Analytica Chimica Acta 559 (2006) 97–104

Use of solid phase microextraction in diffusive sampling of theatmosphere generated by different essential oils

P. Lopez, M.A. Huerga, R. Batlle∗, C. NerinDepartment of Analytical Chemistry, Aragon Institute of Engineering Research (I3A), CPS-University of Zaragoza, Spain

Received 3 October 2005; received in revised form 17 November 2005; accepted 18 November 2005Available online 27 December 2005

Abstract

This work describes the development and optimisation of a complete headspace-solid phase microextraction (HS-SPME) procedure for qualitativeand quantitative analysis of the equilibrium headspace generated by a number of essential oils (EOs) with potential applications in active packaging,including basil (Ocinum basilicum), clove (Sygyzium aromaticum), rosemary (Rosmarinus officinalis), citronella (Melissa officinalis), and cinnamon(Cinnamonum zeylanicum). The method consists of a combination of fully exposed HS-SPME for qualitative analysis and diffusive HS-SPME forquantitative determination.

screeninge e EOs and tos

d extractionv vere ere furtherv d between0 erved despitec all the testa lower than9©

K

1

edeptccap

n-file.ings caneasetivev-entssedtime

d intech-

thee been

0d

First, complete optimisation of a fully exposed HS-SPME procedure was carried out by means of a combination of a Plackett–Burmanxperimental design and response surface modelling (RSM). The results were used to fully describe the atmosphere generated by thelect the most relevant compounds for further consideration.The fibres were then calibrated (i.e. the uptake rate was calculated) by exposing them to known concentrations of terpenes in close

ials. With a sampling time of 30 min at 20◦C, uptake rates ranged from 2.2 to 3.3 pg (min ppbv)−1. Results were checked by sampling oxtended periods of times, with the observed variation being less than 5%, despite a 10-fold increase in extraction time. The results walidated by comparing the calculated diffusion coefficients with theoretical data. The ratios of experimental:theoretical values varie.85 and 1.05. The sensitivity of the uptake rate to headspace concentration was also investigated; variation of less than 10% was obshanges in concentration of four orders of magnitude. The new diffusive sampling method proved to give robust determinations ofnalytes (by contrast, HS-SPME failed for camphene, camphor and cinnamaldehyde), providing repetitivity and intermediate precision% (the values for HS-SPME were 10 and 12%, respectively).2005 Elsevier B.V. All rights reserved.

eywords: Essential oils; Terpenes; Diffusive (passive) sampling; Headspace-solid phase microextraction; Active packaging

. Introduction

Packaging material in immediate contact with food has sev-ral purposes. It encloses the food, protecting it from mechanicalamage, and acts as a barrier between the food and the externalnvironment. Thus, packaging maintains product integrity byreventing or delaying detrimental changes in the food’s nutri-

ional values and sensorial or health characteristics, all of whichontribute both to its objective quality and to the consumer’s per-eption of its quality (its “subjective” quality). Modern thinkingbout food packaging has centered on the development of activeackaging materials (as opposed to conventional passive pack-

∗ Corresponding author. Tel.: +34 976 761000x5296; fax: +34 976 762388.E-mail address: r13bat@unizar.es (R. Batlle).

aging)[1–3], which interact directly with the food or the enviroment in which it is stored, to enhance its quality/safety proWithin this field, the development of antimicrobial packagmaterials has received significant attention. Such materialdelay or even prevent microbial proliferation and thus incrthe shelf-life of packaged goods by improving their objecand subjective quality[4]. One very promising way of achieing this goal involves the use of natural antimicrobial agsuch as essential oils (EOs)[5–10], but such species can be uin food packaging only if the compounds they release overand their effects on food are well-known and understood.

