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Comprehensive two-dimensional
gas chromatography for theanalysis of organohalogenatedmicro-contaminantsPeter Korytar, Peter Haglund, Jacob de Boer, Udo A.Th. Brinkman
We explain the principles of comprehensive two-dimensional gas chroma-
tography (GC GC), and discuss key instrumental aspects with emphasison column combinations and mass spectrometric detection. As the main item
of interest, we review the potential of GC GC for the analysis of organo-
halogenated micro-contaminants, and highlight its superiority over conven-
tional 1D-GC. We present results for 12 compound classes, including
polychlorinated biphenyls, dibenzo-p-dioxins and furans, and n-alkanes,
toxaphene and polybrominated diphenyl ethers. We draw attention to target
analysis as well as within-class and between-class separations.
2006 Elsevier Ltd. All rights reserved.
Keywords: Comprehensive two-dimensional gas chromatography; GC GC; Mass
spectrometry; Organohalogenated compound; Separation
1. Introduction
In 1966, the Swedish chemist Jensen [1]
announced that he had identified a group
of organochlorine compounds that was
accumulated to toxic concentrations in
nature. Unlike the closely related chlori-
nated pesticides, such as DDT and the
various drins which had, so far,
attracted most attention of environmental
chemists the newly detected compounds,
the polychlorinated biphenyls (PCBs), had
entered the environment essentially
unintentionally. Since that time, these twoclasses of persistent organic pollutants,
which shared high annual production,
widespread usage, long persistence and
serious toxic effects, have been the subject
of a rapidly increasing number of funda-
mental as well as applied studies. Over the
years, many related classes of compounds
have been added to the list; Table 1 gives
an overview, and also lists the number of
theoretically possible congeners, which
gives an impression of the degree of com-
plexity that can be expected in technical
mixtures and, thus, in environmental and
food samples. For the rest, we assume that
the general reader will be familiar with the
main characteristics and usage of the
various classes of organohalogens and, at
least in some cases, with the catastrophes
or otherwise, which led to their being
considered priority pollutants by many
governments and international (UN, EU)
bodies.
During the entire past half-century, gas
chromatography with electron-capture
Peter Korytar*
Netherlands Institute for Fisheries Research,
P.O. Box 68, NL-1970 AB IJmuiden,
The Netherlands
Free University, Department of Analytical Chemistry and Applied Spectroscopy,
de Boelelaan 1083, NL-1081 HV Amsterdam,
The Netherlands
Peter Haglund
Umea University, Department of Chemistry, Environmental Chemistry,
SE-901 87 Umea, Sweden
Jacob de BoerNetherlands Institute for Fisheries Research,
P.O. Box 68, NL-1970 AB IJmuiden,
The Netherlands
Udo A.Th. Brinkman
Free University, Department of Analytical Chemistry and Applied Spectroscopy,
de Boelelaan 1083, NL-1081 HV Amsterdam,
The Netherlands
*Corresponding author.
E-mail: [email protected]
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detection (GCECD) was the predominant analytical
technique for selective and sensitive determination of
organohalogens. In the early part of that period, when
packed-column GC was the only means of separation
available, total-PCB determination (based on comparing
an environmental sample with a technical PCB mixture)
was all that could be achieved. The introduction of
(fused-silica) capillary columns was a real breakthrough,
which, all of a sudden, enabled congener-specific deter-
mination of the PCBs and, of course, also of the other
classes of organohalogens. However, it also became clear,
after this major step forward, that capillary GC was notthe solution for all, or even most, problems. Admittedly,
single-column (or one-dimensional, 1D) GC can provide
the required resolution when the number of target ana-
lytes is restricted (e.g., with the organochlorine pesticides
(OCPs) or polychlorinated naphthalenes (PCNs)).
However, even for the PCBs with their still moderate
number of 209 congeners, or for the about 140 CB
congeners present in a technical mixture, no satisfactory
1D-GC solution could be found. And, not surprisingly,
correspondingly more serious problems were encoun-
tered with complicated mixtures, such as toxaphene,
polychlorinated terphenyls (PCTs) and, specifically,
polychlorinated alkanes (PCAs); the PCAs typically show
up in 1D-GC as an essentially unresolved broad band
covering a major part of the baseline. The magnitude of
the problem becomes even clearer when one considers
that the present discussion is about within-class separa-
tions only: interferences caused by other organohalogens
and/or matrix constituents or problems due to widely
different concentrations of (partly) co-eluting congeners
have not been taken into account.
Over the years, several approaches have been used to
alleviate the problems. One of these was sample frac-
tionation (e.g., by size-exclusion or adsorption liquid
chromatography). Another was to filter out interferences
by selective mass spectrometric (MS) detection, which is
powerful but fails to resolve co-elutants with closely
similar mass spectra, as is often the case for congeners of
the same compound class, but also of different classes.
On the GC side, one way to go was the parallel use of
several stationary phases and the combination of infor-mation from the GC runs on different columns to solve
specific separation problems. It was a step further to
combine two different columns in a single set-up: in so-
called multi-dimensional GC (MDGC), selected fractions
from the first column are subjected to a second GC run
that uses a completely different separation mechanism.
Many successful applications have been reported, but one
should keep in mind that, in essentially all cases, they are
of a heart-cutting nature: only a single, or at best a few,
small fractions are transferred to the second column for
further separation. That is, it is an excellent solution
when information is required about a few target analytes,
but not about the entire sample. In the latter instance (i.e.with complex samples and/or when unknowns have to be
traced), MDGC becomes much too complicated and time-
consuming. It is in these situations and their number
can confidently be said to be increasing daily that a so-
called comprehensive approach is needed: MDGC, or GC
GC, is now replaced by GC GC, with which, instead of a
few selected fractions, the entire sample is subjected to
separation on two different columns. One immediate
advantage is that the information content is much
greater, and another is that the GC GC run is ready once
the first-dimension run is finished; that is, GC GC is
much more efficient than MDGC.It is the goal of this review briefly to explain the
principle of GC GC and to discuss key aspects, such as
column-to-column interfacing or modulation, detection
and detector requirements (including a comparison of
time-of-flight MS (TOF-MS) and fast-scanning quadru-
pole MS (qMS) instruments) and the selection of properly
matched column combinations.
We will devote most attention to applications in the field
of organohalogenated micro-contaminants, which will
also be used to highlight the added value of GC GC
compared with 1D-GC. Readers who are interested in a
more detailed review of the various aspects of GC GC and
in applications to compound classes other than organo-
halogens, should consult two extensive reviews [2,3].
2. GC GC: general principle
In GC GC, the entire sample is subjected to two GC
separations that are based on different separation
mechanisms. Fig. 1 shows a schematic of a GC GC
system. In most instances, the sample is first separated
on a high-resolution capillary GC column typically a
1530 0.250.32 mm ID, 0.11 lm df column
Table 1. Main classes of polyhalogenated micro-contaminants
Name Acronym Maximumnumber ofcongenersa
Polychlorinated biphenyls PCBs 209Polychlorinated dibenzo-p-dioxins PCDDs 75
Polychlorinated dibenzofurans PCDFs 135Toxaphene components Toxaphene 61 696
bornane congeners 16 640camphene congeners 12 288dihydrocamphene congeners 32 768
Organohalogenated pesticides OCPs ca. 300Polychlorinated terphenyls PCTs 8149Polychlorinated diphenylethers PCDEs 209Polychlorinated naphthalenes PCNs 75Polychlorinated alkanes PCAs Very highPolybrominated biphenyls PBBs 209Polybrominated diphenylethers PBDEs 209
aEnantiomers not included.
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containing a non-polar stationary phase. As will be
explained in Section 3, an interface called a modulator is
used to separate the first-column eluate into a very large
number of adjacent small fractions. To maintain the
first-column separation, these fractions should be no
larger than one quarter of the peak width, or r, in that
dimension. In order to meet this so-called modulation
criterion, temperature programming in GC GC is
slower than in 1D-GC, and typically occurs at a rate of
13C/min. Each individual fraction is trapped, re-
focused and, next, launched into the second GC column,
which is much shorter and narrower than the first one
typical dimensions are 12 m 0.1 mm ID 0.1 lm df.
The second-column separation generally is of a polar
or shape-selective nature. That is, the separation
mechanisms are indeed different or, in other words,
orthogonal separation conditions have been created. The
separation in the second column is extremely fast and
usually takes only 28 s as against 45120 min for the
first-dimension separation. Consequently, it is performed
under essentially isothermal conditions. The fast sepa-
ration in the second dimension causes the analyte peaks
to be very narrow with widths of, typically, 100600 ms
at the baseline. These narrow peaks require fast detectors
with a small internal volume and a short rise time in
order to achieve a proper reconstruction of the (second-
dimension) chromatograms. In some systems, the second
column is housed in a separate oven to allow more
flexible, independent temperature programming.
The outcome of a GC GC run is a large series of high-
speed, second-dimension chromatograms, which are
usually stacked side by side to form a two-dimensional
Figure 1. Schematic of a GC GC system with different modulator types.
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(2D) chromatogram with one dimension representing the
retention time on the first column and the other the
retention time on the second column. The most conve-
nient way to visualize these chromatograms is as contour
plots, where peaks are displayed as spots in a 2D plane
using colors and/or shading to indicate signal intensities.
