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mass spectrometry
Journal of Chromatography A, 1141 (2007) 279–286
Odour fingerprint acquisition by means of comprehensive two-dimensional gas chromatography-olfactometry and comprehensive two-dimensional gas chromatography/mass spectrometry Barbara d’Acampora Zellner a , Alessandro Casilli a , Paola Dugo b , Giovanni Dugo a , Luigi Mondello a,∗ b
a Dipartimento Farmaco-Chimico, Facolt` a di Farmacia, Universit`a di Messina, viale Annunziata, 98168 Messina, Italy Dipartimento di Chimica Organica e Biologica, Facolt`a di Scienze, Universit`a di Messina, Contrada Papardo, 98166 Messina, Italy
Received 4 October 2006; received in revised form 4 December 2006; accepted 8 December 2006 Available online 16 December 2006
Abstract The analysis of complex matrices, such as perfumes, by means of gas chromatography-olfactometry (GC-O) can be rather imprecise due to the co-elutions, leading to a possible masking of odour-active trace-level compounds by major interferences or agglomeration of olfactive impressions resulting in unreliable olfactive characterization. To overcome these limits an innovative technique, comprehensive two-dimensional gas chromatography-olfactometry (GC × GC-O), was applied, revealing several relevant co-elutions, as in the linalool and linalyl acetate zones. A total of 177 compounds, out of these 135 odour-active, were detected by GC-O, while about 481 out of 818 compounds presented odour-activity through GC × GC-O analyses. In addition, GC/mass spectrometry (GC/MS) and GC × GC/MS analyses were also performed. Peak assignment was achieved by means of different information sources, such as GC/MS, GC × GC/MS, LRI, injection of standards and olfactive impressions. © 2006 Elsevier B.V. All rights reserved. Keywords: Comprehensive two-dimensional gas chromatography-olfactometry (GC × GC-O); GC-O; GC × GC/MS; GC/MS; Perfume analysis
1. Introduction Fragrance materials have their use traced to early antiquity, when spices and resins from animal and plant sources were used for the purpose of perfumery. Perfumes have been intimately associated with human history and are comprised by complex mixtures of odorant materials, the blending of which is a good example of product engineering. Today, perfumers work with a total of several thousand ingredients. On average, 30 to 50 (and sometimes up to 200) ingredients, synthetically manufactured, as well as natural fragrances, are used to create a fragrance composition [1]. Modern methods of analysis have not only enhanced the acquisition of a higher perfumery raw material knowledge, but also brought a greater emphasis on perfume creation. The combination of market forces with gas chromatography (GC) and its features resulted in an explosive accelera-
∗
Corresponding author. Tel.: +39 090 676 6536; fax: +39 090 676 6532. E-mail address: [email protected] (L. Mondello).
0021-9673/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2006.12.035
tion of the evolution rate of flavour and fragrance materials [2]. For decades monodimensional GC and GC/mass spectrometry (GC/MS), as well as GC-olfactometry (GC-O), have been commonly employed in perfume industries. GC-O is a wellknown standard technique which enables the assessment of odour-active components in complex mixtures, based on the correlation between the chromatographic peaks of the eluted substances perceived simultaneously by two detectors, one of them being the human olfactory system. This valuable tool for the investigation of flavour and fragrance matrices is applied for a wide range of samples by using different assessment methods [3]; enantioselective GC-O for the determination of sensory properties of enantiomers has also been carried out [4]. However, considering that perfume raw materials are characterized by a wide variety of components belonging to several chemical classes, extensive co-elutions may occur both on non-polar and polar stationary phases leading to inaccurate identification of odour-active compounds. Moreover numerous compounds, present at trace-level concentrations, can still exert an important
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olfactive or economical impact [5,6]. Since higher chromatographic separation capacities are required and can be hardly achieved by a single capillary column, the demand for more powerful techniques has emerged. Comprehensive two-dimensional gas chromatography (GC × GC) appears to be the most appropriate choice to fulfil the request for enhanced separation and better sensitivity. This orthogonal two-column separation, with complete sample transfer executed by means of a modulator able to trap, refocus and release fractions of the GC effluent from the first dimension (1 D, column 1), and periodically introduce them onto the second dimension (2 D, column 2), enables an accurate screening of complex matrices, offering very high resolution and enhanced detection sensitivity [7,8]. The features of GC × GC are of great usefulness for the perfumery industry, in the determination of individual raw materials quality and authenticity, the detection of essential oils in perfumes by establishing specific patterns, or even the possibility of an automated perfume formulation without unwanted co-eluting components and precise quantification of each raw material [5]. Since GC-O is a technique of great importance in the fragrance industry, the hyphenation of GC × GC to olfactometry (GC × GC-O), providing a bidimensional separation and simultaneous olfactive characterization of components in a complex matrix, likewise represents to be an outstanding approach to suppress the need for co-elution free and more reliable olfactive analysis. This novel technique associates the resolution power of GC × GC with the selectivity and sensitivity of the human olfactory system, enabling the olfactive analysis of congested chromatographic areas. To the author’s knowledge the application of GC × GC-O as a screening procedure for establishing the odour fingerprint of a perfume has not previously been reported in literature. The purpose of this research was to investigate the application of GC × GC-O to a perfume analysis and to obtain the odour fingerprint by means of a complete qualitative characterization of this complex sample. The magnitude of interferences and their influence on the performance of olfactometric analysis was also investigated. Additionally, aspects regarding the panelists, or assessors, observed during the performance of the analysis will also be discussed. 2. Experimental 2.1. Samples A commercial perfume (eau de toilette) was purchased in a local perfumery and stored at 4 ◦ C. That perfume, a male fragrance with predominantly citric fresh and herbaceous notes, used in all analyses has already been analysed and reported by the research group [9]. The following standard compounds were purchased from Sigma–Aldrich (Milan, Italy): linalool, tetrahydrolinalool, n-octenyl acetate, hexyl isovalerate, linalyl acetate, carvone, cuminaldehyde, pentadecane and geraniol; while Sandalore® was purchased from Givaudan (D¨ubendorf, Switzerland). Hotrienol and neral were confirmed by means of the analysis of essential oils containing these compounds, Ho and Bergamot oil, respectively.
2.2. Sample preparation The commercial perfume was analysed undiluted. Each standard component was diluted with n-hexane (1:10, 1:50, 1:100, 1:500 and 1:1000 v/v) prior to injection. 2.3. GC × GC-O analyses GC × GC-O analyses were carried out on a Shimadzu GC2010 gas chromatograph equipped with an AOC-20i autoinjector and an AOC-20s autosampler (Shimadzu, Milan, Italy). The GC was retrofitted with an Everest Longitudinally Modulating Cryogenic System (LMCS) (Chromatography Concepts, Doncaster, Australia) and hyphenated to the Phaser - Sniffing Port OP275 (ATAS GL International B.V., Veldhoven, The Netherlands). The mechanical stepper motor drive responsible for the movement of the cryotrap was initiated by an electronic device via the GC Solution software (Shimadzu) programmed external event, while the cryotrap temperature was regulated by a temperature control system through the Comprehensive Chromatography System Control–CCSC software (Shimadzu). The latter enables the programming of the cryogenic temperature maintaining a defined temperature difference between the cryotrap and the GC oven temperature. A modulation period of 0.125 Hz (8 s cycle) was applied to all GC × GC analyses and the cryogenic trap was maintained at a temperature of 150 ◦ C below the prevailing GC oven temperature for the first 15 min, and at 100 ◦ C below the GC oven until the end of the analysis. Furthermore, a modified carbon dioxide (CO2 ) cylinder, as described elsewhere [10], delivered the gas at a pressure of 125 bar to the LMCS system. The supplied CO2 cools the cryotrap by its expansion, while a small internal flow of nitrogen (N2 ) gas (approximately 10 mL/min) was applied to prevent ice formation inside the trap. Data were collected by the GC Solution software, and by using its export function converted to ASCII data, which were then processed by the Comprehensive Chromatography Manager ver. 1.1 (Shimadzu) achieving a contour representation of the two-dimensional chromatograms. The olfactometry system consisted of a four-port splitter stand located in the GC oven; two ports were connected to the second dimension column outlet and an auxiliary gas outlet; the two remaining ports were connected to the flame ionization detector (FID) and the transfer line (sniffing port) by means of retention gaps (Fig. 1). A control module regulates the transfer line heater, the auxiliary gas supply and the nasal mucosa humidifier (for further information refer to [4]). The transfer line, maintained under constant temperature of 250 ◦ C, ends up in an ergonomic glass nose cone. An auxiliary gas (He) flow of 5 ml/min was kept constant during analyses. Considering the demand for fast response of the evaluators during GC × GC-O analysis, the OdoChronometer software (Shimadzu) has been developed, this is integrated to the GC Solution software, being initiated with the start out of the GC. The software runs along with the chromatographic analysis and registers the time whenever the panelist presses a push-button; the signals are compiled and data output is easily opened as a text file presenting a retention time registration table.