Solid phase microextraction (SPME) was first introduce1989[11] and can now be considered a mature extractionnique that has been extensively reviewed[12–14]. A number ofmethods for air analysis using SPME (HS-SPME), in whichexposed extraction fibre is suspended over the sample, hav

003-2670/$ – see front matter © 2005 Elsevier B.V. All rights reserved.oi:10.1016/j.aca.2005.11.056

98 P. Lopez et al. / Analytica Chimica Acta 559 (2006) 97–104

reported[15–19]. It has been demonstrated that the volume ofthe sample (i.e. the vial or package in our application) has a sig-nificant effect on the results, mainly because it affects the relativerates of depletion of individual species in the headspace[20,21].On the other hand, headspace depletion also affects the compo-sition of the atmosphere since the fibre will act as a very efficientsink for compounds with large partition coefficients (K), mean-ing that the headspace will be enriched in compounds with lowK values. Clearly, there will be less extensive depletion if shortersampling times are used. However, overly short sampling timescould also result in inaccurate determination of the headspacecomposition because of the effects of other potentially relevantfactors, such as differences in release kinetics, re-sorption of theanalytes by the packaging or the packed foodstuff, and degrada-tion or cross-reaction over time.

One method that can reportedly solve these problems is diffu-sive SPME sampling[22,23]. Diffusive (passive) samplers, firstintroduced in the 1970s[24], concentrate chemicals via diffu-sion. They have a number of noteworthy characteristics: theydo not require electricity or any other power source, they donot introduce external physical disruption (i.e. turbulence due toforced flow) to the atmosphere to be sampled or have a majorinfluence on headspace composition; and the process can bemathematically understood or modelled in a relatively simpleway [25–28]. Their principal drawback is that their uptake rateis much lower than that of the active samplers, so their sensitivityi

n bec sidet d foa

pri-m fora obiac scof lingo stemt ling.

2

2

.O ork:C ce,C -2n8 xic)s thisw

asss bas( GCa undsT rich

Madrid, Spain); borneol (464-43-7, Aldrich); camphor (76-22-2,Aldrich); camphene (79-92-5, Aldrich); 1,8-cineole (470-82-6, Aldrich); �-caryophyllene (87-44-5, Sigma, Madrid, Spain);p-cymene (99-87-6, Aldrich); eugenol (97-53-0, Aldrich); thy-mol (89-83-8, Aldrich); estragol (140-67-0, Aldrich);trans-cinnamaldehyde (104-55-2, Aldrich);�-phellandrene (4221-98-1, Fluka, Barcelona, Spain);�-humulene (116-04-1, Sigma);limonene (5989-27-5, Aldrich); linalool (78-70-6, Aldrich);�-pinene (80-56-8, Aldrich);�-pinene (18172-67-5, Aldrich);�-terpinene (99-86-5, Fluka);�-terpinene (99-85-4, Fluka);�-terpinolene (586-62-9, Fluka) and�-terpineol (98-55-5, Fluka).

The SPME fibre holder and different types of SPME fibres(85�m polyacrylate, PA; 100�m carbowax/divinylbenzene,CW/DVB; and 100�m polydimethylsiloxane, PDMS) wereobtained from Supelco (Bellefonte, PA, USA). Fibres were con-ditioned prior to their first use according to the manufacturer’srecommendations.

2.2. Instrumental analysis

Analyses were conducted using a Hewlett Packard gas chro-matograph (GC, HP 5890 series II) in conjunction with a massspectrometer (MS, HP 5971) (Agilent Technologies, Palo Alto,CA, USA). The split/splitless injection port was operated at dif-ferent desorption temperatures (see Section2) with the purgev lockf atilec

HP-6 lox-a nt ini-tf beens wasd

•

•

•

•

•

2

-t ast for atl wasp d thefi

s lower.When using SPME, diffusive sampling determinations ca

arried out by simply retracting the fibre a known distance inhe needle, then placing it in the environment to be samplen appropriate period of time.

