CHAPTER NINETEEN

Green Sample-Preparation
Techniques in Comprehensive
Two-Dimensional
Chromatography
Francesco Cacciola1, Mariarosa Maimone1, Paola Dugo1, 2 and
Luigi Mondello1, 2, *
1
University of Messina, Messina, Italy
2
University Campus Bio-Medico of Rome, Rome, Italy
*Corresponding author: E-mail: lmondello@[Link]

Contents
1. Introduction 601
2. Green Sample-Preparation Methods Prior to Two-Dimensional Gas 605
Chromatography Analysis
2.1 Solid-phase microextraction analysis 605
2.1.1 Untargeted solid-phase microextraction 605
2.1.2 Targeted solid-phase microextraction 609
2.2 Stir-bar sorptive extraction 609
2.3 Solid-phase extraction 610
2.4 Other green sample-preparation techniques 613
3. Capillary Comprehensive Two-Dimensional Liquid Chromatography 615
4. Conclusions 616
References 618
Further Reading 622

1. INTRODUCTION
A consolidated definition for ‘green analytical chemistry’ is the
employment of techniques and methodologies: (1) capable of reducing or
eliminating chemicals hazardous to human health or the environment and
(2) may enable faster and more energy-efficient analysis, without compro-
mising performance criteria [1].
Gas chromatography (GC) is generally accepted as a relatively green
technique, while liquid chromatography (LC) which is not ‘green’ itself

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© 2017 Elsevier B.V.
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ISSN 0166-526X
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602 Francesco Cacciola et al.

can be considered as such only by reducing the amounts of solvents

consumed e.g., nano and capillary LC or using more environmentally
friendly solvents, e.g., superheated water.
When developing a ‘green analysis’, the most critical step is represented
by sample preparation, which is often a quite solvent-consuming process.
Sample preparation has two main objectives, viz., analyte enrichment and
removal of interfering matrix components.
Truly green sample-preparation techniques have received great attention
in the last two decades aiming to combining sample extraction, purification
and enrichment, by using approaches such as solid-phase microextraction
(SPME), stir-bar sorptive extraction (SBSE) and solid-phase extraction
(SPE). Unlike SPME, SBME and SPE, other miniaturized systems e.g.,
single-drop microextraction and microSPE have not yet been investigated
in comprehensive two-dimensional GC (GC  GC). Other approaches,
e.g., supercritical fluid extraction (SFE), pressurized liquid extraction
(PLE), matrix solid-phase dispersion (MSPD), ultrasound-assisted extraction
(UAE), microwave-assisted extraction (MAE) and dispersive liquideliquid
microextraction (DLLME), despite considered greener than the traditional
Soxhlet extraction, still involve the use of solvents and will be only briefly
described in the present chapter [2e4].
It is worth noting that thanks to the rapid evolution and diffusion of
advanced mass spectrometry (MS), e.g., triple-quadrupole, high-resolution
MS with enhanced selectivity and sensitivity, sample preparation can be
simplified and miniaturized. Furthermore, the use of high-resolution chro-
matography techniques, e.g., GC  GC, can also reduce the requirements
of time-consuming sample preparation through an expanded separation
space.
One of the most significant developments in the sample preparation field
has been represented by SPME, introduced by Arthur and Pawliszyn in
1990 [5]. SPME is currently the most popular green sample-preparation
technique and is commercialized in a wide variety of forms (in terms of
selectivity and sensitivity). The most revolutionary aspect of SPME is that,
in many cases, the need of organic solvents is completely eliminated [5e7].
Liu and Phillips introduced just a year after, GC  GC that is considered
one of the most powerful GC approaches available nowadays [8]. GC  GC
is characterized by a plethora of advantages over conventional 1D-GC
mainly represented by higher separation power, enhanced selectivity and
sensitivity and formation of group-type chemical patterns. Another
Green Sample-Preparation Techniques 603

