Sampling and sample preparation for analysis of aromas and fragrances

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Trends in Analytical Chemistry, Vol. 22, No. 3, 2003
Sampling and sample preparation
for analysis of aromas and
fragrances
Fabio Augusto, Alexandre Leite e Lopes, Cla´udia Alcaraz Zini
Some recent advances in sampling and sample-preparation technologies
for fragrance analysis are addressed in this review. Procedures, such as
analytical distillation (vapor distillation and simultaneous distillationextraction), headspace-manipulation methods (static and dynamic headspace analysis and headspace solid-phase microextraction) and direct
extraction methods (such as liquid-liquid, solid-phase and supercritical
fluid), will be discussed and critically evaluated. Contemporary applications of these techniques to the study of natural and synthetic aromas will
be presented.
# 2003 Published by Elsevier Science B.V.
Keywords: Aroma and fragrance analysis; Sample-preparation techniques
Fabio Augusto*,
Alexandre Leite e Lopes
Institute of Chemistry – State
University of Campinas
(Unicamp), C.P. 6154, 13083907 Campinas, Sa˜o Paulo,
Brazil
Cla´udia Alcaraz Zini
Institute of Chemistry –
Universidade Federal do Rio
Grande do Sul (UFRGS),
90040-060 Porto Alegre, Rio
Grande do Sul - Brazil
*Corresponding author. Tel.:
+55-19-3788-3057; Fax: +5519-3788-3023 (faculty);
E-mail: augusto@iqm.unicamp.
br
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1. Introduction
The term ‘‘odor’’ refers to biological, physical and psychological e¡ects caused by
the interaction between chemical stimulants ^ aromas and fragrances ^ and
olfactive systems of living creatures [1].
The study of the composition of fragrances and aromas (F&A) is relevant in
several ¢elds, as follows.
In food science, chemicals or their
blends associated with odors discerned as desirable or agreeable are
related to a multivariate set of sensorial responses know as ‘‘£avor’’
[2]. Flavors are the predominant
elements in the perception of food
and beverage quality [3], and,
therefore, its acceptance by consumers. Also, ‘‘o¡-£avors’’ ^ connected to volatile substances with
unpleasant odors ^ may be caused
by microbial contamination of foodstu¡s [4]. Investigation of these
substances can be important in
food-safety studies.
Odor can be a signi¢cant environmental parameter in some situations, since it can be related to the
human perception of comfort and to
the sanitary conditions of an indoor
atmosphere [5], to the contamination of air resulting from agricultural
[6] and industrial [7] activities, as
well as to the quality of natural and
treated water [8].
Chemical characterization of fragrances released by vegetables and
animals is important to several
branches of industry and science.
Natural essential oils from plants are
traditional base ingredients for perfumes and related toiletry products.
However, the rarity of some plants
and the possible dermatotoxic e¡ects
caused by some natural products
create demand for synthetic alternative mixtures. The design of these
‘‘synthetic’’ essential ingredients
depends fundamentally on reliable
analytical data on the composition of
their natural counterparts [9].
Moreover, knowledge about the
composition and emission dynamics
of £oral scents and similar biogenic
mixtures of volatile organic compounds is fundamental in several
biological studies because of their
many di¡erent roles in plant reproductive processes, defense against
predators
and
intra-species
communication [10].
The development and application of
methodologies for the determination of
the chemical composition of aromas and
similar mixtures is a challenging task.
0165-9936/03/$ - see front matter # 2003 Published by Elsevier Science B.V. doi:10.1016/S0165-9936(03)00304-2
Trends in Analytical Chemistry, Vol. 22, No. 3, 2003
This is a consequence of some inherent general properties of such samples:
(a) The concentration of relevant analytes in fragrance samples can be extremely low. For example, the
literature values for low-odor threshold concentrations
(which can be de¢ned the minimum concentration for
an odor-producing substance that has a 50% probability to be perceived as a faint odor by a human subject [1]) for selected substances important in food,
biological and environmental aromas, such as ethyl
4-methyl-4-pentenoate, 2-iodophenol, butyric acid
and 2-ethyl-3,5-dimethylpyrazine, are respectively
0.06 nL/L, 1 mg/L, 240 nL/L, 0.007 nL/L [11^13].
The typical perceptible concentration for some odorants can, as shown, be less than 1 ng/L of the substance. As a result, the corresponding analytical
procedures must provide extremely high sensitivities,
adequate for detection and quanti¢cation of these species at such low levels.
(b) Apart from the typically low concentration of the
odor-active analytes, most aromas are extremely complex blends of substances. Kotserides and Baumes [14]
reported 48 di¡erent organic volatile substances as
odorants with impact in Bordeaux wines, Cabernet
Sauvignon and Merlot. The same complexity can be
found in £oral scents; Bayrak and Akgu«l [15] identi¢ed
more than 60 volatile organic compounds in samples of
essential oils from Turkish rose (Rosa damascena,
Miller).