The work described herein was conducted with twoary aims. The first was to develop a SPME procedurenalysing EOs’ headspace, focusing on potential antimicrompounds, i.e. terpenes. The second was to assess theor using the developed method for diffusive SPME sampf interesting terpenes, evaluating all the features of the sy

hat could influence the accuracy and reliability of the samp

. Experimental

.1. Chemicals and materials

Essential oils were supplied by Artibal (Sabinanigo, Spain)ils from the following plant species were tested in this winnamon zeylanicum (cinnamon, Chemical Abstract ServiAS, number: 805-91-6),Sygyzium aromaticum (clove, 801-985), Ocimum basilicum (basil, 8015-73-4),Rosmarinus offici-alis (rosemary, 8000-25-7), andMelissa officinalis (citronella,000-29-1). Citronella EO has been classified as harmful (too it cannot be used in food packaging; it was included inork for analytical purposes only.EOs’ constituents were identified by comparing their m

pectra with those reported in the GC/MS computer dataWiley 275). These identifications were confirmed bynalysis, comparing the analytes to reference compohe compounds used were: bornyl acetate (76-49-3, Ald

r

lpe

,

e

.,

alve closed for 120 s. The fibre was kept in the injection bor an additional 8 min to ensure total removal of less volontaminants.

Chromatographic separations were performed using an24 column (94% polydimethylsiloxane:6% cyanopropylsine), 25 m× 0.32 mm i.d., 1.8�m film thickness, and the ove

emperature program for fibre screening was as follows:ial temperature 35◦C, ramped at 3◦C min−1 to 210◦C, heldor 15 min. Once the most appropriate SPME fibre hadelected for each essential oil, an optimised oven programeveloped for each EO:

Basil: 40◦C for 2 min, 10◦C min−1 to 80◦C, 3◦C min−1 to140◦C, 10◦C min−1 to 210◦C, held 15 min.Rosemary: 40◦C for 2 min, 10◦C min−1 to 112◦C,3◦C min−1 to 127◦C, 10◦C min−1 to 210◦C, held 2 min.Clove: 40◦C for 2 min, 10◦C min−1 to 160◦C, 3◦C min−1 to172◦C, 10◦C min−1 to 210◦C, held for 2 min.Citronella: 50◦C for 2 min, 13◦C min−1 to 116◦C,6◦C min−1 to 210◦C, held for 2 min.Cinnamon: 70◦C for 1 min, 10◦C min−1 to 80◦C, 3◦C min−1

to 176◦C, 40◦C min−1 to 210◦C, held for 2 min.

.3. SPME sampling procedure

In the headspace screening experiment, 5.0�L of each essenial oil was injected into a 20 mL SPME vial. The vial when crimped and incubated at the appropriate temperatureeast 60 min before analysis, after which the SPME needleunched through the cap into the headspace of the vial anbre was pushed out to commence sampling.

P. Lopez et al. / Analytica Chimica Acta 559 (2006) 97–104 99

Samples were calibrated using a synthetic standard headspaceof known concentration. A known amount (gravimetrically con-trolled, approximately 0.01 g) of a diethyl ether solution wasplaced in a 20 mL vial capped with a PTFE-faced septum. Thevial was then incubated at 80◦C for 15 min to ensure com-plete evaporation of the solvent, and the fibre, retracted to theappropriate length, was inserted into the vial. At the end of thesampling period, the fibre was retracted and removed from thesampling vial. The needle was then immediately inserted intothe GC injection port for GC/MS analysis.

Quality control was performed using a Shewhart chart[29]for the whole experimental period (estimated at ca. 8 months).The concentration of two different standards (eugenol and cam-phor) in seven samples was determined weekly. The followingequations were then applied:

WL = µ ± 2σ√n

(1)

AL = µ ± 3σ√n

(2)

where WL stands for warning lines (sample means above orbelow this range indicate that the fibre has deteriorated and thatprecautionary action should be taken), AL means action lines(sample means above or below this range indicate that the fibrehas undergone total deterioration and must be replaced),µ rep-r n),t ent cont eent dif-f se ot

3

3

suit-a erenfii m-

ber of potentially interesting compounds (i.e. those described inthe literature as potential antimicrobial agents) detected usingthe fibre, and the relative strength of the signal detected witheach fibre. PDMS gave a more complete headspace profile forbasil, rosemary, and cinnamon, whereas PA and PDMS gavecomparable profiles for clove and citronella. Using the secondcriterion (relative signal strength) the PA fibre was selected forclove analysis and PDMS for citronella.