important aspect pertinent to the employment of such an innovative tech-

nique is the possibility to reduce the need for laborious sample preparation
[9,10].
The first paper combining a green sample-preparation step and
GC  GC was published in 1994 and was based on SFE applied to human
serum [11]: 10 mL of human serum spiked with pesticides was passed
through a C18 SPE cartridge, then purged with N2 for drying. Afterwards,
the SPE packing was treated with supercritical CO2 for 70 min, using
trimethylsilylimidazol as modifier. Detection was performed with a flame
ionization detector (FID), the most common detector employed in the first
10 years of GC  GC applications. Being one of the early GC  GC
papers, the authors focused their attention much more on the separation
step, rather than on the sample treatment: in fact the extract was collected
in 10 mL of dichloromethane thus decreasing the ‘green’ nature of the
experiment.
The second paper combining a green sample-preparation step and
GC  GC was published four years later and was based on SPME prior
to GC  GC-FID analysis of benzene, toluene, ethyl benzene and xylene
isomers (BTEX), along with methyl-tert-butyl ether and ethyl butyl ether,
in groundwater [12]. In that study [12], two different fibre coatings were
evaluated, namely 100 mm polydimethylsiloxane (PDMS) and 75 mm
Carboxen (CAR)/PDMS, the latter showed a higher sensitivity. All the
SPME steps were performed manually, while the ‘target’ analytes were
separated on a rather unorthodox column combination (first dimension,
1
D: apolar 2.0 m  0.10 mm I.D.  5 mm; second dimension, 2D: polar
1.0 m  0.10 mm I.D.  0.14 mm). Analyte transfer between the two
dimensions was carried out by using a thermal sweeper. Method detection
limits were in the 0.36e0.63 ppb (v/v) range, while precision data were
satisfactory with relative standard deviation (RSD) lower than 10.2%.
Unlike this first work, nowadays the entire SPME process is commonly
performed in an automated manner and a much wider availability of fibres
with different selectivity can be found into the market. The modulators
employed in GC  GC setup are now more robust and are normally
operated with a cryogenic fluid. Also, the coupling of GC  GC to MS
became very popular since its first application reported in 1999 [13].
Apart from SPME and SPE, only a few other green sample-preparation
methods coupled to GC  GC have been applied, highlighting a certain gap
between the two research fields. The total number of papers showing the
combination of green sample preparation and GC  GC separation
604 Francesco Cacciola et al.

published in the span of 12 years (1994eOctober 2016) is pretty high

(207 references), and this number raises a bit if it is considered that in
some papers more than one green sample-preparation method was adopted
(218). The most striking aspect is the application of SPME, which has been
by far the most applied green sample-preparation method in the GC  GC
field (over 50% of the total).
In general, GC  GC methods (with or without a green sample-
preparation method ahead) are developed for ‘untargeted’ or (pre- or
post-) ‘targeted’ analysis. The former applications were ‘untargeted’ and
directed to the identification of as many compounds as possible occurring
in a sample, e.g., food aroma. ‘Pretargeted’ experiments were based on
the determination of a limited number of ‘known’ compounds, e.g., petro-
leum contaminants in groundwater, while ‘posttargeted’ analyses were
carried out later and performed through a new investigation on the acquired
data analysis and subsequent analysis in a ‘targeted’ way based on the
‘posttargeted’ data [14].
The possible combinations of green sample preparation, GC  GC and
MS can generate methods of high sensitivity, selectivity and separation
power. The green sample-preparation step prior to GC  GC analysis is
typically based on the use of cryogenic modulation, and mainly on MS
detection. Most applications have been performed in the food research field,
e.g., beverage and flavour (over 40%), followed by environmental and plant-
based investigations.
Benefits of reduced column inner diameter (I.D.) in the field of LC were
proposed a long time ago [15e17]. Surely, the major advantage of low I.D.
columns is their ability to work with reduced sample volumes at low flow
rates. As a consequence, nano or capillary LC offers enhanced mass
sensitivity over ‘conventional’ LC. Other benefits arising from the reduced
column I.D. are the minimal solvent and additive volume requirements for
elution and also easy separation temperature control due to the fast and
effective heat transfer on low I.D. columns. Another key parameter
subjected to miniaturization is the diameter of sorbent particles of LC
columns. Gradual decrease in the sorbent particle diameter allows reaching
extremely high separation efficiencies and peak capacities, comparable to
those in GC [18]. However, the price to be paid is an increase in the pressure
drop across the column, which is inversely proportional to the square of the
sorbent particle diameter. Another problem connected with the progressing
miniaturization of column and sorbent dimensions is the extra-column band
broadening: in fact from a practical point of view, it means that all the flow
Green Sample-Preparation Techniques 605

through spaces (capillary connections, detector cell volume) must be

miniaturized and data acquisition rate should be maximized [19]. Consid-
ering nomenclature of miniaturized LC columns, the situation is quite
complicated and in this chapter only ‘nano’ and ‘capillary’ terms will be
used: as column designation typical I.D. are in the range 0.1e0.5 mm for
the later, whereas 0.01e0.1 mm for the former [20]. An environmentally
friendly sample preparation followed by hyphenation of a nano/capillary
LC  LC and MS can be considered a method of high sensitivity and
notably also a ‘green analytical technique’ whenever limited quantity of
organic solvents are used by the analyst.

