CHAPTER 6
COMPREHENSIVE TWO-DIMENSIONAL GAS
CHROMATOGRAPHY ( GC × GC )
Tadeusz Górecki, James Harynuk and Ognjen Panić
Department of Chemistry, University of Waterloo, Waterloo, Ontario, N2L 3G1 Canada
ABSTRACT
Comprehensive two-dimensional gas chromatography (GC × GC) is one of the most
powerful analytical tools for the analysis of organic compounds in complex matrices.
The technique is based on continuous collection of the effluent from a GC column and
periodic reinjection of small portions of the effluent to a second column of different
properties. The process is repeated at a rate fast enough that each peak eluting from the
first column is sampled at least three times. In this way, the separation achieved in the
first column is preserved, and additional separation in the second column is
accomplished. At the heart of any GC × GC system is an interface, which physically
connects the primary and the secondary columns and allows the periodic
collection/injection to occur. Raw data obtained from a GCxGC run have to be
converted from the linear form to a 2-D representation using special software
algorithms. GC × GC finds applications in many areas, including petroleum analysis,
environmental analysis, forensics, atmospheric chemistry, etc.
Chapter 6
1 INTRODUCTION
Chromatography is a technique used to separate mixtures of compounds, so that the
individual components can be identified and/or quantified. Among the different
chromatographic techniques, gas chromatography (GC) occupies a particularly
prominent role due to its great separating power, flexibility, widespread applications
and relative simplicity. While GC can solve many problems, the technique often fails
when samples are very complex. This is caused by the limited peak capacity of any
chromatographic column. Bands travelling along the column undergo broadening. As a
result, the number of individual bands that can be fully resolved at the outlet of the
column is finite, even if the initial injection band width is infinitely small. This
fundamental limitation cannot be overcome by simply modifying the chromatographic
parameters. The only solution to the problem is to subject the sample separated by the
GC column to additional separation based on a diferent mechanism, which results in
two-dimensional separation.
The idea of subjecting a sample to multiple types of separations to get improved
resolution and separation power was discussed at length by Giddings [1]. In his paper,
he explained the basic criteria for defining separation dimensions and how to best
combine two separation dimensions into a multidimensional technique. For discrete
separations, in which the sample is first subject to separation in one dimension,
followed by the separation in the second dimension, the best results are always obtained
when the two separation mechanisms are independent. However, within a class of
similar compounds there is often some correlation between the two separation
mechanisms, giving rise to diagonal lines on the retention plane [1]. Many readers are
most likely familiar with at least some multidimensional techniques such as twodimensional thin layer chromatography (2-D-TLC) and polyacrylamide gel
electrophoresis (PAGE).
Up until recently, two-dimensional GC separations were carried out only in the
form of heart-cuts. A portion of the effluent from the main GC column was collected
and injected to a second column with a different stationary phase. While this approach
helped solve many problems (for example the determination of oxygenates in gasoline),
it lacked the power of a comprehensive two-dimensional technique, in which the entire
sample is subjected to all dimensions of the separation and any subsequent separation
dimension preserves the separation achieved in all previous dimensions.
Linking two separation techniques in a manner conceptually equivalent to 2-DTLC would be the ideal solution to increase peak capacity and separation power in GC.
This concept was initially difficult to implement from a practical point of view. It is
easy to meet the requirements of a comprehensive 2-D-TLC separation, where the TLC
plate is physically rotated 90 degrees after the first separation and then developed with a
second, different solvent. However, for comprehensive two-dimensional gas
chromatography (GC × GC), one must find a way to complete the first separation on
one column, and then perform a second separation on a different column while
preserving the separation from the first dimension. This requires more than just two
columns coupled together. It was not until 1991 that the late John Phillips realized what
was required to create a GC × GC system and successfully implement the technique [2].
Several reviews of GC × GC have been published in the literature (e.g. [3], [4],
[5]). This paper is based on the most recent review of the technique by Górecki et al.
[6].
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Chapter 6
2 PRINCIPLES OF GC × GC SEPARATION
In a true multidimensional separation, the two separation methods must be independent
of one another; that is, they must be orthogonal [5]. In the case of GC × GC, this implies
that the two columns must be operated in a way that they retain compounds based on
different mechanisms. The very idea of GC × GC is in principle an extension of
conventional two-dimensional gas chromatography (heart-cut GC), as illustrated in
Figure 1. In conventional heart-cut GC (Figure 1a), two separation columns are used,
but only a small portion of the material exiting the first column is introduced to the
second column and subjected to both separation dimensions. Figure 1b illustrates an
extension of heart-cut GC, in which multiple heartcuts are taken from a single
chromatogram. Obviously, less time is allowed for any individual cut fraction to be
separated in this case. In the limiting case when the number of heart-cuts gets high
enough and the time for the heart-cut separation gets short enough, one accomplishes a
comprehensive two-dimensional GC separation (Figure 1c). Provided that the material
exiting the first dimension is sampled frequently enough, the separation in the first
dimension is preserved, and all of the compounds in the sample are subjected to both
separation dimensions. Thus, GC × GC is in effect a two-dimensional separation
method in which very many sequential heart-cuts are taken.
Several basic requirements for a GC × GC system can be defined in a broad
sense. First and foremost, there must be two orthogonal GC columns in the system.