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The breathing pattern also had to be adapted to GC × GC-O, suggested was abdominal breathing which uses primarily the diaphragm, and is congruent with the shape of the lungs and the capacities of the breathing muscles [12]. It processes the most air with the least effort, and is associated with mental stability and concentration, avoiding both dizziness and hyperventilation. 2.6. Detection frequency method
Fig. 1. Schematic representation of the comprehensive two-dimensional gas chromatography-olfactometry instrumentation.
The column set consisted of a SLB-5ms (5% diphenyl95% dimethylpolysiloxane) 30 m × 0.25 mm i.d. × 0.25 m df , serially connected by a zero-dead-volume union (Supelco, Milan, Italy) to a Supelcowax 10 (polyethylene glycol) 0.75 m × 0.1 mm i.d. × 0.10 m df (both columns were purchased from Supelco). The GC was operated under temperature-programmed conditions, from 50 ◦ C to 200 ◦ C at 1.5 ◦ C/min, to 250 ◦ C (20 min) at 20 ◦ C/min. The GC-2010 was equipped with a split/splitless injector (290 ◦ C); injection volume: 0.3 L, in splitless mode injection (1 min sampling time); initial inlet pressure: 130.0 kPa; carrier gas: H2 ; detector: FID (260 ◦ C); H2 flow: 50.0 mL/min; air flow: 400.0 mL/min; make up flow (N2 ): 50.0 mL/min; sampling rate: 8 ms. The sniffing procedure was divided in 5 min sessions with 10 min intervals in order to avoid lassitude. In three analyses the entire chromatographic run was covered by one panelist; each one performed the analyses six times in distinct days. 2.4. GC-O analyses The analyses were carried out by using the GC × GC-O system without cryogenic modulation. The sniffing procedure was divided in 20 min sessions with 15 min interval and the analyses were carried out in triplicate. 2.5. Panel GC × GC-O and GC-O analyses were performed by two evaluators with previous experience in GC-O, both were screened for specific anosmia using the standard solution set proposed by Friedrich et al. [11]; no insensitivities were identified. However, GC × GC-O requests a sharp discerning capacity, utmost concentration and fast response of the panelists, so that the demand for further and accurate training turned out to be indispensable. The training consisted of GC-O, GC × GC-O and direct olfactive analyses of several co-eluting standard mixtures, neat and dilutions, since some odorants are perceived as having distinct odour quality at different concentrations. In each analysis the panelists were asked to describe the odour quality of the perceived impressions according to a glossary of olfactive descriptors suggested by Curtis and Williams [1] and adopted by the group.
Sensorial evaluations were performed using an adaptation of the method proposed by Linssen et al. [13]. The adaptation consisted on the detection of odour-active compounds by two assessors, accompanied by the qualitative description of that odour according to the glossary of olfactive descriptors adopted by the group. 2.7. GC × GC/MS analyses GC × GC/MS analyses were carried out on a Shimadzu GCMS-QP2010 gas chromatograph mass spectrometer equipped with an AOC-20i autoinjector and the Flavour and Fragrance Natural and Synthetic Compounds (FFNSC) ver. 1.2 MS Library (Shimadzu). The GC was retrofitted with the same aforementioned modulator, applying identical modulation period and cryogenic trap temperature program. Data were collected by the GCMS Solution software and likewise converted to ASCII data. Contour representations of the two-dimensional chromatograms were achieved through the same aforementioned Comprehensive Chromatography Manager ver. 1.1 software. The column set and oven temperature program were identical to the GC × GC-O analyses. The GCMS-QP2010 was equipped with a split/splitless injector (250 ◦ C); injection volume: 1.0 L, in split mode (10:1); initial inlet pressure: 119.2 kPa; carrier gas: He; interface temperature 250 ◦ C; MS ionization mode: electron ionization; detector voltage: 0.9 kV; acquisition mass range: 40–400 amu; scan speed 10000 amu/s; acquisition mode: full scan; scan interval: 0.05 s (20 Hz); solvent delay: 5 min. Analyses were carried out in triplicate. 2.8. GC/MS analyses The above-described GC × GC/MS system was used without cryogenic modulation. Analyses were carried out in triplicate. 2.9. First dimension linear retention index (LRI) The first dimension LRI determinations, in both described GC × GC systems, were carried out by injecting a homologous series of n-alkanes containing 24 n-hydrocarbons (C7-C30) (Supelco, Milan, Italy), at a 1000 ppm concentration in hexane. The indices were calculated according to the equation proposed by van den Dool and Kratz [14]. The LRI for GC/MS and GC-O analyses were likewise established. 2.10. Statistical evaluation Data obtained from the GC-O and GC × GC-O analyses were subjected to one way ANOVA analysis (significance level of 5%)
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by using SigmaStat–Statistical Software (Jandel Corporation, San Rafael, CA). 3. Results and discussion 3.1. Aspects of instrumentation Prior to the GC × GC-O analyses, several aspects of the system were investigated, mainly due to the presence of the retention gaps with rather large dimensions, connecting the final part of the 2 D column to the FID and to the sniffing port. Experiments showed that the presence of both retention gaps lead to excessive peak tailing and 2 D resolution loss, so that the previously achieved separation was not preserved. Furthermore, the initial hydrogen linear velocity applied to the system, caused the retention of polar perfume compounds in the 2 D column, which were released in subsequent elution sequences (occurrence of wraparound). The optimization of instrumental parameters turned out to be indispensable. It must be emphasized that the GC2010 gas chromatograph possesses an innovative flow control system which maintains constant linear velocity during the analysis; however, this kind of option may be easily adopted when a single column is installed, whereas for GC × GC-O applications, the use of two columns, characterized by different internal diameters, connected in series, and further extended by means of two retention gaps, is not recognized by the GC software. In order to overcome this limit the equivalent dimensions of the GC × GC-O columns [15] and retention gap set, are determined by using standard flow relationships. Following this, the different pressure values at specific points of interest (Pz ) were derived, providing an estimation of the head pressure applied in the 2 D column and retention gaps. Hence, the average gas linear velocities were estimated in all parts of the set-up via the calculated pressure drops. Another important aspect is the high volatility which characterizes many analytes present in perfumes. In preliminary experiments the modulation temperature in the first 15 min of analysis proved to be insufficient for the entrapment of these substances, which as a consequence of the lack in zone compression were not separated due to peak broadening. The problem was overcome with a decrease, during the initial part of the analysis of the oven/cryotrap temperature difference (−150 ◦ C), enabling efficient compression, and therefore an improved peak capacity. Moreover a slow temperature program (1.5 ◦ C/min temperature program rate) was applied, so that the increase in oven temperature between the first and last accumulated pulses for a given compound was negligible. 3.2. A challenge for the human olfactive system The discriminatory capacity of mammalian olfactory system is such that thousands of volatile chemicals are perceived as having distinct odours. Due to the great efforts over the last decade using various experimental approaches [16–19], the complex pathways of this intriguing system are emerging, including the mechanisms through which the brain decodes and discriminates odorants.
