Optimization of a system for determining volatile
odorants by gas chromatography-olfactometry
M. Tomás, A. Escudero, V. Ferreira, J. Cacho
Laboratory for flavour analysis and enology
Aragon Institute of Engineering Research (I3A).
Faculty of Sciences. University of Zaragoza, Pedro Cerbuna, 12. 50009, Zaragoza, Spain
Tel. +34-976 761290 e-mail: [email protected]
Abstract
The main aim of the present work was to develop a method to quantify very volatile compounds
(acetaldehyde, dimethyl sulphide, butyraldehyde, ethyl acetate, ethyl propionate, diacetyl and
ethyl thioacetate) in wine. In an attempt to simulate the perception of volatiles in a wine glass,
the volatiles present in the headspace of a wine sample were trapped in a sorbent and further
analyzed by gas chromatography-olfactometry (GC-O). Within this work, special attention has
also been paid to optimize two parameters of capital importance in gas-chromatography: the
solvent and the injection mode. Different extraction techniques have been studied to obtain a
headspace extract whose aroma represents the original aroma of wine: dynamic headspace
coupled to solid-phase extraction (DHS-SPE), solid-phase microextraction of headspace (HS-
SPME), static headspace (SHS) and single-drop extraction (SDME). Nowadays, DHS-SPE is
the most widely used extraction technique in the aroma of wine, however, this technique is not
appropriate to determine very volatile odorants. For this reason, other extraction techniques
(HS-SPME, SHS and SDME) have been studied. The best results have been obtained by HS-
SPME and SDME. Ethyl acetate has not been detected in any of the olfactometries.
Keywords: gas chromatography-olfactometry, volatile compound, headspace, aroma extract,
sensory analysis.
1 Introduction
Analytic chemistry of the aroma has an important challenge which is being able to analyze the
responsible compound in the perception of the aromas and flavors released into the nose and
mouth. It means, valuing the compound which cause these feelings and the importance of each
one in these feelings.
The aroma of foods, in general, is a very important organoleptic characteristic to distinguish and
value them. Specifically, in wine, the aroma determines the quality of this product. The analytic
chemistry of wine is very complex. First, because there are many different wines showing quite
different aromas, Second, because the aroma of even a single wine change with time, while it is
stored in the bottle and while it is waiting in the glass to be consumed. Finally, because in most
cases wine do not have a simple characteristic aroma; rather have a palette of subtle aromas
which are very difficult to define and which surely are perceived differently by the different
people. Wine contains thousands of different molecules, and a number approaching one
thousand is formed by volatile molecules. Many of these volatile molecules have some aroma,
but only a limited number of volatiles can be found in wines at concentrations high enough to be
Optimization of a system for determining volatile odorants by gas chromatography-olfactometry
2 PYR-001-11-ART
perceived. Obviously, these compounds are the ones which will have a major influence on the
aroma characteristics of wine and, therefore, are the ones which will have to be analyzed (target
compounds) and considered further in sensory studies (Ferreira and Cacho, 2009).
One of the characteristics that a chemical compound must have to be considered important as
aroma is its volatility, which is the capacity of a substance to evaporate at a determinate
temperature and pressure. A substance is more volatile when its vaporization point is lower.
Molecular weight and polarity determine volatility. Most of the compounds responsible for the
aroma of wine evaporate at room temperature.
Gas chromatography (GC) is the most commonly used procedure for aroma analysis, with
different injectors and detectors. For the analysis of aroma compounds the best GC detector is
the human nose (sniffing port) because only with our senses we can affirm that a compound
smells more than other. For this reason, in the last decades, gas chromatography and
olfactometry have been used for determining the volatile odorants potentially involved in the
aroma of a product (Campo et al, 2005; Ferreira et al, 2002). There are three important
olfactometry (GC-O) techniques: dilution methods, techniques based on frequency and
intensity.
Dilution methods consist on the olfactometry analysis of successive solutions of extracts (1:2,
1:5, 1:10, …) until no odorants are detected in the sniffing port. It is based on the threshold
perception of a compound in air and not on an intensity measurement. Two techniques can be
distinguished, AEDA and CHARM, which main difference is how data is recorded.
Techniques based on the measurement of intensity, consist of preparing only an extract and then
let the judges evaluate the intensity of the odor eluting out of the column. Two different
alternatives for the evaluation of the magnitude of the odor intensity have been proposed. The
first one, time-intensity technique, is based on the presence or absence of the odorant and its
intensity. In the second one, post-intensity, the judges give a numerical measurement of the
overall intensity of an odor eluted out of the column. The intensity of one odorant is obtained
from the average of the intensity given by the judges. The main limitation is the different
utilization of the intensity scale by judges.