2. GREEN SAMPLE-PREPARATION METHODS PRIOR

TO TWO-DIMENSIONAL GAS CHROMATOGRAPHY
ANALYSIS
2.1 Solid-phase microextraction analysis
In general, untargeted metabolomics involves the analysis of all, or as
many as possible, metabolites in a biological system, being ‘ideal’ samples for
SPME GC  GC-MS analysis. The latter can be considered a very exciting
‘green’ approach, capable of providing very high sensitivity, selectivity,
separation power and identification potential [21e29]. On the other
hand, the requirement of a high-resolution GC approach is greatly reduced
in ‘targeted’ analyses, e.g., pesticides in real-world food samples. In the latter
case, it is mainly the selectivity of mass spectrometry, e.g., extraction-ion-
chromatograms (EIC), selected-ion-monitoring (SIM), MS/MS processes,
accurate MS data, which decreases or eliminates background noise and
resolves cases of coelutions, between target analytes and matrix constituents
[30e35]. However, it is noteworthy that an adequate sample preparation
ahead is deemed as essential since interferences, nonvolatile components
and by-products are still present into the analytical system.

2.1.1 Untargeted solid-phase microextraction

The first work describing an ‘untargeted’ SPME step prior to GC  GC
analysis was reported by Chaintreau and coworkers for the elucidation of
sulphur compounds from the in-oven roast beef aroma [36]. More than
70 sulphur compounds were found by SPME (a PDMS fibre was used)
followed by GC  GC hyphenated to time-of-flight (ToF)-MS, and 50
out of them were positively identified. To overcome the absence of many
retention indices in databases, the missing values were simulated using a
606 Francesco Cacciola et al.

multiple linear regression to help the peak identification. The selection of

the most important sulphur odorants from this list was achieved by GC-
olfactometry, using the GC-‘SNIF’ technique. Seven compounds were
detected for the first time in beef aroma, of which only one had been
previously found in nature. Afterwards, Tranchida et al. [37] and Cordero
et al. [38] reported an head space (HS)-SPME-GC  GC employing a
divinylbenzene (DVB)/CAR/PDMS phase, combined to a single quad
MS (qMS) for the analysis of the volatile fraction of roasted coffee and
roasted hazelnuts, respectively. In the first work [37], a high resolution
0.05 mm I.D. capillary, as well as a 0.25 mm I.D. capillary, was used for
the 2D in an optimized GC  GC instrument for the first time. The second
work [38], employed a similar setup aiming to create GC  GC fingerprints
relative to roasted hazelnuts from different cultivars/varieties and geograph-
ical origins. Bean et al. reported a typical example of the enhanced sensi-
tivity, selectivity and separation power of HS-SPME-GC  GC,
combined with a low resolution (LR) ToF-MS [39]. In particular the
qualitative profile of volatiles released from a bacterium (Pseudomonas aerugi-
nosa) after 24 h of growth was studied. The fibre employed for extracting
bacterial volatiles was the same reported in [37,38] operated at a temperature
of 50 C for 10 min. The GC  GC method enabled the detection of
roughly 500 peaks with a signal-to-noise ratio (s/n) higher than 250. After
the peak deconvolution process, through MS database search it was possible
to tentatively identify roughly 40 compounds, belonging to various
chemical classes: aliphatic and aromatic hydrocarbons, alcohols, aldehydes,
ketones, acids, esters and heteroaromatics. Detailed sample discrimination
was performed by using specific GC  GC software functions. A similar
approach was used by Purcaro et al. to correlate the HS-SPME-GC  GC
data with the sensorial quality of virgin olive oil [25]. In that study, and
thanks to the use of specific GC  GC software functions combined with
statistical analysis, the authors extrapolated the ‘chemical blueprint’ relative
to a combination of olive oil defects. The chemical blueprint of a food prod-
uct relates specific volatiles to a particular food property, in this case sensorial
quality. Very interesting metabolomics-related researches were carried out
by Risticevic et al. [21,22,27]. In their former work [21], an optimized
HS-SPME-GC  GC coupled to ToF-MS method for apple metabolite
profiling was employed. The investigation involved the testing of seven
fibres, namely PDMS, polyacrylate (PA), Carbowax, DVB/CAR/PDMS,
PDMS/DVB, CAR/PDMS and Carbopack Z/PDMS, resulting in the
detection of 549, 977, 897, 1163, 1053, 1167 and 745 metabolites,
Green Sample-Preparation Techniques 607