The time period for the odour perception transduction process to occur is still an untouched field of the olfactive system research, according to McGinley et al. (2000) [20] it can last 500 ms. Considering this period of time, one might consider an olfactive analysis with an applied modulation period of 8 s cycle feasible when a single compound or a simple matrix is analysed, but on the other hand too brief for the human brain to decode a high number of olfactive information arising when complex sample analyses are employed. From a panelist for GC × GC-O analysis a high olfactive accuracy, as also exacerbated concentration power and dynamism, are required. Furthermore the breathing pattern during analysis has also a critical function (see Section 2). 3.3. GC-O and GC × GC-O analyses The commercial perfume sample was initially analysed by means of GC-O, and then through GC × GC-O, with the aim of deriving an olfactive fingerprint of this complex matrix. GC-O analyses enabled the detection of 177 compounds and among these 135 presented odour-activity. On the other hand, by means of GC × GC-O about 481 compounds out of 818 were considered to be odour-active. The GC-O chromatogram of the commercial perfume presents a multitude of components, mainly represented by linalool, limonene, linalyl acetate -pinene, geranial, ␣-isomethyl ionone, ␣-hexyl cinnamaldehyde, neral, (Z)methyl-dihydrojasmonate, estragole and eugenol. On the other hand the analogous result, obtained with a 8 s modulation period for the cryogenic trap, is characterized by an increased number of peaks, and thus a more thorough olfactive analysis can be performed. The GC-O chromatogram and GC × GC-O 2 D plot (A and B, respectively) are presented in Fig. 2. In general, the overlap of two or more compounds in monodimensional GC can represent a serious problem for olfactometry. Major problems arise when small amounts of analyte are to be determined in the presence of greater interferences. In aroma research it is well known that potent odour-active compounds are often present as small peaks. Compared to monodimensional GC-O analysis the increase in sensitivity by zone compression in the modulator observed in GC × GC-O leads to the detection of several trace-level compounds. Odours can be quantified by distinct parameters; one of the used terms is threshold concentration, which can be further described at four levels, namely detection, recognition, difference and terminal thresholds. However, only detection and recognition thresholds were considered to be of relevance for the present work. Detection threshold is defined as the lowest concentration or intensity that is perceived by the panelist, while recognition threshold is the lowest concentration or intensity at which a substance or an olfactive quality attribute can be identified and described [21]. Taking in consideration the diverse chemical nature of the sample, the differences in threshold values and the complexity of the “pulsed sniffing”, some segments of the chromatogram were selected to be highlighted in the present work, such as the 2 D chromatogram expansion showed in Fig. 3.
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Fig. 2. GC-O chromatogram (A) and GC × GC-O 2 D plot (B) of the commercial perfume achieved on identical columns and retention gap set without (A) and with (B) cryogenic modulation. For analysis conditions refer to Section 2.
It is worthwhile to point out that the excessive tailing, which can be observed in this figure, is relative to the two most abundant compounds of this perfume formulation, linalool (spot 1) and linalyl acetate (spot 9). Their excessive concentration leads to
a severe overloading of the 1 D column resulting in its bleeding, which results in curved streaks across the 2 D plane. These, however, do not interfere in the determination of odour-active compounds.
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Fig. 3. Expansion of the linalool (spot 1) and linalyl acetate (spot 9) zones in the 2 D plot of the commercial perfume. The numbers refer to those in Table 1.
The zone, with its largest peak identified as linalool (spot 1), by means of GC × GC/MS data, 1 D LRI match, standard injection, and olfactive description, presented to be an outstanding example for the power of GC × GC-O. Through GC-O analysis the single peak was clearly identified as linalool, even though it consisted also of trace-level compounds eliciting similar olfactive impression revealed by GC × GC-O. Although tetrahydrolinalool is commonly applied in floral perfume formulations to enhance the effect of linalool [1], taking in consideration that both the aforementioned compounds possess extremely close LRIs, and that linalool presents to be the major compound in this perfume, it was considered of relevance to investigate the influence of this linalool co-adjuvant on the formulation, establishing its fingerprint. In this specific case, the distinction was not a simple task for the assessors, since linalool was described to
present floral, woody, citric, sweet and fresh notes, whereas spot 2, identified as tetrahydrolinalool, elicited linalool-like impressions with an enhanced powdery note. It has to be pointed out that tetrahydrolinalool, even present at a trace-level concentration, was detected and recognized due to its low threshold value. Even though the threshold of the latter compound is slightly higher than that of linalool [22]. At a first glance, due to the similar olfactive impressions elicited by both the compounds, it resembled the sniffing of a compound in wrap-around effect. As could be observed during the olfactive analyses, in GC × GC-O the wrap-around effect can be perceived and distinguished by the evaluator due to the fact that the noise generated by the mechanical stepper motor drive responsible for the movement of the cryotrap is used as a reference. So an odour-active compound in wrap-around would result in an olfactive sensation interrupted by the moving of the cryotrap. However, this possibility was excluded by the consideration that both impressions not only presented a slight difference, but were also distinctly detected in a single modulation period. A non-symmetrical peak-pulse distribution has also been considered, though observing the pulsed 1 D chromatogram and the 2 D plot, the presence of the trace component, tetrahydrolinalool, hidden behind the interference, linalool, has been confirmed. Linalool was positively matched in seven sequential pulses, at retention times of 17.96, 18.09, 18.22, 18.36, 18.49, 18.62 and 18.75 min, while tetrahydrolinalool presented four pulses at 17.92, 18.05, 18.18 and 18.31 min. For both compounds an approximate 8s-interval was maintained between each successive pulse, corresponding to the modulation frequency. Since linalool is the most abundant component of the sample, and so its 2 D chromatogram consists of a greater number of pulses, other co-elutions could be detected by means of olfactometry; such as n-octenyl acetate (spot 3) described as eliciting green notes, and hotrienol (spot 4) presenting a floral impression (refer to Table 1).
Table 1 Some odour descriptions achieved by means of GC × GC-O and GC-O analysis of a commercial perfumea Spotb
Compound
GC × GC-O LRI
1 2 3 4 5 6 7 8 9 10 11 12 13 14 a b c d
Linalool Tetrahydrolinalool n-Octenyl acetate Hotrienol Hexyl isovalerate Unidentified Neral Unidentified Linalyl acetate Carvone Cuminaldehyde Unidentified Geraniol Unidentified
ref.c
1101 1102 1109 1109 1243 – 1238 – 1250 1246 1247 – 1255 –
GC-O Odour description
LRI exp.d
Odour description
floral, citric, sweet, fresh linalool-like, powdery green floral green, fruity, unripe detected, not recognized citric citric, fresh floral, sweet, citric minty, herbaceous sharp, woody, caraway slightly citric floral, geranium-like fruity
1101 – 1109 – – – 1238 – 1249 – – – 1254 –
floral, citric, powdery, sweet – green, floral undertone – – – citric, green – floral, minty, slightly caraway – – – floral, fruity –
Peak assignment was achieved by means of different information sources: GC/MS, GC × GC/MS, LRI, injection of standards and olfactive impressions. The spot numbers refer to Fig. 3. Linear retention indices available in the FFNSC ver. 1.2 MS Library (Shimadzu, Milan, Italy). Calculated linear retention indices.