In techniques based on frequency, the judges only indicate the presence or absence of the
odorant during the olfactometry. The total intensity is expressed like NIF value (Nasal Impact
Frequency) and this represents the time the odorant has been detected (Campo, 2006).
Extract employed for GC-O have to be representative of product´s aroma. Different techniques
allow isolating the volatile compounds of wine to carry out its evaluation. Some of them are
based on an extraction with an organic solvent, solid-phase extraction and techniques based on
headspace. The extracts should reflect as closely as possible the composition of vapor reaching
the pituitary during the olfaction process and not the composition in volatile compound in the
original sample. It means that the classical approach in which the extract contains quantitatively
all the volatiles present in the product is very far from the optimal solution. The major problem
with these extracts is that they ideally contain 100 % of all the aroma molecules of wine and
hence GC-O experiment will detect 100 % of all the aroma molecules originally present in the
samples. However, during the normal olfaction some aroma molecules are transported very
Optimization of a system for determining volatile odorants by gas chromatography-olfactometry
3 PYR-001-11-ART
efficiently from the liquid wine in the glass or in our mouth to the pituitary while others are
transported just in a negligible proportion because they are strongly retained by the wine
hydroalcholic matrix (Ferreira and Cacho, 2009).
For all these reasons, headspace techniques are the most appropriate to analyze volatile
compounds and headspace techniques can be divided into dynamics and statics. Static
headspace sampling consist of inject directly the vapor phase into the chromatograph. Dynamic
headspace techniques are more sensitive because the vapor phase is renewed due to an inert gas
which is passing continuously, so that, more quantity of volatile compound is trapped.
Dynamic headspace techniques are more appropriate to analyze the volatile compound when the
concentration is in the range between ppm and ppt. If the capture is with LiChrolut EN resins
through SPE, the reconcentration stage is guaranteed because few organic solvent well be used.
Ultimately, in order to obtain a headspace extract, the SPE seems an optimal solution due to its
versatility, automation and the small request of solvent (Ferriera et al, 2004; San Juan et al,
2010).
During the last decade, our research group has employed on “artificial throat” in order to extract
volatile compounds from wine within this approach, solid-phase extraction and dynamic
headspace are combined. Volatile compounds are dragged by a stream of nitrogen and are
trapped in the resins. After that, these are desorbed with an organic solvent in which there are
dissolved and this extract is analyzed by gas chromatograph-olfactometry. Lately, this system
has been studied and optimized (San Juan, 2006).
Another form to introduce the volatile compounds into GC-O is headspace solid-phase
microextraction (SPME). In this case, a polymeric microfiber is used for extracting and
concentrating the analytes directly from the matrix. This technique has being used from the last
two decades because it is easy to automatize and environment friendly (organic solvents are not
used). In this work, SPME is used because it is a suitable alternative, because organic solvents
are not used and the analytes are not masked by solvent (López et al, 2007; Matero-Vivaracho et
al, 2006).
Once the extract has been obtained, the injection system and the solvent play an important role.
The common injector in GC-O is the split/splitless injector. If we work in the splitless mode
with dichloromethane as solvent, analytes and the solvent are eluted together and our analytes
can´t be detected. Some alternatives have been proposed: working with a solvent less volatile
than the analytes, so the solvent is eluted after them; working on split mode as an amount of
vapor is taken out or working on “on-column”, as in this mode the sample is introduced as
liquid into the column or pre-column without previously vaporization.
Analytic chemistry of wine aroma, analyzes qualitatively and quantitatively volatile compounds
which are in this product. But, do these results reproduce the sensory perception when a wine is
tasted? Is coherent the amount extracted with our feeling? For that, in this work, the main
objective is compare the results obtained in the sensory analysis and in the different extraction
techniques and in the olfactometry.
Optimization of a system for determining volatile odorants by gas chromatography-olfactometry
4 PYR-001-11-ART
2 Material and Methods
2.1 Reagents and standards
Dichloromethane and methanol of SupraSolv quality, ethanol of LiChroSolv quality (GC) and
n-hexane of UniSolv quality were obtained from Merk; 1-butanol (≥ 99.5 %, GC) and
isobutanol (PA) were purchased from Panreac, and pure water was obtained from a Milli-Q
purification system. Acetaldehyde (≥ 99.5 %), ethyl acetate (≥99.7 %) and ethyl thioacetate (≥
99.5 %) were supplied by Sigma-Aldrich. Dimethyl sulphide (DMS) (≥ 99 %), butyraldehyde (≥
99 %), ethyl propionate (≥99.5 %) and diacetyl (≥ 99.5 %) were supplied by Fluka. Sodium
sulphate anhydrous and L-tartaric acid were obtained from Panreac; ammonium sulphate and
sodium chloride were supplied by Sigma-Aldrich.