(c) For aromas generated by biological sources, such
as plants and animals, further analytical di⁄culties
arise from the dynamic nature of such systems. For
example, the composition of samples obtained from
detached parts of plants may not correspond to the mixture released by undisturbed live organisms [16,17];
this creates a demand for methods allowing in-vivo sampling. Also, the fact that production and emission of
plant volatiles can be a¡ected or triggered by factors,
such as light, environmental temperature, stress and
the presence of trace atmospheric pollutants [18,19],
can introduce further complications.
(d) Some odorants have limited chemical stability, as
a result of photolysis, oxidation and other reactions;
e.g., the atmospheric chemical lifetime of monoterpenes
under daylight conditions was estimated to range from
less than 5 min (a-terpinene) to 3 hours (a- and
b-pinene, sabinene) [18].
As a result, chemical characterization of F&A ordinarily demands state-of-art techniques for sampling and
sample preparation, analyte separation, detection and
quantitation. Usually, the application of an analytical
separation technique in the ¢nal steps [2] is involved.
Gas chromatography coupled to mass spectrometry
(GC-MS) and other similar detection schemes, such as
infrared absorption spectrometry [20], are the techniques normally employed for F&A chemical analyses.
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The coupling of olfactometric detection to gas chromatography (GC-O) is also extremely relevant for qualitative and quantitative chemical analysis of fragrances
[21]; several instrumental and methodological variants
of GC-O are described in the literature, such as CHARMAnalysis and aroma extraction dilution analysis
(AEDA) [22].
Along with the chromatographic techniques, the use
of ‘‘electronic noses’’ (arrays of electrochemical sensors
that generate an electric signal that emulates the
expected response from the human olfactory system)
has been growing in recent years. The development of
these fascinating devices involves the arrival of suitable
new materials, such as piezoelectric crystals and synthetic conductive polymers, as well as sophisticated
data-processing and chemometric techniques [23].
Their more remarkable features, from the analytical
standpoint, are the possibility of fast, direct, qualitative
and quantitative evaluation of F&A with limited or no
preliminary sample-preparation procedures ^ although
the low sensitivities provided by the devices presently
available still prevent their use in solving most F&A
analytical problems.
Apart from applications involving measurement with
the above-mentioned electronic noses and similar techniques, adequate isolation and pre-concentration of the
odor-active analytes are mandatory and critical steps in
the methodologies for chemical characterization of F&A
[2]. Several sorbent-extraction approaches ^ classical
liquid-liquid extraction (LLE), solid-phase extraction
(SPE), solid-phase microextraction (SPME) and supercritical £uid extraction (SFE) ^ have been employed for
sample preparation in F&A analyses, as well as
dynamic headspace (DHS) and static headspace (SHS)
methods and procedures based on analytical distillation. In the following sections, the application of some
of these techniques to procedures for chemical characterization of £avors and F&A will be addressed.
2. Analytical distillation procedures
Distillation is usually carried out in two slightly di¡erent ways, as follows.
In the ¢rst, the matrix to be extracted is mixed or
suspended with water in a suitable vessel ¢tted
with a condenser and, while the mixture is
boiled, a condensate phase is collected (hydrodistillation). After the process, an organic,
water-insoluble fraction (for vegetable materials, the essential oil) can be separated from the
water.
In the second procedure, steam is passed through
a vessel containing the matrix-water mixture
(steam distillation) to yield a similar condensate.
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2.1. Vapor distillation
Steam-assisted distillation and hydrodistillation are
traditional procedures for isolation of volatile aromarelated compounds from odoriferous samples, such as
food and detached parts of plants. Being simple and
straightforward procedures, they are still extensively
applied for F&A characterization either alone or combined with other sample-preparation procedures. For
example, Saritas et al. [24] isolated the essential oils
from aromatic lichens of various genera (Mnium, Plagiomnium and others) by hydrodistillation of fresh and
dried plant parts. Using GC-MS combined with
13
C-NMR and other techniques, they were able to identify several volatile terpenoid and aliphatic compounds
in these hydrodistillates, including two unreported
volatile sesquiterpenes ((+)-10-epi-muurola-4,11diene and 10,11-dihydro-a-cuparenone).
2.2. Simultaneous distillation-extraction (SDE)
A widespread distillation-based sample-preparation
method for chemical analysis of F&A is simultaneous
distillation-extraction (SDE), also known as the Lickens-Nickerson method. Fig. 1 shows one of the proposed designs for the Lickens-Nickerson SDE apparatus.
An amount of the fragrance-generating sample (food,
beverage, sliced plant tissue, etc.), along with distilled
water for dry samples, is contained in £ask 2, and £ask
3 receives a suitable volume of an extracting solvent
denser than water (dichloromethane, chloroform, etc.).