They must be coupled by a special interface (modulator) that is capable of either
sampling or collecting the effluent from the first column and periodically introducing it
to the second column at a rate that allows the original first dimension separation to be
preserved. Figure 2 presents a conceptual diagram of a GC × GC system. A sample
injected into the system is first subjected to chromatographic separation in the first
column (primary dimension), identical to one-dimensional GC. However, rather than
reach a detector, the effluent from the primary column enters the modulator. This
modulator collects material for a certain period of time, and then injects the entire
fraction that it has collected into the second dimension column as a short
chromatographic pulse. It then collects another fraction of the effluent from the first
column, while the previous fraction is being separated on the second dimension column.
This process of effluent collection and injection repeats itself throughout the entire
analysis. [5] The secondary column then performs another separation of this material
independently of the separation in the first dimension [7]. Finally, the material exiting
the second dimension column is passed to the detector to obtain a series of short second
dimension chromatograms, one after another.
63
Chapter 6
A
Retention Time (min)
2° Dimension Chromatogram
B
Retention Time (min)
2° Dimension Chromatograms
C
Retention Time (min)
Figure 1. The concept of multidimensional GC. (A) single heart-cut GC analysis, in
which a portion of the effluent from the primary column containing analytes
of interest is diverted to the second dimension column and subjected to
additional separation over an extended period of time. (B) dual heart-cut GC
analysis, in which two regions with coelutions are diverted to the second
dimension column, with less time to perform each separation. (C)
comprehensive two-dimensional GC analysis, in which the sizes of the
sequential heart-cut fractions are very small, and the time to develop each
sequential second dimension chromatogram is very short [6].
64
Chapter 6
(f)
(a)
(g)
(c)
(c)
(d)
(e)
(b)
Figure 2. Conceptual diagram of a GC × GC system. (a) injector; (b) first dimension
column; (c) column connector(s); (d) modulator (GC × GC interface); (e)
second dimension column; (f) detector; (g) secondary column oven (optional)
[6].
The need for the modulator is explained in Figure 3. Connecting two columns with
different stationary phases in series without an interface achieves a one-dimensional
separation equivalent to that one would obtain with a stationary phase being a mixture
of the two phases used in the two columns. Even though two analytes may be separated
at the end of the first column, without the modulator to sample the first dimension
periodically, the bands may recombine in the second column and coelute at the detector
(Figure 3b). Another possibility is that the bands will change their elution order when
they flow unchecked from one column to the next (Figure 3c). This instrumental setup
therefore does not preserve the separation achieved in the primary column, violating one
of the conditions for a GC × GC experiment.
With the addition of a properly configured modulator, the effluent from the
primary column can be prevented from continually entering the second dimension
column. If the material the modulator contains is only periodically allowed to enter into
the second dimension column, the primary column separation will be preserved and a
GC × GC separation will be possible. In Figure 3d, the same separation is proceeding in
the primary column, but there is now a modulator between the two columns. It traps and
focuses the black band (Figure 3e), and then injects it into the second dimension
column, while collecting the grey band in the interface (Figure 3f). The grey band is
injected to the second dimension column after the black band has eluted from it. The
grey band can now be separated into two bands, while the spotted band is held in the
modulator (Figure 3g). In this way, the separation achieved in the primary column is
preserved, and additional separation in the secondary column is made possible.
65
Chapter 6
Carrier gas flow
Carrier gas flow
1° Column
1° Column
2° Column
A
D
B
E
C
F
Interface 2° Column
G
Figure 3. The need for the GC × GC interface. (A – C) illustrate how bands separated
on one column can recombine or change elution order on the second column if
they flow uncontrolled from one to the other. (D – G) illustrate how the
interface traps material from the primary column, and then allows discrete
bands to pass to the second dimension column while trapping other fractions
[6].
Preservation of the first dimension chromatogram is achieved through the appropriate
choice of the sampling frequency. In order to preserve the separation achieved in the
first dimension, each peak eluting from this dimension should be sampled at least three
times across its width [8]. Figure 4 depicts a model of the effect of the modulation
period on the preservation of the primary dimension separation. For the model, it was
assumed that the modulator is capable of collecting all of the material that enters it, and
then periodically releasing its entire contents as a narrow band to the second dimension
column. No separation in the second dimension was assumed.
The upper row of Figure 4 shows chromatographic bands as they elute from the
first dimension column. The peaks have base widths of 24 s, and are centered at 15, 55,
56, and 70 s. The modulation frequencies chosen for examination were 6 s (sampling of
the first dimension 4 times per peak; Figure 4b and c) and 12 s (2 times per peak; Figure
4e and f). Modulation in Figures 4b and e was started simultaneously with the beginning
of the run (in-phase with the run; what is called phasing of 0 s). In Figures 4c and f,
modulation was started 3 s after the run has started (phasing of 3 s). Phasing is defined
as the position of the pulses with respect to the position of peaks eluting from the
primary dimension [9]. If two analyses of the same sample are performed with all
conditions the same except that in one case the modulator starts its modulation cycle
immediately when the injection is made, and in the other case the modulator starts its
cycle after a brief delay, two slightly different patterns of peaks will be seen in the final
chromatogram.