Synthetic wine samples were prepared with a 12 % (v/v) ethanolic solution containing 5 gL-1
of
tartaric acid and the pH was adjusted to 3.4 with NaOH diluted.
2.2 Gas chromatography
All analysis were carried out using a Trace (ThermoQuest) gas chromatograph equipped with a
flame ionization detector (FID) and a sniffing port. The column used was a DB-WAX, 30 m x
0.32 mm with 0.5 µm film thickness, hydrogen as the carrier gas (3.5 mL/min), split injection,
injection volume, 1 µL. Injector and detector were both kept at 250 ºC. The temperature
program was the following: 40ºC for 5 minutes, then raised at 2ºC/min up to 150ºC.
2.3 Sensory Analysis
2.3.1 Participants
A panel of seven judges, between the ages of 22 and 40 years old, belonging to the laboratory
staff members participated in this study.
2.3.2 Triangular Test (UNE 87-006-92)
A triangular test was performed to determine the odor threshold of the compounds. Each
panelist was asked to smell three samples, two of which were identical, and asked to indicate
which sample differed from the others. The number of correct responses was compared with a
1/3 probability table to show if there were significant differences between the samples (UNE
87-006-92).
2.3.3 Intermediate intensity determination
In order to carry out the calibration of the results obtained by GC-O a comparison between with
the perception in a glass is needed. Therefore, the concentrations of which supply an
intermediate orthonasal intensity in a glass are looked for. The panelists received previous
training to get familiar with the odor of each compound. In this first session, judges were asked
to rank, according to perceived orthonasal intensity, four glasses with different concentrations
(0, 10, 100 and 1000 times higher than its odor threshold) of each in synthetic wine. Following
the initial training period, various synthetic wines were prepared, containing each compound to
Optimization of a system for determining volatile odorants by gas chromatography-olfactometry
5 PYR-001-11-ART
be studied in varying concentrations. Orthonasal intensity, in sensory analysis and olfactometry,
was evaluated with a 3-point scale (0 = no odour, 1 = weak odour, 2 = clear perception of odour,
strong intensity; 3 = extremely strong intensity of odour; intermediate values of 0.5; 1.5; 2.5
being allowed). Panelist evaluated the orthonasal intensity of a solution containing an unknown
concentration by comparing it with the two references (0 = synthetic wine, 3 = maximum
orthonasal intensity) (San Juan, 2010).
2.4 DHS-SPE extraction
A standard SPE cartridge (0.8 cm internal diameter, 3 mL internal volume) filled with 400 mg
of LiChrolut EN resins was first washed with 20 mL of
dichloromethane and then dried by letting air pass through
it. The cartridge was placed on top of a bubbler flask
(Figure 1) containing 80 mL of wine. A controlled stream
of nitrogen (500 mL/min) was passed through the sample
for 100 min, the cartridge was removed and dried by
letting N2 pass through; then analytes were eluted with 3.2
mL of dichloromethane with 5% ethanol. After this, the
extract was concentrated under a stream of pure N2 to a
final volume of 200 µL. This extract was injected in the
gas chromatograph (San Juan, 2010).
2.5 Solid-phase microextraction (HS-SPME)
Headspace sampling of analytes was carried out with a CombiPal autosampler
equipped with a polymeric fiber which was conditioned by a stream of N2 for
60 minutes at 250 ºC. The extraction was performed with agitation at 250 rpm
in cycles of 5 s on and 2 s off. Twenty milliliter vials were used for headspace
sampling. Desorption took place in the injection port at 250 ºC for 10 minutes.
Previously, the glass liner was changed by a specific to SPME.
2.6 Static Headspace Analysis (SHS)
The synthetic wine samples (15 mL) were pipetted into a vessel (volume 20 ml), sealed with a
septum, and equilibrated for 5 minutes at 40 ºC. 250 µL were drawn by a gastight syringe and
injected in GC-O (Guth, 1997).