Flasks 2 and 3 are heated; the water and solvent vapors
are conducted to the extractor body, where they are
allowed to condense over the surface of the cold tube 4.
Figure 1. Likens-Nickerson simultaneous distillation-extraction apparatus (modified from Perpe`te et al. [26]): 1 – body; 2=sample flask;
3=extracting solvent flask; 4=cold tube; 5=inlet for purge gas;
6=cold water inlet and outlet.
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In this operation, aroma analytes are removed from the
matrix by the water vapor and transferred to the
organic phase when the liquids condense together on
the cold tube. Both water and solvent are collected in
the extractor body after their condensation and return
to the corresponding £asks, allowing continuous
re£ux. A modi¢ed apparatus allows use of solvents
lighter than water, such as pentane or ethyl acetate, as
extractors. Among others, this device was employed by
Schlotzhauer et al. [25] to study insect attractors in the
fragrance of £owers from Japanese honeysuckle (Lonicera japonica). Large amounts of linalool and a-farnesene were found in samples collected during daylight
periods. Perpe'te et al. [26] combined LLE and SDE to
¢nd the key compound responsible for the unpleasant
o¡-£avor in alcohol-free beers (1-methylpropionaldehyde), which was not previously found in reports using
other sample-preparation techniques, such as DHS
analysis.
2.3. Other distillation methods
Atmospheric-pressure distillation-based sample-preparation methods may have some serious disadvantages when applied to F&A characterization,
since the temperatures usually necessary for the operation can lead to degradation of some analytes. For
example, after studying the aroma from sa¡ron (Crocus
sativus L.), Tarantilis and Polissou [27] found that the
most important odorant in these samples, safranal, is
oxidized during conventional steam distillation, forming artifacts, such as 2,6,6-trimethyl-1,3-cyclohexadien-1-carboxylic acid. Other compounds found in
these distillates were also determined as artifacts resulting from the thermal degradation and oxidation of carotenoids from the sa¡ron matrix. The same problems
can be observed in SDE: Siegmund et al. [28] discovered
that 5,6-dihydro-2,4,6-trimethyl-4H-1,3,5-dithiazine,
usually pointed to as an important aroma-active compound in cooked and cured meat aromas, is an artifact
formed during the distillations performed according the
Lickens-Nickerson SDE method.
Vacuum distillation, which can be performed under
milder conditions, can overcome these problems and is
an alternative to regular distillation methods. Moio et
al. [29] combined vacuum distillation and LLE to identify the key odorants from Gorgonzola cheese. After
GC-MS and GC-O analysis of the distillates/extracts,
2-nonanone, 1-octen-3-ol, 2-heptanol, ethyl hexanoate, methylanisole and 2-heptanone were determined to be the most relevant odorants in the aromas of
natural and creamy Gorgonzola cheeses.
Another interesting application of low-pressure distillation was described by Bouchilloux et al. [30]. Using highvacuum distillation (temperatures between 35 C to 45 C
and pressures between 0.5 Pa and 1.5 Pa) and simultaneous derivatization with p-hydroxymercuribenzoic
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acid, the authors were able to determine trace levels of
some powerful odorous thiols in red wines of Bordeaux.
The analytes were identi¢ed and quanti¢ed in the samples as their volatile organomercury derivatives, in
concentrations ranging from 0.25 mg/L to 10 mg/L
(3-mercapto-2-methylpropanol), 10 ng/L to 5 mg/L
(mercaptohexanol) and 1 ng/L to 200 ng/L (3-mercaptohexyl acetate).
3. Headspace methods
Procedures based on the manipulation of the headspace
(HS) in contact with odorous materials are popular and
very suitable for chemical analysis of F&A [31]. Di¡erent approaches have been employed, either using direct
HS analysis or by collection of the odorants in the HS
using sorbent devices or cold traps. These techniques
and their applications for the chemical characterization
of F&A are discussed in the following paragraphs.
3.1. SHS
The simplest way to assess the chemical composition of
an aroma is direct analysis (by GC or another convenient technique) of a portion of the air in contact
with the odor source, without any other sample-treatment step. However, since little or no pre-concentration
of the analytes is involved and, considering the typical
trace or ultratrace levels of the components, it is not
usually feasible to apply SHS to chemical-fragrance
analysis [32]. For example, of 118 references indexed in
an extensive literature review on HS analysis of £oral
fragrances [33], only nine employed SHS as the sampling or sample-preparation procedure. Nonetheless,
when techniques and devices with adequate detection
limits are available, and depending on the concentration of the analytes, SHS can be particularly suitable
because of its inherent simplicity. For example, Clarkson and Cooke [34] employed GC-MS and GC-AED
(atomic emission detection) systems equipped with
high-volume injection ports to analyze aroma compounds in the HS of commercial cigarettes. Injecting 1
mL of the HS air in contact with the samples, the
authors pointed out that, for these samples, both analysis time and detection limits were better than those
obtained using a commercial DHS analyzer.