Apex plots, which use specific symbols to indicate theposition of the peak apexes, are another frequently used
means to display GC GC chromatograms; the overall
presentation becomes much simpler, and that is espe-
cially advantageous when ordered structures are studied
(in this review, apex plots are used (e.g., Figs. 4 and 8)
and are combined with contour plots (e.g., Figs. 9 and
10); contour plots are also displayed (e.g., Figs. 2 and 5).
There is general agreement regarding the main
advantages of GC GC over 1D-GC. Most strikingly, the
peak capacity is much higher, and that yields a dramat-
ically improved separation of the analytes of interest from
each other but also and this is often even more impor-
tant from interfering matrix constituents. In addition, amain benefit of the trapping-plus-refocusing occurring
during modulation is, typically, a 310-fold improved
signal-to-noise ratio compared with 1D-GC. Finally,
compound identification is more reliable in GC GC be-
cause each substance now has two identifying retention
values rather than one. Specifically, when orthogonal
conditions are used, chemically related compounds show
up as so-called ordered structures (i.e. as clusters or
bands). This phenomenon greatly facilitates group-typeanalysis, fingerprinting studies and the provisional clas-
sification of unknowns. It is particularly important in the
study of classes of organohalogens, as will become clear
from most of the figures included in Sections 6 and 7.
In the following sections, we will discuss in some detail
three topics of general analytical interest: modulation;
detection and analytical performance; and, column
selection.
3. Modulation
The key component of a GC GC instrument is themodulator that joins the two columns. It serves three
main goals:
Figure 2. GC GClECD chromatograms of PCA-60 technical mixture on DB-1 in the first dimension and each of the six columns indicated inthe second dimension [24].
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(i) collecting and focusing of each of the fractions elut-
ing from the first-dimension column;
(ii) re-injecting/launching of each collected fraction
into the second-dimension column; and,
(iii) trapping of the next eluent fraction from the first
column during the launch of the preceding
fraction.Over the years, different ways of modulating the first-
column effluent have been reported (Fig. 1). Initially,
heating was preferred, with a rotating slotted heater, the
sweeper, rapidly moving over a thick-film modulation
capillary, heating it locally. Despite its frequent use in
the early years of GC GC, because of the vulnerability
of the set-up, the time-consuming optimization of the
multi-parameter design and the restricted application
range, the sweeper and related modulator types are
obsolete today.
The first major step forward was the introduction of
the longitudinally modulating cryogenic system (LMCS).
This modulator uses expanding CO2 (liquid) for trappingof the analytes at the top of the second-dimension col-
umn. By subsequently moving the trap rapidly to an
upstream position, the re-focused zone is exposed to the
GC oven air and instantaneously volatilized and laun-
ched. Today, jet-based modulators with no moving
parts at all with either CO2 or liquid N2 for cooling
are generally preferred. Single-, dual- and quad-jet
modulators have been introduced, and several of these
have been extensively compared in a recent study [4].
The main conclusion was that all cryogenic modulators,
if properly optimized, can satisfactorily be used for most
applications, and certainly for those in the field oforganohalogen analysis. One caveat should be added. In
order to ensure proper modulation of high-boiling com-
pounds (i.e. highly substituted congeners), cooling
should be just enough to trap the target compounds
safely. Cooling that is too strong can cause remobiliza-
tion to be inefficient and may lead to distorted and/or
tailing peaks [5]. In addition, severe cooling will lead to
more bleed from the first-dimension column being
accumulated and that, in turn, will yield noisier second-
dimension chromatograms. Some studies have indicated
that modulation of high-boiling compounds can easily be
achieved by air cooling [6]. If found to be true in further
studies, this will make GC GC of organohalogens less
expensive and simpler to operate.
4. Detection
As briefly mentioned in Section 2, detectors with a high
data-acquisition rate and a negligible internal volume
are required to describe properly the very narrow peaks
that are the outcome of a GC GC run. Flame ionization
detectors (FIDs) have data-acquisition rates up to 200 Hz
and dead volumes that are effectively zero. It does not
therefore come as a surprise that virtually all early
GC GC studies were carried out with an FID, including
those dealing with organohalogens [7]. However, as
soon as real-life studies and analyte detectability became
an issue of interest, it became clear that FID would have
to be replaced by ECD to obtain sufficient selectivity and
sensitivity. Conventional ECDs have data-acquisitionrates up to 50 Hz, but the main problem is their 1.5-ml
cell volume, which causes severe peak broadening [8].
Kristenson et al. [4] showed that, from amongst the
miniaturized ECDs marketed in recent years, only the
Agilent micro-ECD (lECD) with an internal volume of
150 ll gives acceptable peak widths. The best results
were obtained when working at the maximum flow of
make-up gas (150 ml/min) and at temperatures above
300C. However, as is to be expected, even under opti-
mum conditions, the lECD delivers about 2-fold broader
peaks than an FID [9]. All recent GC GClECD studies
use an Agilent detector.
The main problem of element-selective detection ingeneral and, therefore, also of ECD is that no struc-
tural information is provided. In other words, MS is
indispensable for identification/confirmation of the
numerous separated compounds, whether target ana-
lytes or unknowns. Today, the preferred choice is a TOF-
MS that can, typically, acquire up to 50500 mass
spectra per second (with unit mass resolution). The
coupling of GC GC separation and TOF-MS detection
presents no difficulties, and there is no additional peak
broadening. This is true for both conventional electron
impact (EI) ionization and the recently introduced elec-
tron-capture negative ionization (ECNI) mode. An addi-tional advantage of a TOF-MS is that the sensitivity is
higher than that of the full-scan mode of conventional
scanning MS detectors, while the high acquisition rate
prevents spectral skewing, and deconvolution conse-
quently becomes a very powerful tool.
Unfortunately, TOF-MS instruments are very expen-
sive. From the start therefore, the potential and limita-
tions of qMS instruments, which are available in
essentially all GC laboratories, were investigated by
several groups of workers [1013]. Briefly, the outcome
of these studies was that, for restricted mass ranges of
150200 Da, acquisition rates of 2030 Hz can be ob-
tained, and that tentative identification is then indeed
often possible, but that proper quantification cannot be
achieved on the basis of the 35 data points registered
across a peak. Recently, the situation has improved (i.e.
when two rapid-scanning qMSs were marketed, the
Shimadzu QP 2010 and the Perkin Elmer Clarus 500,
which allow scanning up to 10,000 Da/s [14,15]).
Table 2 provides an illustrative comparison of these two
instruments and a state-of-the-art conventional qMS. A
clear indication of the improved performance is that, if
the lower limit of the peak width at the baseline is set at
300 ms, the conventional qMS is restricted to a mass
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range of about 100 Da, whilst 23-fold larger ranges can
be handled by rapid scanning detectors. This is fullysatisfactory for many, and certainly for most, organo-
halogen applications. In the selected ion monitoring
(SIM) mode, acquisition rates of up to 90 Hz can be used
when only one ion is monitored (i.e. seven data points
can be collected even for peaks having a baseline width
of only 80 ms). While the present conclusions are grat-
ifying, they do not imply that rapid-scanning qMS
instruments can replace TOF-MS in all instances, as,
particularly when a wide mass range has to be covered
(e.g., in searching for unknowns), TOF-MS has to be
used.
4.1. Analytical performance data
Analyte detectability and linearity are influenced by
quite a number of parameters such as GC conditions
and set-up, and the number of modulations but the
key factor is the detector used. Table 3 shows instru-mental limits of detection (iLODs) and dynamic ranges
for various detectors and compound classes quantified
so far by GC GC. The data can be called fully
satisfactory, specifically the iLODs, which are 35-fold
lower than in 1D-GC due to peak re-focusing in the
modulator.
5. Columns and column combinations
To summarize, and simultaneously extend, what has so
far been said about columns or, more appropriatelycolumn combinations to be used in GC GC, in
essentially all early studies, a conventional 1530 m
non-polar first-dimension column was combined with a
Table 3. Analytical performance data for various detectors and classes of organohalogens
Detector (mode) Class/compound iLOD [pg] Linearity* Reference
Range [pg] R 2
lECD PCBs 0.010.07 1400 >0.999 [9,19,20]PCDD/Fs 0.040.15 0.1200 >0.998 [9,19]2,3,7,8-TCDD 0.09 0.140 >0.998 [9,19]
TOF-MS (EI) PCBs 0.110 0.51000 >0.993 [22,37]PCDD/Fs n.a. 0.2500 >0.996 [36,38]2,3,7,8-TCDD 0.20.5 0.5200 >0.996 [36,38,63]PBDEs 5 0.22000 >0.994 [37]OCPs 510 51000 >0.991 [37]
qMS (EI, scan 50 Da) PCBs 12 101000 >0.997 [15]
qMS (ECNI, SIM) PBDEs 1040 n.a. n.a. [14]PCDD/Fs 10710 n.a. n.a. [14]2,3,7,8-TCDD 710 n.a. n.a. [14]
*n.a., not available.
