From the representativeness of sampled odors to an olfactive camera

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Chemistry and Biology of Aroma and Taste

From the representativeness of sampled odors to an olfactive camera Alain Chaintreau, Urs Keller, Barbara Zellner, Kelly Nguy, Sabine Leocata, and Frederic Begnaud J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b06383 • Publication Date (Web): 19 Jan 2019 Downloaded from http://pubs.acs.org on January 20, 2019

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Journal of Agricultural and Food Chemistry

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From the Representativeness of Sampled Odors to an Olfactive Camera

Alain Chaintreau, Urs Keller, Barbara Zellner, Kelly Nguy, Sabine Leocata, Frédéric Begnaud* Firmenich SA, Corporate R&D Division, Route des Jeunes 1, CH-1211 Geneva 8

*Corresponding author. Firmenich SA, Corporate R&D Division, Route des Jeunes 1, CH-1211 Geneva 8 Tel: +41227803031 E-mail address: [email protected] (F. Begnaud)

ACS Paragon Plus Environment


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ABSTRACT

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Although representativeness is often a prerequisite when sampling odors, the methods

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used have never been assessed from the analytical and the sensory perspective

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simultaneously. We validate several critical innovations in the methods used to sample

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odors, starting with a previously developed static-and-trapped headspace (S&T-HS)

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cell, to minimize sorptive biases and allow thermodesorption of trapped odors. The

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addition of a desorption oven allows restoration and testing of odors sampled not only

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by S&T-HS, but also by other techniques (solid-phase microextraction, HS sorptive

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extract, purge-and-trap HS). The S&T-HS cell exhibits satisfactory representativeness,

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much higher than the other three techniques. This allows, for the first time, a proposal to

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use this technology as an olfactive camera to capture and restore an odor. The method

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was tested on a sample of a complex fresh ashtray odor.

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Keywords

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Odor; Aroma; Fragrance; Static-and-trapped headspace; Quantitation;

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Representativeness; Olfactive camera

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INTRODUCTION

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Analytical representativeness

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The recoveries of volatile ingredients present in a solid or liquid matrix have been

20

investigated in many studies. This is notably the case for liquid/liquid extraction,1

21

accelerated solvent extraction,2, 3 simultaneous distillation-extraction,4, 5 high vacuum

22

transfer, solvent-assisted flavor evaporation,6 and stir bar sorptive extraction.7 These

23

recoveries often differ from one constituent to another, and so the collected sample is

24

usually not representative of the relative proportions of the volatile constituents in the

25

matrix. In contrast, recoveries in the gas phase collected from a headspace (HS) have

26

been little investigated despite of the fact that the HS represents the odor of the sample.

27

Validating such an approach requires exact knowledge of the initial composition of the

28

HS to be sampled, since the balance between the odor’s constituents is directly

29

correlated with the global odor perceived. To meet this objective, one has to generate a

30

controlled gas flow with a standard composition, e.g., from permeation tubes.8 This

31

setup allows the measurement of recoveries only from an atmosphere with a stable

32

composition of odorants, or from a closed HS free of any smelling source capable of

33

renewing the odorants in the gas phase. As a consequence, the representativeness of

34

odors collected from the HS around a smelling source remains questionable. Sampling

35

techniques that can be applied to the HS9, 10 are (1) direct sampling of an aliquot of the

36

HS: static HS (S-HS); (2) static partition between the gas and a sorptive phase: HS

37

solid-phase microextraction (HS-SPME) and HS sorptive extraction (HSSE); (3)

38

dynamic desorption of volatiles from the matrix by using a stripping gas and subsequent



4 39

trapping into a sorptive material: purge-and-trap HS (P&T-HS); and (4) concentration of

40

odorants from a static HS into a sorbent: static-and-trapped HS (S&T-HS).11, 12

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The fourth technique is less common than the others, and its principle is described

42

hereafter. A liquid or solid sample is put into the cavity in the base of a tubular container

43

closed by a piston (the cell, Figure 1A). Its volatile constituents then equilibrate with the

44

HS (Figure 1B) and a Tenax trap is connected to the outlet of the piston. Afterwards, the

45

HS is evacuated through the trap by pressing the piston (Figure 1C) to load the Tenax

46

cartridge with the retained volatiles (Figure 1D).

47

The S&T-HS cell was initially developed for physico-chemical applications, and so its

48

inner volume, piston motion, and working temperature were accurately controlled,11, 12

49

The technique was proposed for odor sampling prior to the use of gas chromatography-

50

olfactometry (GC-O)13 and applied to yogurt14 and coffee.15

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Because many authors assume that HS-SPME and P&T-HS representatively sample

52

odors, these techniques are widely used in the scientific literature reporting aroma and

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fragrance compositions. Figure S1 (Supporting Information) compares these two

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techniques with S-HS and S&T-HS. Starting from the equations previously published for

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these methods,10 the proportions of the different analytes recovered from the HS above

56

a low-concentration aqueous solution can be easily calculated. S-HS represents the

57

unmodified composition of the odor above the sample and was taken as the reference.

