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Talanta 70 (2006) 595–601

Application of zNoseTM for the analysis of selectedgrape aroma compounds

Peter Watkins ∗, Chakra WijesunderaCSIRO Food Futures Flagship and Food Science Australia, Private Bag 16, Werribee, Vic. 3030, Australia

Received 24 November 2005; received in revised form 17 January 2006; accepted 17 January 2006Available online 28 February 2006

bstract

A novel and portable gas chromatograph (GC, zNoseTM) has been evaluated for the measurement in grape berries of selected six-carbonompounds; namely, hexanal, cis-2-hexen-1-ol, cis-3-hexen-1-ol and trans-2-hexenal. The zNoseTM is a handheld GC which uses purge and trapor concentration, and has a surface acoustic wave (SAW) sensor as a detector. Operation of the zNoseTM using direct aspiration of the sample

ailed to detect the compounds at the reported odour threshold values. Pre-concentration by Tenax® trapping and solid-phase microextractionSPME) were investigated to improve the zNoseTM sensitivity. Use of a Tenax® pre-trap with the zNoseTM allowed detection of the compounds atoncentration levels in the order of their threshold values. Excessive bleed from the SPME fibre prevented the use of SPME with zNoseTM.

2006 Elsevier B.V. All rights reserved.

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eywords: zNoseTM; Grape; Aroma compounds; SPME; GC/MS; Wine; Purge

. Introduction

Six-carbon (C6) compounds are found in abundance in grapeeaves and contribute to the “grassy” note found within a grapeeaf [1]. They also contribute to grape juice aroma. Trans-2-exenal and hexanal have also been reported to contribute to thegrassy” odour in grape juice [2]. The presence of the C6 volatileompounds enhances the overall grape berry flavour. Some ofhese compounds have low odour threshold values. Hexanal has

threshold value of 4.5 �g L−1 in water [3] while the corre-ponding values for trans-2-hexenal and cis-3-hexen-1-ol are7 and 70 �g L−1 in water [3]. However, using simultaneousistillation and extraction (SDE) and gas chromatography/masspectrometry (GC/MS), the minimum detection limits (MDL’s)or hexanal, trans-2-hexenal, and cis-3-hexen-1-ol were as highs 1.3, 1.4, and 0.8 mg L−1, respectively [3]. These values areigher than the odour threshold values so there is a need for aensitive analytical method that can measure these compounds

t concentrations below their odour threshold.

Since 1998, a novel and portable GC has been available forhe measurement of volatile and semi-volatile compounds. The

∗ Corresponding author. Tel.: +61 3 97313467; fax: +61 3 97313250.E-mail address: Peter.Watkins@csiro.au (P. Watkins).

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039-9140/$ – see front matter © 2006 Elsevier B.V. All rights reserved.oi:10.1016/j.talanta.2006.01.032

rap

NoseTM is a hand-held fast GC that uses purge and trap foroncentration, and a surface acoustic wave (SAW) sensor foretection [4,5]. The zNoseTM has been used for a number ofood analyses; for honey classification [6], honey adulteration7], measurement of plant volatile compounds [8], characterisa-ion of vegetable oils [9] and detection of palm olein adulteration10]. Part-per-billion sensitivity has been reported with thisnstrument for volatile compounds and part-per-trillion sensi-ivity for semi-volatile compounds [4].

In this study, we evaluated the use of the zNoseTM for theeasurement in grape berries of selected C6 volatile compounds;

amely, hexanal, cis-2-hexen-1-ol, cis-3-hexen-1-ol and trans--hexenal. These compounds are present in grape berries in theange of mg L−1 [11,12] and, under normal operating condi-ions, the zNoseTM can measure these compounds at such levelsy direct aspiration of the sample headspace. This means thathe zNoseTM could be used to measure these selected volatileompounds in the field due to the instrument’s portability. Detec-ion limits for these compounds though are higher than thedour threshold values. Thus, the presence of related compoundshich impact on the flavour at levels close to the odour threshold

alues would not be detected. We therefore examined the use ofampling techniques that could increase the sensitivity of theNoseTM. Tenax®-TA is one suitable adsorbent that can be usedor sample enrichment [13]. Here, we report on the evaluation

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dwsg3str(tattsa2wro3zthtemperature for a further 10 s. The SAW crystal detector tem-perature was held at 30 ◦C. Data was analysed using Microsensesoftware (version 4.42, EST). All samples were analysed in trip-licate.