SPME optimisation was then performed, using a combi-nation of a Plackett–Burman screening design (comprisingeight experiments plus three replicates of the central point,all performed in duplicate[31,32]) to determine the relevantfactors, and response surface modelling (CCF,n = 11) withmultiple linear regression (MLR) data analysis.Table 1showsthe factors considered, the ranges within which they werevaried and the optimum values found. Different optimumconditions were identified for each sample matrix.Fig. 1(a)–(e)shows a comparison of typical chromatograms obtained fromeach of the essential oils (EOs) andTable 2lists the analytesidentified.

The fictitious variable is a non-existent factor, and has noeffect on experimental conditions. It was included to detectmodel malfunction or anomalous bias. It was not relevant inany case studied, indicating that the selected variables describethe data adequately. Five factors were found to be relevant in theP–B design (the relevance criterion being that the variables’ errorb n cri-t rest).A orre-l notf amer volumer onlyr latedp icals hoseu theu allerv . Thes walls nds tot was

TH and

F RSM

on

S YS YD NCb

D NCb

H Nb

H b

F NCb

R

foun

esents the target value (standard headspace concentratioσ ishe standard deviation andn is the number of replicates. Givhe selected coefficients, if the process is being adequatelyrolled, approximately 95% of the sample means will lie betwhe warning lines, and 99.7% within the action lines. Threeerent PDMS fibres and one PA fibre were used in the courhis work.

. Results and discussion

.1. Headspace screening experiments

The first stage of the experiments involved selectingble fibres. On the basis of previous experiments, three diffbres (100�m PDMS, 85�m PA, and 100�m CW/DVB) were

nvestigated[30]. Two selection criteria were applied: the nu

able 1S-SPME experimental designs used for optimisation: Plackett–Burman

actor Variation range P&Ba

orption time (min) 10–30 Y(±)orption temperature (◦C) 25–80 Y(±)esorption time (min) 0.1–3.0 NRb

esorption temperature (◦C) Tmin + 10 toTmax− 10 Y(+)eadspace volume (mL) 2–20 Y(+)eadspace to sample ratio (AU) 20–20000 Y(+)ictitious variable (AU) NAb NRb

2 (RSM)

a Y: relevant factor; (+): positive effect, without a maximum; (±): maximumb NA: not applicable; NR: not relevant; NC: not considered.

-

f

t

ars cross the coefficient horizontal axis and the optimisatioerion being the average signal of the compounds of intelthough the desorption temperature of the compounds c

ated positively with the signal strength, this variable wasurther optimised since it cannot be altered at will. For the season, the headspace volume and headspace to sampleatio were not optimised. The former of these variables waselevant for basil, clove, and rosemary, and always correositively with signal strength. Optimisation was impractince the smallest commercially available vial larger than tsed held a volume of 50 mL, and so would have requiredse of too much essential oil. It was found that the use of smials significantly reduced the strength of the analyte signalmaller the vial, the smaller the ratio of the headspace to theurface area. As this ratio increases, adsorption of compouhe vial wall becomes more significant. Another factor that

response surface modelling

a EOs

Clove Basil Rosemary Citronella Cinnam

30 30 30 30 3060 30 50 25 552.0 2.0 2.0 2.0 2.0300 270 270 270 270

C 20 20 20 20 20NC 20000 20000 20000 20000 20000

– – – – –0.940 0.950 0.975 0.970 0.900

d within the experimental range. For detailed discussion, see text.

100 P. Lopez et al. / Analytica Chimica Acta 559 (2006) 97–104

mentioned in Section1, the absolute volume of the headspace,also has a significant effect on signal strength, although its effectis substantially weaker for compounds with comparatively lowpartition coefficients.