66
Chapter 6
y( t )
A
20
16.359
Signal Intensity
Signal Intensity
16.359
y( t )
15
y 1( t )
y 2( t )
y 2( t )
10
y 4( t )
5
0
0
0.2
0.4
0.8
1
1.2
1.4
Time (min)
Signal Intensity
500
0.4
0
0.8
1
1.2
1.4
Time (min)
0
Signal Intensity
0
0.05
0.2
0.4
0.6
0.8
T
1
Time (min)
1.2
1.4
1.55
1.6
1.5
0.2
0.4
0.6
0.8
1
1.2
T
1.4
1.4
Time (min)
F
1000
500
0
1.6
1.4
1500
H
500
1.2
Time (min)
0
2000
2000
1000
1
t
0
1500
0.8
500
1.5
T
0.6
1000
1.6
C
2000
2000
Signal Intensity
0.6
0.4
1500
H
0.2
0.2
E
2000
2000
1000
0
0
0
1.5
1500
0
5
0
1.6
B
2000
2000
Signal Intensity
0.6
t
0
0
10
y 3( t )
y 4( t )
H
15
y 1( t )
y 3( t )
H
D
20
0
0.05
0.2
0.4
0.6
0.8
T
1
Time (min)
1.2
1.4
1.6
1.45
Figure 4. The effect of the modulation period on the preservation of the first dimension
separation. (A) and (D) - hypothetical first dimension separation of four
components (shown in dotted lines), each peak having a base width of 24s. (B)
reconstructed 1-D chromatogram obtained with the modulator operating with a
period of 6 s and phasing of 0 s (see text); (C) reconstructed 1-D
chromatogram obtained with the modulator operating with a period of 6 s and
phasing of 3 s; (E) reconstructed 1-D chromatogram obtained with the
modulator operating with a period of 12 s and phasing of 0 s; (F) reconstructed
1-D chromatogram obtained with the modulator operating with a period of 12
s and phasing of 3 s [6].
67
Chapter 6
Figures 4b and c show that the 6 s modulation period yields a reconstructed 1-D
chromatogram which preserves the most important features of the original
chromatogram (see the well-preserved partial separation between the second and the
third bands). On the other hand, Figure 4d shows that with a 12 s modulation period,
some of the separation achieved in the first dimension is lost (the second and third
bands in Figure 4d are fused into a single band independently of the phase).
Another important feature is the width of peaks in the second dimension of a GC
× GC separation. The narrower the peaks are, the more of them can fit side by side in
the limited amount of space in the second dimension chromatogram, which results in
increased peak capacity. Secondly, if two peaks have the same area, the narrower peak
will be taller, sometimes quite significantly. Thus, the signal intensity is increased, and
the sensitivity of the system can be potentially improved. The magnitude of the gain in
sensitivity will depend primarily on the modulation type and period. Increased signal is
observed when the modulator collects rather than samples the eluent from the first
column. When the modulation period decreases, resolution in the first dimension is
degraded, but the primary dimension peaks are also collected in fewer, larger fractions,
leading to more intense peaks in the second dimension. The effect of second dimension
peak width on peak capacity and signal intensity is shown in Figure 5. In Figure 5a, the
widths of the injection bands in the second dimension at the base are 180ms. In Figure
5b they have been broadened to 300ms. The heights of the peaks in Figure 5b are much
smaller because of their increased width, which also limits the peak capacity in the
second dimension and reduces any potential gains in sensitivity. Interested readers
seeking further and a more in-depth analysis of modulation are referred to papers by
Marriott et al. [9] and by Lee et al. [10].
500
A
500
P( t )
Signal Intensity
Signal Intensity
400
300
y( t )
y( t ) .10 200
100
0
Signal Intensity
500
P( t )
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
t
Time
Retention(min)
Time (min)
0.4
0.45
0.5
0.55
0.6
B
400
300
y( t )
y( t ) .10 200
0
0
500
100
0
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0.45
0.5
0.55
t
Time
Retention(min)
Time (min)
Figure 5. Demonstration of the effect of second dimension peak width on signal
intensity and second dimension peak capacity. (A) second dimension peak
width of 180 ms at the base; (B) second dimension peak width of 300 ms. The
original primary dimension chromatogram is plotted as a solid line, and
magnified 10x (dotted line) in each pane to facilitate visual comparisons [6].
68
0.6
Chapter 6
3 DATA INTERPRETATION
In a GC × GC experiment, the material eluting from the primary column is sampled and
injected in the form of narrow, periodic pulses onto the secondary column. The detector
records a continuous linear signal, which is in fact a series of second-dimension
chromatograms that elute one after another from the secondary column (see Figure 6a).
As each peak from the primary column is sampled at least three times in a proper GC ×
GC experiment, it will show up in at least three consecutive second-dimension
chromatograms. The resulting chromatogram is very complex and difficult to interpret.
It is not immediately obvious which peaks in a series of second dimension
chromatograms originate from the same compound, and which are different compounds.