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From this simulation, only S&T-HS appears to be identical to S-HS, which is logical

59

because the techniques differ only by the size of the HS volume that is sampled and

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consequently by their sensitivities. In contrast, SPME, which is based on a phase


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partition, overcollects most of the HS constituents. This is inherent in the constant

62

release of compounds from the solution to compensate for the amount trapped on the

63

fiber until equilibrium between the fiber and the liquid phase is reached (and not

64

between the fiber and the HS, as is frequently assumed16). Conversely, P&T-HS mostly

65

distorts the balance in the HS between hydrophobic and hydrophilic constituents (See

66

Equation 2 and Figure S1).

67

As a conclusion, the simulated recoveries suggest that S-HS and S&T-HS are the most

68

representative sampling means. Unfortunately, S-HS collects only a small HS volume,

69

which jeopardizes the sensitivity of the GC analysis. In contrast, P&T-HS and HS-

70

SPME distort the original headspace composition and so the perceived odor. However,

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these observations have never been confirmed by an experimental assessment.

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Therefore, the present work aims to analytically and sensorially evaluate these sampling

73

methods and to develop S&T-HS technology to ensure robust analytical

74

representativeness.

75

Sensory representativeness

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The analytical representativeness of the sampled HS does not mean that it is

77

representative of the original odor. Minor highly potent constituents may be missed by

78

the usual detectors (FID, MS) because they occur below the detection limit of

79

instruments, or are hidden because they coelute with major peaks, although they

80

significantly contribute to the overall scent.17 Such constituents are sometimes detected

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only with a multidimensional GC system and/or by olfactive detection. To evaluate the

82

representativeness of the odor collected by various means (e.g., vacuum

83

hydrodistillation, gas syringe, dynamic HS sampler), we can inject the sample into a gas



6 84

chromatograph equipped with a capillary or a deactivated column. The outlet is then

85

inserted in the septum of (1) an airtight glass syringe18 or (2) a glass flask containing a

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small amount of water to trap the volatiles19 or (3) a Teflon bag.19 All of these HS

87

collection methods have major drawbacks: glass devices are prone to adsorptions,20

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even after silanization21; HS trapping in water creates a partition between the flask HS

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and the solution, which leads to a distortion of the original HS composition22; and Teflon

90

is a permeable material. None of these systems allows for control of the HS

91

temperature and volume.

92

So far, the most reliable technique, free of sorption risks, seems to be direct GC-O (D-

93

GC-O).23 It consists of injecting the sample into a GC injector connected to a short

94

deactivated column, the outlet of which is connected to a sniffing port. However, it

95

delivers a brief olfactory stimulus (a few seconds), which does not allow comparison

96

with the original odor under identical conditions or its evaluation by several panelists in

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one session.

98

Still, only the sensory representativeness of the above-mentioned techniques has been

99

tested. Flavorists and perfumers well know that an identical odor may result from

100

different chemical compositions, and so both the sensory and the analytical

101

representativeness are investigated in this work.

102

The concept of an olfactive camera

103

To our knowledge, the concept of an olfactive camera has never been proposed in the

104

scientific literature. It consists of capturing and restoring an odor with minimal distortion,

105

similar to the capture and restoration of an image by a camera. The English word


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“camera” comes from Latin and means “chamber, room.” This implies that, after these

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two steps, the restored odor remains representative from both the analytical and

108

sensory sides. Although none of the existing sampling techniques have ever

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demonstrated such capabilities, the S&T-HS cell can fill this gap.

110

EXPERIMENTAL SECTION

111

Chemicals

112

The constituents of the malodor model came from Sigma-Aldrich (Darmstadt,

113

Germany), except 1-octen-3-ol (Bedoukian, Danbury, CO), Cetalox® (= [(1–)-8,12-

114

epoxy-13,14,15,16-tetranorlabdane], Firmenich, Geneva, Switzerland), and

115

diisopropylene glycol (BASF, Ludwigshafen, Germany). The composition of the malodor

116

model is given in Table S1.

117

HS cell

118

The previously described HS cell12 was equipped with a movable oven that was put in

119

contact with the trapping cartridge only for the duration of the desorption step (details

120

in24; scheme recalled in Figure S2 of the Supporting Information). All stainless steel

121

surfaces in contact with the HS were passivated with a Sulfinert treatment (Restek, now

122

SilcoNert 2000, Silcotek Corporation, Bellefonte, USA) as a result of a performance

123

comparison between different materials and surface treatments (Table S2 and Figure

124

S2). HS cell were operated under controlled temperature as indicated in the text and

125

ambient pressure.



8 126

Prior to use, all parts of the HS cell in contact with the HS or the liquid sample were

127

disconnected and successively rinsed with deionized water and ethanol before being

128

dried out at 600 °C, 20 mbar, for at least 2 h.

129

Tenax cartridges

130

Tenax cartridges were made of silanized Pyrex tubes (89 mm x 6.35 mm OD), and

131

packed with 100 mg of Tenax TA (35/60 mesh, Agilent Technologies, Basel,

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Switzerland) previously washed with acetone and dried at 40 °C under vacuum. The

133

adsorbent was maintained in the tube between two silanized glass wool plugs. The

134

traps were cleaned overnight at 240 °C under nitrogen flow. Before each use, the traps

135

were cleaned again in the thermal desorber at 260 °C for 10 min under a 40 mL/min

136

nitrogen flow, and a blank analysis was performed. To avoid any degradation of

137

samples, we loaded and desorbed Tenax traps within a maximum of 4 h.