Optimal results were obtained at high headspace to samplevolume ratios. The lower limits of this variable were dictatedby practical considerations. Given the vial volume of 20 mL,

a ratio of 20,000 would have required an EO volume of 1�L.Due to the viscosity of some EOs, handling volumes smallerthan this is difficult, so 1�L was selected as the optimumvalue.

Thus, the only factors used in the RSM design were sorptiontime and temperature. For the sake of clarity, the following dis-cussion will be framed with reference to clove EO, which gave

F11�

2151

ig. 1. Typical chromatograms obtained, and peak identities, for the essentia, �-pinene; 2, camphene; 3,�-pinene; 4, myrcene; 5, limonene; 6, 1,8-cineole;3, estragol; 14, bergamotene. (c) Rosemary: 1,�-pinene; 2, camphene; 3,�-pinene-terpinene; 10, terpinolene; 11, linalool; 12, camphor; 13, borneol; 14,�-terpineol; 1, myrcene; 3, limonene; 4, sabinene; 5,�-terpinene; 6, linalool; 7, citronellal; 8, is4,�-copaene; 15, geranyl acetate; 16, eugenol; 17, caryophyllene; 18, humule, �-phellandrene; 6,�-terpinene; 7, limonene; 8, cymene; 9, 1,8-cineole; 10,�-terp6, cinnamaldehyde; 17,�-copaene; 18, eugenol; 19, caryophyllene; 20, humul

l oils (EOs) studied. (a) Clove: 1, eugenol; 2, caryophyllene; 3, humelene. (b) Basil:7, cymene; 8,�-terpinolene; 9, fenchone; 10, linalool; 11, camphor; 12, menthol;; 4,�-phellandrene; 5,�-terpinene; 6, limonene; 7, cymene; 8, 1,8-cineole; 9,5, bornyl acetate; 16, caryophyllene; 17, humulene. (d) Citronella: 1,�-pinene;opulegol; 9, citronellol; 10, geraniol; 11, citral; 12,�-terpinene; 13,�-cubebene;ne; 19,�-cadinene. (e) Cinnamon: 1,�-thujene; 2,�-pinene; 3, camphene; 4,�-pinene;inene; 11, terpinolene; 12, linalool; 13, camphor; 14, borneol; 15,�-terpineol;ene; 21,�-cadinene; 22, calamene; 23, eugenol acetate.

P. Lopez et al. / Analytica Chimica Acta 559 (2006) 97–104 101

Fig. 1 (Continued ).

Table 2Characterization of the headspace generated by the different EOs: peaks identified

Clove Basil Rosemary Citronella Cinnamon

# Analyte # Analyte # Analyte # Analyte # Analyte

1 Eugenol l �-Pinene l �-Pinene l �-Pinene l �-Thujene2 Caryophyllene 2 Camphene 2 Camphene 2 Myrcene 2 �-Pinene3 Humulene 3 �-Pinene 3 �-Pinene 3 Limonene 3 Camphene

4 Myrcene 4 �-Phellandrene 4 Sabinene 4 �-Pinene5 Limonene 5 �-Terpinene 5 �-Terpinene 5 �-Phellandrene6 1,8-Cineole 6 Limonene 6 Linalool 6 �-Terpinene7 Cymene 7 Cymene 7 Citronellal 7 Limonene8 �-Terpinolene 8 1,8-Cineole 8 Isopulegol 8 Cymene9 Fenchone 9 �-Terpinene 9 Citronellol 9 1,8-Cineole10 Linalool 10 Terpinolene 10 Geraniol 10 �-Terpinene11 Camphor 11 Linalool 11 Citral 11 Terpinolene12 Menthol 12 Camphor 12 �-Terpinene 12 Linalool13 Estragol 13 Borneol 13 �-Cubebene 13 Camphor14 Bergamotene 14 �-Terpineol 14 �-Copaene 14 Borneol