2.7 Single-Drop Microextraction (SDME)
Prior to each extraction the microsyringe was rinsed at least 20 times with acetone followed by
10 times with 2-octanol. The plunger was then placed at the 1 µL mark and the tip of the syringe
needle was placed into contact with the extracting solvent. Two µL were withdrawn from the
solvent vial into the syringe. The syringe needle then pierces the sample vial septum (always in
the same position) and the drop of solvent is exposed to the sample headspace (figure 2). After
the extraction period, the solvent drop was withdrawn back into the syringe and directly injected
Figure 2: HS-SPMe system
Figure 1: DHS-SPE system
Optimization of a system for determining volatile odorants by gas chromatography-olfactometry
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into the chromatographic system. The drop volume (~2 µL) was kept constant
for all experiments. 10 mL of synthetic wine and 1.5 g of NaCl were added to
a vessel (20 mL), sealed with a septum, and equilibrated for 25 minutes at 48
ºC (Martenda, Budziak and Carasek, 2007)
3 Results and discussion
3.1 Sensory analysis
Table 1 shows the concentrations which provided an intermediate intensity in the glass. These
values were used for calibrating the different extraction techniques and gas chromatography-
olfactometry.
Table 1: Thresholds, concentrations and orthonasal intensity of studied compounds
a: n = 7; b: n = 6
The concentrations which provide an intermediate intensity in the glass are different for each
compound, from 20 times the odor threshold (ethyl acetate) to 1000 times (diacetyl). This
difference could appear as a consequence of a matrix effect and the specific odor of the
compounds. Additionally, the interactions between diacetyl and different matrixes are already
described (Martineau, Acree and Henick-Kling, 1995). Ethyl propionate has a similar odor than
the hydroalcoholic solution and therefore its concentration must be higher than the other
compounds to detect.
3.2 Optimization of injection system
3.2.1 Direct injection
Initially, the lineal retention indexes were calculated from the chromatogram. For that, very
concentrated solutions were prepared and from these other solutions with lower concentration
Odor
threshold
(µg L-1
)
Concentration
in reference
with maximum
intensity
(mg L-1
)
Concentration
in synthetic
wine
(mg L-1
)
Orthonasal
intensity in
synthetic
wine
Error
(s/n1/2
)
Acetaldehyde 500 5000 75 1.14 0.09a
DMS 17-25 20 5 1.87 0.09b
Butyraldehyde 75 750 75 1.25 0.21b
Ethyl acetate 12264 1200 250 1.36 0.14a
Ethyl
propionate 10 1000 100 1.33 0.17
b
Diacetyl 100 10000 100 1.25 0.28b
Ethyl
thioacetate 25 25 1.5 1.83 0.21
b
Figure 3: SDME system
Optimization of a system for determining volatile odorants by gas chromatography-olfactometry
7 PYR-001-11-ART
(~10 mg L-1
) were obtained. Figure 4, shows the chromatogram obtained when 1 µL was
injected in the split mode (1:10) with the conditions indicated in the paragraph 2.2.
Figure 4: Split injection
3.3 Splitless injection
In order to get the maximum sensitivity, the extracts were preferred to be injected in the splitless
mode. As butyraldehyde, ethyl acetate, ethyl propionate and diacetyl were overlapped by the
solvent (dichloromethane) so that these could not be detected. For this reason, butanol and
isobutanol were used as solvents because they are less volatile than dichloromethane and they
are eluted after the analytes. The chromatography performed under these conditions was not
satisfactory as reconcentration into the column was not achieved. As a result, some
modifications about the initial injection (splitless time: 1 min, injection volume: 1 µL) were
carried out:
- Injection volume: 0.5 µL.
- Splitless time: 0.5 min.
- Working with an isothermal program of temperature (40 ºC).
- Working with pulse pressure (400 kPa)
Figure 5 shows the chromatogram obtained with the new conditions. Again, the results were not
satisfactory enough. The peaks form indicates that there is no solvent effect, the sample doesn´t
wet the pre-column homogeneously, therefore, the pre-column was changed by a polarly off
pre-column with higher internal diameter. Initially, the solution was injected “on-column”
because it is thought that if we could not wet the precolumn with that method, it would be
possible neither with any of the splitless conditions.
Optimization of a system for determining volatile odorants by gas chromatography-olfactometry
8 PYR-001-11-ART
Figure 5: Splitless injection
3.4 “On-column” injection
“On-column” injection consists on introducing the sample as a liquid into the column directly
without previous vaporization (Ferreira, 1997). The chromatography was not improved with
this approach, it means it is not possible to the analytes reconcentrate with butanol or
isobutanol. In this experience, a polar off pre-column with 0.53 mm of internal diameter was
used for injecting 1 µL of liquid sample directly. The chromatographic conditions are indicated
in the paragraph 2.2. Figure 6 shows these unsatisfactory results.