Additionally, it is impossible to plot the entire raw chromatogram in a way that shows
any details. As a result, the data is usually converted into a three-dimensional plot, and
displayed as a top-down view in the form of a contour plot with primary retention
plotted along the ×-axis and secondary retention plotted along the Y-axis. The peaks
appear on such chromatograms as spots of varying colour or contour lines. The
construction of such a plot is outlined in Figure 6. The software uses the modulation
period of the interface and the times at which the pulses to the second dimension
column occur (t1, t2, t3 in Figure 6a-b) to slice the original chromatographic signal into
its component second dimension chromatograms. These chromatograms are then
aligned side-by-side to form GC × GC retention plane (Figure 6c) which is then plotted
top-down as in Figure 6d. The time at which a modulation pulse occurs provides the
primary retention time for all of the peaks that elute between that pulse and the
following pulse. The second dimension retention time of a peak is then its original (1-D)
retention time minus the primary dimension retention time. In order to account for the
dead time in the second dimension column and generate plots that are easier to interpret,
the modulation pulse times are usually increased by the second dimension dead time.
Readers interested in a more in-depth discussion of how GC × GC data is presented and
the different effects that can be seen by altering such parameters as contour intervals
and their spacing, are referred to a paper by Harynuk et al. [11].
To quantify the data in a GC × GC analysis, the peaks in each second dimension
chromatogram that correspond to the analyte of interest are integrated using the
common routines and the raw data file, and then the areas of all these peaks are
summed. Techniques for quantifying the data were introduced by Beens and co-workers
in 1998 [12] and have been improving steadily since. It is important to emphasize that
while peak integration in GCxGC may be more dificult than in conventional 1-D GC,
comprehensive two-dimensional gas chromatography is a fully quantitative technique
that produces accurate and reproducible results. In fact, because of the dramatically
increased resolution between the peaks and signal enhancement due to peak
compression, quantitative results obtained by GCxGC are often more accurate and
precise than results obtained by conventional 1-D-GC.
69
Chapter 6
A
A
t3
t2
Signal
t1
Time
B
B
t1
t2
t3
C
C
1°
t1
t2
ret
e
nti
on
n
entio
t
e
r
2°
t3
t1 t2 t3
2° retention
Signal
D
D
1° retention
Figure 6. The interpretation of GC × GC data and generation of contour plots. (A) Raw
GC × GC chromatogram consisting of a series of short second dimension
chromatograms; t1, t2, and t3 indicate the times when injections to the second
dimension column occurred. (B) The computer uses these injection times to
slice the original signal into the individual second dimension chromatograms.
(C) The second dimension chromatograms are aligned on a two-dimensional
plane with primary retention time and secondary retention time as the × and Y
axes, respectively, and signal intensity as the Z-axis. (D) When viewed from
above, the peaks appear as rings of contour lines or colour-coded spots [6].
A problem with data interpretation can arise if a peak has a retention time in the second
dimension that is longer than the modulation period. This peak will elute with
compounds that are introduced to the second dimension column during the following
modulation cycle, possibly causing coelution problems. As such, it will have an
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Chapter 6
apparent secondary retention time that is shorter than its actual retention time by the
length of the modulation period. Such a peak is termed a wraparound peak. Wraparound
peaks can cause problems if they elute in a region where other peaks of interest elute.
These wraparound peaks are broader than the peaks from the current modulation
period, as they have spent a longer time in the second dimension column. This, in fact,
facilitates recognition of such peaks in a GC × GC cromatogram. Wraparound peaks
interfere with the ordered nature of GC × GC chromatograms and may cause problems
with quantitation if they happen to co-elute with analytes of interest.
One of the great advantages to performing GC × GC separations is that every
component in a mixture has two retention times that can be used for identification
purposes. In effect, this is nearly equivalent to two separate 1-D GC separations, one on
a primary column, and one on a confirmation column, as required by some analytical
methods. The identification by two-dimensional retention coordinates has been likened
to the identification by a 1-D retention time and a mass spectral match [4].
Another interesting feature of GC × GC is the ordered nature of the
chromatograms. Peaks belonging to homologous series of compounds are usually
positioned along straight lines on the 2-D retention plane. This order allows the
researcher to assign an unknown peak to a compound class with no information other
than where the peak lies on the 2-D retention plane. For example, most researchers in
the field can look at a GC × GC chromatogram of gasoline and immediately identify
groups of peaks that represent alkanes, alkenes, various alkyl-substituted benzenes and
napthalenes. Such a feat is not possible with conventional 1-D chromatograms. More
information on the ordered nature of GC × GC chromatograms and their usefulness for
providing rapid identification of compounds in complex hydrocarbon mixtures can be
found in a paper by Schoenmakers et al. [13].
The ordered chromatograms and the highly reproducible retention coordinates
for compounds on the retention plane make the technique very useful for fields such as
forensics. Forensic identification often relies on subjecting a pair of samples to the same
analytical technique and then comparing the pattern of peaks or bands from one sample
to that of the other. GC × GC offers a much more detailed pattern, which makes it easier
to draw firm conclusions on the identity or lack thereof of the suspected sample and the
reference sample. Gaines et al. demonstrated one of the first applications of GC × GC to
pattern matching and forensics [14].
4 INSTRUMENTATION
Much of the basic instrumentation that is used for 1-D-GC has been carried over to GC
× GC. When considering injectors and injection techniques, any technique that is used
for conventional GC can in principle also be applied for GC × GC analyses.
Primary columns are generally 15 – 30 m long, with internal diameter of 0.25
mm and film thickness in the range of 0.25 – 1.0µm. These columns allow for the
generation of peak widths in the second dimension on the order of 10 – 20 s, which are
required for typical modulation periods (3 to 6 s). The first dimension columns typically
have a non-polar stationary phase, either 100% polydimethylsiloxane or 95/5%
methyl/phenyl siloxane. The second dimension separation must be very fast and
performed with a stationary phase that is different from that used in the primary column.