15 Bornyl acetate 15 Geranyl acetate 15 �-Terpineol16 Caryophyllene 16 Eugenol 16 Cinnamaldehyde17 Humulene 17 Caryophyllene 17 �-Copaene

18 Humulene 18 Eugenol19 �-Cadinene 19 Caryophyllene

20 Humulene21 �-Cadinene22 Calamene23 Eugenol acetate

the simplest chromatogram (Fig. 1a). Sorption temperature (Tshereafter) had a positive effect on the extraction of compounds,but the negative coefficient of the quadratic term implies theexistence of a maximum in the range investigated. The influ-ence of sorption time (tso) was also positive in all cases, but inthis case, the quadratic coefficient was also positive, so there wasno maximum in the experimental range. The effects of increas-ing the extraction time (to 40 min) were also investigated, but the

increase in signal strength thus obtained (<5%, data not shown)did not compensate for the longer extraction time. The signifi-cance of the interaction term was extremely low, indicating thatthe variables do not interact to any appreciable degree.Table 1also shows the optimum values obtained for each EO tested. Asan indication of the validity of the method, the averageR2 value(indicating the descriptive power of the model) for each EO isalso included in the table.

102 P. Lopez et al. / Analytica Chimica Acta 559 (2006) 97–104

3.2. SPME diffusive sampling

The theory of diffusive sampling has been fully describedelsewhere[18,24] and will not be discussed in detail here.Briefly, however, the kinetics of the procedure are governed byFick’s law of diffusion

K = M

CHt(3)

whereK is the uptake rate, which can be regarded as an indicationof loading “speed” for different analytes, expressed as a the ratioof the actual mass of analyte loaded in the sampler (i.e. the fibre,M) to the product of the sampling time (t) and the concentrationof the analyte in the headspace to be determined (CH). From thisequation, it follows that onceK is known for a specific chemicalin a sampling device, it can be directly used to quantify unknownvapour-phase concentrations (CH).

The most interesting point about diffusive sampling is thatthe uptake rate for a given sampler geometry is dependent onlyon the diffusion of the analyte

K = DgA

L(4)

As can be seen, the uptake rate is proportional to the dif-fusion coefficient of the analyte in the gas phase (D , which isd reat Efi lings plyr

Thus, the influence of this variable was investigated by vary-ing the path length between 0.5 and 2.5 cm, with the SPME fibreplaced in a 20-mL vial containing 1�L of each EO. As expected,shorter diffusive path lengths resulted in greater amounts of theanalyte reaching the sorbent and thus in stronger signals. How-ever, the use of shorter path lengths also resulted in increases inthe standard deviation, presumably as a result of non-diffusiveeffects. Consequently, a path length of 1 cm was adopted forfurther experiments.

The uptake rate,K, was then calculated. Retracted SPMEfibres were exposed to known concentrations (n = 10) of the ana-lytes in the vapour phase.Table 3shows the results obtained.The first conclusion from the data is that the process is subjectto air-side control rather than sampler control[28], i.e. the ratelimiting step is the diffusion of the analytes to the air/samplerboundary. This means that the uptake rate for a specific chemicalis directly related not to its partition coefficient (K) but insteadto its diffusion coefficient (Dg). The data inTable 3supportthis conclusion. According to Ref.[34] the estimated distribu-tion coefficients between air and a PDMS fibre for�-pinene andlinalool are 4571 and 38,020, respectively. However, despite thisdifference, the observed uptake rates for the two compoundswere calculated to be 2.9 and 2.6 pg (ppbv min)−1, respectively,supporting the air-limitation hypothesis.