Figure 6: “on-column” injection
In conclusion, the solutions can only be injected in the split mode with the conditions indicated
in the paragraph 2.2.
3.5 DHS-SPE extraction
The intensity of the extracts obtained was assessed with 3 judges (“sniffers”). In this case, two
extracts were obtained. According to a t-test, no significant differences were perceived between
the two kinds of extract. As a result, it was decided to work with 6 values of intensity. The
sniffers used a 3-point scale (0 = no odour, 1 = weak odour, 2 = clear perception of odour,
strong intensity; 3 = extremely strong intensity of odour; intermediate values of 0.5; 1.5; 2.5
being allowed) to evaluate the intensity of each compound. The results obtained were compared
to the results obtained in the sensory analysis. Figure 7 shows Iolf – Iglass to evaluate the
approximation of this system to the glass perception.
Optimization of a system for determining volatile odorants by gas chromatography-olfactometry
9 PYR-001-11-ART
Figure 7: DHS-SPE extraction
In the figure 7, an ideal situation should show Iolf – Iglass = 0, which would mean that all
compounds had been retained and had been detected correctly in the olfactometry. On the
contrary, if Iolf – Iglass > 0, the compounds had been overevaluated and if Iolf – Iglass < 0, the
compounds had been underevaluated. Figure 8 shows the chromatogram obtained when 1 µL of
the extract obtained with the conditions indicated in the paragraph 2.4 (wine doped with the
intermediate concentrations indicated in table 1) was injected into the GC-O-FID in the
conditions indicated in the paragraph 2.2.
Figure 8: DHS-SPE chromatogram
Optimization of a system for determining volatile odorants by gas chromatography-olfactometry
10 PYR-001-11-ART
On the other hand, in order to study if there were significant differences between olfactometry
and glass intensity, a t-test (P<0,05) was performed. First, a F-test was carried out to check if
the variances were similar. The t-value was calculated and finally compared with the critic value
with a confidence level of 95 %. The results are shown in the following table.
Table 2: Results of DHS-SPE.
Acetaldehyde DMS Butyraldehyde Ethyl
acetate
Ethyl
propionate Diacetyl
Ethyl
thioacetate
CG-O
DHS
SPE
Iaverage 0.08 0 1.70 0 1.67 2.17 1.42
s 0.20 0 0.69 0 0.41 0.26 0.58
Glass
Iaverage 1.14a
1.87b 1.25
b 1.34
a 1.33
b 1.25
b 1.83
b
s 0.24 0.21 0.52 0.38 0.41 0.69 0.52
tcalculated 8.40 21.96 1.41 5.46 -1.41 -3.05 1.31
tcritic 2.20 2.23 2.23 2.20 2.23 2.23 2.23
a: n = 7; b: n = 6
The results show that there are significant differences for acetaldehyde, dimethylsulphide, ethyl
acetate and diacetyl. Looking at both the chromatogram and the olfactometry results, it can be
seen that acetaldehyde, dimethylsulphide and ethyl acetate are under-evaluated. The main
explanation to this could be that the breakthrough volume (maximum volume of sample and of
rinsing solvent that can be passed through the SPE bed without losses of analyte) and, therefore
the recovery rate in the resin, was short. It is important to point out that, although ethyl acetate
presents a big peak in the chromatograph, it was not detected in the olfactometry because it
exhibits a high odor threshold. On the other hand, diacetyl is over-evaluated in spite of having a
short breakthrough volume. This fact was observed when the nose was pulled up on the top of
the cartridge.
For all this reasons, the following modifications have been carried out in order to optimize a
method based on the dynamic headspace and solid-phase extraction:
1. Decreasing the temperature in the retention of the analytes in the resin by placing ice
around the cartridge. The method proposed in the paragraph 2.4 was used for obtaining
the extract and this was injected with the chromatographic conditions described in the
paragraph 2.2
2. Concentrating under stream of pure N2 to a final volume of 100 µL.
3. Decreasing the temperature of analytes in the trap and concentrating to a final volume of
100 µL.
4. Injecting 2 µL.
5. Increasing the amount of resin. The cartridge was filled with 600 mg of LiChrolut EN.
After 100 minutes, the analytes were eluted with 4.5 mL of dichloromethane with 5 %
methanol.