96 P. Watkins, C. Wijesunder

f external Tenax® trapping and solid-phase microextractionSPME) for sample pre-concentration to improve the detectionimits for grape volatiles by zNoseTM technology.

. Experimental

.1. Materials

Cis-2-hexen-1-ol (>90%), cis-3-hexen-1-ol (>98%) andrans-2-hexenal (>95%) were purchased from Sigma–AldrichSydney, Australia) while hexanal was obtained from MerckGermany). d(+)-Glucose was obtained from Fluka (Stennheim,witzerland). Potassium dihydrogen phosphate was obtainedrom BDH (Kilsyth, Australia). l(−)-Malic acid, l(−)-tartariccid, (d)-fructose, diammonium hydrogen phosphate and potas-ium hydroxide were purchased from Sigma–Aldrich (Syd-ey, Australia). Tenax®-TA was purchased from Alltech Asso-iates (Sydney, Australia). Carboxen®/polydimethyldisiloxaneCar/PDMS) and divinylbenzene/PDMS (DVB/PDMS) SPMEbres were obtained from Supelco Inc. (Sydney, Australia). ThePME fibres were conditioned according to the manufacturer’sirections.

.2. Preparation of stock solutions

A stock solution of cis-2-hexen-1-ol, cis-3-hexen-1-ol andexanal was made by diluting 30 �L of each compound to5 mL with ethanol. A stock solution of trans-2-hexenal wasbtained by diluting 30 �L of the neat compound to 25 mLith ethanol. Subsequent intermediate solutions were preparedy dilution with ethanol. Working standard solutions were pre-ared by diluting the intermediate solution with either deionised,istilled water or synthetic grape juice (SGJ). Preparation ofhe SGJ has been described elsewhere [14]. Briefly, 70.0 g glu-ose, 30.0 g fructose, 7.0 g tartaric acid, 10.0 g malic acid, 0.67 giammonium hydrogen phosphate and 0.67 g potassium dihy-rogen phosphate were dissolved in 1 L deionised, distilledater and the pH was adjusted to 3.8 with 0.2 M potassiumydroxide.

.3. zNoseTM analysis

.3.1. Direct aspirationMeasurements were performed with a zNoseTM Model 4100

apour analysis system (Electronic Sensor Technology—EST,ewbury Park, CA) fitted with a Tenax® trap (approximatelymg) for sample pre-concentration, a miniature DB-5 fusedilica capillary column for separation of components, and ascillating surface acoustic wave (SAW) detector for detec-ion and quantification of the separated components. Sampleliquots (2.0 mL) were sealed in 10 mL glass headspace vialsith PTFE/silicone septa and heated at 40 ◦C for 10 min. The

eadspace volatiles were swept into the trap for 10 s. The trapas then heated to 250 ◦C to desorb and transfer the volatiles

o the analytical column which was heated from 40 to 125 ◦Ct a rate of 3 ◦C s−1 and held at this temperature for a further

FT

lanta 70 (2006) 595–601

0 s. The SAW crystal detector temperature was held at 30 ◦C.ata was analysed using Microsense software (version 4.42,ST). All samples were analysed in triplicate. The detection

imit was defined as the concentration which corresponded tohree times the signal-to-noise ratio. After inspection of outputrom the zNoseTM, noise was arbitrarily chosen as 50. Higherurge times (>10 s) were found to overload the internal Tenax®

rap.

.3.2. Tenax® pre-trapTenax®-TA (0.26 g) was placed in a glass tube (6.25 mm outer

iameter) (referred to as ‘pre-trap’) and held in place by glassool plugs. A Model 3100 high temperature desorption acces-

