Complex sample mixtures have always been a challenge for the analytical chemist. Introduced almost a decade ago, comprehensive two-dimensional gas chromatography (GC×GC) uses two columns connected in series by means of a modulator to greatly increase the peak capacity available from the chromatographic system.1 With the two columns having different separation mechanisms, often the GC×GC analysis results in highly organized chromatograms with the compounds clustered by carbon number and functionality. Position in a particular cluster (class) also gives the analyst some information about the compound’s chemical structure. While in theory this sounds like the perfect situation, in reality, even with the high peak capacity available from the GC×GC systems, some compound and/or class coelutions will occur.
Using a mass spectrometer as the detector of choice for GC×GC technology allows the analyst to further solve the puzzle of the complex sample. Display of characteristic m/z values for each class gives the ability to visually position even the overlapping classes. But to transform this information into at least a semiquantitative result, a more sophisticated tool is needed.
GC×GC technology
Figure 1 - Simple schematic of a GC×GC system operated with a dual-stage thermal modulator.
Figure 1 shows a simple schematic of a GC×GC system employing a dual-stage thermal modulator. While the first column is a typical, long GC column (most of the times coated with a nonpolar stationary phase), the secondary column is a short, narrow-bore capillary (usually coated with a polar or selective stationary phase) chosen to ensure fast separation in the second dimension. As compounds elute out the first column they are focused in the modulator and reinjected into the secondary column as sharp, narrow pulses. Due to the refocusing mechanism, typical widths for the peaks eluting out of the secondary column are in the 50–250 msec range and require fast detectors for accurate characterization. The time-of-flight mass spectrometer (TOFMS) is the only mass spectrometer that has the capability to acquire at fast enough acquisition rates to define even the narrow chromatographic peaks produced by GC×GC technology.
Classifications
In a typical GC×GC system, the separation in the first dimension is based mainly on boiling point, while separation in the second dimension is determined by the polarity of the compounds. This mechanism generates highly structured two-dimensional chromatograms (especially for petroleum-type samples) with chemical classes arranged in what is known as a “roof tile” arrangement. Figure 2 shows an example of such a chromatogram obtained from the analysis of a gas oil sample. The system used to generate the data was a GC×GC-TOFMS system (Pegasus 4D, Leco Corp., St. Joseph, MI). Retention time on the first dimension is represented on the x-axis; retention time in the second dimension is represented on the y-axis. Peak intensity is color scaled, with blue representing the baseline and red representing the most intense peaks. Regions for chemical class elution are enclosed in elliptical shapes with each class shown in a different color. The shapes were directly built onto the chromatogram using drawing tools available in ChromaTOF software (Leco). The template created using one sample can be applied to other samples analyzed subsequently under the same chromatographic conditions. This allows easy visual comparison of data from sample to sample. Peak tables obtained after processing of the data can be filtered to display only one class at a time.
Figure 2 - Two-dimensional chromatogram of a gas oil sample presented as a contour plot. Some of the chemical class regions present are defined using classifications and are shown in various colors.
Figure 3 - Reduced scale of the substituted benzothiophenes (red line) and substituted naphthalenes (yellow line) region from Figure 2. TIC (a), as well as characteristic m/z values for C2-/C3-substituted benzothiophenes (b), and C2-/C3-substituted naphthalenes (c), are shown in the same region of the chromatogram.
While separation of most of the classes is achieved, some partial overlap of the classes is still present. Figure 3 is an example of class overlap in a selected region of the chromatogram presented in Figure 2. Visual examination of the total ion current (TIC) chromatogram only reveals the presence of two chemical classes (clusters) in this region of the chromatogram. Using a mass spectrometer as the detector of choice permits the display of m/z values that are characteristic to specific chemical classes. Figure 3b and c are examples of this ability when GC×GC technology is coupled with TOFMS. When a sum of masses 162 and 176 is plotted, the regions in which C2- and C3-substituted benzothiophenes elute are very easily defined. The same thing happens when the sum of masses 156 and 170 is plotted to define the C2- and C3-substituted naphthalenes. Therefore, we can now visually identify the position in the two-dimensional space of the chemical classes, but can this information be transformed into a more meaningful result (area of the class, % of the class in the sample, etc.)?
Mass spectral filters
Building elliptical shapes around chemical classes allows the analytical chemist to better identify the position of a chemical class “geographically” (as a function of retention times in both the first and second dimension). Extra caution is needed when building classifications in regions of the chromatograms in which overlap of multiple classes occurs (see Figure 3). Adding the spectral information as another level of constraints enables the analyst to obtain better results without the sometimes tedious work of drawing perfect shapes around the chemical classes.
Figure 4 - Reduced scale of the C2-/C3-substituted phenanthrenes/dibenzothiophenes region from Figure 2. The TIC chromatogram is shown with drawn classification regions (a), and with bubble plots applied without (b) and with (c) mass spectral scripts. Bubbles for peaks in overlapping regions are striped, with the color of the stripes taken from each of the overlapping classes.
Figure 4b illustrates the results obtained when classifications without scripts were applied to the C2-/C3-substituted phenanthrenes/dibenzothiophenes region of the chromatogram presented in Figure 2. Display of the chromatogram in a bubble plot format was chosen for this figure to better illustrate the results. In a bubble plot format, peaks are represented as bubbles, with the color of the bubble taken from the color of the classification the respective peak belongs to. Peak area is directly proportional to the radius of the bubble. If a peak belongs to multiple classes, the bubble representing that peak will take the color from the multiple classes and appear striped. This is the case for some of the peaks in the overlapping regions in Figure 4. Peaks that belong to both substituted phenanthrene and substituted dibenzothiophene regions are colored with stripes (yellow and red for the C2 region and pink and blue for the C3 region).
Visual Basic Script language (Microsoft, Redmond, WA) is used in conjunction with ChromaTOF software to create the mass spectral filters (scripts). Arithmetic, comparison, and logistical operators as well as conditional statements and mass spectral criteria are used to create the script functions. A simple example of a script function is illustrated below:
FUNCTION C2dibenzothiophene ()
C2dibenzothiophene=(Rank(1)=212)
END FUNCTION
The above function identifies the C2-substituted dibenzothiophenes in a predefined region of the chromatogram by selecting only the found peaks that have m/z 212 as the most intense mass in the spectrum (base ion). The same type of constraints can be applied for the other three regions presented in Figure 4.
Figure 4c shows the results obtained after classifications with scripts were applied to the region of the chromatogram presented in Figure 3. It is easy to see that all found peaks are now represented by single-color bubbles since only peaks with spectral characteristics of a particular chemical class were able to pass the script constraints. Peak tables can be filtered by classification, and area as well as % area for one particular class can be displayed, allowing the analyst rapid access to quantitative results.
Conclusion
Comprehensive two-dimensional gas chromatography (GC×GC) is a very powerful tool that enables the analyst to obtain a tremendous increase in separation to benefit in the analysis of complex samples. Combining this technique with mass spectrometry gives the ability to further improve the analytical results. Through the use of classifications and scripts, access to both qualitative and quantitative results is easily available.
Reference
- Dalluge, J.; Beens, J.; Brinkman, U. J. Chromatogr. A 2003, 1000, 69–108.
Dr. Veriotti is Applications Chemist, and Mr. Hilton is Product Specialist, Leco Corp., 3000 Lakeview Ave., St. Joseph, MI 49085, U.S.A.; tel.: 269-985-5729; fax: 269-983-7150; e-mail: [email protected].