respectively (minimum s/n and database match: 50 and 750). The peak table
was postprocessed, through the application of a minimum database match
value of 800, to reduce potential misinterpretation of selectivity correlations.
After a very detailed description of the fibre coating selectivities, the authors
reported that the DVB/CAR/PDMS phase gave the most balanced analyte
coverage and the highest number of extracted metabolites (830). Some very
crowded peak apex plots and chromatograms were shown, highlighting the
need for the GC  GC separation. Moreover, since the GC  GC analysis
can generate very detailed separations, the technique is capable to provide
more thorough information on SPME analyte coverage, desorption effi-
ciency, dynamic range and displacement effects [22]. In their most recent
work [27], an in vivo sampling mode of direct immersionesolid-phase
microextraction (DI-SPME) was employed to capture the metabolome of
living plant specimens, using apple (Malus  domestica Borkh.) as a model
system for the first time. Metabolites were extracted from apple tissues
and introduced by thermal desorption into a GC  GC-ToF-MS. The
feasibility of this sampling approach, based on exploitation of microextrac-
tion principles, including negligible depletion of free analyte concentrations,
solventless sampling and sample preparation, and on-site compatibility, was
determined in global metabolite analysis. Rather than adopting an approach
of traditional sample preparation, requiring metabolism quenching and labo-
rious sample preparation, the objective of the study was to capture the
metabolome in vivo, evaluate the feasibility of the approach to provide
unbiased extraction coverage and compare analytical precision when
different SPME sampling modes are employed. The potential of in vivo
DI-SPME in quantitative plant metabolomics was assessed by evaluating
changes in metabolic fingerprints in response to fruit maturation. Very
recently, a comprehensive mapping of volatile compounds in 70 wines,
from 48 wineries and 6 vintages, representative of the two main production
areas for Italian sparkling wines, by HS-SPME-GC  GC-ToF-MS and
multivariate analysis, was carried out by Carlin et al. [28]. The final scope
was to describe the metabolomics space of these wines, and to verify
whether the grape cultivar signature, the pedoclimatic influence of the
production area and the complex technology were measurable in the final
product. The wine chromatograms provided a wealth of information,
with 1695 compounds being found. In an attempt to understand common
metabolic shifts in the young and aged wine datasets, correlation-based
network analysis (CNA) was employed. The relationships for a-isophorone
and safranal were in particular scrutinized as shown in Fig. 1, highlighting
 608
Francesco Cacciola et al.
Figure 1 Network analysis, symmetric difference plus intersect network of a-isophorone and safranal, compared in young and aged Tren-
todoc sparkling wines. Reprinted from S. Carlin, U. Vrhovsek, P. Franceschi, C. Lotti, L. Bontempo, F. Camin, D. Toubian, F. Zottele, G. Toller, A. Fait,
F. Mattivi, Regional features of northern Italian sparkling wines, identified using solid-phase micro extraction and comprehensive two-dimensional
gas chromatography coupled with time-of-flight mass spectrometry, Food Chem. 208 (2016) 68e80. Copyright 2016, with permission from
Elsevier.
Green Sample-Preparation Techniques 609

how CNA can be used as a tool to detect differences in compound behav-

iour based on external/environmental influences.

2.1.2 Targeted solid-phase microextraction

SPME-GC  GC-MS methods have been proposed for ‘targeted’ analyses
in a variety of samples. In 2008, Schurek et al. used HS-SPME-GC  GC-
ToF-MS for the determination of 36 pesticides in green, black and fruit tea
[30]. As 1D of the GC  GC set up an apolar (40 m  0.18 mm
I.D.  0.18 mm df) column was employed whereas in the 2D a medium po-
larity (2.5 m  0.1 mm I.D.  0.1 mm df) column was used. The authors
performed a comparison between the results attained using 1D and 2D
GC processes. LoQ values were in the 1e28 mg/kg range when using
HS-SPME-GC  GC-ToF-MS, and, as expected, were always lower
than those attained through HS-SPME-GC-ToF-MS. Finally, HS-SPME
was compared with a traditional sample preparation process, involving ethyl
acetate extraction and high-performance gel permeation chromatography.
In general, HS-SPME gave a better performance in terms of sensitivity
and reduction of matrix interferences, while the conventional sample
preparation approach was preferred for linearity (over the analyzed concen-
tration range) and repeatability. One year later, Purcaro et al. exploited the
enrichment capability of SPME (a 100 mm PDMS fibre was used) coupled to
a GC  GC combined with a rapid-scanning qMS instrument, for the
determination of pesticides in drinking water [31]. The method limits of
quantification (LoQs) were in the 3 ppte0.084 ppb range, and in any case
always lower than the maximum residue limits imposed by European
legislation. A very interesting work was recently reported by Sulej-
Suchomska et al. dealing with the pollution caused by airport runoff waters
[35]. Polycyclic aromatic hydrocarbons (PAHs) are in fact one of the most
important groups of xenobiotics which are commonly found in runoff water
originating from airports. Regardless of the airport location, chrysene,
phenanthrene and pyrene were the most abundant PAH compounds
detected in all analyzed 34 samples (1.8e26.3 mg/L) by the HS-
GC  GC-ToF-MS method. Such a methodology can be used for tracking
the environmental occurrence of PAHs and assessing the impact of airports
on the environment.