6. Extracting the compounds of the headspace into butanol, without resin in the cartridge
and after 100 minutes extracting the analytes into dichloromethane in order to inject
these in the chromatograph: a standard SPE cartridge 0.8 cm internal diameter, 3 mL
internal volume) filled with 3.2 mL of butanol. The cartridge was placed on top of a
Optimization of a system for determining volatile odorants by gas chromatography-olfactometry
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bubbler flask (figure 1) containing 80 mL of wine. A controlled stream of nitrogen (100
mL/min) was passed through the sample for 100 minutes, the butanol was removed and
mixed with 100 mL of water. The hydroalcoholic phase was dropped into a decantation
funnel and 6 mL of dichloromethane and 20 g of ammonium sulphate were added to
improve the demixing (Cacho, Meléndez and Ferreira, 1992; Cacho, Ferreira and
Fernández, 1992). After that, the mixture was shaken and when two phases were
separated, dichloromethane phase, denser, was dropped into a vial. Sodium sulphate
was added to remove water. This extract was concentrated under a stream of pure N2 to
a final volume of 200 µL. This extract was injected in the gas chromatograph.
The following table shows the results obtained after applying these modifications:
Table 3: Results of the DHS-SPE modifications
Acetaldehyde DMS Butyraldehyde
Ethyl
acetate
Ethyl
propionate Diacetyl
Ethyl
thioacetate
DHS-SPE Iaverage 0.08 0.00 1,75 0.00 1.67 2.17 1.42
s 0.20 0.00 0,69 0.00 0.41 0.26 0.58
Reduce
temperature
Iaverage 0.50 0.00 1,67 0.00 1.50 2.00 0.67
s 0.29 0.00 0,33 0.00 0.29 0.00 0.33
tcritic -1.85 - 0,18 0.68 0.54 1.08 1.82
Reduce T
concentrate
to 100 µL
Iaverage 0.33 0.00 2,00 0.00 1.50 2.00 0.50
s 0.17 0.00 0,00 0.00 0.00 0.00 0.29
tcritic -1.53 - -0,61 0.68 0.68 1.08 2.30
Concentrate
to 100 µL
Iaverage 0.33 0.00 1,83 0.00 1.67 2.17 0.83
s 0.33 0.00 0,17 0.00 0.33 0.17 0.60
tcritic -1.53 - 0,20 0.68 0.00 0.00 1.11
Volume
injection
2 µL
Iaverage 0.33 0.00 2,00 0.00 1.83 2.33 1.67
s 0.17 0.00 0,29 0.00 0.17 0.17 0.33
tcritic -1.53 - -0,52 0.68 -0.62 -0.88 -0.61
600 mg
of resin
Iaverage 0.33 0.00 1,17 0.00 1.67 2.33 1.33
s 0.33 0.00 0,17 0.00 0.17 0.17 0.17
tcritic -1.00 - 1,37 0.68 0.00 -0.88 0.23
Butanol
extraction
Iaverage 0.17 0.00 0,00 0.00 0.50 2.00 0.50
s 0.17 0.00 0,00 0.00 0.50 0.00 0.50
tcritic -0.51 - 4,25 0.68 2.20 1.08 1.91
All these modifications were compared with DHS-SPE extraction by means of a t-test. Statistic
t-value is calculated with a confidence level of 95 %. Previously, the variances were compared
by test F to see if there were differences between them. Results show that there are not
significant differences (tcritic = 2.36), which mean that no modifications provide improvement,
even in butanol extraction the result by butyraldehyde is worst.
In conclusion, DHS-SPE does not provide good results in the study of the very volatile
compounds.
Optimization of a system for determining volatile odorants by gas chromatography-olfactometry
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3.6 Solid-phase microextraction (HS-SPME)
In this kind of extraction different conditions (extraction time, volume of sample, salt) and
polymeric fibers were evaluated in order to choose the optimal conditions to determine the
analytes in a synthetic wine. Table 5 summarizes the different experiences carried out:
Table 4: Different conditions of HS-SPME
A B C D E
Volume of
sample (mL) 5 10 10 10 10
Salt (NaCl)
(g) 1,5 3 3 3 -
Extraction
time (min) 10 10 20 20 40
Temperature
(ºC) 40 40 40 40 40
Polymeric
Fiber DVB/Car/PDMS DVB/Car/PDMS DVB/Car/PDMS Car/PDMS Car/PDMS
The extracts were injected into the GC-O system and the olfactometries were performed by a
trained sniffer. All of these were compared with the intensity in the glass. The results are
presented in figure 9 and in table 5.