3.2. DHS
A large number of reports describing the use of DHS
methods can be found in the literature regarding the
chemical characterization of F&A. Because of the constant depletion of the analytes from the sample or from
the adjacent atmosphere, the general approach on DHS
potentially provides improved analytical sensitivity
when compared to SHS and other equilibrium extraction procedures [2]. Desorption of trapped analytes for
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subsequent analysis can be performed either with small
volumes of adequate solvents [35] or using on-line
automated thermal desorption (ATD) devices [36], the
later being eminently suitable for routine procedures.
Experimental set-ups such as that depicted in Fig. 2
[37] can be used, either in the laboratory or under ¢eld
conditions, to collect fragrance compounds emitted by
live plants in dynamic conditions. A vase containing a
live plant or some parts of it can be isolated from the
environment by a glass bell or similar device. A controlled £ow of puri¢ed air is passed through the chamber, carrying the aroma compounds to a tube
containing a sorbent or to a cold tube, where the volatiles are accumulated for further desorption and analysis. Using a similar apparatus with polyvinyl-acetate
bags as the isolating device instead of the glass bell to
emulate the usual procedure employed in ¢eld sampling
of £oral scents, Raguso and Pellmyr [38] performed
an extensive study of the methodological aspects of
sampling and pre-concentration of £oral fragrances by
DHS with solvent desorption and chromatographic
analysis. Live £owers of Clarkia breweri were used as
fragrance source, as well as synthetic mixtures of
typical £ower odorants. It was found that Porapak Q
was more e⁄cient than Tenax TA or Tenax TA/charcoal mixtures as sorbent for the evaluated fragrances.
However, the relative abundance of the detected compounds ^ a useful parameter in biochemical and ecochemical studies ^ did not depend on the composition of
the sorbent. The trapped compounds were desorbed
using either diethyl ether, dichloromethane or hexane;
the last solvent provided better recoveries. In the range
studied (0.5^1.0 L/min), the e¡ect of the stripping air£ow rate on the recovery of the trapped analytes
generally was not signi¢cant when compared to the
Figure 2. Chamber for DHS isolation and collection of volatiles emitted
by living plants (modified from Vercammen [37]).
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e¡ects of other sources of variation on the results
between experiments, such as variation in the composition of the volatile emissions between di¡erent
specimens of the same plant.
Although it can require more complex and expensive
hardware (prices up to US$ 15,000) and cannot be
applied to fragrances containing temperature-sensitive
compounds, DHS with TD (DHS-TD) has several advantages over solvent desorption: simpler procedure;
improved detection limits; and; no interference of solvent peaks in the chromatograms. Ageloupoulos et al.
[39] monitored the temporal emission pattern of the
volatile compounds (unsaturated C6-alyphatic alcohols
and aldehydes) released by single intact and damaged
leaves of potato (Solanum tuberosum) and broad bean
(Vicia faba) by DHS (using a specially designed sampling
chamber) coupled to GC-MS. The volatiles were trapped
in Tenax TA cartridges and desorbed either thermally
(inserting the cartridges inside the programmed-temperature injector of the GC-MS system) or by a solvent
(diethyl ether). It was determined that TD, which provided better detectability and therefore required
reduced sampling times, allowed the time-emission pro¢les to be assessed for the monitored compounds. Vercammen et al. [37] also employed a commercial on-line
ATD apparatus coupled to a GC-MS to compare sorption
tubes packed with Tenax TA and with pure grinded
polydimethylsiloxane (PDMS), as well as SHS SPME, for
DHS collection of fragrance compounds released by live
roses and jasmine. PDMS was found to be more e⁄cient
for the isolation of volatile odorants released by these
plants, resulting also in cleaner chromatograms.
Analytical methodologies involving DHS-TD for
characterization of the aroma of foods and beverages
are also common. To study the release of volatile £avor
compounds by leaves of Japanese pepper (Xanthoxylum
piperitum), a spice employed in traditional oriental
cuisine, Jiang and Kubota [40] collected the volatiles in
the HS of crushed, mechanically disturbed or intact
leaves in Tenax TA cartridges. The analytes were thermally desorbed in the injector port of a GC-MS, where
they were cryofocused, separated, detected and quanti¢ed (Fig. 3). It was determined that the number of
detected volatile compounds increased from 12 (from
intact leaves) to 22 (mechanically disturbed leaves) and
then to 36 (crushed leaves). For the disturbed leaves, it
was found that the main compounds responsible for the
characteristic aroma were limonene (0.56 mg/g) and
(Z)-3-hexen-1-ol (0.63 mg/g). For crushed leaves,
higher concentrations of C6-aldehydes, especially
(Z)-3-hexenal (17.88 mg/g), and hexanal (6.77 mg/g),
imparted an undesirable grassy odor to the samples.
