intechopen.87199

GC-MS chromatogram of ylang-ylang essential oil isolated from flowers by hydro-distillation and analyzed by GC-MS on a polar column (DB-WAX, 60 m). A. Co-injection of the essential oil and n-paraffin mixture to calculate retention indices (RI). B. RIs of germacrene D, benzyl acetate, and (E,E)-α-farnesene measured in

Figure 5.

127

Study of Essential Oils Obtained from Tropical Plants Grown in Colombia DOI: http://dx.doi.org/10.5772/intechopen.87199

#### Figure 5.

by GC-MS (qualitative analysis), in two columns, with polar and nonpolar stationary phases. In columns with the nonpolar stationary phase, poly(dimethylsiloxane), PDMS, or 5% phenyl-PDMS, the elution of components happens depending on their boiling temperatures (or volatilities), that is, the retention times, tR, increase with the decrease of the volatility and with the increase of the molecular masses and boiling points of the components (Figure 5). The compounds reach the end of the column in the increasing order of their boiling points. In the polar column, poly (ethylene glycol), the elution order of the components is more difficult to predict, because it is related to the intermolecular forces between the analyte and the stationary phase and depends both on the dipole moment of the molecule (the polarity) and on the possibility of hydrogen bond formation between the substance

The elution order of some compounds in columns with different stationary phases may be reversed. This often helps, together with the mass spectra and the fragmentation pattern study, to differentiate, for example, terpene alcohols from their acetates, since the latter sometimes do not exhibit molecular ions, M+• in their mass spectra. When the chromatographic parameters (tR, tRR, or retention indices) and spectroscopic parameters, i.e., mass spectra (characteristic fragmentation pattern; see Figure 6) of the analyte and reference substance (certified standard) coincide, a complete or confirmatory compound identification is achieved. However, when retention indexes and mass spectra are used, extracted from the specialized literature [6, 7] or from the databases (e.g., spectral libraries, NIST, WILEY, Adams [7], others), and compared with the spectroscopic and chromatographic parameters of the EO component, their coincidence leads only to a recognition of the chemical structure, but not to its unambiguous, absolute identification, which requires the use of a standard compound, a pure substance with certified chemical structure. Frequently, it is necessary to isolate the compound from the mixture and purify it for further characterization through the UV, IR, MS, X-ray diffraction, NMR, elemental analysis, or high-resolution mass spectrometry (HRMS). Each one of the mentioned spectroscopic techniques contributes with some structural information, but the combined results allow to assemble the puzzle

The biggest challenge in EO analysis is the complete separation of its components (Figure 7) because their frequent coelution occurs due to their very close or equal distribution constants. Some conventional strategies, e.g., change of the column (polarity), temperature program, use of selective detectors, etc., can often fail or be insufficient to determine all the compounds present in the oil. Multidimensional chromatography makes it possible to separate the peaks of partially or totally co-eluted substances. For this, it uses a second column, usually orthogonal, through the "heart-cutting" operation by means of pneumatic switching valves—today with the micro-fluidic technology, between the two columns and diverting part of the eluent from the first to the second column. This method has played an important role in the development of separation techniques for complex mixtures, including EOs [8, 9]. Multidimensional chromatography requires at least two detectors and can have configurations of up to three columns in the same oven or in separate chromatographic ovens. Along with this, today one of the most modern, complete solutions for the separation of multicomponent mixtures—although not very affordable for most laboratories because of its high price—is comprehensive or total gas chromatography (Comprehensive GC GC), whose applications and developments have grown day after day for more than two

In comprehensive gas chromatography (GC x GC), two columns are used, linked together by means of a modulator. In contrast to conventional multidimensional gas

and the stationary phase.

Essential Oils - Oils of Nature

decades [10, 11].

126

and elucidate the chemical structure unequivocally.

GC-MS chromatogram of ylang-ylang essential oil isolated from flowers by hydro-distillation and analyzed by GC-MS on a polar column (DB-WAX, 60 m). A. Co-injection of the essential oil and n-paraffin mixture to calculate retention indices (RI). B. RIs of germacrene D, benzyl acetate, and (E,E)-α-farnesene measured in nonpolar [poly(dimethylsiloxane)] and polar [poly(ethylenglycol), PEG] columns.

chromatography, the GC x GC requires a single detector with high processing frequency; both columns can be in the same oven or in two separate ovens. There are different types of modulators, e.g., rotary thermal modulator ("sweeper"), cryogenic "jet" system, modulators of valves, or longitudinal cryogenic modulator, among others, which also vary in the cryogenic agent employed; more modern modulators are not cryogenic in nature. The eluent of the first column, by means of the modulator, is "split" into very small "slices," which, one after the other, enter the second column from the first column. The first column (1D) is a conventional column, with length of 25 or 30 m, and the second column (2D) is of rapid chromatography, that is, short and with a very thin internal diameter (0.1 mm or less). The stationary phases in both columns are "orthogonal," i.e., if the first is nonpolar,

Figure 6.

129

[M–CH2=CH2]

through benzylic excision.