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In the case of a commercial perfume, the co-elution of linalool and tetrahydrolinalool is not of economical relevance, but of formulation importance. Nevertheless, in the case of an essential oil, since tetrahydrolinalool is a synthetic material, it would confirm an adulteration. This case confirms the superiority of GC × GC-O to complex sample analysis when compared to GC-O investigations. A further 2 D plot zone, worth of mention, was characterized in GC-O analyses as a single abundant peak, reported as an agglomeration of impressions, such as floral, minty with caraway notes. Although GC/MS analysis identified this peak as linalyl acetate, those olfactive descriptions could not be related to a single compound. In the GC × GC-O evaluations, on the other hand, this zone (Fig. 3) presented a series of co-elutions in the 1 D column, particularly due to the high concentration of linalyl acetate (spot 9), characterized by seven sequential pulses at 29.92, 30.05, 30.19, 30.32, 30.45, 30.58 and 30.71 min, causing interferences with other substances, mainly carvone (29.96, 30.09, 30.23, 30.36, 30.49 and 30.62 min) and cuminaldehyde (29.98 and 30.11 min) (spots 10 and 11, respectively). The aforementioned compounds, as also other minor ones are presented in Table 1, accompanied by their olfactive description. This zone presented to be of relevance for the quality tracing of essential oils used as raw materials in perfume formulations. Linalyl acetate is often used as a reference compound for the quality control of some citrus essential oils, e.g. bitter orange petitgrain oil. Furthermore, it is worthwhile to point out that this 2 D zone exhibits a plurality of co-elutions, represented in Fig. 3 as spots numbered from 5 to 7 and 8 to 12, as well as 13 and 14. Once more it has to be pointed out that the detection and recognition of an odour-active compound is concentration-dependent, and for this reason the analyses of standard compounds were used as reference. It is worthwhile to emphasize that cuminaldehyde, although present in trace level concentration, elicited a relatively strong caraway-like odour, surpassing that of the more abundant linalyl acetate. Notable is also the richness in odour diversities which can be identified through the very fast, and due to the sequential pulses, repetitive olfactive impressions, so that the olfactive quality registered by the assessor in a previous modulation is repeated, and can be thereby confirmed. Several studies consider that the intermittency of the appearance of odour-active compounds also plays an important role reducing sensory adaptation, thus maximizing the amount of olfactory information available to the brain for subsequent analysis [23]. It has been observed, with a certain frequency, that some compounds could be detected but not have their olfactive impression recognized, probably due to their presence in ultra-trace level concentrations, combined with a high recognition threshold value, and being sampled only once. The partial co-elution of Sandalore® and pentadecane illustrates this case. Sandalore® elicits a sandalwood-like note, and presents to have a low threshold value [19], while pentadecane, exhibits a comparable higher odour threshold and elicits an odour characteristic for alkanes. Performing GC-O a single note of sandalwood was elicited, while in GC × GC-O, pentadecane could not be described, but detected, so that the stimulus has been distinguished from an odour-free situation, and the identity afterwards assigned by
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means of 1 D LRI calculation, GC × GC/MS data correlation, and confirmed by standard compounds analysis. In case of a GCO analysis and quantification by GC-FID, the concentration of Sandalore® would be over-estimated, leading to an inaccurate quantitative determination of the perfume formulation. An important aspect to be highlighted regards the analysis time required to complete characterize a sample by GC-O and GC × GC-O; respectively, two and three chromatographic runs. The increment in analysis time consumption by GC × GC-O is related to the demand on efficient olfactometric detection, and is compensated by a more sophisticated distinction of the odour-active compounds present in the perfume. 3.4. GC/MS and GC × GC/MS analyses Mass spectrometric analyses were exploited for peak assignment. GC × GC/MS and GC × GC-O chromatograms of the commercial perfume and standard compounds presented very good correspondence in terms of peak retention. The aforementioned compound identities were confirmed by 1 D LRI, MS data correlation with those present in the FFNSC ver. 1.2 MS Library, chromatogram profile comparison, and injection of standards, supported by the obtained olfactive information. As published elsewhere [9], in GC/MS analysis a total of 186 compounds were detected in the perfume, amongst these 58 were positively identified, while in GC × GC/MS analysis 169 compounds were identified out of 866 detected. With regards to GC-O and GC × GC-O analyses, 177 and 818 compounds were detected, respectively. 4. Conclusions According to the trends of the age and the development of novel derivatives of fragrant compounds, yielding a wide variety of new odorants, techniques regarding their analysis have also to be improved and optimized. In this respect, the above investigation demonstrated that GC × GC-O fulfils this purpose, enabling the bidimensional separation and identification of odour-active compounds, even when present in trace-level concentrations, or when eliciting a rather confused odour perception, comparable to an agglomerate of olfactive impressions. However, during GC × GC-O analyses emphasis must be placed on the compounds odour threshold values, an important aspect that rules olfactive analysis. Furthermore, the assessors shall be trained with emphasis on the odours behaviour on dilution. The odour fingerprint determination by means of GC × GCO, demonstrates that commercial perfume is indeed a very complex sample, and conventional GC-O analyses would fail to adequately record the presence of all constituents. GC × GCO may be considered an unprecedented odour fingerprint acquisition technique, of great use for perfume and fragrance industries, being implemented in quality control or in research and development departments. Representing not only a promising development of olfactometry, as also a new feature of GC × GC. Future research will be devoted to the development of a GC × GC/MS-O system.
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Acknowledgements The Authors gratefully acknowledge Shimadzu Corporation and Supelco Corporation for the continuous support. References [1] T. Curtis, D.G. Williams, Introduction to Perfumery, Micelle Press, New York, 2001. [2] R.R. Calkin, J.S. Jellinek, Perfumery: Practice and Principles, WileyInterscience, New York, 1994. [3] S.M. van Ruth, Biomolec. Eng. 17 (2001) 121. [4] B. d’Acampora Zellner, M. Lo Presti, L.E.S. Barata, P. Dugo, G. Dugo, L. Mondello, Anal. Chem. 78 (2006) 883. [5] A. van Asten, TrAC-Trends Anal. Chem. 21 (2002) 698. [6] C. Sell, in: D.H. Pybus, C.S. Sell (Eds.), The Chemistry of Fragrances, Royal Society of Chemistry, Cambridge, 1999, p. 24. [7] Z. Liu, J.B. Phillips, J. Chromatogr. Sci. 1067 (1991) 227. [8] J.B. Phillips, J. Beens, J. Chromatogr. A 856 (1999) 331. [9] L. Mondello, A. Casilli, P.Q. Tranchida, G. Dugo, P. Dugo, J. Chromatogr. A 1067 (2005) 235. [10] L. Mondello, A. Casilli, P.Q. Tranchida, P. Dugo, S. Festa, G. Dugo, J. Sep. Sci. 27 (2004) 442.
[11] J.E. Friedrich, T.E. Acree, E.H. Lavin, in: J.V. Leland, P. Schieberle, A. Buettner, T.E. Acree (Eds.), Gas Chromatography-Olfactometry: The State of the Art, ACS, Washington, DC, 2001, p. 149. [12] A.C. Guyton, J.E. Hall, Textbook of Medical Physiology, W.B. Saunders Company, Philadelphia, 1996. [13] J.P.H. Linssen, J.L.G.M. Janssens, J.P. Roozen, M.A. Posthumus, Food Chem. 8 (1993) 1. [14] H. van den Dool, P.D. Kratz, J. Chromatogr. 11 (1963) 463. [15] R. Shellie, P. Marriott, P. Morrison, L. Mondello, J. Sep. Sci. 27 (2004) 504. [16] L. Buck, R. Axel, Cell 65 (1991) 175. [17] B. Malnic, J. Hirono, T. Sato, Cell 96 (1999) 713. [18] G. Sicard, A.C. Holley, Brain Res. 292 (1984) 283. [19] S. Bieri, K. Monastyrskaia, B. Schilling, Chem. Senses 29 (2004) 483. [20] C.M. McGinley, M.A. McGinley, D.L. McGinley, Odor Basics, Understanding and Using Odor Testing. Presented at the 22nd Annual Hawaii Water Environment Association Conference, Honolulu, Hawaii, 6th–7th June, 2000. [21] A. Richardson, in: D.H. Pybus, C.S. Sell (Eds.), The Chemistry of Fragrances, Royal Society of Chemistry, Cambridge, 1999, p. 145. [22] K. Bauer, D. Garbe, H. Surburg, Common Fragrance and Flavor Materials, VCH Verlagsgesellschaft, Weinheim, 1990. [23] T.A. Christensen, T. Heinbockel, J.G. Hildebrand, J. Neurobiol. 30 (1996) 82.