ory (EST, Newbury Park, CA) was used to heat the Tenax® andlass tube. Prior to daily use, the tube and Tenax® were purged at00 ◦C for 30 min with nitrogen. An ‘in-house’ syringe was con-tructed to allow transfer of the sample headspace from the vialo the ‘pre-trap’. The sampler was made from a 6.25 to 3.13 mmeducing stainless steel Swagelok fitting. A length of 3.13 mmouter diameter) stainless steel tubing was bent and connectedo the 3.13 mm fitting end while the 6.25 mm fitting end wasttached to the Tenax® tube using PTFE ferrules. Fig. 1 showshe syringe attached to the glass tube with Tenax® connected tohe zNoseTM prior to analysis. Sample aliquots (2.0 mL) wereealed in 10 mL glass headspace vials with PTFE/silicone septand heated at 40 ◦C for 10 min. The sample was then purged formin. The sampler was removed from the ‘pre-trap’ while airas purged through the Tenax® for 0.5 min. This allowed the

emoval of moisture from the ‘pre-trap’. The volatiles were des-rbed from the ‘pre-trap’ at 220 ◦C for 2 min using the Model100 desorption accessory and were transferred to the internalNoseTM trap which was then heated to 250 ◦C to desorb andransfer the volatiles to the analytical column. The oven waseated from 40 to 125 ◦C at a rate of 3 ◦C s−1 and held at this

ig. 1. The zNoseTM with ‘in-house’ sampler attached to glass tube containingenax® and the Model 3100 desorption heating collar.

a / Talanta 70 (2006) 595–601 597

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Table 1Box-Benkhen experimental design for the optimisation of SPME sampling timeand sampling temperature

Experiment Samplingtemperature(◦C)

Samplingtime (min)

Temperature(encoded), tea

Time(encoded), tib

1 35 5 −1 −12 35 15 −1 03 35 25 −1 14 45 5 0 −15 45 15 0 06 45 15 0 07 45 15 0 08 45 25 0 19 55 5 1 −1

10 55 15 1 011 55 25 1 1

a te = (sampling temperature − 45)/10 where 45 is the midpoint temperatureand 10 is the temperature interval.

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P. Watkins, C. Wijesunder

.3.3. SPME transfer tubeThe Model 3100 high temperature accessory was used to des-

rb the volatile compounds from the Car/PDMS SPME fibre. Alass tube with an outer diameter of 6.25 mm and an inner diame-er of 0.7 mm was constructed (Monash Scientific Glassblowing,andenong, Australia). This allowed the insertion of the SPMEbre into the tube and also the use of the Model 3100 heatingollar to heat the glass tube and SPME fibre.

.4. Solid-phase microextraction and gas chromatographyass spectrometry

Headspace SPME–GC/MS was performed using an AgilentC/MS system (Palo Alto, CA, USA) comprising a Model 6890as chromatograph and a Model 5973 mass selective detector, aOC fused silica capillary column (60 m, 0.32 mm i.d., 1.8 �mlm thickness, Agilent) and a CombiPAL SPME autosamplerCTC, Switzerland). Aliquots (2.0 mL) of the standard solutionere placed in 10 mL glass headspace vials and sealed withTFE/silicone septa and steel seals. The vials and their contentsere pre-heated at the selected sampling temperature (35, 45, or5 ◦C) for 2 min prior to the insertion of the Car/PDMS SPMEbre into the headspace where it was held at the selected sam-ling temperature for a period of 5, 15 or 25 min. At the endf the sampling time, the autosampler withdrew the fibre andnserted it into the GC injector (220 ◦C) to desorb the adsorbedompounds for transfer to the analytical column. The fibre waseld in the injector for 7 min.

The GC oven temperature was initially held at −20 ◦C formin, increased to 60 ◦C at a rate of 120 ◦C min−1 and then to20 ◦C at a rate of 6 ◦C min−1 where it was held for a furthermin. The injector was held in the splitless mode for the firstmin of the analysis and then in the split mode (1:20) for the

emainder of the analysis. Helium was used as the carrier gas atflowrate of 1.0 mL min−1.

The MS was operated in electron ionisation mode (70 eV) andata acquired in the full scan mode for the range of 35–250 Da.he detector was maintained at the autotune value. The tem-erature of the source and the detector were 150 and 230 ◦C,espectively, while the MS transfer line was 280 ◦C. The ana-yte response was quantified by measuring the abundance of aharacteristic target ion using Chemstation software. A quali-ying ion was also used to confirm the analyte’s identification.he target and qualifying ions were as follows: hexanal, m/z = 56nd m/z = 82; cis-3-hexen-1-ol, m/z = 67 and m/z = 82; trans-2-exenal, m/z = 69 and m/z = 82; cis-2-hexen-1-ol, m/z = 67 and/z = 82.

