2334 J. Sep. Sci.

2010, 33, 2334–2340

Giorgia Purcaro1,2 Research Article

Peter Quinto Tranchida1
Paola Dugo1,3
Erminia La Camera4 Characterization of bacterial lipid profiles by
Giuseppe Bisignano4
Lanfranco Conte2
using rapid sample preparation and fast
Luigi Mondello1,3 comprehensive two-dimensional gas
1
Dipartimento Farmaco-chimico, chromatography in combination with mass
Facoltà di Farmacia, Università
2
di Messina, Messina, Italy spectrometry
Dipartimento Scienze degli
Alimenti, Università di Udine,
Udine, Italy The bacteria fatty acid profile has been extensively studied for taxonomic classification
3
Campus-Biomedico, Roma, Italy purposes, since bacteria, in general, contain particular and rare fatty acids, compared with
4
Dipartimento Farmaco- animal and plant tissues. As for any real-world sample type, the development of rapid and
biologico, Facoltà di Farmacia,
Università di Messina, Messina,
reliable methods for (i) sample identification (in this case, bacterium type), and (ii)
Italy constituent identification (in this instance, the fatty acid profile) is desirable. In this
research, a half-an-hour procedure, to analyze bacteria, was developed: a 2-min one-step
sample preparation step was followed by a relatively fast comprehensive 2D GC-MS
Received March 4, 2010
separation (25 min). Furthermore, dedicated MS libraries were constructed for the iden-
Revised April 28, 2010
Accepted May 16, 2010 tification of bacteria and fatty acids. Finally, data processing, only qualitative at this stage,
was carried out with the support of a novel comprehensive 2D GC software.

Keywords: Bacterial fatty acid methyl esters / Comprehensive 2-D GC / Lipids /

MS / Split flow
DOI 10.1002/jssc.201000160

1 Introduction Comprehensive 2D GC has been applied successfully in

Comprehensive 2D GC (GC  GC) was introduced in 1991,
and can be certainly considered as one of the most
important inventions in the GC field [1]. The peak capacity
(the number of peaks that can be potentially resolved in the
chromatography space) gain, compared with conventional
GC, is greater than that between the packed and the open-
tubular capillary column.
The main advantages of comprehensive 2D GC, over
conventional GC, are (i) selectivity (two separation dimen-
sions, related to volatility and polarity); (ii) sensitivity (band
compression); (iii) separation power; (iv) structure (forma-
tion of group-type patterns on the 2D plane); (v) speed (in
terms of number of peaks resolved per unit of time).
The hyphenation of GC  GC with an MS detector,
which is a third analytical dimension, generates the most
powerful tool available today for volatile analysis [2].
Correspondence: Luigi Mondello, Dipartimento Farmaco-chimi-
co, Facoltà di Farmacia, Università di Messina, viale Annunziata,
98168, Messina, Italy
E-mail: lmondello@unime.it
Fax: 139-090-388220
Abbreviations: BAME, bacterial fatty acid methyl ester; FA,
fatty acid; FAME, fatty acid methyl ester; LRI, linear retention
index
many research areas. In particular, enhanced sensitivity and
formation of structured chromatograms have proven
to be useful for the analysis of fatty acid methyl esters
(FAMEs) contained in fish and vegetable oils [3–5], milk
fat [6], plasma [7], micro-algae and aquatic meiofauna
species [8].
Recently, David et al. [9] exploited GC  GC-FID for the
study of bacterial fatty acids (FAs). Precious information was
attained through highly structured chromatograms, and
the data derived were compared with that attained by
using the standardized Sherlock MIDI system [10]
(www.midi-inc.com). The latter process is based on the
careful control of growth conditions (24 h), before harvest-
ing the bacterial cells, and processing them through sapo-
nification, methylation and extraction (the procedure takes
about 1 h). Bacterial fatty acid methyl esters (BAMEs) were
identified on the 2D space plane by using two parameters,
namely equivalent chain length in the first dimension and
relative polarity value in the second dimension.
Bacterial lipids are characterized by uncommon
FAs, such as odd-chain, branched-chain (mainly iso- and
anteiso-), cyclopropane and hydroxy FAs; furthermore, also
the more common FAs, such as for instance C18:1,
differ from those of vegetable origin (e.g. cis-vaccenic
acid (C18:1o7) instead of oleic acid (C18:1o9)). The
exploitation of the characteristics of GC  GC, for the
elucidation of such particular FA profiles, appears to be
interesting.

& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jss-journal.com
J. Sep. Sci. 2010, 33, 2334–2340
Since 1963, when Abel et al. first described the
GC analysis of BAMEs, FA profiles have been widely
used for the taxonomic classification of microorganisms
[11–13]. It is important to note that the FA composition,
both in qualitative and in quantitative terms, is markedly
affected by the nature of the medium, by the growth
conditions, as well as by the age of the culture when
harvested. Therefore, the knowledge and the standardiza-
tion of these conditions are essential for valid and repeatable
studies.
The aim of this research is the development of a rapid
approach to identify bacteria, and elucidate BAME profiles.
A single-step, rapid methylation procedure (2–3 min), using
a trimethylsulfonium hydroxide methanol solution, was
employed [14], followed by a relatively rapid GC  GC-MS
analysis.
A typical GC  GC system consists of a primary
conventional column (e.g. 30 m  0.25 mm id  0.25 mm df)
and secondary column, usually a short micro-bore column
(e.g. 1 m  0.10 mm id  0.10 mm df) linked in series, and
with independent selectivities. However, it has been
demonstrated that the potential of GC  GC is only partially
expressed under these conditions, and even worse when
narrower columns (namely 0.05 id) are employed, because
gas linear velocities are generally ideal (or near to optimum)
in the first dimension but far from ideal in the second one.
Tranchida et al. [15–18] have reported the use of a split flow
system to obtain close-to-optimal gas linear velocities in
both dimensions.
In this study, a split flow, twin-oven GC  GC-MS
system was employed, enabling the use of a micro-bore
column set (apolar (0.1 mm id)  polar (0.05 mm id)), under
close-to-optimal gas linear velocity conditions. Furthermore,
dedicated GC  GC-MS libraries were developed to obtain
unambiguous bacteria and BAME identification. Data
processing was carried out with the support of a recently
developed comprehensive chromatography software,
described in more detail elsewhere [18].
2 Materials and methods
2.1 Chemicals and samples
All the reagents employed were purchased from Sigma-
Aldrich (Milano, Italy). The BAMEs mixture was kindly
provided by Supelco (Bellefonte, PA, USA) and was used to
optimize the GC  GC-MS method. The FAMEs used to
construct the GC  GC BAME library, and the GC-MS
FAME library, were from Supelco and Larodan (Malmö,
Sweden).
Five different strains of bacteria were studied in this
work, namely Pseudomonas fluorescens, Bacillus subtilis,
Staphylococcus aureus, Escherichia coli and P. aeruginosa and
were supplied by the Laboratory of Microbiology of the
University of Messina.
& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Gas Chromatography 2335
2.2 Sample preparation
The bacteria were grown on R2A Agar at 281C for 24 h. Lysis
of bacteria and transesterification of the FAs were carried
out in a rapid single step, according to Müller et al. [14].
Briefly, a few colonies of bacteria were harvested and
suspended in 10 mL of distilled water, and then 30 mL of
methanolic trimethylsulfonium hydroxide solution (0.25 M)
were added. The reaction mixture was dried under nitrogen
stream and then redissolved in 200 mL of tert-butyl-methyl
ether/methanol (MeOH) mixture (10:1 v/v) and directly
injected into the GC  GC-MS system.
2.3 Instrumentation
The GC  GC-MS applications were carried out on a
Shimadzu GC  GC-MS system, consisting of two indepen-
dent GC2010 gas chromatographs, connected through a
heated (2801C) transfer line, and a QP2010 Plus quadrupole
mass spectrometer (Shimadzu, Kyoto, Japan) linked to the
primary GC through a cable extension (due to the presence
of the second oven).
The GC  GC-MS system was equipped with an AOC-
20i auto-injector, a split–splitless injector (2501C). The