Odour fingerprint acquisition by means of comprehensive two-dimensional gas chromatography-olfactometry and comprehensive two-dimensional gas chromatography/mass spectrometry Barbara d’Acampora Zellner a , Alessandro Casilli a , Paola Dugo b , Giovanni Dugo a , Luigi Mondello a,∗ b
a Dipartimento Farmaco-Chimico, Facolt` a di Farmacia, Universit`a di Messina, viale Annunziata, 98168 Messina, Italy Dipartimento di Chimica Organica e Biologica, Facolt`a di Scienze, Universit`a di Messina, Contrada Papardo, 98166 Messina, Italy
Received 4 October 2006; received in revised form 4 December 2006; accepted 8 December 2006 Available online 16 December 2006
Abstract The analysis of complex matrices, such as perfumes, by means of gas chromatography-olfactometry (GC-O) can be rather imprecise due to the co-elutions, leading to a possible masking of odour-active trace-level compounds by major interferences or agglomeration of olfactive impressions resulting in unreliable olfactive characterization. To overcome these limits an innovative technique, comprehensive two-dimensional gas chromatography-olfactometry (GC × GC-O), was applied, revealing several relevant co-elutions, as in the linalool and linalyl acetate zones. A total of 177 compounds, out of these 135 odour-active, were detected by GC-O, while about 481 out of 818 compounds presented odour-activity through GC × GC-O analyses. In addition, GC/mass spectrometry (GC/MS) and GC × GC/MS analyses were also performed. Peak assignment was achieved by means of different information sources, such as GC/MS, GC × GC/MS, LRI, injection of standards and olfactive impressions. © 2006 Elsevier B.V. All rights reserved. Keywords: Comprehensive two-dimensional gas chromatography-olfactometry (GC × GC-O); GC-O; GC × GC/MS; GC/MS; Perfume analysis
1. Introduction Fragrance materials have their use traced to early antiquity, when spices and resins from animal and plant sources were used for the purpose of perfumery. Perfumes have been intimately associated with human history and are comprised by complex mixtures of odorant materials, the blending of which is a good example of product engineering. Today, perfumers work with a total of several thousand ingredients. On average, 30 to 50 (and sometimes up to 200) ingredients, synthetically manufactured, as well as natural fragrances, are used to create a fragrance composition [1]. Modern methods of analysis have not only enhanced the acquisition of a higher perfumery raw material knowledge, but also brought a greater emphasis on perfume creation. The combination of market forces with gas chromatography (GC) and its features resulted in an explosive accelera-
∗
Corresponding author. Tel.: +39 090 676 6536; fax: +39 090 676 6532. E-mail address: [email protected] (L. Mondello).
0021-9673/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2006.12.035
tion of the evolution rate of flavour and fragrance materials [2]. For decades monodimensional GC and GC/mass spectrometry (GC/MS), as well as GC-olfactometry (GC-O), have been commonly employed in perfume industries. GC-O is a wellknown standard technique which enables the assessment of odour-active components in complex mixtures, based on the correlation between the chromatographic peaks of the eluted substances perceived simultaneously by two detectors, one of them being the human olfactory system. This valuable tool for the investigation of flavour and fragrance matrices is applied for a wide range of samples by using different assessment methods [3]; enantioselective GC-O for the determination of sensory properties of enantiomers has also been carried out [4]. However, considering that perfume raw materials are characterized by a wide variety of components belonging to several chemical classes, extensive co-elutions may occur both on non-polar and polar stationary phases leading to inaccurate identification of odour-active compounds. Moreover numerous compounds, present at trace-level concentrations, can still exert an important
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olfactive or economical impact [5,6]. Since higher chromatographic separation capacities are required and can be hardly achieved by a single capillary column, the demand for more powerful techniques has emerged. Comprehensive two-dimensional gas chromatography (GC × GC) appears to be the most appropriate choice to fulfil the request for enhanced separation and better sensitivity. This orthogonal two-column separation, with complete sample transfer executed by means of a modulator able to trap, refocus and release fractions of the GC effluent from the first dimension (1 D, column 1), and periodically introduce them onto the second dimension (2 D, column 2), enables an accurate screening of complex matrices, offering very high resolution and enhanced detection sensitivity [7,8]. The features of GC × GC are of great usefulness for the perfumery industry, in the determination of individual raw materials quality and authenticity, the detection of essential oils in perfumes by establishing specific patterns, or even the possibility of an automated perfume formulation without unwanted co-eluting components and precise quantification of each raw material [5]. Since GC-O is a technique of great importance in the fragrance industry, the hyphenation of GC × GC to olfactometry (GC × GC-O), providing a bidimensional separation and simultaneous olfactive characterization of components in a complex matrix, likewise represents to be an outstanding approach to suppress the need for co-elution free and more reliable olfactive analysis. This novel technique associates the resolution power of GC × GC with the selectivity and sensitivity of the human olfactory system, enabling the olfactive analysis of congested chromatographic areas. To the author’s knowledge the application of GC × GC-O as a screening procedure for establishing the odour fingerprint of a perfume has not previously been reported in literature. The purpose of this research was to investigate the application of GC × GC-O to a perfume analysis and to obtain the odour fingerprint by means of a complete qualitative characterization of this complex sample. The magnitude of interferences and their influence on the performance of olfactometric analysis was also investigated. Additionally, aspects regarding the panelists, or assessors, observed during the performance of the analysis will also be discussed. 2. Experimental 2.1. Samples A commercial perfume (eau de toilette) was purchased in a local perfumery and stored at 4 ◦ C. That perfume, a male fragrance with predominantly citric fresh and herbaceous notes, used in all analyses has already been analysed and reported by the research group [9]. The following standard compounds were purchased from Sigma–Aldrich (Milan, Italy): linalool, tetrahydrolinalool, n-octenyl acetate, hexyl isovalerate, linalyl acetate, carvone, cuminaldehyde, pentadecane and geraniol; while Sandalore® was purchased from Givaudan (D¨ubendorf, Switzerland). Hotrienol and neral were confirmed by means of the analysis of essential oils containing these compounds, Ho and Bergamot oil, respectively.
2.2. Sample preparation The commercial perfume was analysed undiluted. Each standard component was diluted with n-hexane (1:10, 1:50, 1:100, 1:500 and 1:1000 v/v) prior to injection. 2.3. GC × GC-O analyses GC × GC-O analyses were carried out on a Shimadzu GC2010 gas chromatograph equipped with an AOC-20i autoinjector and an AOC-20s autosampler (Shimadzu, Milan, Italy). The GC was retrofitted with an Everest Longitudinally Modulating Cryogenic System (LMCS) (Chromatography Concepts, Doncaster, Australia) and hyphenated to the Phaser - Sniffing Port OP275 (ATAS GL International B.V., Veldhoven, The Netherlands). The mechanical stepper motor drive responsible for the movement of the cryotrap was initiated by an electronic device via the GC Solution software (Shimadzu) programmed external event, while the cryotrap temperature was regulated by a temperature control system through the Comprehensive Chromatography System Control–CCSC software (Shimadzu). The latter enables the programming of the cryogenic temperature maintaining a defined temperature difference between the cryotrap and the GC oven temperature. A modulation period of 0.125 Hz (8 s cycle) was applied to all GC × GC analyses and the cryogenic trap was maintained at a temperature of 150 ◦ C below the prevailing GC oven temperature for the first 15 min, and at 100 ◦ C below the GC oven until the end of the analysis. Furthermore, a modified carbon dioxide (CO2 ) cylinder, as described elsewhere [10], delivered the gas at a pressure of 125 bar to the LMCS system. The supplied CO2 cools the cryotrap by its expansion, while a small internal flow of nitrogen (N2 ) gas (approximately 10 mL/min) was applied to prevent ice formation inside the trap. Data were collected by the GC Solution software, and by using its export function converted to ASCII data, which were then processed by the Comprehensive Chromatography Manager ver. 1.1 (Shimadzu) achieving a contour representation of the two-dimensional chromatograms. The olfactometry system consisted of a four-port splitter stand located in the GC oven; two ports were connected to the second dimension column outlet and an auxiliary gas outlet; the two remaining ports were connected to the flame ionization detector (FID) and the transfer line (sniffing port) by means of retention gaps (Fig. 1). A control module regulates the transfer line heater, the auxiliary gas supply and the nasal mucosa humidifier (for further information refer to [4]). The transfer line, maintained under constant temperature of 250 ◦ C, ends up in an ergonomic glass nose cone. An auxiliary gas (He) flow of 5 ml/min was kept constant during analyses. Considering the demand for fast response of the evaluators during GC × GC-O analysis, the OdoChronometer software (Shimadzu) has been developed, this is integrated to the GC Solution software, being initiated with the start out of the GC. The software runs along with the chromatographic analysis and registers the time whenever the panelist presses a push-button; the signals are compiled and data output is easily opened as a text file presenting a retention time registration table.