.5. Response surface methodology (RSM)

The best combination of sampling temperature and time forhe highest sensitivity for the selected compounds by SPMEombined with GC–MS was determined by RSM. A Box-

ehnken [15] experimental design matrix consisting of a setf 11 experiments was used (Table 1). While the Box-Behnkenesign is used less frequently than other designs such as cen-ral composite designs, it is particularly suitable for use with

tmrc

ti = (sampling time − 15)/5 where 15 is the midpoint time and 5 is the timenterval.

esponse surface optimisations. The central point was replicatedhree times to estimate the experimental reproducibility. The

easured analyte responses were used to estimate the coeffi-ients (ai) of a second-degree polynomial model as representedy the equation:

= a0 + a1te + a2ti + a3t2e + a4t

2i + a5teti (1)

here R is the measured analyte ion abundance, te and ti are thencoded variables for sampling temperature and sampling time,espectively [16]. The coefficient estimation was performedsing multi-linear regression with the “lm” command in R, anpen source implementation of the S statistical language [17].he coefficient estimates were then used to generate plots to

dentify the optimum conditions for both sampling temperaturend time.

.6. Analysis of grape berry by zNoseTM andPME/GC–MS

Six Cabernet Sauvignon grape berry samples were providedy CSIRO Plant Industry, Adelaide. The ripe samples had beenacerated prior to arrival and were a non-homogenous mixture

f grape and juice. The samples were stored at −80 ◦C untilequired for analysis. The zNoseTM analysis was performed byirect aspiration of the sample headspace using the conditionsiven in Section 2.3.1. Aliquots (2.0 g) of the macerate wereealed in 10 mL glass headspace vials with PTFE/silicone septa.he vials and contents were heated at 40 ◦C for 10 min prior

o measurement by the zNoseTM. Note that a higher samplingemperature than 40 ◦C for the macerate could increase the riskf introducing moisture to the zNoseTM. Moisture can be swept

hrough the analytical column to the detector which can compro-

ise the analysis. The analyte concentration was determined byeference to an internal standard, 3-octanol (0.47 mg L−1). Foralibration, the standard concentration range for hexanal and

5 a / Talanta 70 (2006) 595–601

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Table 2Calculated least square coefficients for linear regression for direct aspiration

Compound Slope Intercept DLa Odour thresholdb

Hexanal 826.3 −4.7 0.2 4.5Trans-2-hexenal 482.1 −77.7 0.2 47Cis-2-hexen-1-ol 145.4 −21.7 1.2 –Cis-3-hexen-1-ol 283.5 −22.5 0.2 17

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ptslwTtvalue. A desorption temperature of 220 ◦C or higher was alsonecessary as lower temperatures did not provide reproducibleresults.

98 P. Watkins, C. Wijesunder

rans-2-hexenal was 1.0–5.0 mg L−1 while, for cis-2-hexen-1-l, the standard range was 1.0–20.4 mg L−1.

The SPME–GC/MS analysis was performed by heatinghe vials and its contents by sampling the headspace with aar/PDMS fibre at 55 ◦C for 25 min. Separation and detection of

he volatile compounds was done using the conditions given inection 2.4. The injector was held in the split mode (50:1). Theata analysis was performed with the total ion chromatogram.his was done to reflect the total mass response of the SAWetector of the zNoseTM. As for the zNoseTM analysis, the ana-yte concentration was determined using an internal standard,-octanol (4.7 mg L−1). For calibration, the analyte standardoncentration range was 1.0–20.0 mg L−1.