2.2 Stir-bar sorptive extraction

Based on the same concept as SPME, SBSE was first reported in 1999 [40],
with the main difference related to the presence of a greater amount of
610 Francesco Cacciola et al.

extraction phase accounting for a greater sensitivity. However, unlike

SPME, there is a much more limited variety of SBSE sorbents (PDMS
and PDMS/ethylene glycol), and the SBSE thermal desorption process
requires a dedicated GC injector. The combination of SBSE and GC  GC
has been rarely reported, and it has been mainly adopted for highly diluted
samples in ‘targeted’ mode. The ‘untargeted’ mode presents as main incon-
venience the extraction of excessive amounts of solute by the large amounts
of stationary phase, giving rise to ‘overloading’ phenomena especially in the
2
D column. Ochiai et al. used SBSE GC  GC, combined with an HR
ToF-MS system, with the objective of analyzing ‘pretargeted’ (organochlo-
rine pesticides) and ‘untargeted’ contaminants in river water [41]. The stir
bar was characterized by a length of 20 mm and a 0.5 mm layer of PDMS
(phase volume ¼ 47 mL). Method sensitivity was satisfactory, with limits
of detection (LoDs) in the 10e44 pg/L range. The authors also performed
an ‘untargeted’ search, and identified further 20 contaminants in river water.
In a posterior ‘pretargeted’ GC  GC study on river water and wastewater,
involving the use of an LR ToF-MS system, the enhanced sensitivity of
SBSE was exploited for the extraction of 13 phencyclidines (PCPs), 15
PAHs and 27 pesticides [42]. Method detection limits ranged from 0.01
to 2.15 ng/L in river water (in the 0.02e2.5 ng/L range for wastewater).
As an example, Fig. 2A and Fig. 2B show the GC  GC-ToF-MS contour
plots of the ‘target’ and ‘untargeted’ compounds, respectively, identified in a
river water sample collected 20 m downstream of the Alcal
a de Henares
wastewater treatment plant (WWTP) emission point. In Fig. 2B over
2700 peaks at S/N ¼ 10 are show. For the comparison of the two cited
works [41,42], the results attained by Ochiai et al. [41] show higher sensi-
tivity for the selected pesticides probably due to the different type of
ToF-MS used.

2.3 Solid-phase extraction

After SPME, SPE can be considered the most widely applied sample prep-
aration technique used as enrichment and cleanup method for the isolation
of analytes from liquid samples. There are several sorbents which may be
used for this task according to the different chemistry of the analytes e.g.,
C18, ion-exchange, molecularly imprinted polymers, immunosorbents
and so on [42]. The first application was published in 2010 by Matamoros
et al. [43] who investigated SPE-GC  GC combined to LR ToF-MS
for the determination of 97 contaminants in river water. The cartridge
used for the SPE process was packed with a styrene-divinylbenzene sorbent.
Green Sample-Preparation Techniques 611

Figure 2 GC  GC-ToF-MS contour plot and peak table of a river water sample
collected at 20 m downstream of the point of emission of the Alcala de Henares waste-
water treatment plant. Analysis of target (A) and nontarget (B) compounds. Reproduced
from M.J. Go
mez, S. Herrera, D. Solé, E. García-Calvo, A.R. Ferna

ndez-Alba, Automatic
searching and evaluation of priority and emerging contaminants in wastewater and river
water by stir bar sorptive extraction followed by comprehensive two-dimensional gas
chromatography-time-of-flight mass spectrometry, Anal. Chem. 83 (2011) 2638e2647.
Copyright 2011 American Chemical Society.
612 Francesco Cacciola et al.