It is desirable for compounds from a modulator pulse to elute from the second
dimension with a range of second dimension retention times that is less than or equal to
the modulation period. This puts large constraints on the choices for second dimension
columns. Typical dimensions for these columns range from 0.5 to 1.5 m in length and
0.1 mm in diameter, though some researchers prefer to use 0.25 mm diameter columns.
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Chapter 6
Thinner films of stationary phase are also typically used (0.1 - 0.25 µm) as they offer
higher efficiencies and are not as retentive as thicker-filmed columns. The stationary
phase chosen for the second dimension must offer a different separation mechanism
than the primary column. Typically, wax-based phases, or 50/50 phenyl/methyl phases
are used.
Both columns are usually placed in the same GC oven. In some solutions, a
separate oven for the second dimension column is used (see Figure 2). The application
of a second oven provides more flexibility in the control over the secondary separation,
but at the same time it complicates the design of the system and adds one more variable
to the already long list of method parameters.
The most important requirement for the detectors used for GC × GC is that they
should be fast. Peaks eluting from GC × GC systems are very narrow (typically 150 –
400 ms at the base). Thus, to get a sufficient number of data points to accurately
describe the shape of a peak (at least 10 points per peak), a detector that can collect data
at a rate of at least 50 Hz is required. Few GC detectors fulfil this requirement. Most
research is done with Flame Ionization Detectors (FID). Some research has also been
conducted using a micro Electron Capture Detector (µ-ECD) [15], and more recently
with Atomic Emission Detector (AED) [16] and Sulphur Chemiluminescence Detector
(SCD) [17]. Data acquisition rate in the scanning mode is too slow for most mass
spectrometers to handle the narrow peaks obtained from a GC × GC system. The only
exception is the Time-of-Flight Mass Spectrometer (TOF-MS) [5]. GC × GC-TOF-MS
systems are now commercially available, though their cost is daunting to many
laboratories. The other difficulty with handling such a system is the sheer size of the
data files that are generated over the course of an analysis, which must then be
interpreted if they are to be of any use. The challenge of handling GC × GC-TOF-MS
data was discussed recently by Dallüge et al. [18].
The heart of any GC × GC system is the modulator. In general, most designs can
be classified into two main categories: thermal modulators and valve-based modulators.
Thermal modulation is the more commonly applied modulation technique. Thermal
modulators can be subdivided into two categories: heater-based (applying an increase in
temperatue) and cryogenic (applying a decrease in temperature). Following is a brief
description of selected representative modulator designs.
Thermal Modulators
Heater-based modulators
Phillips’ first modulator, introduced in 1991, employed a segment of thick-filmed
capillary column coated with a layer of gold paint as the interface between the primary
and the secondary columns [2]. As a chromatographic band from the primary column
entered the thick-filmed segment of the column, it partitioned into the stationary phase
and slowed down, focusing into a narrow band. The analytes trapped in this way were
periodicaly released by thermal desorption caused by passing electrical current through
the metallic coating on the capillary. Trapping was restored again once the capillary
cooled down to the oven temperature.
It was quickly realized that any compounds arriving at the modulator while it
was hot would simply pass directly through it without being focused. This resulted in
broad injection pulses onto the second dimension column. To solve this problem, a
dual-stage trap was proposed. In this design, the two traps operated alternately, so that
when one was hot, the other was cold, and vice versa. With the first trap collecting
material while the second trap was launching its contents to the second column, no
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Chapter 6
breakthrough could occur. The dual stage design is one of the main features of nearly all
thermal modulators in use today.
The original GC × GC interface had many weaknesses, mostly due to the fact
that the modulator capillaries and the paint coating were not very robust or reproducible.
The first reliable heated modulator was also developed by Phillips and co-workers [19].
The rotating thermal modulator, shown in Figure 7, accomplished trapping, focusing
and re-injection through the use of a rotating slotted heater that periodically passed
along a segment of thick-film GC column acting as the trap. When analytes entered the
interface, they were trapped by partitioning into the stationary phase as in the original
modulator. Desorption was accomplished through the use of a mechanical heater that
was held at an elevated temperature (typically 100°C higher than the final oven
temperature). As the heater passed over the column moving in the same direction as the
carrier gas, it caused any material sorbed in the heated region to partition into the gas
phase where it was simultaneously “swept” towards the end of the modulator and
focused into a narrow band before entering the second column. This modulator was the
first to become commercially available. It performed very well for many applications,
but it also had its limitations due to the use of moving parts, which sometimes caused
problems.
Both modulator designs had significant disadvantages. The trapping mechanism
made collecting volatile compounds at conventional oven temperatures practically
impossible. The modulators could be used for GC × GC analysis of volatiles only with
the oven at subambient temperatures. Another limitation was related to the use of
elevated temperature to mobilize the trapped analytes. To avoid thermal decomposition
of the stationary phase in the trapping capillary, the final oven temperature had to be
lower by at least 100°C than the thermal limit of the stationary phase. This significantly
limited the range of the boiling points of compounds amenable to GC × GC analysis
with these modulators.
Figure 7. Schematic diagram of the rotating thermal modulator [6].