. Results and discussion

.1. zNoseTM measurements in the standard mode (directspiration)

Method optimisation experiments were performed with stan-ard solutions of hexanal, trans-2-hexenal, cis-2-hexen-1-ol andis-3-hexen-1-ol; all of which are volatile components naturallyresent in grape berries. Initially, the solutions were prepared inoth water and a synthetic grape juice. However, as the zNoseTM

esponse for the analytes was not notably different for the twoedia, all subsequent method optimisation experiments were

erformed using standard solutions prepared in water.The zNoseTM responses for each volatile compound in the

tandard mixture were measured using a direct aspiration pro-edure recommended by the instrument manufacturer (EST) foreasuring 2,4,6-trichloranisole in wine. This method did not

chieve complete separation of all components in the mixture.emperature programming was used in an attempt to improve

he component resolution. Oven ramp rates from 2 to 5 ◦C s−1

ere evaluated, and the best separation was obtained with theamp rate of 3 ◦C s−1. With this ramp rate, the componentsere baseline-resolved with the exception of trans-2-hexenal

nd cis-3-hexen-1-ol which co-eluted. The zNoseTM manufac-urers also supply a more polar column (DB-624) which couldave provided better separation of the last two compounds buthis column was not available to us when this work was carriedut.

The zNoseTM responses for the standard mixture were mea-ured for the concentration range of 1–20 mg L−1. The responseas found to be linear over the range of 1–4 mg L−1 but non-

inear from 4 to 20 mg L−1. The calculated linear regressionoefficients (slope and intercept) for the range of 1–4 mg L−1

re shown in Table 2; these were used to determine each ana-yte’s detection limit.

Young and Suffet [3] using SDE–GC/MS reported detec-ion limits of 11.3 mg L−1 for hexanal and trans-2-hexenal,nd 0.8 mg L−1 for cis-3-hexen-1-ol. Better sensitivity has been

chieved by pre-concentration of the volatiles using SPME prioro GC/MS analysis. For example, Sanchez-Palomo and co-orkers [12], using SPME–GC/MS, reported detection limits

or hexanal, trans-2-hexenal, cis-2-hexen-1-ol and cis-3-hexen-Fp

a DL (detection limit (mg L−1)) = 3 × signal-to-noise.b Odour threshold value (�g L−1) from [3].

-ol of 0.071, 0.022, 0.089, and 0.021 mg L−1, respectively.he detection limits obtained by SPME–GC/MS for the grapeolatiles approach their odour threshold values. Initial experi-ents with the zNoseTM operating the instrument in the standard

perating mode gave detection limits higher than their odourhreshold values. Thus, there is a need to improve the sensitivityf zNoseTM prior to its use for detecting these compounds inrapes at levels which impact on odour and flavour.

.2. zNoseTM measurements using external Tenax®

‘pre-trap’) for sample pre-concentration

The effect of pre-concentrating the volatiles using a Tenax®

re-trap prior to the zNoseTM was investigated. First, the effect ofhe purge time was examined using a 1 mg L−1 trans-2-hexenalolution at room temperature. The zNoseTM response increasedinearly with the headspace purge time (Fig. 2). Similar resultsere found with hexanal, cis-2-hexen-1-ol and cis-3-hexen-1-ol.he headspace purge time was set to 30 s as poor repeatability of

he measurement was observed at purge times higher than this

ig. 2. Plot of the zNoseTM response for trans-2-hexenal against the headspaceurge time using Tenax® ‘pre-trap’.

a / Talanta 70 (2006) 595–601 599

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Table 4Calculated regression coefficients for zNoseTM response for standard range of30–500 �g L−1 using Tenax® for pre-concentration

Compound Quadratic regression Log vs. log

a0a a1 a2 DLb c0

c c1 DL

Hexanal −0.9 6.6 −0.0054 23 2.40 0.85 21Trans-2-hexenal 214.5 92.8 −0.0035 2 3.56 0.76 7Cis-2-hexen-1-ol −2.0 4.0 −0.0036 39 2.01 0.82 39Cis-3-hexen-1-ol 135.6 6.2 −0.0054 25 3.49 0.66 10

