Microsoft Word - 3debree.docx CHEMICAL ENGINEERING TRANSACTIONS VOL. 54, 2016 A publication of The Italian Association of Chemical Engineering Online at www.aidic.it/cet Guest Editors: Selena Sironi, Laura Capelli Copyright © 2016, AIDIC Servizi S.r.l., ISBN 978-88-95608-45-7; ISSN 2283-9216 Comparative Study of Odours Present in Twin Fragrances by GC-sniffing-ToFMS Carmen Villatoroa, Luciano Vera*a, Hansruedi Gygaxb a Odournet SL. Av. de Can Domenech. Parc de Recerca UAB. 08193 Bellaterra – Cerdanyola del Vallès – Barcelona -Spain b Gygarome Consulting. Marktbündtenstrasse 8. 7310 Bad Ragaz, Switzerland lvera@odournet.com Fragrances have the power to generate an identity and evoke memories and sensations based on their characteristic raw materials. These raw materials correspond to volatile organic compounds (VOCs) present at concentrations above their odour threshold value in gaseous phase. Knowledge about the fragrance formula allows create modifications of the fragrance or to launch an imitation. These products try to evoke the smell of the original fragrance whereas being commercialized at more affordable prices to the end consumer. Consequently, it is important to establish limits of sensory identity, or put in another way, to quantify the limits where the imitation fragrance does not jeopardize the identity of the original. Defining sensory delineations is a difficult task because of the subjective nature of sensorial experience. However, it is becoming increasingly possible to devise unambiguous methods due to recent technological advancements in the sensitivity of instruments. It can be done by means of instruments capable of acting as human noses, or rather, acting as a powerful combination of nose-instrument complexes. These complexes take advantage of the complementary capacities existing between the high sensitivity of the human olfactory system and robust instrumental analysis techniques. GC-Sniffing, a human-instrument complex, is the most powerful analytical technique for odour identification. It can obtain chemical and sensory information in a single chromatographic analysis, with an experimented analyst detecting odours at the same instant in which the chemicals responsible for those odours are being chemically detected by the instrument. This study analyses two similar samples by GC-Sniffing-ToFMS, one of them commercialized as an imitation of the other, with the purpose of finding those sensory and chemical differences that make each one of them unique. The results show different concentrations of odour compounds and sensory perception by GC- Sniffing-ToFMS in the original and its perfume-twin. Some odour compounds were identified as key markers to understand sensory differences between the twin fragrances under study. 1. Introduction The global market for Fragrances and Perfume is forecast to exceed US$40 billion by 2020 (GIA, 2015). 83% of women wear perfume occasionally and 36% daily. The successful of this powerful industry is due to its extensive marketing, high profit margins and a careful choice of the target audience. Retail price of perfumes give to the company a 95% profit and only 3% of the cost spent on the production and ingredients. Because perfumes can be associated with a brand, large establishments (clothing stores try that people can recognize its own perfume to enter) or companies with environmental awareness, want to strengthen their image by creating a perfume for their consumers without animal testing or synthetic chemicals as part of their business strategy. Increasingly, applications multiply and the market increases, so it becomes more necessary the existence of protocols to describe uniquely fragrances of each brand, and even to patent its formulation, and also to differentiate between an original perfumes and its copy. Determination of the chemical composition of a fragrance is a complex task. On average, 30 to 50 (and sometimes up to 200) ingredients, synthetically manufactured, as well as natural extracts or essences, are used to create a fragrance composition (Curtis, et al., 2001). Fragrance analysis has radically changed over the last 15–20 years, this is mainly to the rapid evolution of analysis techniques and the fast increase in the number of chemical analyses