138

Direct HS (D-HS) sampling

139

The sample (1 mL) was put in the silanized glass cup together with a silanized glass stir

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bar. This cup was placed in the bottom part of the HS cell and the cell was closed with

141

the piston in the bottom position. Temperature regulation of the whole cell was started

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by using the double jacket (30 °C). The piston was then moved to its top position, driven

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by a PC-controlled motor, to fill the HS chamber with 250 mL of purified ambient air

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through an open split made of a T-union (Figure 1B-D). After equilibration under stirring

145

(1 h), a Tenax trap (100 mg) was connected between the piston and an open split fed

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by pure air to prevent the cartridge from external pollutant retrodiffusion. The piston was

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pressed down to evacuate the gas phase at 40 mL/min (Figure 1C). All traps were


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desorbed within 3-4 h after loading. Alternatively, instead of a Tenax cartridge, a sniffing

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port was connected to the piston outlet for olfactive evaluation of the HS (Figure 1D).

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Sampling of the fresh ashtray odor

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A Tenax trap connected to a gas pump (100 mL/min) was left for 1 h 30 min next to an

152

ashtray located in a smoking room. It was then desorbed into a clean S&T-HS cell

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according to the restored HS (R-HS) protocol. All panelists participating in the D-HS or

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R-HS evaluation were smokers and signed a consent form describing the sensory

155

protocol.

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Restored HS (R-HS)

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The empty HS cell was assembled with the piston in its lowest position and its

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temperature was regulated with a double jacket. Prior to starting any R-HS assay, we

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heated the desorption oven to 240 °C. The loaded trap was then inserted between the

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piston outlet and an open split maintained under nitrogen flow (Figure 1E). When

161

necessary, N2 was previously moistened by bubbling it into water. The piston was

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moved up at a controlled rate for about 1 min to remove oxygen from the Tenax trap

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maintained at ambient temperature (Figure 1F). The oven (240 °C) was then put in

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contact with the cartridge to start the desorption (Figure 1G,H) and to transfer the

165

volatiles into the HS chamber. At the end of desorption, the oven was removed and the

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trap was replaced with either a clean cartridge to recollect the R-HS (Figure 1I) or a sniff

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port to smell the restored odor. To allow desorption in backflush, we had to connect the

168

trap in the opposite position to that of the trapping step. Because the same gas volume

169

was used during the trapping and desorption steps, the odorants were restored at the

170

same HS concentration.



10 171

The desorption of the SPME fiber and the Twister used for HS-SPME and HSSE,

172

respectively (vide infra) were achieved similarly. An empty glass cartridge was equipped

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with a septum and a nitrogen inlet at its upper end. Its other end was connected to the

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top of the piston. The SPME fiber was inserted in the septum or the Twister was hung in

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the middle of the glass tube. The moist N2 flow entering the open split was 80 mL/min.

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The oven was rotated to heat the glass tube at 240 °C for 6 min for the fiber or 10 min

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for the Twister.

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Thermodesorption-GC-FID

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Loaded Tenax traps were thermally desorbed with a TurboMatrix (Perkin-Elmer, Basel,

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Switzerland) hyphenated to a GC QP3100 (Shimadzu) equipped with an FID. The GC

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column was a DB-17ms, 30 m x 0.25 mm ID, 0.25 μm df (Agilent Technologies, Basel,

182

Switzerland).

183

The Tenax trap was heated for 3 min at 220 °C and the thermodesorbed volatiles were

184

swept by a flow of helium (30 mL/min) and reconcentrated in an internal Tenax cold trap

185

(–30 °C). Both steps were in splitless mode. The cold trap was then rapidly heated to

186

240 °C to transfer the analytes into the GC column. Helium was used as carrier gas

187

(180 kPa). The oven temperature was started at 50 °C for 5 min, increased by 5 °C/min

188

to 120 °C, maintained for 2 min, and then increased by 10 °C/min to 240 °C and

189

maintained for 5 min.

190

SPME and HSSE sampling

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Either a 100 µm PDMS fiber (red hub, Supelco, Darmstadt, Germany) or a PDMS

192

Twister (0.5 mm wide x 10 mm long) was used. The former was conditioned at 250 °C


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for 30 min and the latter at 240 °C overnight. The fiber or the Twister were exposed to

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the HS of the malodor solution (1 mL) in a closed 100 mL glass flask that was equipped

195

with a stir bar, a double-jacket, and a Teflon-lined septum to insert the fiber or hang the

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Twister. The typical exposure times to the HS were 30 min for the fiber and 100 min for

197

the Twister, both at 30 °C. A blank analysis was performed before each use.

198

P&T-HS sampling

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The malodor solution (1 mL) was put in a silanized glass tube (5 x 150 mm) equipped

200

with a sintered glass disk at the bottom and a double jacket (T = 30 °C). A Tenax

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cartridge was connected to the outlet and pure nitrogen was bubbled through the

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sample from the sintered glass (40 mL/min), typically for 10 min.

203

Metrics

204

An analytical comparison between two samples was made on the basis of Euclidean

205

distance: 𝑛

206

𝐸𝑢𝑐𝑙𝑖𝑑𝑒𝑎𝑛 𝑑𝑖𝑠𝑡𝑎𝑛𝑐𝑒 =

∑ (𝑥 ― 𝑦 )² 𝑖

𝑖

𝑖 = 1

207

where xi and yi are the relative proportions (mass percentages) of compound i in the

208

measured sample and the reference HS, respectively, the reference being D-HS in this

209

study.