Table 2. Comparison of performance of two rapid-scanning and a conventional qMS in GC GC [11,1315,62]
Mass range (Da) ornumber of ions monitored
Perkin Elmer Clarus 500 Shimadzu QP 2010 Agilent MSD HP 5973
maximumacquisition (Hz)
minimumpeak widtha (ms)
maximumacquisition (Hz)
minimumpeak widtha(ms)
maximumacquisition (Hz)
minimumpeak widtha(ms)
Full scan
400 17 410 20 350 12 540300 23 300 25 280 15 470200 31 230 33 210 20 350100 63 110 50 140 n.a. n.a.
SIM1 ion 91 80 n.a. 33 2102 ions 45 160 n.a. 18 3903 ions 30 230 n.a. 12 580
n.a., not available.aCalculated for seven points per peak from: minimum peak width = 7/maximum acquisition.
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much shorter, narrower (medium-)polar or shape-
selective second-dimension column. Using such a
non-polar more polar column set has three distinct
advantages:
A wealth of information on the 1D-GC separation of
the compound classes of interest is available in the
literature and can be used for the first-dimensionseparation.
The non-polar columnsalso have a high thermal stabil-
ity and, consequently, cause little bleeding. As was
shown in one study [19], use of some polar columns
in the first dimension (e.g., an LC-50or a DB-Dioxin sta-
tionary phase) can cause LODs to be 10 times higher.
While in the first dimension there is a truly volatility-
based boiling-point separation, for all other, more
selective columns, separation will indeed be mainly
governed by specific analyte/stationary phase interac-
tions with, however, also a volatility-based contribution.
Fortunately, the extremely rapid second-dimension
separation will be essentially isothermal. This impliesthat, for sample constituents present in each individual
first-dimension effluent fraction (which will have clo-
sely similar boiling points), there will be no volatility
contribution. In other words, the two separation
mechanisms are indeed independent, and the condi-
tions are orthogonal.
In the early literature on GC GC, most of the above
was already clearly understood and, possibly because of
this and also because of a rather limited availability of
short and narrow-bore second-dimension columns, not
too much attention was devoted to optimizing the
selection of column combinations. In recent years, this
attitude has changed and, today, several interesting
studies on, specifically, second-dimension column selec-
tion are available notably in the context of organo-halogen analysis [1923]. We include illustrative
examples in Sections 6 and 7 below. The present dis-
cussion is therefore limited to two comments:
(i) when aspects such as nature of substituents (Cl vs.
Br), molecular shape (planar vs. non-planar) or
parent-compound nature (aromatic vs. non-
aromatic) play a role, even a brief study of
second-column characteristics can be most reward-
ing; and,
(ii) the column combination providing the best struc-
tural ordering (often considered the Number
One criterion) does not always offer the best
overall resolution of a class of compounds (highlyrelevant when unraveling the composition of rela-
tively unknown mixtures such as toxaphene or
PCAs).
As an example, Fig. 2 shows what is found when a
non-polar DB-1 first-dimension column is combined with
six different second-dimension columns for the study of
PCAs [24]. In this case, using 007-65HT as the second
column yielded optimal structural ordering (less band
Table 4. First- and second-dimension columns used for GC GC of organohalogens
Code Phase References
First-dimension columnsDB-1, HP-1, VF-1ms 100% methylpolysiloxane [19,20,2224,33,35,37,54]Rtx-Dioxin 2 Proprietary [36]Rtx-500 Proprietary (carborane) [38]HT-5 5% phenyl-methylpolysiloxane (carborane) [19]HT-8 8% phenyl-methylpolysiloxane (carborane) [22]DB-XLB Proprietary [9,19,21,22]DB-Dioxin Proprietary (44% methyl, 28% phenyl, 20% cyanopropyl,
8% polyoxyethylene-polysiloxane)[19]
LC-50 50% liquid crystalline-methylpolysiloxane [7,19]Chirasil-Dex 2,3,6-tri-O-methyl-b-cyclodextrin [2527]BGB-172 25% 2,3,6-tert.-butyldimethylsilyl-b-cyclodextrin [28]BGB-176SE 20% 2,3-di-O-methyl-6-O-tert.-butyldimethyl-b-cyclodextrin [28]
Second-dimension columnsBPX-5 5% phenyl-methylsilphenylene [7]HT-8 8% phenyl-methylpolysiloxane (carborane) [19,20,2224,35,37,54]Rtx-500 Proprietary (carborane) [36]DB-17 50% phenyl-methylpolysiloxane [35]BPX-50 50% phenyl-methylpolysiloxane (silphenylene) [19,22,38]007-65HT 65% phenyl-methylpolysiloxane [19,23,24]OV 1701, DB-1701 14% cyanopropyl-phenyl-methylpolysiloxane [19,35]BPX-70 70% cyanopropyl polysilphenylene-siloxane [21]SP-2340 100% biscyanopropyl polysiloxane [21]VF-23ms Proprietary (7090% cyano-containing polymer) [19,23,24,26,27]007-210 50% trifluoropropyl-methylpolysiloxane [19,23,24]LC-50 50% liquid crystalline-methylpolysiloxane [9,19,21,2326]SupelcoWax-10, CP-WAX-52CB Polyethylene glycol [19,23,24,27,33,35]
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overlap than with SupelcoWax-10 and LC-50), but, with
VF-23ms, a much larger part of the 2D-separation space
was used and overall resolution was, consequently,
better.
The only exception to what was said above is in the
area of chiral separations. Since separations such as
those of the pairs of atropisomeric PCBs require longcolumns and long run times for these applications,
the more discriminating (i.e. in this case shape-selective)
column is used in the first dimension [2528]. As can be
seen in Table 4, substituted b-cyclodextrins were the
preferred stationary phases in these studies. Relevant
examples are included in Section 6 below.
6. Within-class separations
6.1. Polychlorinated biphenyls (PCBs)
PCBs are products of anthropogenic activity and com-
prise a class of chlorinated aromatic compounds with110 chlorine atoms attached to a biphenyl backbone.
In total, there are 209 CB congeners; the presence of
about 140 of these has been confirmed in technical
formulations, such as Aroclors and Clophens, and also in
environmental samples.
For regulatory and monitoring purposes, seven CBs
(CBs 28, 52, 101, 118, 138, 153 and 180) have been
selected because of their abundance in humans and in
foodstuff of animal origin, and they are often called the
EU indicator CBs.
In the past two decades, much attention has been paid
to the toxicology of PCBs, particularly to the congenersthat show the same type of toxicity as polychlorinated
dibenzo-p-dioxins and dibenzofurans (PCDD/Fs), the
so-called dioxin-like or WHO CBs. They include four
non-ortho (CBs 77, 81, 126 and 169) and eight mono-
ortho (CBs 105, 114, 118, 123, 156, 157, 167 and 189)
substituted CBs.
Due to restricted rotation around the central CC bond
of biphenyl, some CB congeners occur as enantiomers.
Separation of such enantiomer pairs is interesting, since
it enables the study of enantioselective bioaccumulation
and biodegradation. There are 19 such pairs (CBs 45,
84, 88, 91, 95, 131, 132, 135, 136, 139, 144, 149,
171, 174, 175, 176, 183, 196 and 197), which exist as
stable atropisomers at ambient or physiological temper-
atures.
The separation of any of the groups of CBs listed above
and, specifically, of all CB congeners from each other and
from the plethora of matrix constituents is a challenging
task. Even though 1D-GC (with ECD or MS detection)
can separate some 100150 CB congeners, there still is
no unambiguous chromatographic separation of the 12
WHO CBs, or even the seven EU indicator CBs, from the
209 congeners or, even, the 140 present in technical
formulations [29]. To achieve the intended goal, it is
necessary to use sample fractionation, multiple injec-
tions on different stationary phases, heart-cut multidi-
mensional GC or columns coupled in series. It should,
therefore, come as no surprise that, since the introduc-
tion of GC GC, there have been many attempts to use
this technique for improving separation of PCBs.
There are over 20 papers [4,5,79,15,1923,2528,3038] that discuss PCB separation by GC GC.
However, most of these use PCBs merely as test analytes
to optimize and/or demonstrate modulator tuning,
proper modulator or detector performance, and instru-
ment set-up. These studies have clearly contributed to
the development of robust GC GC procedures but
provide little information on the separation itself. The
present review therefore mainly discusses papers aimed
at improving separation and/or detection of CB cong-
eners [7,2022,32] and the atropisomeric CB pairs
[2528].
The first study on the GC GC separation of PCBs was
presented by Phillips and Xu [32], who used theretention database of all 209 CBs published for 20 sta-
tionary phases [39] to construct 2D gas chromatograms.
The authors did not discuss the separation of individual
congeners, but they predicted structured chromato-
grams for the non-polar semi-polar DB-1 CNBP
column combination. The CBs in the 2D plane were
grouped together according to the number of chlorine
substituents, and the position of the congeners within a
homologue group was determined by the number of
ortho chlorines, with the retention sequence being:
4 < 3 < 2 < 1 < 0.
In 2001, Haglund et al. [7] analyzed PCBs in technicalClophen A50. The authors used a rather unconventional
column set-up, with a highly shape-selective liquid
crystal LC-50 (10 m 0.15 mm 0.1 lm) column in the
first dimension and a non-polar BPX-5 (0.25 m 0.1
mm 0.1 lm) column in the second dimension. The
non-ortho CBs were most strongly retained by the LC-50
column, followed by the mono-, di- and multi-ortho CBs.