CH2=C=O neutral fragment and formation of [M—42]+•

Fragmentation pattern in mass spectra (electron impact, 70 eV) of some essential oil components. A. Ethyl benzoate mass spectrum. B. Methyl m-methyl benzoate mass spectrum. C. α-Rupture and typical benzoyl ion (m/z 105, 119) formation. D. McLafferty transposition of ethyl benzoate molecular ion M+• and formation of

Study of Essential Oils Obtained from Tropical Plants Grown in Colombia

DOI: http://dx.doi.org/10.5772/intechopen.87199

spectrum. H. Methyl 2-phenylacetate mass spectrum and the formation of tropylium ion (m/z 91) generated

<sup>+</sup>• (m/z 122) fragment. E. p-Methylphenyl acetate mass spectrum. F. Elimination of

, diagnostic ion for acetates. G. Benzyl acetate mass

Study of Essential Oils Obtained from Tropical Plants Grown in Colombia DOI: http://dx.doi.org/10.5772/intechopen.87199

#### Figure 6.

among others, which also vary in the cryogenic agent employed; more modern modulators are not cryogenic in nature. The eluent of the first column, by means of the modulator, is "split" into very small "slices," which, one after the other, enter the second column from the first column. The first column (1D) is a conventional column, with length of 25 or 30 m, and the second column (2D) is of rapid chromatography, that is, short and with a very thin internal diameter (0.1 mm or less). The stationary phases in both columns are "orthogonal," i.e., if the first is nonpolar,

Essential Oils - Oils of Nature

128

Fragmentation pattern in mass spectra (electron impact, 70 eV) of some essential oil components. A. Ethyl benzoate mass spectrum. B. Methyl m-methyl benzoate mass spectrum. C. α-Rupture and typical benzoyl ion (m/z 105, 119) formation. D. McLafferty transposition of ethyl benzoate molecular ion M+• and formation of [M–CH2=CH2] <sup>+</sup>• (m/z 122) fragment. E. p-Methylphenyl acetate mass spectrum. F. Elimination of CH2=C=O neutral fragment and formation of [M—42]+• , diagnostic ion for acetates. G. Benzyl acetate mass spectrum. H. Methyl 2-phenylacetate mass spectrum and the formation of tropylium ion (m/z 91) generated through benzylic excision.

component in the second column. The second column, therefore, must be short and very thin and separate the components in just a few seconds. Since the second column is connected to the detector (MSD, FID, or ECD), it must have a very-highreading and signal-processing frequency. In most cases, a time-of-flight (TOF) mass detector is used, which is the best option—though expensive—to make a quantitative analysis and identification of compounds in such complex mixtures, as

Study of Essential Oils Obtained from Tropical Plants Grown in Colombia

Further technical details for EO chemical characterization and that of their components can be found elsewhere [12, 13]. In summary, EO characterization necessary for its quality control and the determination of authenticity, as part of a technical data sheet necessary for its commercialization, can be divided into four main stages or areas: (1) organoleptic properties, (2) physicochemical determinations, (3) qualitative and quantitative analysis of the components present in the oil (chemical composition), and, finally, (4) some other determinations, e.g., pesticide

3.3 Chemical compositions of essential oils obtained from tropical plants grown

CENIVAM has studied Colombian plants widely used in popular medicine or in

culinary, for example, anise [14], oregano [15], rue [16, 17], and other species introduced from Asia, such as lemongrass, citronella, ginger, citrics [18–20], vetiver, and ylang-ylang [21–23], as well as several native species, among others, Copaifera officinalis [24], Spilanthes americana [25], Lepechinia schiedeana [26], Lippia alba [27], Xylopia americana [28], Hyptis umbrosa [29], Callistemon speciosus

(sims) DC. [30], Swinglea glutinosa [31], Satureja viminea [32], and Lippia origanoides [33], with emphasis on the comparative study of extraction methods [34–40]. Table 1 summarizes the composition of several Lippia EOs, according to compound families. The knowledge of the chemical composition has been the basis for the interpretation of the results of bioactivity assays such as genotoxicity

Fragment of GCxGC-HRMS-TOF chromatogram of ylang-ylang essential oil contaminated with plasticizer

(phthalate) traces. m/z 149 is a base peak in alkyl phthalates' mass spectra.

are EOs (Figure 8).

in Colombia

Figure 8.

131

residues, traces of heavy metals, etc.

DOI: http://dx.doi.org/10.5772/intechopen.87199

Figure 7.

Ylang-ylang essential oil chromatogram (GC-MS) fragment. A. Chromatographic peak at tR = 30.76 min is highlighted. B. Mass spectrum corresponded to this peak, identified by the software as ethyl benzoate (Figure 6A); nevertheless, the peak is "contaminated" with other compounds; the presence of typical sesquiterpene hydrocarbon ions (m/z 204, 189, 161,) is observed.

the second column is polar, and vice versa. The modulation time, required for the transfer of a very small portion of eluent from the first column to the second, must be very short and similar but never longer than the elution time of the "slowest"

Study of Essential Oils Obtained from Tropical Plants Grown in Colombia DOI: http://dx.doi.org/10.5772/intechopen.87199

component in the second column. The second column, therefore, must be short and very thin and separate the components in just a few seconds. Since the second column is connected to the detector (MSD, FID, or ECD), it must have a very-highreading and signal-processing frequency. In most cases, a time-of-flight (TOF) mass detector is used, which is the best option—though expensive—to make a quantitative analysis and identification of compounds in such complex mixtures, as are EOs (Figure 8).

Further technical details for EO chemical characterization and that of their components can be found elsewhere [12, 13]. In summary, EO characterization necessary for its quality control and the determination of authenticity, as part of a technical data sheet necessary for its commercialization, can be divided into four main stages or areas: (1) organoleptic properties, (2) physicochemical determinations, (3) qualitative and quantitative analysis of the components present in the oil (chemical composition), and, finally, (4) some other determinations, e.g., pesticide residues, traces of heavy metals, etc.
