optimization of a system for determining volatile odorants...

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


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.


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


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


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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


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(~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.


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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.


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


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


11 PYR-001-11-ART

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.


12 PYR-001-11-ART

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


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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


14 PYR-001-11-ART

Figure 11: HS-SPME chromatogram

Table 6: HS-SPME results


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,


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


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.


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


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


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

References

Cacho, J., Ferreira, V. and Fernandez, P. (1992). Microextraction by demixing for the

determination of volatile compounds in aqueous-solutions. Analytica Chemica Acta. 264(2):

311-317

Cacho, J., Meléndez, J. and Ferreira, V. (1992). Development of a method for analyzing

volatiles from foodstuff matrices, including microextraction by demixture. Application to the

analysis of grapes. Mikrochimica Acta. 108(1-2): 61-72.

Campo, E. (2006). La base química del aroma de vinos blancos de variedades españolas y de

diversos vinos especiales. Evaluación de nuevas técnicas olfatométricas, cuantitativas y

sensoriales para su estudio, identificación de odorantes más importantes y modelización de

notas sensoriales. Tesis Doctoral.


18 PYR-001-11-ART

Campo, E. et al. (2005). Prediction of the wine sensory properties related to grape variety from

dynamic-headspace gas chromatography-olfactometry data. Journal of Agricultural and Food

Chemistry, 53(14): 5682-5690.

Ferreira, V. (1997). Cromatografía: Fundamentos y práctica. Prensas Universitarias de

Zaragoza.

Ferreira, V and Cacho, J. (2009). Identification of Impact Odorants of Wines. In: Wine

Chemistry and Biochemistry. Springer, 393-415.

Ferreira, V. et al. (2002). Chemical charactization of the aroma of Grenache rosé wines: Aroma

Extract Dilution Analysis, quantitative determination and sensory reconstitution studies. Journal

of Agricultural and Food Chemistry, 50(14): 4048-4054.

Ferreira, V. et al. (2004). Simple strategy for the optimization of solid-phase extraction

procedures through the use of solid-liquid distribution coefficients Application to the

determination of aliphatic lactones in wine. Journal of Chromatography A, 1025(2): 147-156.

Guth, H. (1997). Identification of character impact odorants of different white wine varieties.

Journal of Agricultural and Food Chemistry, 45(8): 3022-3026.

López, R. et al. (2007). Quantitative determination of wine highly volatile sulfur compounds by

using automated headspace solid-phase microextraction and gas chromatography-pulsed flame

photometric detection - Critical study and optimization of a new procedure. Journal of

Chromatography A, 1143(1-2): 8-15.

Martendal, E. et al. (2007). Application of fractional factorial experimental and Box-Behnken

designs for optimization of single-drop microextraction of 2,4,6-trichloroanisole and 2,4,6-

tribromoanisole from wine samples. Journal of Chromatography A, 1148(2): 131-136.

Martineau, B., Acree, T. and Henick-Kling, T. (1995). Effect of wine type on the detection

threshold for diacetyl. Food Research International, 28(2): 139-143.

Mateo-Vivaracho, L. et al. (2006). Automated analysis of 2-methyl-3-furanthiol and 3-

mercaptohexyl acetate at ng L-1 level by headspace solid-phase microextracion with on-fiber

derivatization and gas chromatography-negative chemical ionization mass spectrometric

determination. Journal of Chromatography A, 1121(1): 1-9.

Norma UNE 87-008-92.

San Juan, F. (2006). Mejora y caracterización del método de extracción de aromas basado en el

espacio de cabeza dinámico-extracción en fase sólida-cromatografía de gases. Aplicación a

diferentes matrices. Postgrado de Iniciación a la Investigación.

San Juan, F. et al. (2010). Producing headspace extracts for the gas chromatography-

olfactometric evaluation of wine aroma. Food Chemistry, 123(1): 188-195.

optimization of a system for determining volatile odorants...

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