210

The sensory similarity between two HSs was evaluated by panelists on a 5-point scale

211

(Identical/Slightly different/Different/Very different/Completely different). The global

212

similarity was calculated as the weighted average of the 30 panelists’ evaluations:



12 𝑛

213

𝑆=

∑𝑖 = 1𝑚𝑖𝑥𝑖 𝑛

∑𝑖 = 1𝑚𝑖

214 215

RESULTS AND DISCUSSION

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The difficulty when testing the representativeness of a sampled HS lies in two steps: (1)

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generating a long enough gas flow with a defined composition to be either sampled on

218

trapping cartridges or smelt by several panelists, and (2) restoring the trapped odor in a

219

device to ensure a suitable sensory evaluation. The S&T-HS cell represents a potential

220

solution for both steps. It can generate a controlled gas flow with constant composition,

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which fits with step 1. By adding a thermodesorption device, the cartridge loaded from a

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first cell, as shown in Figure 1, can be redesorbed in a second cell. The redesorbed HS

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can then either be retrapped for analytical comparison or evaluated by a sensory panel

224

as required by step 2. We have previously demonstrated that the adsorption in the cell

225

corresponded to a Langmuir isotherm (See ref. 21, Supporting Information), which

226

means that the adsorbed amount is proportional to the amount of volatiles in the HS. In

227

the present work, we will demonstrate that the retained amount in the R-SH is very low,

228

compared to the D-HS taken as a reference. As a consequence, the adsorption of

229

volatiles when preparing the D-HS from the sample may be assumed to also be very

230

low, because in both cases (the walls exposed to the sample odor, or to the restored

231

odor) the cell walls are exposed to similar concentrations of volatiles in the headspace.

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Design of a device for cartridge desorption

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The fact that the oven installed at the top of the piston of the P&T cell was mobile

235

allowed us to maintain it permanently at the desorption temperature to limit its heating

236

inertia. It was kept away from the cartridge until the beginning of its desorption, which

237

raised the inner temperature of the cartridge to 195 °C within 150 s over the whole

238

length of the Tenax packing. To prevent the loaded analytes from oxidation and external

239

pollution from ambient air, we maintained the cartridge to be thermodesorbed under

240

nitrogen through an open split (Figure 1E). Just before heating, the cell piston started to

241

move from the bottom to the upper position at a controlled rate (Figure 1F), ensuring

242

complete sucking of the desorbed volatiles into the HS chamber (this completion was

243

assessed by performing a second desorption after the first one). The desorption started

244

when the oven was rotated to surround and heat the cartridge. The thermodesorbed

245

volatiles were transferred into the receiving cell by the nitrogen flow (Figure 1G) to

246

restore the initial HS (R-HS) (Figure 1G,H). To reanalyze the HS of the cell, we

247

removed the oven, installed a new blank cartridge, and pressed the piston again to trap

248

the volatiles in the Tenax cartridge before transferring the latter in the thermal desorber

249

hyphenated to a GC (Figure 1I). Alternatively, the Tenax cartridge was replaced by a

250

sniff port for evaluation of the restored odor.

251

Improvement of recoveries

252

We tested the desorption performances of the initial stainless steel system by

253

comparing the composition of retrapped volatiles (Figure 1I) to that of the previously

254

loaded trap (Figure 1D), using a malodor model compounded with five odor constituents

255

found in washing machines. The D-HS sampling was achieved by trapping the volatiles



14 256

from the HS in equilibrium with a solution of the model (Figure 1A-D). This trap was then

257

redesorbed in the S&T-HS cell (Figure 1E,F), which was itself recollected in a second

258

trap by pressing the piston again (R-HS). Both traps were thermodesorbed in a GC-FID

259

to compare the resulting chromatograms. None of the R-HS constituents exceeded a

260

recovery of 60% when compared with the composition of D-HS (Figure 2A). These

261

malodorous compounds are commonly known to adsorb on many surfaces despite their

262

moderate hydrophobicity (log P < 3.6). As a consequence, the inertness of the S&T-HS

263

cell had to be improved to optimize its representativeness.

264 265

The improvements in the HS cell started from its initial design, as shown in Figure 2A. It

266

was made of polished stainless steel, with Viton seals coated with Teflon, and the

267

cartridges were desorbed at 25 °C with dry nitrogen. Different improvement steps were

268

tested by using the malodor model: passivation of HS cell surfaces, use of moistened

269

nitrogen, and temperature of the HS cell during thermodesorption of the cartridge.

270

Cell passivation. First, different materials were separately tested against their sorptive

271

properties: polymers (polyether ether ketone, polyether polyurethane,

272

polyfluoroethylene, polycarbonate) and passivated stainless steel (Silcosteel, Sulfinert,

273

Inertium) in comparison to the non-treated stainless steel originally used for the

274

construction of the cell (see Figure S3). The lowest sorption was obtained with Sulfinert,

275

which was then chosen for the S&T cell construction of the HS chamber (cell tube,

276

bottom part, and piston, Figure S2). Recoveries from the malodor model under the

277

same conditions as in Figure 2A were slightly improved (Figure 2B), and then improved


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again (up to 43% to 79%) when the transfer tube of the piston was also passivated

279

(Figure 2C).