As a consequence, the non- and mono-ortho congeners
eluted at the highest temperature within each homo-
logue group, and the toxic non-ortho CBs 77, 126 and
169 and the mono-ortho CBs 105, 118 and 156 all
showed up in the lower right-hand corner of the contour
plot. In order to achieve reasonable second-dimension
elution times (10C/min) had to be used.
Under these conditions, all six planar CBs quoted above
were successfully separated from other CBs present in
the mixture within a run time of only 17 min. In addi-
tion, the seven EU indicator CBs were also adequately
resolved. In our opinion, despite the excellent separation
of the non-ortho CBs from the other congeners, their
quantification may be problematic, because the excessive
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bleed of the LC-50 phase at the elevated temperatures at
which non-ortho CBs elute will also be modulated and
dramatically increase the noise level and, thus, increase
the LODs. In addition, the column combination does not
allow the determination of the many other CBs, because,
due to the fast temperature programme required, the
separation power of the first-dimension column cannotbe used fully and many CBs co-elute.
In 2002, Korytar et al. [20] tested three column
combinations for the separation of 90 CBs with emphasis
on the separation of the 12 WHO CBs. They preferred
a classical set-up, with a non-polar HP-1 (30 m
0.25 mm 0.25 lm) separating solely on the basis of
volatility in the first dimension, and three more polar
phases, BPX-50, HT-8 and SupelcoWax-10 (all 1 m 0.1
mm 0.1 lm), in the second dimension. A complete
separation of the WHO CBs from each other and from the
other CBs was obtained with the latter two columns.
With HP-1
SupelcoWax-10, only six congeners wereinvolved in co-elutions, as against 12 congeners for
HP-1 HT-8. However, the latter combination provided
the highest information content, because structured
chromatograms were obtained. The CBs were found to
be grouped together according to the number of chlorine
Table 5. Co-eluting PCBs on six column combinations [21,22]*,**
DB-1 HT-8 HT-8 BPX-50 DB-XLB BPX-50 DB-XLB SP-2340 DB-XLB LC-50 DB-XLB BPX-70
Non-ortho CB congeners 77/144
Mono-ortho CB congeners 118/131a
Marker CB congeners 52/69 153/168 101/90 101/90 153/168138/163/164
Other CB congeners 23/54a 132/179a 38/47/62a 21/33 4/10 31/53a
16/32 160/175a 20/21/33 47/62/65 20 /33 47/62/6520/21/33 20/33 66/155 42/59 43/69 42/5943/49 47/48 84/89 37/40a 62/65 57 /94a
48/75 93/95/98 90 /101 57/94a 58/67 86/11242/59 112/119 107/123 58/67 63/76 106 /10741/64 97/117 63/76 88 /95 175/18261/70 108/107 86/125 84/8956/60 163/164 107/134a 83/119
98/102 182/187 160/163 86/12588/91 175/182 160 /16383/112 201/204 175/182a
115/116 196/203 201/204108/107 196/203139/149134/143146/165182/187196/203
No. of CBs resolved byGC GC
163 188 191 176 181 194
No. of CBs resolved byGC GCMS
165 192 192/194 184 183 198
Temperature ramp (C/min) 1 1 1 1.5 1.5 0.5
Modulation time (s) 3 3 3 4 5 5
Run time (min) 140 146 144est. 100est. 90 240
, all congeners separated.aResolved by MS.est.Run time not explicitly mentioned in paper; estimated from available information.*CB congener classification according to their presence in any of the Aroclors 1242, 1254 or 1260: Bold, >1.0 wt.%; Bold, 0.051.0 wt.%;italics, trace or undetected.**IUPAC numbering is used. Numbering of [21] was corrected accordingly.
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substituents and, within a group, the number of ortho
chlorines. This is the structure predicted by Phillips and
Xu [32], but now verified experimentally. The ordered
structure is very useful because it helps to predict the
position of other CB congeners in the 2D plane. This
enabled the prediction, on the basis of published 1D-GC
retention data, that the WHO CBs will be separated fromall other congeners present in Aroclor mixtures. In
addition, a large number of unknown peaks detected in a
cod-liver extract could be provisionally identified as CB
congeners.
The effort to analyze PCBs by means of GC GC cul-
minated in two recent studies [21,22], in which attempts
were made to separate all 209 CBs. In these studies,
seven column combinations were evaluated. The sepa-
ration for six of these is visualized in Table 5. The
DB-XLB HT-8 set-up is not included because it pro-
vided a very limited improvement over 1D-GC due to the
very similar separation mechanisms applied in both
dimensions. When comparing the data of Table 5, oneshould keep in mind that the results also depend on run
time or, more precisely, the temperature ramp during
elution, and on modulation time. In general, slower
elution and a shorter modulation time yield more effi-
cient separation. Table 5 therefore includes these two
parameters. As for the DB-1 HT-8 column combina-
tion, the ordered structure quoted above has been con-
firmed for all CBs (Fig. 3). The separation of the
homologue groups was so clear cut that only one
co-elution of two congeners, CBs 23 and 54, was caused
by the overlapping of two (tri- and tetra-substituted)
homologue series. All other co-elutions occurred within
these series. For the rest, this column set performed
much poorer, in terms of number of congeners resolved,
than the other column combinations. Because most
co-eluting compounds have closely similar mass spectra,
additional separation by means of MS detection is pos-sible in a limited number of cases only such as here for
CBs 23 and 54.
As regards the other column sets, the best result was
found for DB-XLB BPX-70, which separated 198
congeners. However, the run time was as high as 4 h, or
almost double that of most other procedures and it is
questionable whether, in several instances, a limited
overall increase of resolution justifies such an excessive
demand of time. In other words, from a practical point of
view, HT-8 or DB-XLB combined with BPX-50 or
DB-XLB LC-50 are to be preferred with the first two
sets separating 192 congeners in 2.5 h, and the latter a
lower number (183) of analytes, but in a very short time(1.5 h). Two of these combinations separate all WHO
CBs and EU indicator CBs. The HT-8 BPX-50 set has
the added advantage of yielding structured chromato-
grams. For the penta-CBs, this is shown in the apex plot
of Fig. 4, which also clearly visualizes the non-ortho-
number-based sub-division.
One should consider that the above separations were
studied with the congener concentrations in the stan-
dard mixture being essentially the same and with
RS > 0.5 as the separation criterion. As is well known,
Figure 3. GC GCTOF-MS chromatogram of all CB congeners on DB-1 HT-8 column set [22].
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this resolution will not be satisfactory for quantifying
partly co-eluting congeners with widely different con-
centrations, as is often true for PCBs and other classes oforganohalogens in real-life samples. Even if RS > 1, the
determination of the WHO CBs in one run together with
predominant congeners may be difficult.
Another problem was encountered when the target
analytes had to be determined in a seal-blubber sample
[21]. After the usual sample treatment, some of them
were found to be present in concentrations too low to be
detected by GC GClECD. One solution would be to
concentrate the extract. However, some of the major
congeners would then overload the second-dimension
column and, in addition, it would be difficult to keep all
congeners within the working range of the detector.Therefore, two separate injections will be required.
6.1.1. Chiral analysis. With atropisomeric CBs, the
preferred method is heart-cut MDGC [29]. Most
reported methods use two-oven systems and comprise
an achiral precolumn and a chiral main column. Heart
cuts (from the precolumn) containing the atropisomers
of interest and, inevitably, other PCBs or interferences
are directed towards the chiral column. With typical
precolumns, such as DB-5 or DB-XLB, three or four out
of the 11 atropisomeric pairs present in technical for-
mulations co-elute with some of the 140 CBs [29] and
must be separated in the second dimension. Chirasil-
Dex (permethylated 2,3,6-tri-O-methyl-b-cyclodextrin)
is the most commonly used chiral column that can
resolve nine out of the 19 stable, and seven out of the
11 interesting pairs of chiral congeners [29]. The
column sequence used in heart-cut MDGC has to be
changed for GC GC, because the second-dimension
column must be short and, if a short chiral column is
used, no separation of the enantiomers would be
achieved (i.e. the chiral column is used in the first
dimension, and a polar or shape-selective column in the
second dimension).
In all published papers [2528], Chirasil-Dex was used
as the first-dimension column; in one study [28],
BGB-172 (25% 2,3,6-tert.-butyldimethylsilyl-b-cyclo-dextrin) and BGB-176SE (20% 2,3-di-O-methyl-6-O-
tert.-butyldimethyl-b-cyclodextrin) were also evaluated.
As second-dimension columns, Bordajandi et al. tested
HT-8, VF-23ms and SupelcoWax-10 for 65 [28] or 90
[27] CBs, whilst Harju et al. [25,26] used VF-23ms and
LC-50 for all 140 Aroclor CBs. The results of the latter,
more comprehensive study are displayed in Table 6. It
shows that all nine atropisomeric pairs, which are
resolved on Chirasil-Dex, partly or completely co-elute
with one or more CBs in 1D-GC, while all but two or
three of the co-elutions are resolved in the second
dimension. As an application, grey-seal tissue was ana-lyzed on both column combinations. Seven out of the
nine atropisomeric pairs were detected, and enantio-
meric fractions could be determined for six of these (CBs
91, 95, 132, 135, 149 and 174) by using lECD and/or
TOF-MS detection; the second eluting enantiomer of CB
84 co-eluted with CB 56. The most abundant
Table 6. Co-elutions for nine atropisomeric CB pairs in 1D-GCand GC GC with 140 Aroclor CBs [26]*
Atropisomeric
CBs
Co-elutants on
Chirasil-Dex (CD) CD VF-23ms CD LC-50
84 56, 90, 99, 101 56 9991 63 95 93 93
132 176, 141 141 141 (0.7)a
135 110, 82 136 115 149 77, 124 174 202 176 132, 141
aRS in second dimension.*For classification code, see Table 5.