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Chapter 6
Another heated modulator was developed in our laboratory. It used a pair of in-line
micro sorbent traps to trap and focus analytes eluting from the primary column. The
sorbent traps were positioned in Silcosteel capillaries that could be resistively heated in
order to desorb the contents of the traps [20]. This modulator design had no moving
parts, and the sorbent bed had a much higher capacity than the thick-filmed modulator
capillaries used in other modulators, resulting in more efficient trapping of volatile
compounds. On the other hand, thermal stability of the sorbent limited the modulator’s
applicability to higher boiling compounds as in the other heated modulator designs.
Cryogenic Modulators:
The first cryogenic modulator, longitudinally modulated cryogenic system (LMCS),
was developed in Australia by Philip Marriott [21]. The design of this interface is
schematically depicted in Figure 8. A segment of a column is cooled with liquid CO2 to
cause analytes to partition into the stationary phase in a small region of the column. To
introduce the material to the second-dimension column, the trap is physically moved to
a region of the column upstream of the trapping position. The cooled region rapidly
heats back up to the oven temperature allowing the trapped analytes to be remobilized.
Additionally, by placing the cryotrap upstream of the desorption point, breakthrough of
analytes from the primary column is prevented. Subsequent relocation of the trap to its
original position traps a new portion of primary column effluent in the downstream
position.
CO2 (l)
T
R
Figure 8. Schematic diagram of the longitudinally modulated cryogenic system
(LMCS). T and R denote the trap and release positions of the cryotrap as it
moves along the column. As an analyte band moves down the column, it
encounters the trap at the T position and is focused and held in place. When
the trap moves to the R position, the band that was trapped at the T position is
released and launched to the second dimension column, while the cryotrap
prevents breakthrough of material from the first column. After the focused
band has left the trapping zone, the cryotrap returns to the T position and the
cycle repeats itself [6].
74
Chapter 6
This modulator was the first that was reliable enough to be used routinely, and it has
been used by many people. One advantage that cryogenic modulation offers over heated
modulation is that the modulator only needs to be raised to the oven temperature for
desorption, not to a temperature above the oven. This allows for higher oven
temperatures to be used in the analysis compared to heated modulators. The main
weakness of this modulator comes from the use of liquid CO2 as the cryogenic agent.
Trapping temperature of approximately –50°C is not sufficiently low to efficiently trap
more volatile analytes. In addidition, the modulator relies on moving parts, which can
potentially cause problems.
It is generally believed that cryogenic modulation has fewer drawbacks than
heated thermal modulation, and cryogenic interface with no moving parts would be
highly beneficial. Two non-moving cryogenic modulators were developed at similar
times. One, developed in our laboratory, used two deactivated stainless steel capillaries
that were connected in series and placed in a cryochamber cooled with liquid nitrogen.
Trapping was effected through freezing rather than partitioning as in the CO2 - cooled
interfaces. To inject the contents of the trap to the second dimension column, an
electrical pulse was delivered to the trap to resistively heat it [20]. This interface could
easily trap most volatile analytes, and that the injection timing could be controlled very
precisely. The main disadvantage with this modulator was the ocasional formation of
cold spots leading to band broadening as a result of wear of the cryochamber seals.
The other non-moving cryogenic interface that was developed used two liquid
CO2 cold jets for trapping and two hot jets for desorption to effect two-stage modulation
[22]. Material eluting from the primary column was trapped by the first cold jet. It was
then desorbed by turning the first cryojet off and the first warm jet on. The material was
re-trapped along with any material arriving at this moment from the primary column by
the second cold jet. Before it was injected to the secondary column (by turning off the
cold jet and turning on the warm jet), the first cryojet was turned back on to prevent
breakthrough of the analytes from the primary column. This modulator generally
performed very well, but was overly complicated with four jets that needed to be
controlled in sequence. However, it did provide the basis for most of the new cryogenic
modulators that have been developed recently, and it has been successfully
commercialized.
Beens et al. developed a simplified version of this modulator that uses only two
cryojets to do the trapping, allowing the GC oven to provide the warm air for heating
the trapping zones as shown in Figure 9 [23]. A further simplification of the modulator
that uses only a single jet to perform modulation by careful control of the timing was
introduced by Adahchour et al. in 2003. This modulator is the simplest cryogenic
modulator developed thus far. Its primary advantage is the simplicity of the
instrumentation. The disadvantage of it is that with only one trapping zone, the timing
of the jet and tuning of the instrument must be done very carefully to avoid excessive
breakthrough of the material from the first column while the trap is hot.
75
Chapter 6
CO2 (l)
D
U
Figure 9. Schematic diagram of the dual cryojet interface. When the downstream jet (D)
is on and and the upstream jet (U) is off, material from the primary column is
trapped as a narrow band within the second dimension column. It is then
released by turning the downstream jet off, and retrapped by the upstream jet.
The downstream jet is turned back on before desorption from the second stage
is effected to prevent breakthrough [6].