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The breathing pattern also had to be adapted to GC × GC-O, suggested was abdominal breathing which uses primarily the diaphragm, and is congruent with the shape of the lungs and the capacities of the breathing muscles [12]. It processes the most air with the least effort, and is associated with mental stability and concentration, avoiding both dizziness and hyperventilation. 2.6. Detection frequency method
Fig. 1. Schematic representation of the comprehensive two-dimensional gas chromatography-olfactometry instrumentation.
The column set consisted of a SLB-5ms (5% diphenyl95% dimethylpolysiloxane) 30 m × 0.25 mm i.d. × 0.25 m df , serially connected by a zero-dead-volume union (Supelco, Milan, Italy) to a Supelcowax 10 (polyethylene glycol) 0.75 m × 0.1 mm i.d. × 0.10 m df (both columns were purchased from Supelco). The GC was operated under temperature-programmed conditions, from 50 ◦ C to 200 ◦ C at 1.5 ◦ C/min, to 250 ◦ C (20 min) at 20 ◦ C/min. The GC-2010 was equipped with a split/splitless injector (290 ◦ C); injection volume: 0.3 L, in splitless mode injection (1 min sampling time); initial inlet pressure: 130.0 kPa; carrier gas: H2 ; detector: FID (260 ◦ C); H2 flow: 50.0 mL/min; air flow: 400.0 mL/min; make up flow (N2 ): 50.0 mL/min; sampling rate: 8 ms. The sniffing procedure was divided in 5 min sessions with 10 min intervals in order to avoid lassitude. In three analyses the entire chromatographic run was covered by one panelist; each one performed the analyses six times in distinct days. 2.4. GC-O analyses The analyses were carried out by using the GC × GC-O system without cryogenic modulation. The sniffing procedure was divided in 20 min sessions with 15 min interval and the analyses were carried out in triplicate. 2.5. Panel GC × GC-O and GC-O analyses were performed by two evaluators with previous experience in GC-O, both were screened for specific anosmia using the standard solution set proposed by Friedrich et al. [11]; no insensitivities were identified. However, GC × GC-O requests a sharp discerning capacity, utmost concentration and fast response of the panelists, so that the demand for further and accurate training turned out to be indispensable. The training consisted of GC-O, GC × GC-O and direct olfactive analyses of several co-eluting standard mixtures, neat and dilutions, since some odorants are perceived as having distinct odour quality at different concentrations. In each analysis the panelists were asked to describe the odour quality of the perceived impressions according to a glossary of olfactive descriptors suggested by Curtis and Williams [1] and adopted by the group.
Sensorial evaluations were performed using an adaptation of the method proposed by Linssen et al. [13]. The adaptation consisted on the detection of odour-active compounds by two assessors, accompanied by the qualitative description of that odour according to the glossary of olfactive descriptors adopted by the group. 2.7. GC × GC/MS analyses GC × GC/MS analyses were carried out on a Shimadzu GCMS-QP2010 gas chromatograph mass spectrometer equipped with an AOC-20i autoinjector and the Flavour and Fragrance Natural and Synthetic Compounds (FFNSC) ver. 1.2 MS Library (Shimadzu). The GC was retrofitted with the same aforementioned modulator, applying identical modulation period and cryogenic trap temperature program. Data were collected by the GCMS Solution software and likewise converted to ASCII data. Contour representations of the two-dimensional chromatograms were achieved through the same aforementioned Comprehensive Chromatography Manager ver. 1.1 software. The column set and oven temperature program were identical to the GC × GC-O analyses. The GCMS-QP2010 was equipped with a split/splitless injector (250 ◦ C); injection volume: 1.0 L, in split mode (10:1); initial inlet pressure: 119.2 kPa; carrier gas: He; interface temperature 250 ◦ C; MS ionization mode: electron ionization; detector voltage: 0.9 kV; acquisition mass range: 40–400 amu; scan speed 10000 amu/s; acquisition mode: full scan; scan interval: 0.05 s (20 Hz); solvent delay: 5 min. Analyses were carried out in triplicate. 2.8. GC/MS analyses The above-described GC × GC/MS system was used without cryogenic modulation. Analyses were carried out in triplicate. 2.9. First dimension linear retention index (LRI) The first dimension LRI determinations, in both described GC × GC systems, were carried out by injecting a homologous series of n-alkanes containing 24 n-hydrocarbons (C7-C30) (Supelco, Milan, Italy), at a 1000 ppm concentration in hexane. The indices were calculated according to the equation proposed by van den Dool and Kratz [14]. The LRI for GC/MS and GC-O analyses were likewise established. 2.10. Statistical evaluation Data obtained from the GC-O and GC × GC-O analyses were subjected to one way ANOVA analysis (significance level of 5%)
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by using SigmaStat–Statistical Software (Jandel Corporation, San Rafael, CA). 3. Results and discussion 3.1. Aspects of instrumentation Prior to the GC × GC-O analyses, several aspects of the system were investigated, mainly due to the presence of the retention gaps with rather large dimensions, connecting the final part of the 2 D column to the FID and to the sniffing port. Experiments showed that the presence of both retention gaps lead to excessive peak tailing and 2 D resolution loss, so that the previously achieved separation was not preserved. Furthermore, the initial hydrogen linear velocity applied to the system, caused the retention of polar perfume compounds in the 2 D column, which were released in subsequent elution sequences (occurrence of wraparound). The optimization of instrumental parameters turned out to be indispensable. It must be emphasized that the GC2010 gas chromatograph possesses an innovative flow control system which maintains constant linear velocity during the analysis; however, this kind of option may be easily adopted when a single column is installed, whereas for GC × GC-O applications, the use of two columns, characterized by different internal diameters, connected in series, and further extended by means of two retention gaps, is not recognized by the GC software. In order to overcome this limit the equivalent dimensions of the GC × GC-O columns [15] and retention gap set, are determined by using standard flow relationships. Following this, the different pressure values at specific points of interest (Pz ) were derived, providing an estimation of the head pressure applied in the 2 D column and retention gaps. Hence, the average gas linear velocities were estimated in all parts of the set-up via the calculated pressure drops. Another important aspect is the high volatility which characterizes many analytes present in perfumes. In preliminary experiments the modulation temperature in the first 15 min of analysis proved to be insufficient for the entrapment of these substances, which as a consequence of the lack in zone compression were not separated due to peak broadening. The problem was overcome with a decrease, during the initial part of the analysis of the oven/cryotrap temperature difference (−150 ◦ C), enabling efficient compression, and therefore an improved peak capacity. Moreover a slow temperature program (1.5 ◦ C/min temperature program rate) was applied, so that the increase in oven temperature between the first and last accumulated pulses for a given compound was negligible. 3.2. A challenge for the human olfactive system The discriminatory capacity of mammalian olfactory system is such that thousands of volatile chemicals are perceived as having distinct odours. Due to the great efforts over the last decade using various experimental approaches [16–19], the complex pathways of this intriguing system are emerging, including the mechanisms through which the brain decodes and discriminates odorants.