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P. Watkins, C. Wijesunder

The zNoseTM results for the standards in the concentrationange of 30–500 �g L−1 was not linear. Additionally, wide dif-erences existed between the volatiles with respect to the preci-ion of measurement under these conditions. For cis-2-hexen-1-l, the relative standard deviation (RSD, for n = 3 measurements)anged from 12.4 to 19.1% while for cis-3-hexen-1-ol, trans-2-exenal and hexanal, the RSD ranged from 1.0 to 10.8, 2.1–14.6,nd 6.6–23.4%, respectively. Inspection of the residual plotsplot of the measured minus expected values versus expectedalues; results not shown) suggested that a quadratic expressionan be used to relate the zNoseTM response to the analyte con-entration. However, exclusion of the measured responses for00 �g L−1 resulted in a linear range for the instrument between0 and 200 �g L−1. The wide differences in the RSD values canither suggest that the instrumental response is not based on aormal (Gaussian) distribution or the presence of outliers in theata [18]. This can influence the result of the linear regressionalculation as it is assumed the error in the measured responses constant [19]. When such a variation in RSD occurs, an alter-ative approach is to use statistical procedures that are robusto outliers or deviations from normality. These ‘robust regres-ion techniques’ can be used when the error is not constant [18].able 3 shows the calculated regression coefficients (ordinary

east squares and robust) for the zNoseTM response and analyteoncentration for the range of 30–200 �g L−1. The linear regres-ion coefficients were used to find the detection limits for eachnalyte.

The detection limits for the C6 compounds improved with these of Tenax® for sample pre-concentration compared to the usef direct aspiration. For hexanal, the detection limit was reducedrom 0.2 mg L−1 to 28 �g L−1, an almost 10-fold increase inensitivity. Similar trends were found for trans-2-hexenal andis-3-hexen-1-ol. The detection limit for cis-2-hexen-1-ol waseduced from 1.2 mg L−1 to 48 �g L−1. These detection limitsre also quite close to the odour threshold values for the com-ounds. This suggests that the zNoseTM with an external Tenax®

pre-trap’ could be used for measuring grape volatile compoundst levels detectable by the human nose. This, of course, wouldeed to be validated by sensory evaluation. Additionally, Tenax®

an also be used for pre-concentration of samples where the

evels are lower than the detection limits found by direct aspi-ation. This was not evaluated in this current study but woulde useful in, say, studies where the development of these com-ounds is monitored at the early stages of berry growth. The

able 3alculated regression coefficients for zNoseTM response for standard range of0–200 �g L−1 using Tenax® for pre-concentration

ompound Ordinary least squares Robust

b0a b1 DLb b0 b1 DL

exanal 42.0 5.4 28 45.9 5.4 28rans-2-hexenal 248.1 8.4 18 242.0 8.5 18is-2-hexen-1-ol 30.2 3.1 48 42.1 2.9 52is-3-hexen-1-ol 186.3 4.9 31 197.3 4.7 32

a b0 = intercept, b1 = slope.b DL = detection limit (�g L−1).

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a y = a0 + a1x + a2x2.b DL = Detection limit (�g L−1).c loge(y) = c0 + c1 loge(x).

evels present in the Cabernet Sauvignon berries were too higho use with Tenax®. Pre-concentration of the sample headspaceith Tenax® would have caused saturation of the zNoseTM’sAW detector. Once this happens, a number of remedial stepsre required prior to the detector functioning properly.

Quadratic regression can be used to relate the instru-ent response with the analyte concentration between 30 and

00 �g L−1. The wide variation in RSD can also influence theesults for the calculation of quadratic regression coefficients.ne way to overcome this is to use a data transformation wherenon-linear relationship is changed to a linear one [19]. A com-only used method is log–log transformations where log(y)

nd log(x) are used to replace y and x where, in this case, ys the instrumental response and x is the analyte concentration.able 4 shows the calculated coefficients (quadratic regressionnd log–log) for the zNoseTM response and analyte concentra-ion for the range of 30–500 �g L−1. As for linear regression,here has also been an improvement in the detection limits forhese compounds.

.3. zNoseTM measurements using SPME for samplere-concentration

To determine the optimal sampling temperature and timeombination for SPME pre-concentration, the headspace oftest solution (100 �g L−1) was sampled using a Car/PDMS

PME fibre at three different extraction times (5, 15 and5 min) and three different temperatures (35, 45 and 55 ◦C)

nd analysed by GC/MS. The MS responses for each analyterom each set of extraction conditions were used to estimatehe coefficients of Eq. (1). The estimated coefficients are shownn Table 5. The RSD value for the experimental reproducibility

able 5alculated coefficient estimates for Eq. (1) from multilinear regression

ompound a0 a1 a2 a3 a4 a5 RSD(%)

exanal 136.0a 8.5 41.4a −3 −14.4 0.7 3.7rans-2-hexenal 42.6a 9.6b 19.0a −2.8 −5.5 1.9 3.7is-2-hexen-1-ol 12.9b 5.4 6.5 −0.3 −2.2 0.8 33.1is-3-hexen-1-ol 16.6c 6.9b 9.7c −2.6 −0.8 2.5 11.5

a p < 0.001.b p < 0.05.c p < 0.01.