LoDs of 2, 3, 3, 3, 5 and 6 ppt were attained for dieldrin, endrin, atrazine,

phenanthrene, aldrin and galaxolide, respectively, and in the range 0.5e
100 ppt for 97 contaminants. One year later, Weldegergis et al. employed
SPE GC  GC-LR-ToF-MS for detailed investigation of volatiles in
South African red wines [44]. It was found that reversed phase (RP)-SPE
(a styrene-divinylbenzene sorbent was used) offered advantages over
HS-SPME employed by the authors in a previous work [18], since a minor
amount of highly polar compounds e.g., acids and alcohols were extracted,
which were the reason of poor chromatographic analysis viz., overloading,
tailing, wrap-around, coelution with other compounds. The SPME fibre
consisting of a microporous carbon suspended into a liquid polymer
(CAR/PDMS) was characterized by a good analyte coverage on a polarity
basis but it showed a high degree of discrimination towards high molecular
weight metabolites [45]. The proposed SPE-GC  GC coupled to LR
ToF-MS approach proved to be beneficial for the detection of terpenes,
phenols, lactones and sulphur-containing solutes, many of which are present
at the mg/L level in wine. Overall, 276 analytes were identified in the three
wines and many of them for the first time, through MS database matching,
linear retention indices and standard compounds, when available [44]. A
very interesting application was recently reported by Dimitriou-Christidis
et al. [46] for the determination of nonpolar halogenated micropollutants
in WWTP influent, effluent, primary sludge and secondary sludge matrices
(including both the liquid and particle phases). The GC  GC-microelec-
tron capture detector (mECD) method investigated allowed them to deter-
mine up to 59 target analytes e.g., toxaphenes, polychlorinated
naphthalenes, organochlorine pesticides, polychlorinated biphenyls, poly-
brominated diphenyl ethers, and emerging persistent and bioaccumulative
chemicals. For most analytes extracted by RP-SPE, recoveries fell between
70% and 130% in all matrix types. The main finding was that sorption onto
dissolved organic carbon can contribute substantially to the apparent solidse
liquid distribution of hydrophobic micropollutants in WWTP streams.
Interesting miniaturized devices which are in between SPE and SPME
are represented by needle traps (NTs), generated by packing adsorbent ma-
terial inside a needle. NTs are employed for analyte extraction, enrichment
and release through thermal desorption. Compared to conventional traps,
NTs are characterized by substantial advantages for on-site sampling. A
typical NT application is that related to the isolation of volatile organic
compounds as reported for alkane mixture (C6-C15) [47] and human
breath [48].
Green Sample-Preparation Techniques 613

2.4 Other green sample-preparation techniques

Apart from SPME, SBSE and SPE that are considered ‘green’, other sample
preparation techniques do imply a certain quantity of solvent extraction and
are thus considered ‘less green’ than the formers but surely ‘greener’ than
traditional approached (e.g., Soxhlet), among them SFE, PLE, MSPD,
UAE, MAE, DLLME can be included.
SFE appears to be an ideal substitute of traditional liquidesolid extrac-
tion techniques since no hazardous wastes are produced. Most SFE processes
have been based on the employment of CO2 because of its specific charac-
teristics, namely critical temperature and pressure of 31 C and 72 bar, as well
as a low toxicity and extraction rapidity. However, supercritical CO2 extrac-
tion do also present a series of limitations, such as the low selectivity towards
polar compounds (selectivity can be increased by using low amounts of an
organic modifier), and the frequent requirement of cleanup [49]. The use
of SFE, prior to GC  GC analysis, has been reported in ‘untargeted’ works.
For example, Pripdeevech et al. used GC  GC-FID and GC  GC-qMS
to characterize a vetiver oil sample attained through different extraction ap-
proaches, namely SFE, MAE, simultaneous distillation and extraction (SDE)
and solvent extraction (SE) [50]. For the three techniques, different amounts
of solvents were employed (SDE, 150 mL of dichloromethane; SFE, 10%
dichloromethane, 10% methanol and 20% toluene; MAE, dichloromethane,
methanol and toluene 30 mL each; SE, dichloromethane, methanol and
toluene 500 mL each). SFE turned out to be the most rapid method but
also the most effective in terms of extraction capacity, providing a clean
extract due to the great reduction of nonvolatile material compared to SE
and MAE. GC  GC-qMS analyses enabled the identification of 245 com-
pounds, considering all the extracts.
PLE is based on the use of solvents in the liquid state, under high
pressures and temperatures. Under such conditions, extraction processes
are performed in a much faster manner, using lower amounts of solvents.
The improved penetration of the solvent into the matrix makes the cleanup
step optional since it could be avoided by adding a specific sorbent into the
extraction-cell [49]. Ong et al. used PLE-GC  GC-FID for the screening
of 24 PAHs in soil [51] using three soil extraction methods, all performed at
150 C and 14 MPa, namely (1) solvents: hexane/acetone (1:1 v/v); a post-
extraction chromatography cleanup step was performed; (2) solvent: hexane;
in-cell cleanup was performed; (3) same as method 2, but with a different
extracting solvent mixture [hexane/dichloromethane (3:1 v/v)]. The
614 Francesco Cacciola et al.