Introduction of artifacts produced by degradation of
the sorbent can be a major di⁄culty in DHS-TD; e.g.,
Canac-Arteaga et al. [41] found that the interaction of
water vapor, volatile compounds and the trapping
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Figure 3. GC-MS chromatographic profiles after DHS-ATD collection of
volatiles released from Japanese pepper (Xanthoxylum piperitum) leaves
(modified from Jiang and Kubota [40]).
material (Tenax TA) can produce artifacts during the
analysis of aromas of dehydrated cheese and Parmesan
cheese. Retention of water by the trapping material can
also be a potential problem, especially when it is necessary to cryofocus the analytes in the chromatographic
column before separation (moisture can freeze and clog
the column). Carbon molecular sieves (Carbosieve, Carboxen) can retain large amounts of water, as opposed
to non-polar polymeric sorbents, such as Tenax and
graphitized carbon blacks. Polar polymers, such as
Porapak T and N, can also hold appreciable amounts of
water, which can, however, be easily removed prior to
analyte desorption by a current of dry air [42].
Introduction of products from the thermal degradation of adsorptive materials can be avoided in DHS procedures by using cryotrapping. Stra¤nsky¤ and Valterova¤
[43] determined the emission pro¢le of compounds related to the putrid fragrance from Hydrosme rivieri £owers
during their £owering period. A single, live specimen
was enclosed in a glass cylinder, which was purged by
15 mL/min of pure air. The compounds released were
trapped in a U-shaped tube containing methanol at
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78 C. It was found that emission of dimethyl di- and
tri-sulphides started after 70 h of £ower enclosure,
peaking at 118 h and ending after 142 h (Fig. 4). Before
70 h of enclosure, only n-alkanes were detected. To
eliminate water co-volatilized with the analytes, Kolb et
al. [44] proposed modi¢cation of a commercial on-line
DHS-cryotrapping device by incorporating a water
trap (glass-lined steel tube packed with 65% LiCl on
Chromosorb W/AW). The authors reported good
results in the application of this technique to several
processes, including the characterization of aroma from
ground co¡ee.
3.3. HS-SPME
SPME, a fast, simple and convenient sample preparation method introduced in 1990 [45], has gained
increasing popularity for F&A analysis, especially as an
alternative to DHS methods. It is specially suitable for
qualitative and quantitative analysis of fragrance compounds released by odorous samples, only requiring
exposure of a ¢ber to the HS above the sample for a
suitable period of time, followed by direct TD on the
heated injection port of a GC [46]. For these reasons,
HS-SPME is being presently considered as the best
available choice for sample preparation in fragrance
and F&A analysis [2].
Because of the small dimensions of the sampling
device and the simplicity and speed of the extraction
procedure, HS-SPME is able to collect fragrances from
live plants with minimum disturbance of the specimen,
under both laboratory and ¢eld conditions. Vereen et
al. [47] employed HS-SPME and GC-MS to study volatile
compounds released by intact and mechanically
damaged leaves of Fraser ¢rs (Abies fraseri) in the ¢eld.
Branches of ¢r were enclosed in 100 mL Tedlar bags
Figure 4. Profile of the emission of volatiles from a single Hydrosme
rivieri flower (modified from Stra´nsky´ and Valterova´ [43]) after
DHS-cryotrapping sampling and GC-FID analysis. Compounds:
*=dimethyl disulphide; &=dimethyl trisulphide; ^=n-decane;
*=n-undecane; &=n-dodecane; ~=n-tridecane.
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and the air inside was extracted with 100 mm PDMS
¢bers over periods of up to 4 h. After 5 min of extraction, monoterpenes such as 3-carene predominate;
after 3 h, the major component in the chromatograms
is bornyl acetate, with minor amounts of heavier compounds (e.g., camphor and borneol). Two species
(b-phellandrene and g-terpinene), detected in the fragrance exhaled from damaged leaves, were assigned as
wound-response compounds. However, inadequate
precision (RSD > 20%) and slow equilibration times for
heavier compounds prevented quantitative application
of HS-SPME to these samples.
Zini et al. [48] employed HS-SPME and GC-ITMS (ion
trap mass spectrometry) to assess the emission pro¢les
from intact and mechanically damaged leaves of living
Eucalyptus citriodora trees, using the sampling chamber
shown in Fig. 5 and extractions with PDMS/DVB ¢bers
performed every 30 min for continuous periods of
between 8 h and 10 h. The main compounds identi¢ed
were isoprene, citronellal, citronellol and b-caryophyllene. Di¡erent patterns of dependence between
extracted amounts and leaf-enclosure times were
observed; e.g., for rose oxide (cis-4-methyl-2-(2-methyl1-propenyl)-tetrahydropyran), a maximum in the area
versus time curves appears after 300-400 min of leaf
enclosure, while, for citronellal, the peak areas decayed
exponentially from the start of the experiments.