0.7
1
1.3
1.6
1.9
4200 4700 5200 5700 6200 67001tR (s)
2tR
(s)
103 100
94102
88
91
84
89989593
127
126
104
96
111 120
124
105
122114
106118
123
108
107
121
92
90
113
99
101
112,119
10983
12587
8611085
115116
97,117
82
0 50 50 5
989593
1098386116
Figure 4. GC GCTOF-MS apexplotof thepenta-CBson HT-8 BPX-50columnset. Pink, tetra-orthoCBs; green, tri-orthoCBs;red,di-orthoCBs;light blue, mono-orthoCBs; dark blue, non-orthoCBs. Boxes represent co-eluting congeners [22].
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atropisomers, CBs 135, 149 and 174, had enantiomeric
fractions (EFs) close to the racemic value, while the EFs
for CBs 91, 95 and 132 invariably deviated significantly
from that value.
6.2. Polychlorinated dibenzo-p-dioxins/furans
(PCDD/Fs)PCDD/Fs are highly toxic compounds formed as
by-products during a variety of chemical and combus-
tion processes [40]. Notorious examples are their (trace-
level) presence in technical PCB mixtures, chlorinated
phenoxyalkanoic acid pesticides and fly ash. The number
of chlorine substituents can vary from one to eight to
produce up to 75 CDD and 135 CDF positional isomers.
There is a pronounced difference in toxic and biological
effects amongst these CDD/F congeners and toxicities
vary 1,00010,000-fold. Seven 2,3,7,8-substituted
CDDs and 10 CDFs are generally considered the most
toxic, since they have toxic properties similar to 2,3,7,8-
tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD), which isthe most toxic congener of the group. Consequently, the
analytical challenge is to detect, and quantify, these 17
priority congeners and, also, the 12 WHO CBs discussed
above (which also show a dioxin-like effect). Such ultra-
trace analyses are expensive, both because of the time-
consuming pre-treatment and clean-up, and the need to
use GChigh-resolution MS (GCHRMS) with its unri-
valled sensitivity and selectivity [18]. In other words,
sample throughput is low and cost is high. Not surpris-
ingly, therefore, the potential of GC GC with its
improved selectivity is eagerly explored to find less
demanding but equally rewarding analytical ap-proaches.
The first attempt to analyze dioxins and dioxin-like
CBs by GC GC was made by Grainger et al. [16,17].
They used an early sweeper for modulation and HRMS
(resolution power 3000) for detection of a 24-compound
mixture containing the non-ortho CBs 77, 81, 126 and
169 and a suite of PCDD/Fs. Rather unconventional
column dimensions were used: a very short first-
dimension DB-5 column (2 m 0.25 mm 0.25 lm)
and a rather long second-dimension OV-1701 column (3
m 0.1 mm 0.05 lm). With this set-up, the separation
took only 18 min, with an impressive iLOD of 335 attog
(S/N 9) for 2,3,7,8-TCDD. Unfortunately, no further
information was provided.
As a part of their PCB study quoted above, Korytar
et al. [20] tried to separate the priority CDD/Fs and WHO
CBs from each other and the bulk of 90 CBs on
HP-1 HT-8. The outcome was successful for all except
one pair of target analytes, 1,2,3,7,8-PeCDD/CB 169.
This stimulated a further search for a column combi-
nation that would separate all target compounds from
each other and, as importantly, from matrix
co-extractants. In a subsequent study [19], therefore,
seven first-dimension and eight second-dimension
columns were tested. With a 100% methylpolysiloxane
stationary phase (DB-1) in the first dimension to create
orthogonal conditions, all congeners with different toxic
equivalency factor (TEF) values could be separated if
VF-23ms or LC-50 were used in the second dimension.
When other types of first-dimension column were used
(and orthogonality was partly sacrificed), a DB-XLBcolumn combined with 007-65HT, VF-23ms or LC-50
gave a complete separation of all 29 priority congeners.
With a spiked, fractionated milk extract, DB-XLB LC-
50 was found to be the most powerful column combi-
nation, because of the good separation of all priority
congeners from each other as well as matrix constitu-
ents. Analytical performance was satisfactory with a
close to 3-order linearity, and iLODs of 30150 fg
injected mass.
Two of the quoted column sets the preferred
DB-XLB LC-50 and also VF-1 LC-50 were used by
Danielsson et al. [9,41], who studied the quantification
of dioxins and dioxin-like CBs by GC GClECD. Fish oilfrom herring, spiked cows milk, vegetable oil and an eel
extract were analyzed by two GC GC and four
GCHRMS laboratories, with the latter serving as refer-
ences. Fig. 5 shows typical GC GClECD chromato-
grams of the mono-ortho-CB, and non-ortho-CB and
CDD/F fraction of herring oil on DB-XLB LC-50, and
clearly demonstrates the efficiency of the LC-50 phase to
separate the target analytes from matrix co-extractants.
The quantification data for WHO CBs obtained on both
GC GClECD systems were closely similar and agreed
very well with the GCHRMS data. As for the dioxins,
data produced on DB-XLB LC-50 also showed goodagreement, but this was not true for VF-1 LC-50 due to
interferences co-eluting with some congeners. For the
rest, the CVs were somewhat higher for GC GC than for
HRMS (540% vs. 227%). The total toxic equivalent
(TEQ) data obtained on the preferred GC GC system
compared well with those obtained by GCHRMS, with
CVs for both techniques being below 10%. These results
show that the proposed method meets the EC require-
ments for a WHO CDD/F-plus-CB screening method,
which should have a false negative rate of less than 1%
and a TEQ CV of less than 30%. However, more samples
need to be analyzed to confirm fully the criterion for the
false negative rate. For the rest, as Danielsson et al. noted
[9,41], before GC GClECD can be considered a con-
firmatory method, the EC Directives will have to be
revised; currently, MS is the only mode of detection
allowed for this purpose.
Focant et al. [36,38] devoted two studies to GC GC
TOF-MS. In their first report [36], Rtx-Dioxin 2 was
employed as the first-dimension column because it is
known for its excellent separation of the priority CDD/Fs
from each other and also from non-2,3,7,8-substituted
congeners. For the second dimension, they selected a
relatively non-polar Rtx-500 column (similar to HT-8),
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because they expected the second dimension to bringmore in terms of signal enhancement after modulation
than added resolution retention on that column
should, consequently, be rather weak. An iLOD of 0.5 pg
was obtained for 2,3,7,8-TCDD. As for the separation of
the 17 CDD/Fs and the four non-ortho CBs analyzed, one
co-elution persisted in both dimensions (viz between CB
126 and 2,3,7,8-TCDD). This co-elution is especially
problematic because CB 126 is usually present in con-
centrations one or two orders of magnitude higher than
2,3,7,8-TCDD, causing a serious over-estimation of the
sample TEQ due to the much higher toxicity of 2,3,7,8-
TCDD. The problem was solved by deconvoluting the
masses involved. The congener-specific data found by
GC GCTOF-MS and GCHRMS for various environ-
mental (sediment, fly ash) and biological (vegetation,
fish) samples showed good agreement for 2,3,7,8-TCDD
and most other congeners.
However, in their next study [38], the authors used
another column combination, Rtx-500 BPX-50. All
priority CDD/Fs and four non-ortho CBs were then
separated and, in a next run, all EU indicator and
mono-ortho CBs were also separated from each other
and other CBs. The iLOD of 2,3,7,8-TCDD improved to
an impressive 0.2 pg. The system was used to analyze
fish, pork, and milk samples and the results werecompared with conventional GCHRMS. Not unex-
pectedly, the earlier conclusions on CBs were con-
firmed. For the CDD/Fs, the results were strongly
concentration dependent. When the average congener
concentrations were rather high (above 0.4 pg/g fat
weight, which corresponds to 1.1 pg of compound in-
jected), as for fish, and a large sample intake was used
(15 g), the two techniques showed good agreement.
But, again, the CVs for GCHRMS were 714%, as
against 1060% for GC GCTOF-MS. With pork and
milk, for which the CDD/F concentrations were much
lower (above 0.030.1 pg/g fat weight), there were
many over-estimations with CVs up to 90%. However,
the congener distribution was still well defined in all
instances and can be used (e.g., for tracking sources of
contamination). Despite the problems, the TEQ results
for the CDD/Fs and CBs compared favorably with those
of GCHRMS because a rather good description of the
main TEQ contributors (2,3,7,8-TCDD, 1,2,3,7,8-
PeCDD and 2,3,4,7,8-PeCDF) was achieved.
