Comprehensive two-dimensional gas chromatography (GC×GC) was introduced two decades ago, but interest in the technique developed only recently. Making use of a modulating device placed between two columns connected in series, GC×GC technology can increase the capacity of a chromatographic system to thousands of peaks. Both thermal and valve-based modulators are reported in the literature, with the first being more widely used and commercially available. In thermal devices, as eluent from a first column enters the modulator, it gets trapped through the use of cold jets and reinjected into a secondary column by rapid heating. The compounds present in the sample are not only subjected to two different separation mechanisms, but the thermal modulator refocuses the bands and can enhance detection limits. On the other hand, the narrow chromatographic peaks obtained from the GC×GC systems limit the choices of detectors that can be used in conjunction with this technology. As typical peak widths in the second dimension are in the range of 50 to 150 msec, detectors with fast acquisition rates capabilities are required for adequate peak definition.
LECO's Corporation’s Time-of-Flight Mass Spectrometer (TOFMS) is capable of acquisition rates up to 500 spectra/sec, adequate for the characterization of peaks as narrow as 20 msec. The high acquisition rate not only provides good chromatographic peak characterization but also, in conjunction with the absence of spectral skewing, enables true spectral deconvolution capabilities. Even with the peak capacity generated by the GC×GC technology, the complexity of real life samples almost always results in peak overlap, and co-elutions become a day-to-day reality. GC×GC-TOFMS technology is a useful tool for complex sample analysis.
Figure 1. Simple schematic of a GC×GC system
operated with a dual-stage thermal modulator. Click
to enlarge.
Figure 1 shows the simple schematic of a GC×GC-TOFMS system equipped with a dual-stage thermal modulator. While the first column is typically a long capillary column (most of the time non-polar), the second column is a short, narrower bore column (most of the time polar). This set-up enables very fast separations in the second dimension. The secondary column can be enclosed in an independently controlled mini-oven (shown in Figure 1) that allows better retention control for the compounds analyzed.
Perfume Sample Analysis
Recent health concerns about allergic reactions caused by chemicals present in fragrances of synthetic or natural origin have lead to an increased interest in the analysis of perfumes. The examples below come from the analysis of a fragrance sample spiked with a standard mixture of 35 compounds: 24 allergens, 3 carcinogens, 2 internal standards and 5 isomers of the targeted compounds. The complexity of the fragrance samples as well as the interference of the ions in the targeted compounds with the ions in the matrix components makes it impossible to perform the analysis on a single one-dimensional GC-MS system.
Due to the nature of the sample, the column combination for the analysis had to provide the maximum separation possible in both the first and the second dimension. To accomplish this, the two columns selected were chosen to be as different in polarity as possible: 30-m × 0.25-mm × 0.25-μm Rtx-5 in the first dimension and a 1-m × 0.1-mm × 0.1-μm DB-Wax in the second dimension. Both ovens were operated under a temperature programming rate of 5 C/min, with the secondary oven offset from the main oven by +10°C. The TOFMS was set to store the data for a continuous mass range from 35 to 350 μ and the acquisition rate was set at 150 spectra/sec.
[Insert figure 2 here.] Figure 2. TIC chromatogram of the standard allergens mixture
zoomed to show the elution region for the compounds of interest. The upper right
corner shows the full scale of the TIC chromatogram.
Figure 2 shows the total ion current (TIC) chromatogram of the standard mixture
obtained with a Pegasus 4D GC×GC-TOFMS system (Leco Corp., St. Joseph, MI).
Click
to enlarge.
The x-axis represents retention time on the first column while the retention time
on the second column is shown on the y-axis. Peak intensity is shown on a color
scale from blue to red, with red showing the most intense peaks. It is easy to
see that the selected column combination provided very good separation for all
the 35 components in the mixture with their retention times in the second dimension
spanning over almost 3.5 sec (from 0.567 to 4.053 sec). Some of the 35 components
present in the mixture have identical or very close retention times in the first
dimension. This indicates that an attempt to separate this mixture by one-dimensional
analysis using a 5% phenyl column will most likely result in co-elution of these
compounds. Some examples of possible co-elutions are pairs 5/7, 12/13, 18/19,
25/26, 29/28.
The bigger challenge, though, comes with the analysis of a spiked perfume sample.
Even though most of the analytes were successfully separated from the matrix in
this case, high matrix interference can almost completely mask the presence of
some of the targeted analytes as presented in part (a) of Figure 3. It can be
seen in this figure that peak 24 (amylcinnamic aldehyde) is almost completely
masked by matrix while peak 25 (8-phenyl-1-octanol) and most of the other allergens
in the figure are lost in the blue background due to the big concentration difference
between the analyte of interest and the matrix. Plots of characteristic ions allow
easier visualization of the targeted compounds in the chromatogram as shown in
part (b) of this figure. Here only ions 91 and 115 are plotted, and this makes
compound 25 very easy to spot and improves visual identification for compound
24 (even though high matrix interference is still present).
Figure 3. Contour plot of a perfume sample spiked
with a standard allergen mixture shown as (a) TIC and as (b) sum of ions 115 and
91. Part (c) of the figure shows the co-elution of one of the targeted analytes
with a matrix peak. Mass spectral information for the targeted peak is also presented
in here.Click
to enlarge.
Even with the great increase in peak capacity obtained by GC×GC, co-elutions
of peaks can still be present, and they present a challenge for the chemist performing
the analysis. The TOFMS has the advantage of spectral continuity across the chromatographic
peak profile (no spectral skewing) that allows the ChromaTOF software's (LECO
Corp.) automated peak find and true signal deconvolution algorithms to correctly
locate and accurately extract spectral information for co-eluting peaks. Part
(c) of Figure 3 shows very good deconvolution results for peak 24. The upper part
of the figure shows the Caliper spectrum (spectrum of the peak at the caliper
position), the middle part shows the Peak True spectrum (spectrum of the peak
after deconvolution), and the lower part shows the Reference spectrum (spectrum
of the standard analyzed individually). A closer look at the three spectra presented
in this part of Figure 3 shows almost complete removal of masses 83 and 84 (very
high intensities in the caliper spectrum), 137, 153, 165, 181 from the Peak true
spectrum. Also, the shared masses in the Caliper spectrum are correctly distributed
in the Peak True spectrum. This deconvolution process generates a match of 924
with the reference standard (with 999 being the perfect match) even though the
intensity of the targeted peak (24) is 56 times smaller than for the matrix peak
and the separation between peak apexes is only 0.1 sec.
Conclusion
The combination of increased peak capacity obtained with GCxGC technology, True Signal Deconvolution facilitated by the TOFMS detector, and the unique algorithms
Tincuta Veriotti, an applications chemist with LECO Corp., may be contacted
at tincuta.veriotti@leco.com
or by phone at 269-985-5730. LECO Corporation 3000 Lakeview Avenue St. Joseph, MI, 49085