primary column (situated in GC1), an SLB-5 ms (silpheny-
lene polymer) 11.4 m  0.1 mm id  0.1 mm df, was
connected to a custom-made Supelcowax-10 1 m  0.05 mm
id  0.05 mm df poly(ethylene glycol) capillary and to a
0.20 m  0.05 mm uncoated capillary (both columns, provi-
ded by Supelco, were located in GC2), by using a fixed outlet
capillary column splitter (SGE, Ringwood, Victoria,
Australia). The uncoated capillary was connected to a
manually controlled valve, namely an OSS-2 outlet splitter
system (SGE).
The final part of the primary column was used to create
a double loop for the cryogenic dual-stage, loop-type
modulator (under license from Zoex, Houston, TX, USA).
The modulation time applied was 4 s and the duration of the
hot pulse (3251C) was 375 ms.
Optimized split flow GC  GC-MS: GC1 temperature
program: 130–2801C at 51C/min. GC2 temperature program:
160–2801C at 51C/min. Initial He pressure (constant linear
velocity): 552.3 kPa, which corresponds to about 45 and
70 cm/s linear velocity in the first and second column,
respectively. Injection volume: 0.1 mL; split ratio: 10:1.
The MS transfer line and ion source were maintained at
250 and 2001C, respectively. The scan range was of m/z
40–360, at a scan speed of 10 000 amu/s, and a sampling
frequency of 25 spectra/s. MS ionization mode: electron
ionization (70 eV).
Data were collected by the GCMS solution software;
bidimensional visualization was carried out by using the
ChromSquare v.1.0s software (Chromaleont, Messina,
Italy). The MS libraries used for spectral matching were
laboratory constructed.
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2336 G. Purcaro et al.
3 Results and discussion
3.1 GC  GC optimization
The GC  GC apolar–polar column combination, for FAME
analysis, enables the attainment of highly structured 2D
chromatograms and has been commonly reported in the
literature [3–5]. The physical dimensions of each column
(11.4 m  0.1 mm id 1 1 m  0.05 mm id) were chosen on
the basis of the necessity to develop a relatively fast
GC  GC-MS technique. Moreover, an uncoated capillary
(0.2 m  0.05 mm id) was used to divert part of the first
dimension effluent to waste, thus enabling the generation of
the desired linear velocities in both dimensions. The
advantages of splitting flows in GC  GC have been
demonstrated using both flame ionization [15–17] and MS
[18] detection, in comparative studies with non-split flow
experiments.
In the present split flow, twin-oven GC  GC-MS
application, various experiments were carried out by
manually regulating the split valve at different stages. The
best operational conditions were attained with the split valve
completely opened: linear velocities of ca. 45 and 70 cm/s
were calculated in the first and second dimensions,
respectively. It must be emphasized that these calculations,
due to nonideal gas behavior, must be considered only as
approximations. The relatively high (but ideal) first-dimen-
sion He velocity was combined with a relatively accelerated
temperature program (130–2801C at 51C/min), to produce a
rather fast GC  GC separation (the last compounds of
interest eluted within 25 min). However, the operational
conditions applied generated rather narrow primary column
chromatography bands (6–9 s) and, hence, a short modula-
tion period (4 s) was necessary to achieve a near-to-sufficient
number of cuts per peak. In fact, each peak was modulated
ca. two times and, hence, a certain degree of resolution was
lost in the first dimension. However, the selectivity of the
primary column is not high toward BAMEs, whereas the
secondary column is much more selective. For this reason
non-ideal modulation conditions were tolerated, and the
attention was directed to the optimization of the separation
in the second dimension.
Initially, a split flow GC  GC-MS experiment was
carried out on a standard solution, containing 26 methyl