6.3. Toxaphene
Toxaphene is an organochlorine pesticide mixture of
complex composition. The major constituents are
Figure 5. GC GClECD of the (A) mono-ortho-CB and (B) non-ortho-CB and CDD/F fractions of a fish oil on DB-XLB LC-50. Assignment for(B): 1, CB 77; 2, CB 126; 3, CB 169; IS1, 1,2,3,4-TCDD; 4F1, 2,3,7,8-TCDF; 4D1, 2,3,7,8-TCDD; 5F1, 1,2,3,7,8-PeCDF; 5F2, 2,3,4,7,8-PeCDF;5D1, 1,2,3,7,8-PeCDD; 6D3, 1,2,3,7,8,9-HxCDD; IS2, 1,2,3,4,6,7,9-HpCDD; 7D1, 1,2,3,4,6,7,8-HpCDD; 8D1, OCDD [9].
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chlorobornanes (ca. 75 wt.%), with chlorocamphenes
in second place, while chlorodihydrocamphenes are
present as minor components [42]. Individual members
of these compound classes have been isolated from the
technical mixture, and they are likely products of
synthesis. The presence of chlorobornenes, which has
also been reported [42] as a possible class of constitu-ents, is discussed at the end of this section. The number
of congeners that can be found theoretically exceeds
60,000 (cf. Table 1). 1D-GC, which is the method of
choice to study the composition of technical toxaphene,
cannot create a satisfactory separation. Typically, about
100 peaks show up in a GCECD chromatogram. Better
results were obtained by combining GC with other
separation techniques, such as adsorption chromatog-
raphy on silica [43] or active carbon [44], normal-
phase LC [45], or heart-cut MDGC [46]. With these
techniques, the number of compounds in technical
toxaphene was estimated to be at least 177 [43], 246
[45], 300 [46], and 675 [44]; all quoted methods werevery time-consuming (e.g., the 675-peak experiment
required pre-fractionation into no less than 160 frac-
tions with a subsequent 30-min GC analysis of each
fraction). In the environment and particularly in higher
organisms, many toxaphene constituents are degraded,
and only a few are bio-accumulated. This leads to a
significantly simpler toxaphene-residue pattern com-
pared to the technical mixture. One problem with the
quantification of individual congeners is the limited
availability of the standards. Today, only 23 standards
are commercially available (Table 7). Since various
nomenclature rules were proposed for the toxaphene
components [4753], each congener in Table 7 has
several names.
In view of the separation problems outlined above,
expectations were high when GC GC became available
as a tool to unravel the composition of complex mixtures
[54]. Indeed, the use of an HP-1
HT-8 column com-bination yielded highly structured chromatograms and
easily revealed a complex mixture of over 1000 com-
pounds in a run of less than 3 h. Subsequent analysis of
the 23-standard mixture and EI-TOF-MS evaluation of
technical toxaphene showed that the 2D chromatogram
is structured according to the number of chlorine sub-
stituents in a molecule (see Fig. 6), with little, if any,
dependence on the class of compounds (bornanes or
camphenes). The range of chlorination found with
GC GCEI-TOF-MS was 511 substituents per mole-
cule. Using home-made software to calculate the total
area for each iso-substitution band, hexa- to nona-
chlorinated compounds were found to be the majorcomponents of toxaphene and represented some 97% of
the total toxaphene mass.
In a more recent study [23], six column combina-
tions were tested for the separation of 12 classes of
organohalogenated compounds (see Section 7); one
of these was technical toxaphene. Ordered structures
were then also found for DB-1 combined with 007-210,
007-65HT or LC-50. Somewhat surprisingly, another
column combination, DB-1 VF-23ms, did not deliver
ordered structures but offered the best overall separa-
tion; the entire GC GC plane was used and visual
Table 7. List of 23 commercially available standards for toxaphene and their various codes
IUPAC name Parlar[47,48]
Nikiforov[49]
Wester et al.[50,51]
AV-code[52]
OK-code[53]
2,2,3-exo,8,9,10-hexachlorocamphene 11 C[032001]-(11)2-exo,3-endo,8,8,9,10-hexachlorocamphene 12 C[021001]-(21)2-exo,3-endo,7,8,9,10-hexachlorocamphene 15 C[021011]-(11)2,2,5,5,9,10,10-heptachlorobornane 21 HpCB-6533 B[30030]-(012) B7-499 99-0132,2,3-exo,8,8,9,10-heptachlorocamphene 25 C[032001]-(21)2-endo,3-exo,5-endo,6-exo,8,8,10,10-octachlorobornane 26 OCB-4921 B[12012]-(202) B8-1413 198-3032,2,3-exo,8,8,9,9,10-octachlorocamphene 31 C[032001]-(22)2,2,5-endo,6-exo,8,9,10-heptachlorobornane 32 HpCB-6452 B[30012]-(111) B 7-515 195-1112,2,5,5,9,9,10,10-octachlorobornane 38 OCB-6535 B[30030]-(022) B8-789 99-033
2,2,3-exo,5-endo,6-exo,8,9,10-octachlorobornane 39 OCB-6964 B[32012]-(111) B8-531 199-1112-endo,3-exo,5-endo,6-exo,8,9,10,10-octachlorobornane 40 OCB-4917 B[12012]-(112) B8-1414 198-1132-exo,3-endo,5-exo,8,9,9,10,10-octachlorobornane 41 OCB-3223 B[21020]-(122) B8-1945 41-1332,2,5-endo,6-exo,8,8,9,10- octachlorobornane 42a OCB-6460 B[30012]-(211) B8-806 195-3112,2,5-endo,6-exo,8,9,9,10-octachlorobornane 42b OCB-6454 B[30012]-(121) B8-809 195-1312-exo,5,5,8,9,9,10,10-octachlorobornane 44 OCB-2455 B[20030]-(122) B8-2229 97-0332-endo,3-exo,5-endo,6-exo,8,8,9,10,10-nonachlorobornane 50 NCB-4925 B[12012]-(212) B9-1679 198-3132,2,5,5,8,9,10,10-octachlorobornane 51 OCB-6549 B[30030]-(112) B8-786 99-1132,2,5-endo,6-exo,8,8,9,10,10-nonachlorobornane 56 NCB-6461 B[30012]-(212) B9-1046 195-3132,2,3-exo,5,5,8,9,10,10-nonachlorobornane 58 NCB-7061 B[32030]-(112) B9-715 103-1132,2,5-endo,6-exo,8,9,9,10,10-nonachlorobornane 59 NCB-6455 B[30012]-(122) B9-1049 195-1332,2,5,5,8,9,9,10,10-nonachlorobornane 62 NCB-6551 B[30030]-(122) B9-1025 99-0332-exo,3-endo,5-exo,6-exo,8,8,9,10,10-nonachlorobornane 63 NCB-3261 B[21022]-(212) B9-2206 169-3132,2,5,5,6-exo,8,9,9,10,10-decachlorobornane 69 DCB-6583 B[30032]-(122) B10-1110 227-133
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inspection showed that, here, the highest number of
congeners was observed. The column sets that yielded
ordered structures indicated the presence of a cluster of
unknown compounds (Fig. 6). This group of com-
pounds was not observed in the earlier study [54],
because the temperature programme then used was
much slower. This caused much more spreading of the
toxaphene congeners in the 2D plane; wrap-around
occurred and led to co-elution with the unknown
compounds. Combined information on the peak shapes
and the ECNI-TOF mass spectra added as inserts dem-
onstrated that the unknowns were decomposition
products formed during the first-dimension GC run (i.e.
chlorinated bornenes, probably formed by HCl elimi-
nation). So far, not a single polychlorinated bornene
has been isolated from technical mixtures and the
assumption about their presence was based only on
GCMS and GCFTIR data [42]. The observations cited
created serious doubts about the presence of bornenes
in technical toxaphene.
6.4. Polychlorinated alkanes (PCAs)
PCAs are complex mixtures with a degree of chlorina-
tion of 3070 wt.%, and carbon chain lengths of
C10C13 (short-chain PCAs), C14C17 (medium-chain
PCAs) or >C17 (long-chain PCAs). They are used as
extreme-pressure additives in industrial cutting fluids,
plasticizers and flame retardants for polyvinyl chloride
(PVC) and other plastics and rubbers, and as additives
in paints and sealants. PCAs are persistent and non-
biodegradable, and they accumulate in the food chain.
The global production of PCAs has been reduced since
the early 1980s [55], but is still in the range of
380,000 tons [56]. Short-chain PCAs cause particular
concern due to the high amounts released into the
environment, and their toxicity, which is higher than
that of other PCAs.
Analysis of PCAs is difficult because of the extreme
complexity of the mixtures and the lack of quantification
standards, and semi-quantification is all that can be
achieved. In many environmental studies dealing with
Figure 6. Total-ion GC GCECNI-TOF-MS chromatogram of technical toxaphene on DB-1 HT-8, with the mass spectra of two peaks asindicated. Lines indicate position of iso-substitution bands [23].