a routine laboratory is required to run. The analysis of aroma compounds in DOI: 10.3303/CET1654023 Please cite this article as: Villatoro C., Vera L., Gigax H., 2016, Comparative study of odours present in twin fragrances by gc-sniffing-tofms, Chemical Engineering Transactions, 54, 133-138 DOI: 10.3303/CET1654023 133 fragrances, based on detection and identification of volatile organic compounds (VOCs) is preferably carried out by gas chromatography (GC) in combination with mass spectrometry (GC-MS). Although GC-MS can provide chemical identification of volatile spices in fragrances, it does not provide qualitative information about sensory perception of the aroma molecules. GC-Olfactometry (GCO) or GC-sniffing technique improves the performance of GC-MS systems in terms of odour analysis because it allows to obtain a sensory description (by a trained assessor) of the each fragrance molecule eluted from a chromatographic run while at the same time the these molecules are identified by a chemical detector, commonly MS (Guichard, et al., 1995; Mayol and Acree, 2001). In this way, a system GCO- MS is probably the most powerful technique for chemical characterization of odorous compounds and fragrance analysis (Bratolli et al., 2013; Delahunty et al., 2006). Sensitivity has a crucial role in this type of analysis. Odours are detected by the human nose at very low concentrations (low ppt), so it is necessary the use of highly sensitive detector in this type of instrumental configuration to avoid, to the extent possible, cases of odour detected by the nose without a spectral signal. Time of Flight-Mass Spectrometer (ToFMS) is the most sensitive chemical detector able to detect molecular traces at concentration levels of a 100 times lower than standard MS detectors (Vera et al., 2013). Linking the molecular information provided by a GC-ToFMS with the perceived intensity and odour description by the sniffing technique, allows detailed understanding of the key odour impact molecules present in the perfume. The main objective of this study is to compare the odours perceived between perfume-twin with their respective chemical identification through TD-GCO-ToFMS (Thermal Desorption-Gas Chromatography- Olfactometry-Time of Flight Mass Spectrometry). 2. Instrumentation The chromatographic analyses were carried out by a thermodesorption unit (TD) coupled to a Gas Chromatograph-Mass Spectrometer (GC-ToFMS) instrument. The instrumental system is composed of a Gas Chromatograph (Agilent 7890 model, Agilent, USA), Time-of-Flight Mass Spectrometer (BenchTOF-dx model, Almsco, Germany) and Thermal Desorption (Unity model, Markes International Limited, Llantrisant, UK). The column used was the mid polar DB-624; (60m, 250µm, 1.4µm; Agilent, USA). The deconvolution process providing the chemical identification from the GC-MS data was carried out by using TargetView V3 (software developed by ALMSCO International, Germany). The sniffing analysis was performed by an olfactory detector port OP275 (GL Sciences Inc., Japan). The type of sorbent tubes used was TenaxTA/Carbograph5 (Markes International Limited, Llantrisant, UK). All tubes, before used were conditioned by TC20 (tube conditioning-20) (Markes International Limited, Llantrisant, UK). A manual pump (EasyVOC, Markes International Limited, Llantrisant, UK) was used to capture the volatile compounds in gas phase to the sorbent tubes 3. Experimental procedures 3.1 Sample preparation For this study, one women perfume-twin was selected (Perfume A and B). In two different samplings for each perfume, 25μl of perfume was direct injected on a Nalophan bag with 1 L of inert gas (nitrogen 5.0). After 10 minutes of equilibration time, 100 ml of the headspace was collected into a sorbent tube through a manual pump. Additionally, one thermodesorption tube without sample and under the same sampling conditions than real samples was collected as a blank. 