Figure 9: Optimization of HS-SPME
Optimization of a system for determining volatile odorants by gas chromatography-olfactometry
13 PYR-001-11-ART
Table 5: Olfactometry results in optimization of HS-SPME
As can be seen, conditions C and D provide more similar results to the intensity in the glass than
the rest of conditions. The only difference between them is the type of polymeric fiber. Finally,
condition D was used for working with several sniffers because if the fiber has divinylbenzene
(DVB), the results are not better. The sample is also injected in GC-O in order to calibrate this
technique. Three sniffers have participated in these experiences. These results are shown in the
following graphic.
Figure 10: HS-SPME results
The following chromatogram shows eight peaks, the analytes and ethanol. The olfactometry
results are compared with the intensity in the glass through a t-test with a confidence level of 95
% and considering the variances homogeneous.
A B C D E GLASS
Acetaldehyde 0 1 1 1 1 1.17
DMS 1 1.5 2 2 1 1.88
Butyraldehyde 1 1.5 1.5 1.5 0 1.25
Ethyl acetate 0 0 0 0 0 1.36
Ethyl propionate 0,5 1.5 1.5 1.5 1.5 1.33
Diacetyl 2 2 2 2 2.5 1.43
Ethyl thioacetate 0.5 0.5 1 1 1 1.83
Optimization of a system for determining volatile odorants by gas chromatography-olfactometry
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Figure 11: HS-SPME chromatogram
Table 6: HS-SPME results
Acetaldehyde DMS Butyraldehyde Ethyl
acetate
Ethyl
propionate Diacetyl
Ethyl
thioacetate
CG-O
HS-SPME
Iaverage 1.00 1.67 1.67 0.00 1.50 1.83 1.33
s 0.00 0.58 0.58 0.00 0.00 0.29 0.58
Glass
Iaverage 1.14a
1.87b 1.25
b 1.34
a 1.33
b 1.25
b 1.83
b
s 0.24 0.21 0.52 0.38 0.41 0.69 0.52
tcalculated -1.08 -0.83 1.09 -6.01 0.68 0.84 -1.32
t critic 2.31 2.36 2.36 2.31 2.36 2.36 2.36
a: n = 7; b: n = 6
The statistic results show that there are not significant differences for acetaldehyde, dimethyl
sulphide, butyraldehyde, ethyl propionate, diacetyl and ethyl thioacetate. Ethyl acetate has not
been detected in the olfactometry, all sniffers gave an intensity of 0, although a peak can be seen
in the chromatogram.
In conclusion, this extraction technique provides good results for six analytes. For this reason,
the method proposed is the following: 10 ml of synthetic wine and 3 g of NaCl are placed in a
20 ml standard headspace vial and sealed. A Carboxen/PDMS (85 µm) polymeric fiber is used
for extracting the analytes, it was conditioned by a stream of N2 for 60 min at 250 ºC. After this,
samples are extracted for 20 min at 40 ºC. Extraction was performed with agitation at 250 rpm
in cycles of 5 s on and 2 s off. Desorption took place in a splitless mode at 250 ºC for 10 min
(splitless time: 3 min). Previously, the glass liner was changed by a specific to SPME.
3.7 Static headspace (SHS)
In this kind of extraction, volatile compounds which are in the headspace are injected into the
column directly. The method which is used for this extraction is indicated in the paragraph 2.5.
Several experiences have been carried out in order to optimize the injection system. First,
Optimization of a system for determining volatile odorants by gas chromatography-olfactometry
15 PYR-001-11-ART
extract was injected in the splitless mode at 250 ºC and 40 ºC, the olfactometry results were
satisfactory, however, the peaks were very wide and the analytes were overlapped. For that, the
extract was injected in split (1:10), several temperatures were tested, the best results were
obtained when the injector was at 150 ºC. The peaks were wider than those observed by other
techniques, but the peak maxima could be correctly identified and there is enough separation for
olfactometry. Figure 3 shows this chromatogram.
Figure 12: SHS chromatogram, split injection at 150 ºC.
Table 7: SHS results
Acetaldehyde DMS Butyraldehyde Ethyl
acetate
Ethyl
propionate Diacetyl
Ethyl
thioacetate
CG-O
SHS
Iaverage 0.17 1.67 1.50 0.00 1.17 1.67 0.67
s 0.29 0.29 0.50 0.00 0.29 0.29 0.29
Glass
Iaverage 1.14a
1.87b 1.25
b 1.34
a 1.33
b 1.25
b 1.83
b
s 0.24 0.21 0.52 0.38 0.41 0.69 0.52
tcalculated -5.29 -1.26 0.68 -6.01 -0.62 0.50 -3.56
t critic 2.31 2.36 2.36 2.31 2.36 2.36 2.36
a: n = 7; b: n = 6
Statistics results indicate that there are significant differences for acetaldehyde, ethyl acetate and
ethyl thioacetate. In comparison with the extraction techniques described previously, this
provides better results than dynamic headspace in the study of very volatile compound. A
disadvantage is the irreproducibility because the amount of gas which was drawn into the
gastight syringe is different in each extraction.