280

Moistened desorption gas. The number of sites occupied by the analytes on the

281

surface of the cell walls was small and obeyed a Langmuir adsorption model.21

282

Therefore, creating competition with an additional volatile compound in the HS could

283

reduce the adsorption of the observed analyte. Water was the best candidate, since it

284

was already present in many samples submitted to D-HS sampling (e.g., aroma from

285

food samples) and would not disturb analyses when present in small amounts.

286

Therefore, in addition to passivation, desorption of traps was achieved with moistened

287

nitrogen in the stainless steel cell. The recoveries of the resulting R-HS (Figure 2D)

288

were significantly improved in comparison with the R-S obtained with dry nitrogen

289

(Figure 2A).

290

HS cell heating. Increasing the cell temperature should minimize the exothermic

291

physisorption. Recoveries were indeed improved by raising the temperature from 25 °C

292

to 70°C (Figure 2E vs 2D). With an additional increase to 80 °C, a recovery decline was

293

observed, possibly from degradation of the malodor constituents (data not shown).

294

Combining these conditions, i.e., passivated HS chamber, trap desorption with moist

295

nitrogen, and cell maintained at 70 °C, led to less dispersed recoveries at close to 80%

296

(Figure 2F).

297

Piston heating. In the experiment reported above, the transfer line connected to the

298

piston was heated only by conduction when the cell was maintained at 70 °C. As a

299

result, its temperature dropped to 30-40 °C at 4 cm above the inlet connected to the



16 300

piston. Therefore, a resistor was added around the transfer tube to maintain a

301

homogeneous temperature of 73-80 °C over its whole length. Combined with the

302

previous improvements (passivation + moist desorption nitrogen + heated cell), this led

303

to quantitative recoveries of the malodor R-HS (Figure 2G).

304

Sampling representativeness of the optimized HS cell

305

The long-lastingness of fragrances notably depends on the hydrophobicity of their

306

constituents, which are supposed to “stick” to the surfaces to which they are applied.

307

Cetalox® well exemplifies this case (log P = 4.76) and was added to the malodor model

308

to extend the tested application range of the HS cell.

309

Analytical representativeness. The D-HS and R-HS of the “extended” washing

310

machine malodor were compared by calculating the Euclidean distance. This R-HS to

311

D-HS distance of 2.1, as well as the distance of 1.3 measured between two identical D-

312

HS samplings, are small compared with the distances observed between other

313

techniques and D-HS samplings (up to 372, see below and Figure 5). This suggests

314

that the R-HS did not significantly differ from the original D-HS. Most of the compounds

315

were repeatably sampled in both cases, despite two successive trapping steps with R-

316

HS (Figure 3). The R-HS recoveries were greater than 92%, except for geosmin.

317

However, none of the recovered proportions significantly differed from those of the D-

318

HS sampling. These observations support good analytical representativeness of the HS

319

cell and its possible use to capture and restore odors.

320


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Sensory representativeness. Because of the constraints of cell volume and piston rate,

322

only 15 panelists per session were able to evaluate the odor, whereas 30 panelists are

323

required to achieve robust results. Therefore, two sessions were needed for a complete

324

evaluation. Surprisingly, the similarity score was only 7.2 out of 10: 17 panelists found

325

that the two cells were “slightly different” and five panelists that they were “identical

326

(Figure 4, R-HS). An edge effect was noticed: panelists were reluctant to rate the

327

similarity at the scale extrema,25 which is a well-known bias in sensory measurements.

328

Moreover, by changing the order of the first smelled cell, we observed that people were

329

influenced by the first odor smelled and that the second one was less powerful because

330

of nose saturation.25 To assess the impact of such biases on the measurement, we

331

performed a similarity test in triplicate by using two cells containing the same D-HS

332

sample (Figure 4, D-HS), which gave a score of 7.0 out of 10 (SD = 0.1). This result

333

demonstrates that 7 is the highest possible score corresponding to identical stimuli

334

using this sensory test and a scale of 10. This value does not significantly differ from the

335

score between the D-HS and R-HS cells, which led us to conclude that the restored

336

odor was similar to the initial odor of the D-HS. This observation confirmed that the HS

337

cell can restore a representative “sensory image” of the captured initial odor and could

338

be used as an olfactive camera. “Camera” comes from Latin and the “camera obscura”

339

(dark chamber) was the ancestor of the modern photographic device. The present

340

olfactive camera restores trapped odors in the same way that a camera allows

341

restoration of captured images. (Figure S2).



18 342

Analytical comparison with other HS sampling methods

343

HS-SPME, HSSE, and P&T-HS are the most frequently used techniques to sample the

344

HS around an odorous sample.26 Despite the recovery differences predicted by the

345

theory and recalled in the introduction (Figure S1), a large number of studies report odor

346

composition by using one of these three sampling methods. Therefore, we tested these

347

methods versus the HS cell by reheating the loaded fiber, the stir bar, or the trapping

348

cartridge, respectively, to restore the HS composition in the HS cell chamber. The

349

resulting odor was compared with the initial one by using the analytical Euclidian

350

distance and the sensory similarity test.