3.2. Instrumental method Desorption of the analytes retained on the TenaxTA/ Carbograph5TD sorbent tubes was carried out in a Unity Thermal Desorption system. In the primary desorption, tubes were heated to 300ºC with a helium flow rate of 50 mL min-1 during 8 min. This was done to desorb the analytes which were refocused on a hydrophobic general purpose cold trap, filled with Tenax TA and a graphitised carbon, cooled at 10ºC. After flash-heating of the cold trap at 320ºC during 5 min, analytes were injected into the chromatographic column. Separation and detection were performed in a 7890N gas chromatograph and Time-of-Flight Mass spectrometer, using a mid-polar DB-624 capillary column (60m, 250μm, 1.4μm) and helium gas (6.0) as the carrier at a flow rate of 1.5 mL min-1. The oven temperature of the GC was initially held at 40ºC for 5 min, then raised to 165ºC at a rate of 13ºC min-1 and then raised again to 230ºC at a rate of 4ºC min-1 and held at that temperature for 5 min. The GC-MS interface was set at 230ºC. The mass spectrometer acquired data in scan mode with an m/z interval from 47 to 400, operating at an electron impact energy of 70 eV. The odour-active VOCs directed to the sniffing port were detected and characterized by two trained assessors at 22ºC. As soon as the assessor detects an odour, attribute, durability and intensity values (from 1 to 5) are 134 assigned (Table 1). On an additional run, the Mass Spectrometer detects the chemical species in the sample by using the same instrumental methodology for the posterior identification of chemical compounds, both odorous and odourless. During the analysis, each assessor takes on the GC-sniffing task for 18 minutes before handing over to the other assessor, to cover the 36 minutes (approx.) of the whole chromatographic process. In this way the sniffing task is divided in 2 periods covering the entire chromatogram. Each assessor covers all chromatographic runs twice, so that each assessor covers the entire chromatogram, to confirm those doubtful odours and /or try to find other additional odours not detected in the previous sniffing. Therefore, two sample injections are necessary to analyse each sample. The analysis of the blank and the real sample were analyzed in duplicate. Table 1: Scale of values used to describe the odours perceived Value Mean Characteristic 1 Faintly perceived Difficult to describe 2 Perceived noticeable Noticeable 3 Strong Very well perceived 4 Very strong Nasal saturation 5 Extremely strong Only extreme cases (cause interruption of sniffing process) 3.2 Data processing All compounds detected by MS were chemically identified using TargetView software referencing to the NIST11 spectral library. Compounds above 80% of similarity were considered to be identified. Odours detected by sniffing only were confirmed after being detected at least twice out of the total 4 analyses of the sample. Those odours identified just once were discarded. Before assigning a chemical compound name (chemical structure) to each of the odours perceived, four checks were performed: 1) direct check through MS identification, 2) Candidates search based on Kovats index and odour descriptor. An additional tube containing C6-C18 hydrocarbons was analysed to find their retention times on the GC method used. These retention times are used as reference to calculate the Kovats index of the rest of compounds, from a defined Kovats index value assigned to hydrocarbons. 3) Automatic scan by using a library prepared exclusively for this project containing all relevant candidate compounds and their retention times. 4) Exhaustive manual searching based on the presence of target ions of candidate compounds in key retention times. 4. Results and discussion In general, the results of the GCO -ToFMS analyses show that the perfume A has a similar chromatographic profile in comparison to the perfume B (Figure 1). 63 and 54 VOCs were identified in perfume A and B respectively. The comparative graph of Figure 2 shows that the highest concentration of compounds identified in the samples were terpenes and esters. Figure 1. GC-MS chromatograms of perfume A and B. 5.00 10.00 15.00 20.00 25.00 30.00 0 1e+07 2e+07 3e+07 4e+07 5e+07 6e+07 7e+07 Time--> Abundance TIC: Perfume_FK_DBC.d\ data.ms 5.00 10.00 15.00 20.00 25.00 30.00 0 1e+07 2e+07 3e+07 4e+07 5e+07 6e+07 7e+07 Time--> Abundance TIC: Perfume_FR_DBC.d\ data.ms Perfume A Perfume B 135 Figure 2 shows a slight difference in the concentration of terpenes for perfume A compared to B. Out of a total of 33 terpenes identified in both samples 32 were identified in perfume A and only 22 in perfume B. Terpenes identified only in perfume A were: α-gurjunene, α-bisabolene, α-guaiene, Longifolene, (E)-beta-Famesene, Germacrene D, alloocimene, Linalool, Carveol, 3-Carene and C10H16 terpene. 