HS-SPME also has been extensively employed in
recent applications related to food £avors. Roberts et al.
[49] studied methodological and operational aspects of
the application of HS-SPME to analysis of volatile £avor
compounds. For co¡ee aroma, the best sensitivity overall was achieved using ¢bers coated with 65 mm PDMSDVB; this ¢ber was also found to be the most adequate
for heavier polar odorants, such as vanillin. CarbowaxDVB ¢bers were found to be suitable for organic acids.
Figure 5. Glass chamber for SPME sampling of volatiles emitted from
single, live leaves (based on Zini et al. [48]). 1=Silanized glass cylindrical
body; 2 – silanized glass lid; 3 – SPME sampling holes topped with silicone septa; 4 – SPME holder+fiber; 5 – DC power supply for microfan;
6 – microfan; 7 – Teflon support for microfan; 8 – Teflon tape seal.
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Non-polar odorants were detectable at the mg/L level,
while polar compounds could generally be detected at
the mg/L level only; e.g., in the extraction of co¡ee
aroma with PDMS-DVB ¢ber for 5 min followed by
GC-MS analysis, they found 7.7 mg/L vanillin, 116 mg/L
furaneol, 2.3 mg/L ethylguaiacol, 5.7 mg/L guaiacol,
40 mg/L 4-vinylguaiacol, 0.2 mg/Lb-damascenone,
0.4 mg/L 2,3-diethyl-5-methylpyrazine and 1 mg/L
2-ethyl-3,5-dimethylpyrazine.
In a similar study conducted by Augusto et al. [50], different SPME ¢bers were compared for the characterization
of the aromas of industrialized pulps of Brazilian tropical
fruits ^ cupuassu (Theobroma grandi£orum, Spreng.), caja¤
(Spondias lutea, L.), siriguela (Spondias purpurea, L.) and
graviola (Anona reticulata, L). The best e⁄ciencies overall
for these aromas were obtained with Carboxen-PDMS
¢bers, and especially for low-molar mass compounds.
Another area where HS-SPME is quickly becoming
favored is in studies of the aroma of alcoholic beverages,
essential in the assessment of the quality of such products and in optimizing their production. Sala et al.
[51] developed a HS-SPME methodology for determination of the aroma-active heterocycles, 3-alkyl-2-methoxypyrazines, in musts from Cabernet Sauvignon and
Merlot grapes. Levels as low as 0.1 ng/L of the analytes
could be detected after extraction of the sample HS with
PDMS-DVB ¢bers and GC-NPD (nitrogen-phosphorous
detection) analysis of the extracts. Weber et al. [52]
showed that chromatographic pro¢les of aroma
obtained by HS-SPME and GC-MS can be useful in classifying samples of wine according to their production
area, grape variety and vintage. Nonato et al. [53]
compared HS-SPME and conventional LLE in the
analysis of secondary aroma compounds (e.g., esters
and heavy alcohols) in Brazilian sugar-cane spirits
(cachacc¸ a). The precision in quantifying the target
analytes with SPME was better than with the standard
LLE procedure.
Over and above these reports, a new HS-SPME
approach for food £avor analysis was recently proposed
by Pe¤re's et al. [54]. To characterize the ripening of
Camembert cheese, the HS in contact with a 2 g sample
contained in 10 mL £asks was exposed for 10 min to a
75 mm Carboxen-PDMS SPME ¢ber. In preliminary
assays, the extracted materials were analyzed by
GC-MS to identify odorant compounds. For subsequent
experiments, the extracted compounds were thermally
desorbed from the ¢ber and directly introduced into the
ionization chamber of a MS without chromatographic
separation. More than 60 compounds were identi¢ed in
the HS-SPME extract by GC-MS; the Carboxen-PDMS
¢ber was found to be especially e⁄cient for collecting
sulfur compounds (e.g., dimethyl sul¢de, methyl
thioacetate), which are important impact odorants for
this cheese variety. The HS-SPME-MS data obtained
without chromatographic separation were statistically
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processed and found to be valuable for qualitative
chemometric classi¢cation of the samples.
4. Direct extraction procedures
Methods involving application of LLE, SPE or SFE
directly to the samples to isolate odorants are found
occasionally in the recent literature. Although
HS-based methods are usually more suitable for fragrance analysis, in some cases, direct extraction of
the target analytes from the matrixes is necessary; e.g.,
species with high odor impact and in extremely
reduced concentrations can be important to some F&A,
but may not be detected using HS methodologies if their
volatility is not high enough to provide a suitable
concentration in the sample HS [1].