GC  GC chromatograms were similar for all the extracts, and hence no

significant differences were observed between in-cell and out-of-cell
cleanup methods. The expanded separation space provided by GC  GC
was so effective that overlapping between the interferences and target
analytes was avoided. The soil concentrations of PAHs (mg/kg amounts)
determined by methods 2 and 3 were lower compared to method 1. It is
highly presumable that analytes were not completely eluted from the sor-
bent used for the in-cell cleanup process.
MSPD enables simple, rapid, low-cost extraction, as well as cleanup, in a
single step, and by using small sample quantities. It was first reported in 1989
by Barker et al., in a research work focused on the isolation of drug residues
from animal tissue [52]. In 2009 Ramos et al. used MSPD-GC 
GC-mECD system for the determination of a variety of organophosphorus
pesticides, triazines and pyrethroids in different fruits (orange, pear, grape,
apple) [53]. The MSPD method consisted in the use of 100-mg of homo-
genised fruit peel, apart from the grapes that were analysed in total, mixed
with the same quantity of C8-bonded silica, and then packed in a 3-mL
SPE cartridge; the SPE device was then washed with 15 mL of water and
elution was performed by using 0.7 mL of ethyl acetate. Method sensitivity
was good, with the LoDs values lower than 1 mg/kg.
UAE uses mechanical energy produced by low-frequency sound waves
to speed up extraction, often with high yields. The two main UAE
approaches are based on the presence of a water bath or probe. Even though
the former option is the most popular one, the ultrasonic probe can be in
direct contact with the sample or can better focus its energy to the sample,
greatly reducing the UAE time [54]. Sometimes the process requires an
additional cleanup step. Morales-Mu~ noz et al. described the use of dynamic
UAE, combined with GC  GC-LR-ToF-MS, for the analysis of contam-
inants in marine sediments [55]. The UAE system was characterized by the
presence of a probe placed at 1 mm from the surface of the extraction cham-
ber and the 6 mL of solvent (hexane) were pumped forwards and backwards
through the chamber for 15 min, prior to collection in a vial. Conventional
SE was also performed, requiring a much higher content of solvent (80 mL
of hexane) and an extraction period of 24 h. From a comparison of the two
methods, UAE recoveries and repeatability values were better than conven-
tional SE.
MAE is based on the use of microwave-generated energy to solvent-
extract analytes from a matrix. The main advantage of MAE is the rapid
heating of the sample-solvent mixture, leading to short extraction times
Green Sample-Preparation Techniques 615

and to the relatively low consumption of organic solvents. When MAE is

performed in a closed vessel, the extraction temperature can exceed the
boiling point, as in PLE, further enhancing extraction efficiency. Addition-
ally, several samples can be subjected to MAE simultaneously, hence
increasing sample throughput [56]. A comparison between MAE, PLE
and UAE was made by Kristenson et al. for the GC  GC-mECD analysis
of polychlorinated biphenyls (PCBs) in sludge [57]: all the three types of
extracts were treated with copper prior to GC analysis to reduce the
amounts of S-containing interferences. UAE was characterized by the best
recoveries (60%e100%) and precision (RSDs ¼ 2%e11%), despite affected
by longer extraction period compared to MAE; on the other hand, PLE
gave the lowest recoveries (45%e75%), probably related to the higher
solventesample ratio (3.3) compared to MAE (2), and to the lower extrac-
tion time compared to UAE.
DLLME is based on the use of small volumes of an extraction and
disperser solvent, in aqueous samples and was first described in 2006 by
Rezaee et al. for the determination of organic compounds in water [58].
To the best of our knowledge, the only DLLME-GC  GC work was
reported by Tugizimana et al. for monitoring of ergosterol-induced metab-
olite changes in Nicotiana tabacum cells [59]. The employed DLLME
GC  GC coupled to LR ToF-MS method enabled a clear differentiation
between control and ergosterol-treated cells.

3. CAPILLARY COMPREHENSIVE TWO-DIMENSIONAL

LIQUID CHROMATOGRAPHY
LC  LC has scattered a great attention in the last two decades due to
its indisputable advantages over conventional 1D-LC [60e62]. LC  LC
performed at nano/capillary scale can greatly increase the sensitivity of the
overall method and can be considered a ‘green’ analytical tool whenever
the sample preparation and separation involve only a limited amount of
organic solvents (<10 mL). Nano/capillary LC  LC also renders the
hyphenation to MS more useful than conventional 1D-LC, and most of
applications have been so far devoted to proteomics, clinical biochemistry,
environmental and food.
Nagele et al. [63] analyzed a yeast proteome on an LC  LC setup
comprised of a capillary strong cation exchange column (35  0.3 mm
I.D.) in the 1D and a nano C18 column (150  0.75 mm I.D.) in the 2D.
In the switching configuration a 6-port and a 10-port switching valve
616 Francesco Cacciola et al.