The original four-jet modulator was also simplified by Ledford and co-workers who
introduced a modification that relies on a single jet of cryogen, and a loop of capillary to
effect dual-stage modulation [24]. In this interface, the single jet cools two segments of
a capillary simultaneously. In the first segment, effluent from the primary column is
collected when the cryojet is turned on. When the cryojet is turned off, the material in
the first stage is introduced to the loop along with breakthrough. The cryojet is then
turned back on to collect a new portion of effluent in the first stage, while the material
in the loop is flushed to the second trapping stage. Then, when the cryojet is turned off,
material from the first stage is injected into the loop while material from the second
stage is injected to the second dimension column. This interface is the simplest dualstage cryogenic modulator developed thus far; however, care must be taken when
setting its operational parameters. The length of the loop, combined with the linear
velocity of the carrier gas through it, put limitations on the modulation periods that can
be used with this type of interface.
Trapping of the most volatile analytes requires that liquid nitrogen (LN2) is used
as the cooling agent. This complicates the design of the system, and significantly
increases the costs of analysis. Consequently, reduction in the consumption of LN2 is
one of the modulator design goals. We have recently developed a cryojet modulator that
uses liquid nitrogen at a rate of approximately 20L/d through the implementation of a
cryogen-saving delivery system [25]. This may aid in making the use of liquid nitrogen
as a cryogen for modulation feasible for more laboratories.
76
Chapter 6
Valve-based GC × GC Interfaces:
The other family of GC × GC interfaces is the valve-based interface group. The initial
attempts at modulation employing valves were accomplished via a diaphragm valve, as
introduced by Bruckner and co-workers [26]. In this design, most of the effluent from
the primary column was vented to the atmosphere. At the same time, carrier gas from an
auxiliary supply was continuously delivered to the secondary column. The effluent from
the primary column was sampled periodically by actuating the valve for a brief period
of time, and the secondary chromatogram was recorded. The process was repeated
periodically for the entire chromatographic run. This method proved to be less sensitive
than thermal modulation because rather than collecting the material from the primary
column and injecting all of it to the second dimension column, only small fractions of
the effluent from the primary column (10-20 %) were periodically taken and separated
[7].
Seeley introduced an alternative configuration of the valve – based interface by
connecting a sample loop to the valve [27]. The differential flow interface is illustrated
in Figure 10. This technique used a primary column operated at a low flow rate, with the
effluent passing into a 20µL sample loop before being vented to the atmosphere. The
second dimension column was operated at a relatively high flow rate, at least 20 times
higher than the flow in the primary column. Thus, when the valve was actuated to inject
a sample to the second dimension column, the gas in the sample loop was physically
compressed and injected as a narrow pulse to the second dimension column. With this
method, approximately 80% of the effluent from the primary column was sampled [27].
With the physical compression into a narrow band, there is some potential for an
increase in sensitivity, though not to the same extent as in thermal modulators.
Sample Loop
Vent
•• •
•• •
Auxiliary gas
supply
Figure 10. The valve-based differential flow modulation GC × GC interface. Flow
through the primary column is slow, and passes through a sample loop
before being vented. The second dimension column is operated at a very
high flow rate by means of the auxiliary gas supply. By switching the
valve, the material in the sample loop is compressed by the high pressure of
the auxiliary gas supply and launched onto the second dimension column
[6].
77
Chapter 6
The partial venting of the primary column effluent by the valve-based modulators has
sparked many debates whether this approach to GC × GC qualifies as truly
“comprehensive”. The issue has been finally settled at the First International
Symposium on Comprehensive Multidimensional Chromatography (March 6-7, 2003,
Volendam, The Netherlands), where a consensus was reached that as long as the valvebased interfaces sample the primary column often enough to faithfully represent the
primary separation, the technique can be considered comprehensive (albeit without the
same sensitivity enhancements as seen with thermal modulators). Applying the valve
interface thus sacrifices sensitivity for a non-thermal approach to modulation. The main
advantages of the valve-based techniques are that there is no possibility of
breakthrough, even for the most volatile analytes, and that there is no reliance on
cryogenics. They can also be used for very fast second dimension separations (1s or
less) due to the fast valve switching times, which make it possible to produce very
narrow injection bands. Their main disadvantage is the presence of a valve in the
chromatographic flow path. Since there are no diaphragm valves that can be heated to a
high enough temperature to allow heavy compounds to pass through them unimpeded,
this modulator cannot be used for applications such as the separation of high molecular
weight PAHs.
With the various types of modulators and the number of combinations of
primary and secondary columns that are available, there may be some confusion as to
what is the best approach for one to take to performing GC × GC separations. The
simple answer is that there is no single best interface or column set for all applications.
For example, for the analysis of mixtures with many high boiling compounds (such as
crude oils), a system that relies on liquid CO2 cryogenic modulation and uses columns
that can withstand high temperatures would be the best. For the analysis of highly
volatile compounds such as VOCs in urban air, a valve-based system or a cryogenic
modulator using liquid nitrogen would be the best choice.
5 APPLICATIONS
GC × GC can provide the user with such possibilities as increased separation power,
increased sensitivity and highly structured, ordered chromatograms that are more
suitable for pattern recognition-based analysis. Either some or all of these characteristics
have been exploited in many different fields. While initially mostly petroleum and
environmental samples were studied, the scope of applications has broadened greatly.
Application areas now also include fields such as food and fragrance, health, and
forensic science. Table 1 gives examples of some of the applications of GC × GC in
various fields. Following is a brief summary of some of the more interesting
applications.