esters and using the same temperature program in both
ovens (data not shown). Although the GC  GC separation
performance was good, wrap around occurred due to the
low primary column elution temperatures. Consequently, a
series of positive temperature offsets were tested in GC2,
with the best result attained through the application of
1301C offset. A bidimensional chromatogram expansion,
relative to the optimized split flow GC  GC-MS application,
is shown in Fig. 1.
As can be observed, the contour plot is characterized by
an ordered structure: saturated BAMEs (C11:0–C20:0) are
located in the non-polar zone of the chromatogram, and are
aligned along a distinct band; methyl esters with one bond
& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
J. Sep. Sci. 2010, 33, 2334–2340
(C16:1–C19:1) are also aligned horizontally and are slightly
more retained than the saturated counterparts. The only
methyl ester with two double bonds (C18:2) is, as expected,
slightly more retained than the monosaturates. The hydroxy
BAMES, characterized by intense H-bond interactions with
the poly(ethylene glycol) phase, are situated in the most
polar zone of the chromatogram.
The rapid-scanning quadrupole mass spectrometer was
operated using an m/z 40–360, a 10 000 amu/s scan speed
and a 25 Hz sampling frequency. Such MS parameters
enabled the attainment of an average six to eight spectra per
peak. The minimum number of data points per peak is
widely discussed in the literature. There is a reasonable
agreement on ten data points per peak, including the base-
line points, for a satisfactory peak reconstruction and
quantification [19]. The six to eight spectra per peak obtained
in the present application enabled reliable peak identifica-
tion but still fell short of the requirements for reliable
quantification.
3.2 GC  GC-MS library construction
Once optimized, the split flow GC  GC-MS method, the
same was used to construct two dedicated MS libraries. The
first, named GC  GC BAME library, contained pure MS
spectra relative to FAs (methyl esters) commonly found in
bacterial lipid extracts, and linear retention index (LRI)
values. Initially, mixtures of standard FAMEs were
subjected to GC  GC-MS analysis and the spectra attained
were included in the library. After, the LRI values of the
methyl esters were automatically calculated by using the
comprehensive chromatography and GC-MS software, on
the basis of the retention times of a C7–C30 alkane series,
coanalyzed with the standard compounds. At present, the
GC  GC BAME library contains MS spectra and LRI values
relative to ca. 80 BAMEs.
The new comprehensive chromatography software is
directly linked with the GC-MS software used for acquisi-
tion. Therefore, it is possible to perform the MS library
search from the former software through the tools of the
latter one (more details about the software are reported in
[18]), which enables the application of a twin-filter:
(i) spectral similarity and (ii) LRI range. The primary filter
eliminates GC  GC BAME library matches with a spectral
similarity (expressed in %) lower than a minimum value set
by the analyst; the other filter deletes GC  GC BAME
library spectra, characterized by an LRI value outside a
predefined range, and with respect to the LRI value of the
unknown compound. The twin-filter is very useful when
classes of structurally similar compounds are analyzed: for
example, FAs such as C16:0, iso-C16:0 and anteiso-C16:0
have very similar mass spectra, and therefore the LRI filter
is necessary for identification (Fig. 2).
The other MS library, named as Bacteria Library, is in a
very early stage of development. The library was constructed
by (i) subjecting real bacterial FA samples to the fast
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J. Sep. Sci. 2010, 33, 2334–2340 Gas Chromatography 2337