351

HS-SPME. The HS-SPME is a three-phase extraction: analyte in solution–air–fiber

352

adsorbent, according to eq 1:

353

𝐾𝑓𝑉𝑓

𝜌 = 𝐾𝑓𝑉𝑓 + 𝐾𝑔𝑙𝑉𝑔 + 𝑉𝑠

(1)

354

where 𝜌 is the recovery of a volatile extracted by the fiber, Kf the fiber-to-sample

355

partition coefficient, Kgl the gas-to-sample partition coefficient, Vg the volume of gas, VS

356

the volume of sample, and Vf the volume of fiber coating.

357

GC-FID analyses of the malodor model were performed after various extraction times:

358

the extraction equilibrium was reached after 20 min, and so an exposure of 30 min was

359

chosen for the sensory test. The higher the hydrophobicity (log P) of constituents, the

360

greater the extraction by the PDMS fiber, such as for geosmin and Cetalox® (data not

361

shown). The recoveries refer to the initial concentrations above the malodor solution as

362

measured by D-HS by using the HS cell. Values above 100% were due to the forced


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19 363

release of volatiles from the liquid mixture triggered by their depletion in the HS because

364

of their adsorption on the SPME fiber. In fact, when the gas-to-sample partition

365

coefficient or the HS volume is low, the term relative to the gas phase (KglVg) is

366

negligible.10, 27 In that case, n depends only on individual fiber-to-sample partition

367

coefficients such as in the case of a direct SPME sampling dipped in an aqueous

368

solution. Even after reaching equilibrium, the Euclidean distance from the SPME

369

composition to that of D-HS was around 50 (Figure 5, blue curve), and so the SPME

370

was not representative of the HS composition at any fiber exposure time.

371 372

HSSE. The sorption mechanism from the HS by using a stir bar (HSSE) is similar to that

373

of HS-SPME and also obeys eq 1. Because of the greater absorbent volume for HSSE

374

(55 µL for HSSE vs 0.6 µL for 100 µm PDMS SPME fiber), the time to equilibrium is

375

longer than with SPME. In the present case, this equilibrium was not reached after 2 h

376

30 min, and so the analytical distance between D-HS and R-HSSE was transiently

377

reasonably short and then increased with exposure time (Figure 5, red curve).

378 379 380

P&T-HS. The recovery ρ of a given volatile from P&T-HS10 can be calculated with eq 2:

𝜌=1―𝑒

―𝑘𝑔𝑙.𝐹.𝑡 𝑉𝑙

(2)

381

where kgl is the gas-to-sample partition coefficient, Vl the volume of the sample, F the

382

flow of stripping gas, and t the extraction time.



20 383

This equation shows that recoveries of volatiles depend on individual partition

384

coefficients and on the stripping time: the more volatile the compound, the better the

385

extraction. Therefore, the balance of the extracted volatiles varies and should not be

386

representative of the initial volatile composition of an odor. No equilibrium was reached

387

with this sampling method. Because the Euclidean distance reached a minimum at 10

388

min (Figure 5), this time was selected to test the sensory similarity.

389

Sensory comparison of HS sampling methods

390

Using the conditions defined in the analytical comparison, we trapped the HS from the

391

malodor standard solution by using the four methods and then desorbed it into a clean

392

S&T-HS cell. An olfactive port was then connected at the top of the piston to smell the

393

expelled air in comparison to that of the D-HS malodor standard from a second HS cell.

394

As was done for the evaluation of HS cell representativeness (see above), the highest

395

possible sensory similarity was determined as the score between the D-HS of two cells

396

containing the same malodor solution sample. SPME, HSSE, and P&T were evaluated

397

by using the optimal sampling times, as determined above. Among the odors restored

398

by using the different techniques, the R-HS from the HS cell exhibited the highest

399

sensory similarity (Figure 6), followed by the R-P&T-HS odor. R-HS-SPME and R-HSSE

400

were the most dissimilar. Results for R-P&T-HS must, however, be cautiously

401

interpreted because the optimum sampling time was selected, temporarily inducing a

402

similar analytical composition to that of D-HS. But this transient state rapidly changed

403

with the stripping time (Figure 5); moreover, this optimum cannot be determined for an

404

unknown odorous sample, which implies a high risk of collecting a distorted HS.


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21 405

Test with a complex odor

406

To pin down the capabilities of the olfactive camera and identify possible areas of

407

improvements, it was used to trap and restore the complex ashtray odor of a smoking

408

room. The short Euclidean distance (5.1) between D-HS and R-HS chromatograms

409

suggested a good similarity of GC profiles compared with the different results in Figure

410

5. However, the early eluting peaks from thermodesorbed traps were poorly resolved

411

and not used in the analytical distance calculation. At this stage, no attempt was made

412

to improve the chromatographic resolution of early eluting peaks because this trial

413

aimed only at evaluating the potential of the olfactive camera for complex odors. The

414

sensory similarity between the cell containing the ashes (D-HS) and the R-HS was 6.9,

415

which indicated a high similarity, despite being slightly lower than that between two

416

identical HS cells (7.2). The panelists noticed that the “freshly smoked cigarette” top

417

note was weaker in the D-HS than in the R-HS odor. This was attributed to the less

418

efficient trapping of highly volatile compounds that were not included in the analytical

419

distance calculation and known to have small breakthrough volumes in Tenax. This

420

result suggests that the small odor bias would not be inherent in the olfactive camera

421

itself, but would presumably be due to the trapping technology that should be further

422

improved.