The time period for the odour perception transduction process to occur is still an untouched field of the olfactive system research, according to McGinley et al. (2000) [20] it can last 500 ms. Considering this period of time, one might consider an olfactive analysis with an applied modulation period of 8 s cycle feasible when a single compound or a simple matrix is analysed, but on the other hand too brief for the human brain to decode a high number of olfactive information arising when complex sample analyses are employed. From a panelist for GC × GC-O analysis a high olfactive accuracy, as also exacerbated concentration power and dynamism, are required. Furthermore the breathing pattern during analysis has also a critical function (see Section 2). 3.3. GC-O and GC × GC-O analyses The commercial perfume sample was initially analysed by means of GC-O, and then through GC × GC-O, with the aim of deriving an olfactive fingerprint of this complex matrix. GC-O analyses enabled the detection of 177 compounds and among these 135 presented odour-activity. On the other hand, by means of GC × GC-O about 481 compounds out of 818 were considered to be odour-active. The GC-O chromatogram of the commercial perfume presents a multitude of components, mainly represented by linalool, limonene, linalyl acetate -pinene, geranial, ␣-isomethyl ionone, ␣-hexyl cinnamaldehyde, neral, (Z)methyl-dihydrojasmonate, estragole and eugenol. On the other hand the analogous result, obtained with a 8 s modulation period for the cryogenic trap, is characterized by an increased number of peaks, and thus a more thorough olfactive analysis can be performed. The GC-O chromatogram and GC × GC-O 2 D plot (A and B, respectively) are presented in Fig. 2. In general, the overlap of two or more compounds in monodimensional GC can represent a serious problem for olfactometry. Major problems arise when small amounts of analyte are to be determined in the presence of greater interferences. In aroma research it is well known that potent odour-active compounds are often present as small peaks. Compared to monodimensional GC-O analysis the increase in sensitivity by zone compression in the modulator observed in GC × GC-O leads to the detection of several trace-level compounds. Odours can be quantified by distinct parameters; one of the used terms is threshold concentration, which can be further described at four levels, namely detection, recognition, difference and terminal thresholds. However, only detection and recognition thresholds were considered to be of relevance for the present work. Detection threshold is defined as the lowest concentration or intensity that is perceived by the panelist, while recognition threshold is the lowest concentration or intensity at which a substance or an olfactive quality attribute can be identified and described [21]. Taking in consideration the diverse chemical nature of the sample, the differences in threshold values and the complexity of the “pulsed sniffing”, some segments of the chromatogram were selected to be highlighted in the present work, such as the 2 D chromatogram expansion showed in Fig. 3.
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Fig. 2. GC-O chromatogram (A) and GC × GC-O 2 D plot (B) of the commercial perfume achieved on identical columns and retention gap set without (A) and with (B) cryogenic modulation. For analysis conditions refer to Section 2.
It is worthwhile to point out that the excessive tailing, which can be observed in this figure, is relative to the two most abundant compounds of this perfume formulation, linalool (spot 1) and linalyl acetate (spot 9). Their excessive concentration leads to
a severe overloading of the 1 D column resulting in its bleeding, which results in curved streaks across the 2 D plane. These, however, do not interfere in the determination of odour-active compounds.
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Fig. 3. Expansion of the linalool (spot 1) and linalyl acetate (spot 9) zones in the 2 D plot of the commercial perfume. The numbers refer to those in Table 1.
The zone, with its largest peak identified as linalool (spot 1), by means of GC × GC/MS data, 1 D LRI match, standard injection, and olfactive description, presented to be an outstanding example for the power of GC × GC-O. Through GC-O analysis the single peak was clearly identified as linalool, even though it consisted also of trace-level compounds eliciting similar olfactive impression revealed by GC × GC-O. Although tetrahydrolinalool is commonly applied in floral perfume formulations to enhance the effect of linalool [1], taking in consideration that both the aforementioned compounds possess extremely close LRIs, and that linalool presents to be the major compound in this perfume, it was considered of relevance to investigate the influence of this linalool co-adjuvant on the formulation, establishing its fingerprint. In this specific case, the distinction was not a simple task for the assessors, since linalool was described to
present floral, woody, citric, sweet and fresh notes, whereas spot 2, identified as tetrahydrolinalool, elicited linalool-like impressions with an enhanced powdery note. It has to be pointed out that tetrahydrolinalool, even present at a trace-level concentration, was detected and recognized due to its low threshold value. Even though the threshold of the latter compound is slightly higher than that of linalool [22]. At a first glance, due to the similar olfactive impressions elicited by both the compounds, it resembled the sniffing of a compound in wrap-around effect. As could be observed during the olfactive analyses, in GC × GC-O the wrap-around effect can be perceived and distinguished by the evaluator due to the fact that the noise generated by the mechanical stepper motor drive responsible for the movement of the cryotrap is used as a reference. So an odour-active compound in wrap-around would result in an olfactive sensation interrupted by the moving of the cryotrap. However, this possibility was excluded by the consideration that both impressions not only presented a slight difference, but were also distinctly detected in a single modulation period. A non-symmetrical peak-pulse distribution has also been considered, though observing the pulsed 1 D chromatogram and the 2 D plot, the presence of the trace component, tetrahydrolinalool, hidden behind the interference, linalool, has been confirmed. Linalool was positively matched in seven sequential pulses, at retention times of 17.96, 18.09, 18.22, 18.36, 18.49, 18.62 and 18.75 min, while tetrahydrolinalool presented four pulses at 17.92, 18.05, 18.18 and 18.31 min. For both compounds an approximate 8s-interval was maintained between each successive pulse, corresponding to the modulation frequency. Since linalool is the most abundant component of the sample, and so its 2 D chromatogram consists of a greater number of pulses, other co-elutions could be detected by means of olfactometry; such as n-octenyl acetate (spot 3) described as eliciting green notes, and hotrienol (spot 4) presenting a floral impression (refer to Table 1).
Table 1 Some odour descriptions achieved by means of GC × GC-O and GC-O analysis of a commercial perfumea Spotb
Compound
GC × GC-O LRI
1 2 3 4 5 6 7 8 9 10 11 12 13 14 a b c d
Linalool Tetrahydrolinalool n-Octenyl acetate Hotrienol Hexyl isovalerate Unidentified Neral Unidentified Linalyl acetate Carvone Cuminaldehyde Unidentified Geraniol Unidentified
ref.c
1101 1102 1109 1109 1243 – 1238 – 1250 1246 1247 – 1255 –
GC-O Odour description
LRI exp.d
Odour description
floral, citric, sweet, fresh linalool-like, powdery green floral green, fruity, unripe detected, not recognized citric citric, fresh floral, sweet, citric minty, herbaceous sharp, woody, caraway slightly citric floral, geranium-like fruity
1101 – 1109 – – – 1238 – 1249 – – – 1254 –
floral, citric, powdery, sweet – green, floral undertone – – – citric, green – floral, minty, slightly caraway – – – floral, fruity –
Peak assignment was achieved by means of different information sources: GC/MS, GC × GC/MS, LRI, injection of standards and olfactive impressions. The spot numbers refer to Fig. 3. Linear retention indices available in the FFNSC ver. 1.2 MS Library (Shimadzu, Milan, Italy). Calculated linear retention indices.