4(10)-thujene was identified only in perfume B. Figure 3 shows the overlapped chromatograms in the range 30-35 minutes containing some terpenes and guaiyl acetate. Figure 2. Comparison based on chemical families for both perfumes (A and B). Figure 3. Overlapped chromatograms showing some of the identified terpenes and guaiyl acetate. Of the total odours obtained in the GC-sniffing, we only show the most relevant odours with its chemical identification (Table 2). According to the information in table 2, important amount of terpenes and esters were identified in both perfumes. On the other hand, isoprenyl acetate, guaiyl acetate (esters), beta.-Myrcene, (-)-β- Pinene, Longifolene, α-Guaiene (terpenes) showed the highest intensity values in the chemically identified odours for perfume A compared to B. The assessors perceived these compounds as woody, green, minty, sweet and floral. Conversely, we have identified the terpene 4(10)-thujene at 19.5 min only in perfume B giving a turpentine and woody notes. Figure 4 shows little differences between perfume A and B in the intensities of sensory profile for floral, woody and citrus notes. These differences are key to the overall smell of each perfume. 0.E+00 1.E+09 2.E+09 Alcohols Aldehydes Aromatic Alcohol Esters Ketones Oxygen-containing compounds Terpenes Perfume A Perfume B 30 .1 g er m ac re ne D 31 .4 (E )- be ta -F ar m es en e 32 .0 C ar yo ph yl le ne 31 .8 L on gi fo le ne + α- G ua ie ne 33 .1 α -b is ab ol en e 33 .7 α -g ur ju ne ne 32 .6 G ua iy la ce ta te 30.00 30.50 31.00 31.50 32.00 32.50 33.00 33.50 34.00 34.50 0 500000 1000000 1500000 2000000 2500000 3000000 3500000 4000000 Time--> Abundance TIC: Perfume A TIC: Perfume B 136 Table 2: Odorants perceived during sniffing process and chemically identified by ToFMS Intensity b IK a RT Odour descriptor Compound CAS Perf A Perf B 734 17.5 Woody, green, minty Isoprenyl acetate 5205-07-2 2 1 760 18.6 Pine, turpentine α-Pinene 80-56-8 3 3 777 19.4 Anise, fruity, green, sweet beta-Myrcene 123-35-3 2 1 781 19.5 Turpentine, woody 4(10)-thujene 3387-41-5 2 783 19.7 Anise, minty (-)-β-pinene 18172-67-3 3 1 795 20.3 Pine, minty beta-Ocimene 13877-91-3 2 2 807 20.8 Minty ß-Phellandrene 555-10-2 2 2 816 21.2 Citrus, herbaceous, turpentine gamma-Terpinene 99-85-4 3 3 851 22.6 Floral Linalyl anthranilate 7149-26-0 3 3 862 23.0 Fruity, herbaceous, sweet Methyl benzoate 93-58-3 3 3 870 23.4 Fruity, sweet Linalyl hexanoate 7779-23-9 2 2 895 24.3 Citrus, floral β-Terpinyl acetate 10198-23-9 3 3 900 24.5 Floral, fruity, sweet Benzyl acetate 140-11-4 3 3 914 25.1 Floral, sweet 4-Terpinenyl acetate 4821-04-9 3 3 924 25.5 Floral, minty, oily α-Terpineol 98-55-5 1 1 941 26.1 Citrus, sweet Nerol 106-25-2 3 3 952 26.7 Sweet, minty Lavandulyl acetate 25905-14-0 3 3 1105 31.8 Woody Longifolene+α-Guaiene 475-20-7 / 3691-12-1 2 1110 32.0 Spicy, woody Caryophyllene 87-44-5 3 3 1127 32.6 Floral, fresh Guaiyl acetate 134-28-1 2 a Kovats index: normalized ratio of the retention time eluting n-alkane. b Scale from 1 to 5: 1, very low; 2, low; 3, medium; 4, strong; 5, extremely strong. Figure 4. Sensory profile (sum of intensity) of perfume A and B based on odor descriptor and intensity. 137 5. Conclusions With the made findings we conclude that GC-Sniffing methodology is a useful and powerful tool to determine differences in perfume twins. Both chemical and sensory results showed clear differences in the original perfume and its twin. Some terpenes (beta.-Myrcene, (-)-β-Pinene, Longifolene, α-Guaiene) and esters (isoprenyl acetate, guaiyl acetate) were identified as key odour impact molecules for the original perfume providing important differences in some notes as anise, floral, fruity and sweet. References Brattoli M., Cisternino E., Dambruoso P.R., de Gennaro G., Giungato P., Mazzone A., Palmisani J., Tutino M., 2013, Gas Chromatography Analysis with Olfactometric Detection (GC-O) as a Useful Methodology for Chemical Characterization of Odorous Compounds, Sensors, 13, pp. 16759-16800. 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