423

The above-reported developments in the S&T-HS cell significantly minimized the

424

adsorptions of volatile compounds on the device’s surfaces. This led to high recoveries

425

of all analytes, including that most prone to adsorption, and good representativeness of

426

sampled odors, both from the analytical and the sensory viewpoint. In comparison with

427

the other HS sampling methods tested here, S&T-HS thus appears to be the most



22 428

representative method. As demonstrated in this work, the S&T-HS cell allows testing of

429

various trapping techniques. It will, a fortiori, be effective to investigate the

430

performances of sorbents in the trapping cartridges in a next development step. This

431

technique represents the first prototype that allows the capture and restoration of a non-

432

distorted odor and constitutes a proof of concept as an olfactive camera.

433 434

ABBREVIATIONS USED

435

D-GC-O = direct GC-O; D-HS = direct headspace; GC-O = gas chromatography-

436

olfactometry; HS = headspace; HSSE = HS sorptive extraction; HS-SPME = HS solid-

437

phase microextraction; P&T-HS = purge-and-trap HS; R-HS = restored HS; R-HSSE =

438

restored HS sorptive extraction; R-HS-SPME = restored HS solid-phase

439

microextraction; R-P&T-HS = restored purge-and-trap HS; S-HS = static HS; S&T-HS =

440

static-and-trapped headspace.

441

ACKNOWLEDGEMENTS

442

The authors wish to acknowledge Dr I. Cayeux for her support in sensory analysis, Mrs

443

A. Hugon and J.P. Probst for cell engineering, all panelists who contributed to these

444

results, and Barbara Every, ELS, of BioMedical Editor, for English language editing.


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23 445

AUTHOR INFORMATION

446

Funding

447

This work was exclusively supported by Firmenich SA.

448

Author contributions

449

A. Chaintreau is the designer of the initial olfactive camera. A. Chaintreau and F.

450

Begnaud alternatively monitored the project and wrote the manuscript through the

451

contributions of all authors. The optimization of the HS-cell representativeness, its

452

extension to the restoration of odors, and its evaluation as an olfactive camera were

453

conducted by B. Zellner, U. Keller, and K. Nguy, respectively.

454 455

Supporting information

456

The Supporting Information is available free of charge on the ACS Publications website

457

at DOI: XXXXXXXXX.

458

Figure S1: Calculated recoveries using various HS methods

459

Figure S2: Scheme of the S&T-HS cell equipped with the trap desorption oven

460

Figure S3: Sorption characteristics of various materials and treated surfaces exposed to

461

the malodor model

462

Figure S4. GC profiles of the D-HS (left) and R-HS (right) of the ashtray malodor

463

Table S1: Composition of the malodor model

464

Table S2: Material and surface treatments tested for their sorptive properties

465 466



24 467

REFERENCES

468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502

1. Reineccius, G., Biases in analytical flavor profiles introduced by isolation method. In Flavor measurement, Ho, C. T.; Manley, C. H., Eds. Marcel Dekker: New-York, 1993; pp 61-75. 2. Richter, B. E.; Jones, B. A.; Ezzell, J. L.; Porter, N. L., Accelerated Solvent Extraction: A Technique for Sample Preparation. Anal. Chem. 1996, 68, 10331039. 3. Cicchetti, E.; Chaintreau, A., Comparison of Extraction Techniques and Modeling of Accelerated Solvent Extraction for the Authentication of Natural Vanilla Flavors. J. Sep. Sci. 2009, 32, 1957-1964. 4. Godefroot, M.; Sandra, P.; Verzele, M., New method for quantitative essential oil analysis. J. Chromatogr. A 1981, 203, 325-335. 5. Maignial, L.; Pibarot, P.; Bonetti, G.; Chaintreau, A.; Marion, J. P., Simultaneous distillation-extraction under static vacuum: isolation of volatiles at room temperature. J. Chromatogr. A 1992, 606, 87-94. 6. Engel, W.; Bahr, W.; Schieberle, P., Solvent assisted flavour evaporation a new and versatile technique for the careful and direct isolation of aroma compounds from complex food matrices. Z. Lebensm. Unters. Forsch. 1999, 209, 237-241. 7. Baltussen, E.; Sandra, P.; David, F.; Cramers, C., Stir bar sorptive extraction (SBSE), a novel extraction technique for aqueous samples: theory and principles. J. Microcolumn Separations 1999, 11, 737-747. 8. Koziel, J. A.; Spinhirne, J. P.; Lloyd, J. D.; Parker, D. B.; Wright, D. W.; Kuhrt, F. W., Evaluation of Sample Recovery of Malodorous Livestock Gases from Air Sampling Bags, Solid-Phase Microextraction Fibers, Tenax TA Sorbent Tubes, and Sampling Canisters. Journal of the Air & Waste Management Association 2005, 55, 1147-1157. 9. Pillonel, L.; Bosset, J. O.; Tabacchi, R., Rapid preconcentration and enrichment techniques for the analysis of food volatile . A review. Lebensm.Wiss. u.-Technol. 2002, 35, 1-14. 10. Chaintreau, A., Sample preparation: headspace techniques. In Encyclopedia of Analytical Chemistry, Meyers, R. A., Ed. Wiley: Chichester, 2000; pp 4229-4246. 11. Chaintreau, A.; Grade, A.; Munoz-Box, R., Determination of partition coefficients and quantitation of headspace volatile compounds. Anal. Chem. 1995, 67, 3300-3304.