4.1. LLE
Despite its simplicity, the modern tendency is to replace
LLE by other techniques, because high-purity solvents
are required for trace analysis and because of the need
to reduce the environmental and health risks associated with their manipulation. Also, in the case of biological F&A, LLE cannot be applied to live samples.
However, there are several contemporary reports on
use of this technique for F&A analysis, especially for
collection of preliminary data [55].
Lo¤pez and Go¤mez [56] addressed some operational
parameters on the application of LLE to extract aroma
compounds from wines. Several solvents ^ diethyl
ether, n-pentane, Freon-11, n-hexane, 1:1 ether/pentane, 1:1 ether/hexane and dichloromethane ^ were
compared for extractions of odoriferous terpenic substances from ‘‘arti¢cial wine’’ (12% v/v ethanol in
water). It was concluded that dichloromethane and 1:1
ether/pentane are the best solvents for extracting these
analytes from wine.
In a related study, continuous LLE was employed by
Rocha et al. [57] to extract free and releasable odor
volatiles from Portuguese Bairrada white grapes
employed in the local wine industry. A volume of
250 mL of raw or enzimatically treated must was continuously extracted for 25 h with 75 mL of dichloromethane at 50 C. The organic phase was dried and
concentrated by vacuum distillation prior to the GC-MS
analysis. Chemometric analysis of the data allowed differentiation of several grape varieties and of the enzyme
treatment studied.
Conventional batch LLE is also still employed occasionally in F&A analysis; recent examples include the
identi¢cation of odorants in sugar-beet juice [58].
4.2. SPE
SPE can be directly applied to isolate odorants from
liquid or lique¢able odoriferous samples, such as
Trends in Analytical Chemistry, Vol. 22, No. 3, 2003
beverages, fruit pulps and tissues. A typical contemporary application of SPE to aroma analysis was
presented by Wada and Shibamoto [59], who studied
the direct extraction of odorants from Chablis red wine
using Porapak Q columns. Samples spiked with selected
aroma analytes (2-methyl-1-propanol, 3-methyl-1butanol, 2-phenylethanol, ethyl hexanoate, ethyl
octanoate, diethyl butanedioate and hexanoic,
octanoic and decanoic acids) were passed through
30-cm glass columns packed with the sorbent. Diethyl
ether, dichloromethane and pentane were evaluated
to desorb the extracted materials, which were analyzed
by GC-FID and GC-MS. Dichloromethane was found
to be the best desorbing solvent, recovering up to
(103.0 3.7) % of the spiked analytes. Analysis of
unspiked samples revealed 67 volatile odor compounds
in this variety of red wine.
SPE has also been applied to characterize butter
aroma. Adahchour et al. [60] assessed the use of SPE
cartridges packed with di¡erent sorbents ^ C18, C8, NH2
and CN-bonded silica, as well as SDB-1 (PS-DVB copolymer) ^ to extract aroma compounds from this material.
Butter samples were melted and the aqueous fraction,
after separation of the fat by centrifugation, was passed
through the SPE cartridges. The cartridges were eluted
by methyl acetate, which was dried and analyzed by
GC-MS. Best overall recoveries were obtained with SDB1 cartridges (average recovery ca. 80%); detection limits for impact odorants, such as vanillin and diacetyl,
were in the low-pg range.
For samples such as fruit pulps, SPE can be combined with isolation techniques, such as distillation,
as exempli¢ed by Boulanger et al. [61]. After distillation of cupuassu pulp and extraction with XAD-2
resin, the main odorants were identi¢ed as linalool,
a-terpineol, 2-phenylethanol, myrcene, limonene,
ethyl 2-methylbutanoate, ethyl hexanoate, butyl
butanoate, 2,6-dimethyl-oct-7-en-2,6-diol, (E)- and
(Z)-2,6-dimethyl-octa-2,7-dien-1,6-diol and methoxy2,5-dimethyl-3(2H)-furanone.
Stan¢ll and Ashley [62] presented an interesting
application of SPE to analysis of £avor-related alkylbenzenes in cigarette smoke. Tobacco from disassembled
cigarettes was ‘‘smoked’’ in a smoking machine and the
particulate material produced collected with ¢lter pads.
The collected particulate was then suspended in hexane
and the target analytes extracted from these solutions
with CN-silica SPE cartridges. After elution with a
hexane/toluene/tetrahydrofuran mixture and concentration of the extract by vacuum evaporation, alkylbenzene odorants were detected and quanti¢ed by GC-MS.
Detection limits per cigarette ranged from 1.1 ng
(methyleugenol) to 27.8 ng (eugenol). This method
was tested to quantify aroma alkylbenzenes in commercial brands sold in the USA, and levels of target analytes
up to several micrograms per cigarette were found.