600 P. Watkins, C. Wijesundera / Ta

Fig. 3. Perspective plot of predicted response for trans-2-hexenal as a functionoS

(tac

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Cab

aa

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4

ymtCewScspoecFment with those from SPME–GC/MS. The zNoseTM results

TC

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f encoded temperature and time. Analysis was performed by GC–MS withPME sampling using Car/PDMS SPME fibre.

from the experimental design centre point) is also shown in theable. The repeatability of the measurement was quite good forldehydes, but this was not the case for cis-2-hexen-1-ol andis-3-hexen-1-ol.

The linear term for te (temperature) was statistically signifi-ant (p < 0.05) for the aldehydes and cis-3-hexen-1-ol while timeti) was only significant for trans-2-hexenal and cis-3-hexen--ol. There was no evidence to suggest that the second orderariables and the interactive term (te × ti) have any statisticalignificance for the model. Empirical models were developedor the analyte response using the Car/PDMS fibre from RSMnd the coefficient estimates. Perspective plots were producedy plotting the predicted analyte response against the coded vari-bles, te and ti. From each plot, the optimal set of parametersas determined.Fig. 3 shows the perspective plot for trans-2-hexenal using the

ar/PDMS SPME fibre. Similar plots were found for the othernalytes. The plot shows that the analyte response increases asoth temperature and time increase. Thus, the best temperature

fSp

able 6oncentrations of selected C6 volatile compounds in six Cabernet Sauvignon grape b

ample Hexanala (�g g−1) Trans-2-hexenal (�g g−1)

zNoseTM SPME–GC/MS zNoseTM SPME–GC/M

2.7 ± 0.5 6.6 ± 0.5 0.2 ± 0.1 0.8 ± 0.12.1 ± 0.2 5.5 ± 0.4 0.4 ± 0.1 0.8 ± 0.21.7 ± 0.1 7.4 ± 0.1 0.2 ± 0.1 0.7 ± 0.11.9 ± 0.3 8.6 ± 1.0 1.5 ± 0.2 3.3 ± 0.62.0 ± 0.3 6.5 ± 2.2 1.3 ± 0.5 2.7 ± 0.90.4 ± 0.2 3.0 ± 0.3 0.3 ± 0.2 1.2 ± 0.1

a Mean ± standard deviation for n = 3 replicates.b Co-eluted with trans-2-hexenal on zNoseTM.

lanta 70 (2006) 595–601

nd time combination for the C6 volatile compounds was 55 ◦Cnd 25 min.

The headspace of a trans-2-hexenal (100 �g L−1) solutionas sampled using the optimised headspace conditions for thear/PDMS SPME fibre, i.e. at 55 ◦C for 25 min. The fibre wasesorbed for 2 min at 220 ◦C and a significant amount of mate-ial was measured with the zNoseTM. The amount of materialdsorbed on the sensor was quite high and prevented the sensorrom properly functioning. A number of remedial steps wereerformed but these were not successful at removing the inter-erence. The interference occurred because of artefacts releasedrom the SPME fibre. The interference was not specific to thear/PDMS fibre because similar interference was found withdivinylbenzene/PDMS fibre. EST, the instrument’s manufac-

urer, also confirmed that artefacts from SPME fibres wereetected by the zNoseTM [20]. GC/MS analysis revealed theresence of residual material from PDMS of the SPME fibres.urther work would be necessary to confirm that this residualaterial was the same as the artefacts detected by the zNoseTM.

n light of these difficulties, it was concluded that SPME cannote readily used with the zNoseTM.