We would like to emphasise that this finding does not neglectthe influence of the partition coefficient in the sampling process.R ling,p ncen-t no hasei vant

TC entra

A Con

1

T 2.9� 2.2L 2.6B 2.C 3.� 2.� 2.C 3� 2� 3.L 2.1 2.� 2.T 2.C 2.B 2.7E 2.C 3.H 2.C 3E 2.

gependent only on the sampling temperature), the surface a

he opening of the sampler (A, 0.00086 cm2 for standard SPMbres[33]), and the distance from the opening to the sampurface (L). In SPME, this last factor can be varied by simetracting the fibre further into its housing needle.

able 3alculated uptake rates, the effects of extending the sampling time, conc

nalyte Uptake rate (pg (ppbv min)−1)

30 min 150 min 300 min

hymol 2.9 2.8 2.9-Terpineol 2.2 2.1 2.1inalool 2.6 2.6 2.7omyl acetate 2.2 2.1 2.1ymene 3.3 3.2 3.3-Pinene 2.9 2.9 2.9-Pinene 2.4 2.4 2.5amphene 3.0 3.1 3.1-Phellandrene 2.9 2.9 2.9-Terpinene 3.2 3.1 3.2imonene 2.7 2.8 2.8,8-Cineole 2.6 2.7 2.6-Terpinene 2.9 2.9 2.9erpinolene 2.8 2.8 2.8amphor 2.6 2.5 2.6omeol 2.7 2.7 2.7ugenol 2.6 2.7 2.8aryophyllene 3.3 3.3 3.2umulene 2.5 2.5 2.6innamaldehyde 3.1 3.0 3.0stragol 2.9 2.9 3.0

a Calculated from Eq.(4).

b From Ref.[34].

ofeverse diffusion can be a major factor in diffusive samparticularly when sharp changes occur in the sample co

ration during the sampling period[35]. Reverse diffusion caccur if the binding of the target compound to the SPME p

s not sufficiently strong, and it has been found to be rele

tion effects, and ratios of observed:theoretical diffusion coefficients (n = 4)

centration effect Diffusion coefficient (10−6) (m2 s−1)

100 10000 Dea Dt

b Ratio

2.9 3.1 5.6 5.4 0.962.3 2.4 4.5 5.3 0.852.7 2.8 5.0 5.2 0.96

2 2.2 2.4 4.3 4.8 0.903 3.3 3.2 6.1 5.8 1.059 3.0 3.0 5.6 5.8 0.974 2.5 2.4 5.2 5.8 0.89.0 3.0 3.2 5.9.9 2.9 3.1 5.72 3.3 3.5 6.17 2.7 2.9 6.06 2.7 2.7 5.29 3.0 3.1 5.88 2.7 2.8 5.76 2.6 2.8 5.1

2.7 2.9 5.46 2.6 2.8 5.13 3.3 3.3 4.65 2.6 2.6 4.8.1 3.2 3.4 6.19 3.0 3.2 5.6

P. Lopez et al. / Analytica Chimica Acta 559 (2006) 97–104 103

Table 4Figures of merit

Analyte HS-SPME Diffusive-SPME

Robustness Repetitivity Intermediate precision Robustness Repetitivity Intermediate precision

Thymol Y 4 5 Y 4 4�-Terpineol Y 4 5 Y 4 4Linalool Y 5 7 Y 5 5Bornyl acetate Y 6 6 Y 6 5Cymene Y 3 6 Y 4 4�-Pinene Y 7 6 Y 6 6�-Pinene Y 6 4 Y 6 5Camphene N 6 8 Y 6 5�-Phellandrene Y 5 9 Y 5 4�-Terpinene Y 4 11 Y 4 4Limonene Y 6 8 Y 4 51,8-Cineole Y 7 7 Y 6 6�-Terpinene Y 9 10 Y 6 5Terpinolene Y 6 8 Y 5 5Camphor N 10 12 Y 6 5Bomeol Y 8 10 Y 5 4Eugenol Y 6 6 Y 5 6Caryophyllene Y 7 9 Y 5 5Humulene Y 3 5 Y 4 3Cinnamaldehyde N 5 10 Y 5 5Estragol Y 6 8 Y 6 7

Average 5.9 7.6 5.1 4.9

Comparison of SPME approaches: full exposure vs. diffusive sampling (n = 6).

for both PDMS and PA fibres that are exposed intermittently toa sample and pure air[36]. Since such sharp changes in analyteconcentration are unlikely to occur in active packaging, reversediffusion is unlikely to have a significant effect. This was veri-fied by using the observed experimental uptake rates to calculateexperimental diffusion coefficients (De), which were then com-pared to available theoretical values[37]. These comparisons(expressed as the ratios of the two values) are also shown inTable 3. As can be seen, the ratios varied between 0.85 and 1.05,supporting the air-limited hypothesis.