Petroleum samples can contain thousands of compounds, and in some situations,
most (if not all) of them can be analytes of interest [28]. Beens et al. provided an
extensive illustration of how GC × GC can be applied to the analysis of petroleum
samples such as non-aromatic hydrocarbon solvents, kerosene, crude oil and olefinic
petroleum fractions [28]. In this application, the structured chromatograms provided an
excellent qualitative overview of the mixture’s chemical composition. Complete
separation of kerosene was also accomplished. The power of GC × GC is shown even
more in the separation of crude oils which are extremely complex mixtures consisting of
thousands of compounds. The structured nature of the GC × GC chromatograms allows
the researcher to easily quantify individual compound classes. The high resolving power
also allows for the quantitation of individual trace components in the matrix, a feat
impossible with other separation techniques.
78
Chapter 6
In environmental analysis, GC × GC offers increased separation power and
sensitivity that is usually greater than in conventional 1-D GC. The increased separation
power often makes it possible to vastly simplify complex sample clean-up procedures or
to eliminate them entriely, leaving the instrument to separate matrix interferences from
the analytes.
GC × GC allowed the determination of many small, highly volatile, polar
compounds in an urban atmosphere [29]. Many of these compounds were not known to
exist in the atmosphere until this study, due to their low concentrations and the complex
nature of the samples resulting in many coelutions when separated in a single
dimension. In a conventional 1-D separation, only 10-20 peaks were found, many of
them with coeluents. In the GC × GC separation, 100-200 compounds could be
observed, some of them being potentially harmful to humans, even in very low
concentrations [29].
Separation of complex mixtures of chlorinated compounds found for example in
polychlorinated biphenyls (PCBs), polychlorinated dibenzo-p-dioxins (PCDDs) and
dibenzofurans (PCDFs) is an area of environmental analysis that has challenged the
researchers for many years. Though relatively few congeners of these compounds are
toxic, they have to be detemined in a mixture of compounds with very similar properties
(e.g. 209 PCB congeners). Conventional analysis of these compounds requires extensive
sample preparation, followed by multiple 1-D GC analyses on different columns to
quantify the analytes. High resolution mass spectrometry often has to be used for
detection and final identification. The problem can be solved in a much more efficient
way by using GC × GC. Several researchers have succeeded in analysing mixtures of
the toxicologically significant congeners of these compounds (along with some other
commonly-found congeners) in a single analysis and with the use of a much simpler
detector (µ-ECD) (see e.g. [15] and [30]).
Dallüge et al. illustrated how the utilization of GC × GC can simplify sample
preparation for the analysis of pesticides in food extracts [31]. To prepare the sample
(celery) for analysis, a sample was taken and chopped, then mixed with ethyl acetate
and sodium sulphate. This mixture was blended, centrifuged, and the ethyl acetate layer
was removed and dried over sodium sulphate before being injected to the GC × GCTOF-MS system for analysis. The analyte peaks were resolved from the matrix peaks,
and all of the 58 target pesticides were identified with a good match. Even though some
of the pesticides did partially coelute with the matrix peaks on the column set that was
used in this work, they could be easily quantified using standard deconvolution routines
because the GC × GC separation provided relatively simple mixtures of compounds to
the mass spectrometer.
GC × GC can also be used to investigate thermodynamic properties of
compounds in a way never before seen in chromatography. Marriott et al. characterised
the E/Z isomer interconversion of oximes by GC × GC [32]. When a mixture of the E/Z
isomers is separated by GC, the outcome depends on temperature. If it is low enough
that the molecules cannot overcome the internal energy barrier to interconversion, two
distinct peaks, one for each isomer, will arise. As the temperature increases, a plateau
region can be observed between the two peaks, representing molecules that have been
converting back and forth between the two isomers. Many researchers have been able to
determine theoretical ratios of the two isomers at any given time during the plateau
region; however, there has been no way to experimentally confirm the theoretical
models. GC × GC made it possible to separate the two coeluting isomers in the plateau
region on the second dimension column. Because the second dimension separation was
very fast, no interconversion between the isomers could occur during the second
79
Chapter 6
dimension separation, and two distinct peaks with no plateau between them were
observed. This was the first demonstration of direct observation and measurement of
such phenomena in GC.
TABLE 1
An overview of some applications and advantages of GC × GC in various fields of
research.
Application area
Advantage over 1-D GC
References
Petrochemical /
Organics
Greatly enhanced separation power
[13], [14], [28], [33],
[34], [35], [36], [37],
[38], [39], [40], [41],
[42]
Foods and Fragrance
Highly ordered structured
chromatograms make group-type
analysis of a sample simple.
Greatly enhanced separation power
Allows determination of trace
analytes not observable in 1-D
separations
Environment and
Health
Simplified sample preparation if the
analytes can be separated from
many large matrix peaks.
Greatly enhanced separation power
Potential gains in sensitivity
Simplified sample preparation may
be possible
Forensics
Multiple 1-D analyses for
determination of analytes can be
replaced with a single GC × GC
analysis (e.g. PCB analysis)
Greatly enhanced separation power
Highly ordered and structured
chromatograms provide more
detailed patterns to be used in
pattern recognition/comparison
studies.
80
[43], [44], [45], [46],
[47], [48], [49], [50],
[51], [52], [53]
[15], [18], [29], [30],
[31], [54], [55], [56],
[57], [58], [59], [60],
[61], [62], [63], [64],
[65], [66]
[14], [67], [68]
Chapter 6
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