. Grape berry analysis

Six Cabernet Sauvignon grape berry macerates were anal-sed by the zNoseTM using the direct aspiration mode. Thisode of operation allows measurement by the zNoseTM of

he macerate headspace at the reported levels for the selected6 compounds in grape berries [11,12]. The grape berry mac-rates were also analysed by SPME–GC/MS; the headspaceas sampled at 55 ◦C for 25 min with a Car/PDMS fibre. ThePME–GC/MS analysis showed that hexanal, trans-2-hexenal,is-2-hexen-1-ol and cis-3-hexen-1-ol were present in the berryamples (Table 6). Cis-3-hexen-1-ol was a relatively minor com-onent in the grape berries, which concurs with the results ofther workers [10,11]. Trans-2-hexenal and cis-3-hexen-1-ol co-luted on the zNoseTM and, because the latter is only a minoromponent, the entire peak was assigned to trans-2-hexenal.or cis-2-hexen-1-ol, the zNoseTM results are in good agree-

or hexanal and trans-2-hexenal are slightly lower than thePME–GC/MS results. Some differences between the two sam-ling techniques can be expected. The zNoseTM uses purge and

erries

Cis-2-hexen-1-ol (�g g−1) Cis-3-hexen-1-olb (�g g−1),SPME–GC/MS

S zNoseTM SPME–GC/MS

15.7 ± 2.6 14.2 ± 0.7 0.2 ± 0.113.3 ± 0.7 13.2 ± 1.1 0.2 ± 0.114.4 ± 1.5 29.5 ± 7.0 0.5 ± 0.120.0 ± 2.1 39.5 ± 3.6 0.7 ± 0.115.7 ± 1.5 20.7 ± 5.5 0.4 ± 0.315.5 ± 3.4 13.7 ± 0.3 0.2 ± 0.1

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rap (P&T) for sample concentration and is dependent on theample purge time, whereas SPME is dependent on the fibrexposure time [21]. Mallia et al. reported differences betweenhe two techniques for the measurement of cheese aroma com-ounds [22]. P&T was better for extracting highly volatile com-ounds while SPME was more effective for extracting mediumnd high boiling compounds. These workers did not report onhe application of these methods to the C6 compounds used inhis study. Therefore, further work would be required to examinehe effect of the sampling technique on the measurement of the

6 compounds. At the measured concentration levels (�g g−1)eported here though, the differences between the results fromhe two methods would not greatly impact on the overall aromaerception.

Hexanal has been reported in grapes used for white wineanufacture at a range of 2.6–8.8 mg L−1 [11]. It has also been

ound in the pulp and skins of Muscat grapes at reported lev-ls of 0.4 and 2.3 �g g−1, respectively [12]. Trans-2-hexenalnd cis-2-hexen-1-ol have also been found in grape berries asell; reported ranges for trans-2-hexenal in white grapes were.8–6.3 mg L−1while, for Muscat pulp and skins, the levels wereeported as 0.3 and 1.5 �g g−1, respectively [11]. Low levels ofis-2-hexen-1-ol (35–54 �g L−1) have been reported for whiterapes [11] while higher levels have been reported for Muscatrape skins (1.1 �g g−1) than for the grape pulp (0.13 �g g−1)12]. For this work, the aldehyde levels found in the Cabernetauvignon grape berries are similar to those found for the Mus-at berries while the levels of cis-2-hexen-1-ol are higher thanhose previously reported. The zNoseTM does show potential forhe measurement of these selected compounds. As a portablenstrument, the zNoseTM offers the possibility that field mea-urements of a particular compound of interest can be made forrape berries. Once a grape volatile compound has been estab-ished as a wine flavour precursor, the zNoseTM can be used aspredictive tool to measure the impact of the compound on theverall wine flavour.

. Conclusion

The zNoseTM has been evaluated to measure selected nat-

rally occurring C6 compounds in Cabernet Sauvignon grapeerries. In the normal mode of operation (direct aspiration), theNoseTM was used to measure the selected compounds in sixrape berry macerates. Detection limits for these compounds

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lanta 70 (2006) 595–601 601

ere higher than the odour threshold values. This could impactn the measurement of related aroma compounds at levels closeo the threshold values. Thus, Tenax® trapping and SPME werevaluated as sample pre-concentration steps for use with theNoseTM. The detection limits with Tenax® trapping were foundo be close to the reported threshold values. SPME, on the otherand, was not suitable for sample pre-concentration due to inter-erence to the zNoseTM from fibre artefacts. The grape berryacerates were also analysed by SPME–GC/MS.

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