Trends
4.3. SFE
Compared to the direct extraction methods discussed
above, SFE presents some advantages for fragrance
analysis. For example, since the critical point for CO 2
(the most popular and most convenient £uid for SFE) is
31.1 C at 7.38 MPa, extractions can be carried out
under milder temperature conditions and without need
of further aggressive procedures, such as distillation of
excess solvent, as is common in LLE and SPE [63].
Volatile compounds from £owers, leaves and stems of
guaca (Spilanthes americana, Mutis ^ a South American
plant with medicinal and insecticide properties) ^ were
isolated by Stashenko et al. [64] using both SFE and
SDE, and analyzed by GC-MS, GC-FID and GC-NPD. SFE
was performed with CO 2 at 40^45 C and 7.24^7.53
MPa, using up to 30 g of vegetable sample. Extraction
time was 2 h and, after depressurization, the collected
analytes were dissolved in 2 mL CH2Cl2. Compared to
SDE, a larger number of compounds could be detected
at levels above 100 ppb in the SFE extracts (e.g., 67
analytes with SFE and 43 analytes with SDE, for £oral
extractions); the overall extraction yield for SFE was up
to twice that for SDE. SFE was found to be especially
e¡ective in isolating sesquiterpenes and nitrogenated
compounds from these samples.
There are also several SFE applications reported for
food-£avor analysis. Leunissen et al. [65] developed a
SFE-GC-MS method for fast analysis of roasted peanut
aroma. For aroma isolation, 2.00^2.50 g of ground,
roasted peanuts were extracted for 10 min with CO2 at
50 C and 9.6 MPa. The conditions were adjusted to
minimize the extraction of greasy materials. The CO2
was passed through a silica trap at 5 C to collect the
extracted materials; the trapped analytes were desorbed by dichloromethane and analyzed by GC-MS. The
characteristic aroma compounds were found to be
methylpyrrole, hexanol, hexanal, 2-furanmethanol,
2-furancarboxyaldehyde, benzeneacetaldehyde and
several alkylpyrazines. Quanti¢cation limits for these
analytes were estimated to be in the range between
80 ng/g (methylpyrrole) and 0.4 ng/g (ethyl- and
2,3,5-trimethylpyrazine).
Supercritical CO2 is a poor extractor for polar substances; therefore, for such analytes, addition of modi¢ers or use of other £uids, such as supercritical water, is
advisable [2]. Kubatova et al. [66] also compared
water^SFE and CO2^SFE with hydrodistillation for the
separation of aromatic essential oils from savory
(Satureja hortensis) and peppermint (Mentha piperita).
Extraction of savory with supercritical water for
40 min removed almost 100% more thymol and
carvacrol and 150% more borneol and linalool than
hydrodistillation. For CO2 extractions, yields of
borneol and linalool were similar than those for
hydrodistillation; however, for thymol and carvacrol
the e⁄ciency of CO2^SFE was only half that of
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167
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Trends in Analytical Chemistry, Vol. 22, No. 3, 2003
hydrodistillation. Water-SFE was found to be highly
selective for polar oxygenated £avor compounds,
when compared with CO2^SFE or hydrodistillation.
5. Conclusion
As in any analytical procedure, a suitable choice of
sample-preparation technique is essential for accurate,
reliable characterization of the chemical composition of
F&A. However, because of the peculiarities of these
samples ^ especially the common presence of thermally
or chemically labile analytes in trace or ultratrace
concentrations ^ selection of the analyte-isolation and
pre-concentration technique, as well as careful optimization of the corresponding operational parameters,
are of paramount importance.
Some of the general techniques discussed in the previous sections ^ namely distillation procedures, LLE and
SPE ^ are still employed occasionally for F&A analysis.
However, for more critical analysis at least, the present
tendency is to replace them with methodologies that
are simultaneously less aggressive to analytes and
capable of dealing with ultra-low concentrations of
analytes in samples. Most of the state-of-the-art,
contemporary applications in F&A analytical chemistry
are focused on several variations of DHS sampling or,
more recently, on HS-SPME. Despite the fact that these
techniques have drawbacks (e.g., expensive hardware
for DHS-ATD, limitations on the extracted masses for
HS-SPME), for nearly all cases in F&A analytical chemistry, either DHS or HS-SPME can obtain dependable
results.
Perhaps the next frontier to be breached in this area
will be ¢eld sampling and analysis. The great majority
of analytical work on plant fragrances is still performed,
totally or in part, in the laboratory and/or under invitro conditions (i.e., using detached parts of the plants
as the source of fragrance). Nonetheless, adequate,
realistic characterization of the composition of organic
volatile emissions from plants can be made only in
their natural habitats, and with minimal biological,
chemical and physical disturbance. There is therefore
a need for portable or semi-portable instruments (such
as GC-MS) and sampling procedures based on small,
rugged hardware. For sample preparation, SPME
seems to be the technique that most closely ¢ts these
demands.
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