In conclusion, this technique is appropriate to determine dimethyl sulphide, butyraldehyde,
ethyl propionate and diacetyl.
3.8 Single-drop microextraction (SDME)
This technique consists of placing a very small amount (drop) of an extracting solvent, on the
tip of a microsyringe needle in direct contact with the headspace. After a certain extraction time,
the solvent is withdrawn into the microsyringe and injected into the chromatographic system for
separation and detection. The method used for this study had already been development, and
only the solvent, the temperature in the injection and the drop volume have been optimized.
Optimization of a system for determining volatile odorants by gas chromatography-olfactometry
16 PYR-001-11-ART
Different solvents were proposed: butanol, propylene glycol and isopropanol, however, these
are volatile and do not stand for 25 min. Therefore, 2-octanol has been used for the extraction
and preconcentration. Initial chromatographic conditions did not guarantee an appropriate
separation, and finally the injection temperature was down at 150 ºC and the chromatography
was better.
Another important question to address is the volume of the drop. First, 2 µÑ were used,
however, it was thought that if the volume of the drop was lower, the analytes would be more
concentrated and the olfactometry results and the chromatography would be better.
Nevertheless, the analytes were not more concentrated and finally the volume of the drop was 2
µL.
Figure 13 shows the SDME chromatogram in which five analytes can be seen. Ethanol is a
major volatile compound in wine, therefore, a big peak appears and ethyl propionate and
diacetyl are overlapped by ethanol. In spite of this, both compounds have been detected in the
olfactometry as they present very specific odors.
Figure 13: SDME chromatogram
Olfactometry results are shown in table 9, these are compared with the intensity in the glass.
Statistic results support the conclusion that there only are significant differences for ethyl
acetate with a confidence level of 95 %. This method has provided good results to analyzed six
analytes.
Table 8: SDME results
Acetaldehyde DMS Butyraldehyde Ethyl
acetate
Ethyl
propionate Diacetyl
Ethyl
thioacetate
CG-O
SDME
Iaverage 1.50 1.67 2.00 0.00 1.42 1.83 1.17
s 050 0.29 0.50 0.00 0.38 0.29 0.29
Glass
Iaverage 114a
1.87b 1.25
b 1.34
a 1.33
b 1.25
b 1.83
b
s 0.24 0.21 0.52 0.38 0.41 0.69 0.52
tcalculated 1.37 -1.26 2.05 -6.01 0.29 0.84 -2.04
t critic 2.31 2.36 2.36 2.31 2.36 2.36 2.36
a: n = 7; b: n = 6
Optimization of a system for determining volatile odorants by gas chromatography-olfactometry
17 PYR-001-11-ART
4 Conclusion
The main aim of the present work was to develop a method to quantify very volatile compounds
(acetaldehyde, dimethyl sulphide, butyraldehyde, ethyl acetate, ethyl propionate, diacetyl and
ethyl thioacetate) in wine.
Splitless injection has not been achieved in the extraction techniques with an organic solvent,
therefore, the extracts were injected in split (1:10). Dynamic headspace sampling coupled to
solid-phase extraction is not an useful technique in order to analyze very volatile compounds.
Other kind of techniques which provide good results, have been optimized.
The best results have been obtained by headspace solid-phase microextraction and single-drop
microextraction, in which acetaldehyde, dimethyl sulphide, butyraldehyde, ethyl propionate,
diacetyl and ethyl thioacetate have been analyzed by gas chromatography-olfactometry
correctly. On the other hand, ethyl acetate has not been detected in any of the olfactometries
performed.
5 Acknowledgments
This research was supported by the I3A Fellowship Program and Laboratory for flavour
analysis and enology of University of Zaragoza.
6 Abbreviations used
DHS-SPE, dynamic headspace and solid-phase extraction; HS-SPME, headspace solid-phase
microextraction; SHS, static headspace; SDME, single-drop microextraction; GC-O, gas
chromatography-olfactometry; FID, flame ionization detector; DMS, dimethyl sulphide; DVB,
divinylbenzecen; PDMS: polydimethylsiloxane
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