A final test of the method was performed by exposing the fibreto the standard headspace for three different durations, from 0.5to 5 h (with five replicates of each duration) and calculatingthe uptake rate for each sampling period.Table 3presents theresults obtained. No statistically significant deviations (p = 0.05)were obtained, supporting the conclusion that short samplingtimes can be used to calculate the uptake rate, making systemoptimisation less tedious and time-consuming.

A very interesting potential feature of diffusive sampling isthat since the method relies purely on diffusion (which onlydepends on sampling temperature), the composition and con-centration of the headspace should not affect the uptake rate(which can be regarded as a measure of the sensitivity of themethod). This was checked by calculating the uptake rates forthe test analytes at three concentration levels (relative concentra-tions of 1, 100 and 10,000). The results of this study are showni t andl hod-o vent

Table 4details the analytical performance of both SPMEapproaches, including their robustness, intermediate precision,and reproducibility[29,38]. The values obtained are compara-ble to and somewhat better than the few examples previouslyreported in the literature for terpene analysis[39,40], highlight-ing the potential of this methodology as a reference method forheadspace calculations.

As might be expected, diffusive sampling gave better resultsfor intermediate precision, due to the inherent characteristics ofthe method. Accordingly, the technique was found to be robustfor all compounds under study, while HS-SPME performedpoorly for determining camphor, camphene, and cinnamalde-hyde. The major limitation of diffusive approaches is relatedto their comparatively limited sensitivity in comparison to fullyexposed HS-SPME, which is estimated to be 10–100 times moresensitive, depending on the compound (data not shown).

4. Conclusions

The combination of complete-exposure SPME for qualitativecharacterization of an unknown atmosphere together with dif-fusive SPME for quantitative determination has great utility inheadspace analysis. The method is simple, robust, easy to opti-mise and has been proven to give good analytical performance.Uptake rates can be determined with short sampling times, thusminimising the time needed for method development.

ivenc con-c s than1 tion.

n Table 3. The variation in uptake rates between the highesowest concentrations was only 10%, implying that this metlogy offers affordable determination of major and minor (e

race) components.

Another interesting finding is that the uptake rate for a gompound is not influenced by the overall composition orentration of the headspace, with observed variations of les0% over four orders of magnitude changes in concentra

104 P. Lopez et al. / Analytica Chimica Acta 559 (2006) 97–104

This is highly useful when major and minor (even trace) com-ponents are of interest and need to be determined in a singleshot.

In terms of its content of potentially useful antimicrobialagents (terpenes), cinnamon essential oil was found to have themost interesting headspace. This makes cinnamon EO the mostpromising alternative for use in active packaging where no directcontact with the packaged foodstuff is needed.

The principal limitation of the technique described hereinarises from the limited number of stationary phases availablefor SPME. In the work presented here, this did not constitute aserious problem, since the analytes of interest were very similarin nature, but experiments designed to extend the applicabilityof the technique to other interesting compounds with differentpolarities failed. This limitation may potentially be overcomethrough the use of binary or even ternary phases.

Acknowledgements

This work was financed by the Spanish Project CAL 03-080-INIA, which is part of the Plan Nacional de Alimentation, Spain.R. Batlle gratefully acknowledges the former Spanish Ministryof Science and Technology for personal funding through theRamon y Cajal program. P. Lopez gratefully acknowledges theRegional Government of Aragon and the European Social Fundf

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