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halogenated micro-contaminants, an essentially
unresolved band covering a large part of the GC baseline
is all that indicates the presence of PCAs. Currently,
some 40 individual standards are available, but most of
them, as will be demonstrated below, are not present in
the technical mixtures. Mixtures of PCAs, with various
chlorine contents and carbon-chain lengths, are used as
reference standards for semi-quantification. Analysis is
usually carried out by GC coupled to HRMS in ECNI
mode. The method is based on monitoring [MCl] ions
to determine the concentrations of individual classes (i.e.
congeners with the same number of carbon and chlorine
atoms). HRMS is highly selective and eliminates inter-
ferences caused by other polychlorinated pollutants and
Figure 7. (A) GC GCECNI-TOF-MS extracted ion chromatogram (m/z 7073) of polychlorinated decanes with 55 wt.% Cl.(B) GC GCECNI-TOF-MS chromatograms of C10C13 technical mixture with 55 wt.% Cl. Colored lines indicate the position of apices withinthe band of polychlorinated (red) decanes, (green) undecanes, (blue) dodecanes and (black) tridecanes. (C) Overlay of GC GCECNI-TOF-MSchromatograms of (red) short-, (green) medium- and (blue) long-chain PCA mixtures with different Cl content. White numbers indicate number of(carbon + chlorine) atoms of the compounds present in the bands [24].
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PCAs with the same nominal mass. However, this
detection method is not available in many laboratories
and is too expensive for routine analysis, so low-resolution
MS (LRMS) is also used for quantification. An interna-
tional inter-comparison study for short-chain PCAs has
shown that the two techniques can give comparable
quantitative results (e.g., in biota [57,58]). Nevertheless,because of the increased risk of interferences, improved
sample clean-up and/or the use of GC GC instead of
1D-GC is urgently required.
The potential of GC GC was investigated in a recent
paper in which six column combinations were tested
[24]. The highest number of separated congeners was
found with the DB-1 VF-23ms combination, but, for
characterization and quantification, DB-1 007-65HT
was preferred because this set provided more informa-
tion in terms of ordered structures (i.e. group and sub-
group separation). As demonstrated for polychlorinated
decanes in Fig. 7A, the separation of PCA congeners
with the same chain length is based on the number ofchlorine substituents. The authors added that the
number of congeners in the technical mixtures (formed
by uncontrolled synthesis) was relatively rather limited.
If a larger number were present, the iso-substitution
bands would become broader and neighboring bands
would start to overlap. This is demonstrated in Fig. 7A
by the outlying positions of several individual congen-
ers, which should be considered unlikely products of
uncontrolled synthesis. With mixtures of PCAs of
varying chain length, the ordered structures comprise
compounds having the same number of carbon-plus-
chlorine atoms for example, C10Cl8 is on the samediagonal line as C11Cl7, C12Cl6 and C13Cl5. This is
elegantly visualized in Fig. 7B. The position of the
various compounds in each diagonal band depends on
the number of carbon atoms: compounds with longer
carbon chains have lower second-dimension retention
times. This carbon-chain-length selectivity creates a
distinct separation of compounds that differ by at least
three carbons; C10 and C13 compounds show no over-
lap in Fig. 7B. For a mixture of short-, medium- and
long-chain PCAs, the (carbon+chlorine)-based ordering
is seen to hold over a summed-number range of at least
14 to 26. In addition, the carbon-chain-length selec-
tivity creates a partial separation of the three PCA
groups (Fig. 7C). Obviously, using GC GC is a major
step forward in PCA analysis, although, simulta-
neously, it is also clear that additional separation power
possibly provided by LC-based sample fractionation
will be needed for a satisfactory overall unraveling of
the composition of PCA mixtures.
Two relevant examples of the added value of GC GC
compared to 1D-GC are as follows. In the quoted study
[24], two dust samples were analyzed, and significantly
different GC GClECD patterns of the short- and med-
ium-chain PCAs were easily observed. Visual evaluation
of the chromatograms showed that one sample con-
tained more medium- than short-chain PCAs and that
these medium-chain PCAs had a relatively low degree of
chlorination, while the other sample contained more
short- than medium-chain PCAs, with the short-chain
PCAs having a higher degree of chlorination. In other
words, pattern recognition based on the visual evalua-tion allowed provisional identification of sample com-
position.
Another advantage of GC GC is the elimination of
LRMS interferences amongst the PCAs. Recently, Reth
and Oehme [56] discussed three critical limitations of
LRMS for the analysis of short- and medium-chain PCAs.
[MCl] ions of a specific PCA (e.g., [MCl + 2] of
C11H17Cl7 with m/z 360.9) interfere with:
(i) the [MCl] ions of a PCA with five carbon atoms
more and two chlorine atoms less (i.e. [MCl]
(m/z 361.1) of C16H29Cl5);
(ii) the [MCl] ions of a PCA with two carbon atoms
more and one chlorine atom less (i.e. [MCl + 8](m/z 361.0) of C13H22Cl6); and,
(iii) with [M + Cl] ions of a PCA with the same number
of carbon atoms and two chlorine atoms less (i.e.
[M + Cl] (m/z 360.9) of C11H19Cl5).
GC GC solves these three problems due to the
enhanced chromatographic resolution.
6.5. Polybrominated diphenylethers (PBDEs)
PBDEs are widely used as flame retardants (e.g., in
polymers, textiles, electronic boards) and, similar to the
PCBs, there are 209 BDE congeners. However, with only
some 2025 congeners, the composition of the technicalPBDE mixtures is rather simple. Most environmental
monitoring programmes focus on the analysis of seven
congeners (BDEs 28, 47, 99, 100, 153, 154 and 209),
which are most abundant in technical mixtures and are
conventionally considered a type of reference set, similar
to the seven EU indicator CBs. However, in environ-
mental and biota samples, many other BDEs are found
than the 2025 technical congeners because of their
photolytic and biological debromination and metabolism
in higher animals.
The analysis of PBDEs is carried out by GCECD or
GCMS (EI or ECNI mode) [59]. Today, 125 BDE cong-
eners are commercially available and their retentioncharacteristics on seven stationary phases have been
reported [60]. No stationary phase separates all cong-
eners and not even the seven reference BDEs from all
others. The best separation was achieved on a DB-XLB
column with which 70 congeners were separated, while
55 congeners were involved in 22 co-elutions. In real-life
samples, the situation is even worse because other bro-
minated compounds (e.g., polybrominated biphenyls
(PBBs), hexabromocyclododecane (HBCD), tetrabromo-
bisphenol-A (TBBP-A) or dimethyl-tetrabromobisphenol-
A (me-TBBP-A)) may be present in the sample extract
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and cause further interferences, which, often, cannot be
solved by means of MS.
GC GC of BDEs is challenging because of the high
boiling points and thermal instability of the higher bro-
minated congeners. In a study to be discussed in more
detail in Section 7, Focant et al. [35,37] found that the
higher brominated congeners were retained too stronglyin the second dimension, with the limited thermal sta-
bility of most stationary phases not allowing a suffi-
ciently high temperature to speed up the separation. In
the end, an HT-8 column was used; it was stable up to
360C. However, even with a DB-1 HT-8 column set,
hepta-BDE 183 and deca-BDE could not be included
because retention was still too great.
Korytar et al. [61] used GC GClECD to separate the
125 BDEs recently marketed. For the first dimension,
DB-XLB was not a good choice because nona- and deca-
substituted congeners were decomposed completely on
this phase and partial decomposition was observed down
to hexa-BDEs. Fortunately, on DB-1, decomposition ofnona-BDEs was negligible and only slight decomposition
of deca-BDE was observed. As for the second dimension,
from amongst six columns tested, 007-65HT was found
to add most to the selectivity of the first-dimension sep-
aration. In contrast with the findings quoted above, no
extreme peak broadening or trapping was observed for
the nona-BDEs 206, 207 and 208, and deca-BDE 209.
Possibly, the band broadening in [35] was caused by a
low temperature of the MS transfer line, which houses a
significant part of the second-dimension column or by
too low a temperature of the cooling gas used for
modulation.Fig. 8 shows the considerably improved separation on
the DB-1 007-65HT column set, with only 17
co-eluting pairs involving 35 congeners. In addition, the
seven reference BDEs were separated from all other BDEs.
When a dust extract was analyzed (insert in Fig. 8), 18
BDE congeners were identified.
Various other brominated flame retardants and a
number of BDE metabolites were all found to elute within
the BDE band, and that caused several more co-elutions
(Fig. 8). Of course, some separations were improved,
notably that of TBBP-A and BDE 153; the latter analyte
can now be quantified even if a high concentration of
TBBP-A is present, as was true for the dust sample.Finally, Fig. 8 shows that second-dimension separation
facilitates the use of fluorinated BDEs as internal stan-
dards, because all F-BDEs that co-elute with the parent
compound in the first dimension are separated in the
second dimension, appearing just below that parent.
Figure 8. GC GClECDof ( ) BDEs,( ) fluorinated BDEs,( ) otherbrominated flame retardants and( ) BDE metabolites on DB-1 007-65HT.Insert: GC GClECD contour plot of dust extract [61].
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7. Between-class separations
For obvious reasons, the common denominator of nearly
all papers devoted to GC GC analysis of organohalo-
gens is the (much) improved within-class resolution that
can be achieved, with the joint addressing of the priority
CDD/Fs and CBs as the only, and logical, exception.Because of the marked successes so obtained, we now see
a gradual shift of interest to the more intriguing
between-class separations [23,35,37].