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In the case of a commercial perfume, the co-elution of linalool and tetrahydrolinalool is not of economical relevance, but of formulation importance. Nevertheless, in the case of an essential oil, since tetrahydrolinalool is a synthetic material, it would confirm an adulteration. This case confirms the superiority of GC × GC-O to complex sample analysis when compared to GC-O investigations. A further 2 D plot zone, worth of mention, was characterized in GC-O analyses as a single abundant peak, reported as an agglomeration of impressions, such as floral, minty with caraway notes. Although GC/MS analysis identified this peak as linalyl acetate, those olfactive descriptions could not be related to a single compound. In the GC × GC-O evaluations, on the other hand, this zone (Fig. 3) presented a series of co-elutions in the 1 D column, particularly due to the high concentration of linalyl acetate (spot 9), characterized by seven sequential pulses at 29.92, 30.05, 30.19, 30.32, 30.45, 30.58 and 30.71 min, causing interferences with other substances, mainly carvone (29.96, 30.09, 30.23, 30.36, 30.49 and 30.62 min) and cuminaldehyde (29.98 and 30.11 min) (spots 10 and 11, respectively). The aforementioned compounds, as also other minor ones are presented in Table 1, accompanied by their olfactive description. This zone presented to be of relevance for the quality tracing of essential oils used as raw materials in perfume formulations. Linalyl acetate is often used as a reference compound for the quality control of some citrus essential oils, e.g. bitter orange petitgrain oil. Furthermore, it is worthwhile to point out that this 2 D zone exhibits a plurality of co-elutions, represented in Fig. 3 as spots numbered from 5 to 7 and 8 to 12, as well as 13 and 14. Once more it has to be pointed out that the detection and recognition of an odour-active compound is concentration-dependent, and for this reason the analyses of standard compounds were used as reference. It is worthwhile to emphasize that cuminaldehyde, although present in trace level concentration, elicited a relatively strong caraway-like odour, surpassing that of the more abundant linalyl acetate. Notable is also the richness in odour diversities which can be identified through the very fast, and due to the sequential pulses, repetitive olfactive impressions, so that the olfactive quality registered by the assessor in a previous modulation is repeated, and can be thereby confirmed. Several studies consider that the intermittency of the appearance of odour-active compounds also plays an important role reducing sensory adaptation, thus maximizing the amount of olfactory information available to the brain for subsequent analysis [23]. It has been observed, with a certain frequency, that some compounds could be detected but not have their olfactive impression recognized, probably due to their presence in ultra-trace level concentrations, combined with a high recognition threshold value, and being sampled only once. The partial co-elution of Sandalore® and pentadecane illustrates this case. Sandalore® elicits a sandalwood-like note, and presents to have a low threshold value [19], while pentadecane, exhibits a comparable higher odour threshold and elicits an odour characteristic for alkanes. Performing GC-O a single note of sandalwood was elicited, while in GC × GC-O, pentadecane could not be described, but detected, so that the stimulus has been distinguished from an odour-free situation, and the identity afterwards assigned by
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means of 1 D LRI calculation, GC × GC/MS data correlation, and confirmed by standard compounds analysis. In case of a GCO analysis and quantification by GC-FID, the concentration of Sandalore® would be over-estimated, leading to an inaccurate quantitative determination of the perfume formulation. An important aspect to be highlighted regards the analysis time required to complete characterize a sample by GC-O and GC × GC-O; respectively, two and three chromatographic runs. The increment in analysis time consumption by GC × GC-O is related to the demand on efficient olfactometric detection, and is compensated by a more sophisticated distinction of the odour-active compounds present in the perfume. 3.4. GC/MS and GC × GC/MS analyses Mass spectrometric analyses were exploited for peak assignment. GC × GC/MS and GC × GC-O chromatograms of the commercial perfume and standard compounds presented very good correspondence in terms of peak retention. The aforementioned compound identities were confirmed by 1 D LRI, MS data correlation with those present in the FFNSC ver. 1.2 MS Library, chromatogram profile comparison, and injection of standards, supported by the obtained olfactive information. As published elsewhere [9], in GC/MS analysis a total of 186 compounds were detected in the perfume, amongst these 58 were positively identified, while in GC × GC/MS analysis 169 compounds were identified out of 866 detected. With regards to GC-O and GC × GC-O analyses, 177 and 818 compounds were detected, respectively. 4. Conclusions According to the trends of the age and the development of novel derivatives of fragrant compounds, yielding a wide variety of new odorants, techniques regarding their analysis have also to be improved and optimized. In this respect, the above investigation demonstrated that GC × GC-O fulfils this purpose, enabling the bidimensional separation and identification of odour-active compounds, even when present in trace-level concentrations, or when eliciting a rather confused odour perception, comparable to an agglomerate of olfactive impressions. However, during GC × GC-O analyses emphasis must be placed on the compounds odour threshold values, an important aspect that rules olfactive analysis. Furthermore, the assessors shall be trained with emphasis on the odours behaviour on dilution. The odour fingerprint determination by means of GC × GCO, demonstrates that commercial perfume is indeed a very complex sample, and conventional GC-O analyses would fail to adequately record the presence of all constituents. GC × GCO may be considered an unprecedented odour fingerprint acquisition technique, of great use for perfume and fragrance industries, being implemented in quality control or in research and development departments. Representing not only a promising development of olfactometry, as also a new feature of GC × GC. Future research will be devoted to the development of a GC × GC/MS-O system.
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Acknowledgements The Authors gratefully acknowledge Shimadzu Corporation and Supelco Corporation for the continuous support. References [1] T. Curtis, D.G. Williams, Introduction to Perfumery, Micelle Press, New York, 2001. [2] R.R. Calkin, J.S. Jellinek, Perfumery: Practice and Principles, WileyInterscience, New York, 1994. [3] S.M. van Ruth, Biomolec. Eng. 17 (2001) 121. [4] B. d’Acampora Zellner, M. Lo Presti, L.E.S. Barata, P. Dugo, G. Dugo, L. Mondello, Anal. Chem. 78 (2006) 883. [5] A. van Asten, TrAC-Trends Anal. Chem. 21 (2002) 698. [6] C. Sell, in: D.H. Pybus, C.S. Sell (Eds.), The Chemistry of Fragrances, Royal Society of Chemistry, Cambridge, 1999, p. 24. [7] Z. Liu, J.B. Phillips, J. Chromatogr. Sci. 1067 (1991) 227. [8] J.B. Phillips, J. Beens, J. Chromatogr. A 856 (1999) 331. [9] L. Mondello, A. Casilli, P.Q. Tranchida, G. Dugo, P. Dugo, J. Chromatogr. A 1067 (2005) 235. [10] L. Mondello, A. Casilli, P.Q. Tranchida, P. Dugo, S. Festa, G. Dugo, J. Sep. Sci. 27 (2004) 442.
[11] J.E. Friedrich, T.E. Acree, E.H. Lavin, in: J.V. Leland, P. Schieberle, A. Buettner, T.E. Acree (Eds.), Gas Chromatography-Olfactometry: The State of the Art, ACS, Washington, DC, 2001, p. 149. [12] A.C. Guyton, J.E. Hall, Textbook of Medical Physiology, W.B. Saunders Company, Philadelphia, 1996. [13] J.P.H. Linssen, J.L.G.M. Janssens, J.P. Roozen, M.A. Posthumus, Food Chem. 8 (1993) 1. [14] H. van den Dool, P.D. Kratz, J. Chromatogr. 11 (1963) 463. [15] R. Shellie, P. Marriott, P. Morrison, L. Mondello, J. Sep. Sci. 27 (2004) 504. [16] L. Buck, R. Axel, Cell 65 (1991) 175. [17] B. Malnic, J. Hirono, T. Sato, Cell 96 (1999) 713. [18] G. Sicard, A.C. Holley, Brain Res. 292 (1984) 283. [19] S. Bieri, K. Monastyrskaia, B. Schilling, Chem. Senses 29 (2004) 483. [20] C.M. McGinley, M.A. McGinley, D.L. McGinley, Odor Basics, Understanding and Using Odor Testing. Presented at the 22nd Annual Hawaii Water Environment Association Conference, Honolulu, Hawaii, 6th–7th June, 2000. [21] A. Richardson, in: D.H. Pybus, C.S. Sell (Eds.), The Chemistry of Fragrances, Royal Society of Chemistry, Cambridge, 1999, p. 145. [22] K. Bauer, D. Garbe, H. Surburg, Common Fragrance and Flavor Materials, VCH Verlagsgesellschaft, Weinheim, 1990. [23] T.A. Christensen, T. Heinbockel, J.G. Hildebrand, J. Neurobiol. 30 (1996) 82.