sec

3.20
3-OH-C14:0
3-OH-C12:0

2-OH-C16:0

2.40
2-OH-C14:0
2-OH-C12:0

c9,10-C19:0
c-C18:1
C16:1 c9 10 C17:0
c9,10-C17:0 C18:2
1.80 2-OH-C10:0

C20:0
C20 0
i-C16:0 C18:0
0.80 C14:0 C19:0
C16:0 i-C17:0 C17:0 tr-C18:1
C13:0
C15:0
C12:0
Figure 1. GC  GC-MS chromatogram of
C11:0 i-C15:0
a standard mixture of BAMEs. i, iso; a,
a-C15:0
0.00 anteiso; tr, trans; c9,10-C17:0, methyl
2.80 5.80 8.40 11.20 14.00 min cis-9,10-methylenehexadecanoate.

Inten.(x10,000)
1.00 74
C16 0:0
0.75
87
0.50 LRI: 1925
43
0.25 55
143 227
97 129 171 185 199 239 270
115 157 213
0.00
50.0 75.0 100.0 125.0 150.0 175.0 200.0 225.0 250.0 m/z

Inten.(x10,000)
1.00 74
iC16:0
0.75
87 LRI: 1888
0.50
43
0.25 55
143 227
97 129 157 171 185 199 270
115 213 241
0.00
50.0 75.0 100.0 125.0 150.0 175.0 200.0 225.0 250.0 m/z

Inten.(x10,000)
1.00 74

aC16:0
0.75
87
0.50 LRI: 1894
43 55
0.25 143
97 115 227 270 Figure 2. MS spectra of C16:0,
111 129 171 185 199 213 241
157
0.00 iso-C16:0, anteiso-C16:0, with LRI
50.0 75.0 100.0 125.0 150.0 175.0 200.0 225.0 250.0 m/z values.

GC  GC-MS method, (ii) deriving a single averaged spec- print, and is altogether comparable (in terms of concept) to
trum, comprising all the methyl esters in the retention time direct electron-ionization-MS analysis. The averaged spec-
range and (iii) finally subtracting the compressed chemical trum was derived from the untransformed GC  GC-MS
noise at three points across the chromatogram. The bacterial chromatogram, using the instrumental GC-MS software. In
spectrum attained can be considered as a sample finger- Fig. 3, there is an example of a bacterial spectrum

& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jss-journal.com
2338 G. Purcaro et al. J. Sep. Sci. 2010, 33, 2334–2340

(P. fluorescens). At present, the library contains only five
spectra (the same bacteria number analyzed in this study),
because it is currently in the testing stage. Consequently,
the results herein reported, using the GC  GC Bacterial
Library, are to be considered as preliminary.
3.3 Real sample analysis
Five different bacteria strains were analyzed by using a rapid
one-step method, previously described by Müller et al. [14].
During the sample preparation process, a C7–C30 alkane
series was subjected to GC  GC-MS analysis. A GC  GC-
MS chromatogram expansion, relative to a bacterial sample,
is shown in Fig. 4. The averaged spectrum of the
untransformed GC  GC-MS chromatogram was subjected
to library matching, after subtraction of the baseline in three
different points: the bacterium was identified as E. coli with
a 98% similarity (Fig. 5). The considerable difference,
between the E. coli spectrum and the other bacteria library
spectra, can be deduced observing the second best match
provided by the GC-MS software (66% similarity).
Once the bacterium was correctly identified, then, the
FAs’ profile was investigated. Peak assignment was auto-
matically achieved by using a recently developed compre-
hensive chromatography (ChromSquare v.1.0) program, in
combination with the GC-MS software, and the GC  GC
BAME library. As aforementioned, the GC-MS software
enabled the application of a dual-filter, allowing the elim-
ination of all matches with an: (i) MS similarity below a
predefined limit (90% in this case); (ii) LRI value outside a
predefined range, and with respect to the unknown
compound (710 LRI units in this case). The research
process was carried out using both the GC  GC BAMEs
library and a GC-MS FAMEs library; the latter was
constructed some time ago, using a conventional SLB-5 ms
capillary and containing more than 200 compounds. The
MS similarity (MS %) and LRI values, for the 16 methyl
esters identified in the E. coli sample, derived from both of
the previously mentioned libraries, as well as the differences
from the experimental LRI values, are listed in Table 1.
Three FAs, viz. C20:1n7, C22:1n10 and C22:0, were identi-
fied by decreasing the MS% filter value to 80%, due to the
low on-column amounts of these constituents. As can be

Inten.(x10,000)
1.00 45
P.fluorescens
0.75 74
87
0.50 Figure 3. MS averaged spec-
55
trum of P. fluorescens, derived
0.25 75 97 from the untransformed
42 111 129 143
171 185 199 207 227 GC  GC-MS chromatogram,
0.00 after subtraction of the base-
50.0 75.0 100.0 125.0 150.0 175.0 200.0 m/z
line in three different points.

sec

3.193
3-OH C14:0

2.395

C16:1n7
C18:1n9
1.596 C17:1n9 C22:1n10
C9,10-C18
C20:1n7

C22:0
C20:0

0.798 C17:0 C18:0

C16:0
C13:0 C15:0
C14:0
C12:0

Figure 4. GC  GC-MS chromatogram of a

0.000 real-world bacteria sample (E. coli). For
3.587 7.173 10.760 14.347 17.933 min peak identification refer to Table 1.

& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jss-journal.com
J. Sep. Sci. 2010, 33, 2334–2340 Gas Chromatography 2339

Figure 5. Bacterium MS search

result for the GC  GC-MS
chromatogram shown in Fig. 4.

strictly necessary. Some characteristic BAMEs were identi-

Table 1. BAMEs identified in the GC  GC-MS chromatogram
shown in Fig. 4, along with library match similarities
(MS %) derived from the GC  GC library and the
monodimensional GC library, average experimental
LRI values (n 5 3), and the differences between the
latter and the LRI values reported in the GC  GC
library and in the monodimensional GC library,
respectively
Compound GC  GC Lib GC Lib
fied, such as hydroxyl (3-OH C14:0) and cyclopropanic (cis-
9,10-C19:0) ones. Three consecutive applications were
carried out on the E. coli sample. The repeatability of the
LRI values, and of the first and second dimension retention
times, were evaluated, in terms of SD. All the 16
compounds identified shown a SD below 0.04 min and
0.05 s in the first and second dimensions, respectively.

MS LRI Diff. MS LRI Diff. 4 Concluding remarks

% Exp (n 5 3) % Exp (n 5 3)
The objective of this phase of research was to determine
Me. C12:0 96 1522 4 96 1523 3
whether the development of a fast GC  GC-MS method,
Me. C13:0 96 1624 2 96 1624 2
Me. C14:0 94 1726 2 96 1724 4
supported by two novel MS libraries, was suitable for the
Me. C15:0 94 1826 4 95 1825 5 rapid characterization of bacteria. The results attained can
Me. 3-OH C14:0 91 1873 6 94 1870 9 be considered as satisfactory. Lysis of the bacteria cells and
Me. C16:1n7 91 1901 7 93 1903 5 methylation of the FAs were carried out in a very brief single
Me. C16:0 94 1923 9 95 1925 7 step and the GC  GC-qMS analysis lasted less than 20 min.
Me. C17:1n7 92 2008 5 94 2009 4 Moreover, retention times both in the first and in the second
Me. C17:0 95 2024 5 95 2026 3 dimension, and the similarity match gave good repeatability
Me. C18:1n9 92 2104 5 94 2105 4 results.
Me. C18:0 95 2125 5 95 2126 4 In truth, the lab-constructed GC  GC BAME library was
Me. c9,10-C17:0 92 2208 5 94 2207 6
not strictly necessary, since good results were obtained by
Me. C20:1n7 87 2301 8 87 2300 9
using the monodimensional GC FAME library. Consequently,
Me. C20:0 90 2329 1 90 2326 4
Me. C22:1n10 87 2510 0 88 2510 0 the FAME library can be used and expanded without the
Me. C22:0 82 2533 -1 82 2528 4 additional costs related to the consumption of liquid nitrogen.
Finally, although the results attained from the Bacteria
Library were satisfactory, they must be considered as preli-
seen, the LRI differences, for all of the compounds identi- minary. Many more bacterial samples must be analyzed, MS
fied, were below 710 units. This good agreement demon- fingerprints derived, using standardized growth and collec-
strates that a dedicated library for GC  GC analysis is not tion conditions.

& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jss-journal.com
2340 G. Purcaro et al.
The authors gratefully acknowledge Shimadzu Corporation
and Supelco Corporation for the continuous support. Giorgia
Purcaro acknowledges Chromaleont for the financial support.
The authors have declared no conflict of interest.
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