As a first attempt, Focant et al. [35] used GC GC
TOF-MS for the simultaneous determination of 38
predominant CBs, 11 persistent halogenated pesticides
(OCPs), one PBB and eight PBDEs. With the DB-1 HT-8
column combination selected, of 58 test compounds,
only one pair of CBs was not resolved.
In their next study [37], the authors determined the
priority compounds in serum and milk. Single-injection
GC
GC was compared with a validated GCHRMSprocedure, which required three separate injections with
three different temperature programmes and was, of
course, much more time-consuming. The data shown for
human serum corresponded very well between the two
techniques. For the milk sample and the more abundant
Figure 9. GC GClECD on DB-1 LC-50 of: (A) PCBs, PBBs, PCDEs, PBDEs, PCDTs,h PCNs, PCDD/Fs, OCPs, individualtoxaphene standards; (B) PCAs (PCA-60) as color contour plot and other classes as black dots; (C) PCTs (Aroclors 5442 + 5460) as color contourplot and dioxin-like CBs (black dots) and planar PCTs (white arrows); (D) PCDD/F fraction of a sediment extract [23].
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congeners (>1 ng/g of lipid), per-cent deviations between
the two methods were below 20%, which is acceptable.
However, for some less-abundant congeners (
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separations were found to strongly depend on the
column combination used.
We summarize a few relevant conclusions here:
On DB-1 HT-8, congener separation on the basis of
the number of halogen substituents, well-known for
PCBs and toxaphene, was also observed for PCTs
and PBDEs. However, there was no group-typeseparation (i.e. although the DB-1 HT-8 set can be
successfully used for within-class separation, there is
a high risk of interferences if congeners from other
compound classes are present).
Group separation based on planarity was obtained
with the DB-1 LC-50 column set. Fig. 9AC shows
that three groups of analytes could be distinguished.
Three-ring planar compounds, such as PCDD/Fs,
PCDTs and planar PCTs, were most strongly retained.
Next in line were two-ring planar compounds, such as
PCNs and planar PCBs. Non-planar analytes showed
least retention and did not interfere with the planar
compounds. The practicability of this column set isdemonstrated in Fig. 9D, which shows a chromato-
gram of the PCDD/F fraction of a sediment sample
after fractionation on a carbon column. Obviously, a
properly tuned GC GC system could accommodate
a very high number of compounds in the 2D plane,
and could separate dioxins (indicated by black acro-
nyms) from co-extractants (white-yellow band along
first-dimension axis).
Group-type separation was also delivered by
DB-1 007-65HT (Fig. 10A-B). Here, the PBDEs were
the strongest retained compounds in the second
dimension and the PCAs are the least retainedorganohalogens. This is a rewarding result because
PCAs and PBDEs are usually present in the same frac-
tion after clean-up. As an example, the analysis of a
dust sample is shown in Fig. 10C: the group separa-
tion is clear-cut. Finally, Fig. 10B displays the overlay
of three mixtures toxaphene, PCA-60 and Aroclors
5442 plus 5460. What shows up in the 2D plane
can be interpreted in two rather different ways:
one is to emphasize that the presence of these three
types of mixtures in any real-life sample will virtu-
ally obscure all other classes of organohalogens,
even in GC GC; but,
it is as interesting to note that there is a striking
separation of the three compound classes of
interest.
In addition, clearly ordered structures are observed for
toxaphene and PCAs.
The last column combination tested, DB-1 VF-
23ms, yielded excellent within-class separation, espe-
cially of non-aromatic compounds OCPs, toxaphene
and PCAs but there was no group separation. This
column set should, therefore, be preferred when
unraveling the composition of, specifically, toxaphene
and the PCAs, is the main aim of a study.
8. Conclusions and perspectives
Accurate, precise congener-specific analysis of organo-
halogenated compounds has been a main goal of ana-
lytical research since the early days of OCP and PCB
determination in environmental samples in the 1960s.
Three areas of interest can be recognized, viz. to ensure/improve:
(i) the separation of the analytes of interest from other
constituents present in a sample to enable their
quantification;
(ii) the sensitivity of the selected procedures and
achieve the required LODs; and,
(iii) the reliability of the identification/confirmation of
the target analytes and/or unknowns.
The common denominator of well-known milestones
of analytical chemistry, such as the introduction of
capillary GC columns, the coupling of GC with selec-
tive detectors, such as the ECD and low- or high-
resolution MS, and the introduction of heart-cut MDGC,is that they all simultaneously improved these three
aspects.
The present review shows that GC GC is effecting a
considerable further improvement and that this tech-
nique will no doubt become another milestone in
improving the analysis of the various classes of
organohalogenated compounds of interest at present. It
is worthwhile adding that the relatively brief introduc-
tory sections on instrumental aspects show that GC GC
can be considered a mature technique (with the excep-
tion of sufficiently rapid and (semi-)automated data
handling and interpretation) and that proper instru-mentation is commercially available.
The degree to which the overall resolution of
organohalogens can be improved by means of GC GC
depends on differences in the physicochemical properties
of the target analytes but, of course, also on the (non)-
complexity of the technical mixtures. The best results for
within-class separations were observed for very complex
mixtures, such as toxaphene, PCAs and PCTs, for which
about 10 times as many peaks can easily be identified.
For less complex mixtures, such as PCBs and PBDEs, the
gain is self-evidently more modest, but still some 50%
more congeners were resolved than by 1D-GC. For the
class of PCNs, with only 75 congeners, the improve-
ment was limited, irrespective of the stationary phase
used. However, one should always consider that the
separation of the target analytes from matrix constitu-
ents is another key issue and, here, GC GC contributes
to a satisfactory overall improvement also in the case of
limited numbers of congeners. Between-class separations
of the various groups of organohalogens come in more
or less the same category, and improvement is pro-
nounced because of the larger differences in physico-
chemical properties between classes than amongst
congeners of the same class. A striking example is the
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essentially complete separation of the technical mixtures
of toxaphene, short-chain PCAs and PCTs in one
GC GC run.
The selection of a suitable column combination is
essential to achieve the desired separation. Certainly for
the fairly hydrophobic and essentially non-polar
organohalogens, the best option is to use a non-polarstationary phase in the first dimension, because then a
truly orthogonal system can be set up. Pure dimethyl-
polysiloxane phases containing 08% diphenyl groups or
those stabilized with polycarborane are preferred and are
easily available from various producers. The separation
mechanism in the second dimension should differ as
much as possible from that in the first dimension. Today,
commercial columns with geometry suitable for use in
the second dimension are coated with one of the fol-
lowing stationary phases polyethylene glycol or dim-
ethylpolysiloxane with up to 70% diphenyl groups, up to
100% cyanopropyl groups, up to 50% trifluoropropyl
groups or up to 50% liquid crystals. Detailed experi-mental work has shown that all of these, except the
trifluoropropyl phase, effect a significantly improved
separation of the organohalogens.
One problem common to all second-dimension sta-
tionary phases, except the phenyl phase, is their low
temperature stability. Further attention needs to be paid
urgently to the production of low-bleeding polar/shape-
selective phases that are stable up to 300C. Another
challenge for the manufacturers is to produce columns
containing two different stationary phases, with a
discrete in-between border. Coupling columns by means
of press-fits, which is a rather laborious and delicate job,will then become superfluous.
A notable exception to the (non-polar) (polar/shape-
selective) set-up is encountered in the analysis of enan-
tiomers such as the 19 pairs of atropisomeric CBs. The
shape-selective (i.e. chiral) column now has to be used in
the first dimension, because the rather marginal sepa-
ration usually obtained on such phases, makes it abso-
lutely necessary to use a long column of, typically,
2060 m: the 12 m length of a second-dimension
column would have little effect.
Analyte detectability can also be affected by the
column combination selected. Low-bleeding columns
should be used in the first dimension to keep noise as low
as possible. This is another reason why non-polar sta-
tionary phases are preferred in the first dimension. In
addition, the second-dimension separation is performed
under isothermal conditions. Consequently, if a study
mainly aims to improve LODs, phases that hardly, or do
not at all, retain the target analytes are preferred in the
second dimension, in order to generate very narrow and,
thus, very high peaks. In this way, an impressive iLOD of
0.2 pg was obtained for 2,3,7,8-TCDD with TOF-MS
detection. However, we would agree that the key aspect
of the analytical approach is now lost it is essentially a
1D-GC operation and there is no or very limited
improvement of the separation.
As regards the detection of organohalogens, today the
lECD is the most sensitive detector available for these
compounds with iLODs typically down to 10 fg. The
data-acquisition rate of 50 Hz is satisfactory for nearly
all applications, but there is considerable peak broad-ening due to the relatively large volume of the detector
cell (150 ll). In other words, the development of a
lECD with a 3050 ll cell volume would be a distinct
bonus.
For the rest, whatever the merits of lECD detection
(and these are significant), for analyte identification/
confirmation, one has to use a mass spectrometer.
Today, the work-horse is the Leco TOF-MS, which can
acquire up to 500 spectra per second over the entire
mass range and is described in the literature for a wide
variety of analytes and sample types. With this instru-
ment, excellent deconvolution of mass spectra can be
obtained. As can be seen from the present review, this isalso true for most organohalogen analyses.
However, it is also clear that, for ultra-trace studies,
satisfactory results cannot always be obtained. In such
instances, the