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12. Brachet, A.; Chaintreau, A., Determination of air-to-water partition coefficients using automated multiple headspace extractions. Anal. Chem. 2005, 77, 3045-3052. 13. Pollien, P.; Ott, A.; Montigon, F.; Baumgartner, M.; Munoz-Box, R.; Chaintreau, A., Hyphenated headspace-gas chromatography-sniffing: screening of impact odorants and quantitative aromagram comparisons. J. Agric. Food Chem. 1997, 45, 2630-2637. 14. Ott, A.; Fay, L. B.; Chaintreau, A., Determination and origin of the aroma impact compounds of yogurt flavor. J. Agric. Food Chem. 1997, 45, 850-858. 15. Pollien, P.; Krebs, Y.; Chaintreau, A., Comparison of a brew and an instant coffee using a new GC-olfactometric method. Assoc. Sci. Int. Cafe 1998, 1997. 16. Pawliszyn, J., Solid phase microextraction. Wiley-VCH: New York, 1997. 17. d'Acampora Zellner, B.; Dugo, P.; Dugo, G.; Mondello, L., Gas chromatography–olfactometry in food flavour analysis. J. Chromatogr. A 2008, 1186, 123-143. 18. Selli, S.; Rannou, C.; Prost, C.; Robin, J.; Serot, T., Characterization of Aroma-Active Compounds in Rainbow Trout (Oncorhynchus mykiss) Eliciting an Off-Odor. J. Agric. Food Chem. 2006, 54, 9496-9502. 19. Hallier, A.; Serot, T.; Prost, C., Odour of cooked silurus (Silurus glanis) flesh: Evaluation by sensory analysis and comparison of collection methods to assess the odour representativeness of extracts obtained by dynamic headspace. J. Sci. Food Agric. 2004, 84, 2113-2122. 20. Shani, A.; Clearwater, J., How efficient are all-glass systems for collection of airborne volatiles? J. Chem. Ecol. 1997, 23, 1621-1633. 21. Brachet, A.; de Saint Laumer, J.-Y.; Chaintreau, A., Multiple headspace extraction procedure: adsorption modeling & determination of air-to-water partition coefficients. Anal. Chem. 2005, 77, 3053-3059. 22. Matsunaga, A.; Ziemann, P. J., Gas-Wall Partitioning of Organic Compounds in a Teflon Film Chamber and Potential Effects on Reaction Product and Aerosol Yield Measurements. Aerosol Science and Technology 2010, 44, 881-892. 23. Rega, B.; Fournier, N.; Guichard, E., Solid phase microextraction (SPME) of orange juice flavor: Odor representativeness by direct gas chromatography olfactometry (D-GC-O). J. Agric. Food Chem. 2003, 51, 7092-7099. 24. Begnaud, F.; Chaintreau, A.; Keller, U., Volatile compounds trap desorption device and method for desorbing volatile compounds from a trap. Patent 2012, WO 2012/031975 A1.



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25. Lawless, H. T.; Heymann, H., Sensory Evalution of Food: Principles and Practices. Springer Science & Business Media: 2010. 26. Rubiolo, P.; Sgorbini, B.; Liberto, E.; Cordero, C.; Bicchi, C., Analysis of the plant volaile fraction. In The Chemistry and Biology of Volatiles, Herrmann, A., Ed. Wiley: Chichester, 2010; pp 49-93. 27. Yang, X.; Peppard, T., Solid phase microextraction for flavor analysis. J. Agric. Food Chem. 1994, 42, 1925-1930.

548 549


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27 550

Figure captions

551

Figure 1. Sampling of the direct headspace (D-HS) (A–D) and redesorption of the

552

loaded Tenax cartridge into an empty HS cell (E-H) to restore the HS (restored HS, R-

553

HS).

554

Figure 2. Constituent recoveries from the malodor model after R-HS sampling,

555

compared to the D-HS sampling, as a function of various improvements. S.S. =

556

stainless steel, Sulf. = Sulfinert, 2-IPMP = 2-isopropyl-3-methoxypyrazine, 2-MIB = 2-

557

methylisoborneol.

558

Figure 3. Relative proportions of the D-HS and the corresponding R-HS of the malodor

559

model (bars) and recovery of the R-HS relative to the D-HS (line).

560

Figure 4. Similarity test by 30 panelists between two identical D-HS cells (blue bars,

561

three replicates) and between D-HS and R-HS cells (red bars).

562

Figure 5. Evolution of the Euclidean distance between HS-SPME, HSSE, and P&T-HS

563

samples and the initial HS composition (logarithmic scales).

564

Figure 6. Sensory comparison between the different sampling methods: panelists’

565

opinions (bars) and sensory similarity scores (brown line).



28


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29

Figure 1



30

Figure 2


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31

Figure 3



32

Figure 4


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33

Figure 5



34

Figure 6


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35 For Table of Contents Only


From the representativeness of sampled odors to an olfactive camera

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