with two C18 trap columns (5  0.3 mm) were used to transfer and precon-
centrate the analytes from the 1D before the RP-LC separation in the 2D. A
similar setup was investigated by Wang et al. for proteome analysis of
hepatocellular carcinoma (HCC) [64], in which 229 proteins of the analyzed
tissue were identified and among them several of them were found to be
related with the development of the HCC disease. An opposite combination
with a nano LC column in the 1D and a capillary in the 2D in a typical
‘continuous flow’ LC  LC arrangement was used by Haunet et al.
(Fig. 3). [65]. The 10-port 2-position switching valve had two loops
alternatively filled at nanoflow rate by the effluent from the 1D: the
two loops were alternatively switched into the 2D capillary flow during a
1-min long modulation cycle. An automated nano LC  LC-MS system
was used by Pinkse et al. [66] for analyzing peptides fractionated on a
TiO2-C18 precolumn in the 1D. Phosphopeptides were retained on the
TiO2 precolumn while nonphosphopeptides were trapped on the C18
phase. First, the linear watereacetonitrile gradient eluted only the nonphos-
phopeptides from the 1D, and it enabled their separation in the 2D on the
nano C18 column. Phosphopeptides were eluted from the TiO2 precolumn
by an ammonium bicarbonate solution and separated in a second-gradient
run. Such configuration works as a vented column where one six-port valve
opens or closes the vent and closes or opens the splitter line. The same prin-
ciple involving a 2D precolumn in a vented column setup was also used in
other more recent works [67e69]. Off-line approaches for protein analysis
based on a combination of hydrophilic interaction liquid chromatography
and RP-LC were used by Boersema [70] and Di Palma [71]. These setups
employed a 0.1-mm precolumn and a nano (0.075-mm I.D.) column.
The fractions were collected on a 96-well plate and subsequently analyzed
by RP-LC. Finally, in 2012 a fully automated capillary LC  LC method
based on the use of high-pH RP with low-pH RP separations, in the 1D
and 2D, respectively, was developed for the first time for proteomic analysis
by Sommella et al. [72]. By means of such an approach despite the use of
identical stationary phases, high peak capacity values were obtained with
negligible mobile-phase consumption and enhanced sensitivity.

4. CONCLUSIONS
Apart from SPME and SPE combined with GC  GC separations,
which are undoubtedly the most established ones (roughly 50% of the total
 Green Sample-Preparation Techniques
Figure 3 (A) and (B) Contour plots of the total ion current chromatograms showing the LC  LC separations of a 99-component standard
mix and a wastewater sample, respectively. (C) and (D) Analyte maps for the standard mix and the targets in the wastewater sample, respec-
tively, based on the analytes’ peak maxima presented as C. 1D double peaks are highlighted by þ and D for first and second maximum,
respectively. Reproduced from J. Haun, J. Leonhardt, C. Portner, T. Hetzel, J. Tuerk, T. Teutenberg, T.C. Schmidt, Online and splitless nano-

617
LC  capillary LC with quadrupole/time-of-flight mass spectrometric detection for comprehensive screening analysis of complex samples, Anal.
Chem. 85 (2013) 10083e10090. Copyright 2013 American Chemical Society.
618 Francesco Cacciola et al.

works present in literature), only a limited part of the other published works
involved the use of other green sample-preparation methods prior to
GC  GC analysis. SPME-GC  GC applications, provide very high
sensitivity, selectivity, separation power and identification potential, and
the majority were carried out in ‘untargeted’ mode as it usual in metabolo-
mics studies; however, the final number of green sample-preparation GC
applications is much higher compared to the ones achieved by GC  GC
ones and, thus it can be concluded that nowadays the greatest amount of
green sample-preparation research is performed in the GC field. Besides,
considering the GC  GC transfer devices, it should be emphasized that
cryogenically modulated GC  GC cannot be considered truly as a green
approach, due to its high operational costs (mainly due to the consumption
of cryogenic fluids); on the other hand, environmentally friendly forms of
GC  GC, e.g., flow-modulation systems, could represent a viable alterna-
tive. On the other hand, LC  LC performed at nano/capillary-scale is a
valuable ‘green’ tool due to enormous reduction of organic solvent
whenever the sample preparation involves only a limited amount of organic
solvents (<10 mL) allowing to greatly increase the sensitivity of the overall
method. It is predictable that both GC  GC and LC  LC applications
performed at nano/capillary scale will grow in the future being further a
great match for straightforward hyphenation to MS methods.

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FURTHER READING
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[2] B. Xu, P. Li, F. Ma, X. Wang, B. Matthaus, R. Chen, Q. Yang, W. Zhang, Q. Zhang,
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