Figure 1. Schematic of the selectable 1D/2D-GC-MS system. Interesting sections of the standard 1D chromatogram can be heartcut for separation on the 2D column. Analytes can be cryofocused using a GERSTEL CryoTrap System (CTS) positioned between the 1D and 2D columns. Heart-cut fractions from multiple injections can be cryofocused and combined into one 2D-separation for enhanced sensitivity. The 1D and 2D chromatograms and olfactograms are acquired on the same MSD and ODP.
Analytical conditions
TDS Splitless
30 °C – 60 °C/min – 250 °C (5 min)
CIS Liner packed with glass wool
Solvent venting (50 mL/min)
Peach flavor: Split (10:1)
Gin: Splitless
-150 °C – 12 °C/s – 280 °C (3 min)
Pneumatics Constant pressure

GC oven

250 °C isothermal
Column 1 10 m Rtx-5 (Restek), LTM configuration,
0.18 mm ID, 0.18 μm film thickness (df)
Peach flavor:
40 °C (1 min) – 10 °C/min – 260 °C (0.8 min) – 100 °C/min – 40 °C
Gin:
40 °C (1 min) – 10 °C/min – 160 °C (0.8 min) – 140 °C/min – 300 °C
CTS Peach flavor:
-50 °C (11.2 min) – 20 °C/s – 240 °C (2 min)
Gin:
-50 °C (17 min) – 20 °C/s – 240 °C (2 min)
Column 2 10 m DB-Wax (Agilent), LTM configuration, 0.18 mm ID, 0.18 μm film thickness (df)
Peach flavor:
40 °C (11.2 min) – 20 °C/min – 230 °C (1.5 min) – 50 °C/min – 40 °C
Gin:
40 °C (17 min) – 10 °C/min – 210 °C – 170 °C/min – 40 °C
MSD Full scan, 40-350 amu
Figure 2. Stacked view TICs of the 1st dimension chromatogram (A) and the combined 1st and 2nd dimension chromatogram (B) that results from heart-cut from 10–11 mins. Sample: 0.1 μg/mL bucchu ketone in water.
Figure 3. Stacked view peach flavor sample TICs of the 1st dimension chromatogram (A) and the combined 1st and 2nd dimension chromatogram that results from a heart-cut from 10–11 mins. (B). The absence of peaks in the 1D chromatogram during the heart-cut period is evidence that eluting compounds are transferred efficiently to the 2D column.
Figure 4. Stacked view peach flavor sample TICs resulting from the desorption of 1 (A) and 5 (B) Twisters respectively. Heart-cuts were performed between the 1st and 2nd column from 10–11 mins. The combined 1st and 2nd dimension (1D/2D) chromatograms are shown. In the bottom trace, the initial 1D part is from the 5th Twister desorption whereas the 2D chromatogram part results from accumulated heart-cuts from all five 1D runs. Analytes were focused on the GERSTEL CryoTrap System (CTS) between the 1D and 2D columns.
Figure 5. 1st dimension TIC of gin sample.
Figure 6. TIC of gin sample: Combined 1st and 2nd dimension chromatogram that results from a heart-cut from 9.36-10.35 mins.
Figure 7. Stacked view gin sample TICs resulting from the desorption of 1 (A) and 5 Twisters (B) respectively. Heart-cuts were performed between the 1st and 2nd column from 9.36-10.35 mins. The combined 1st and 2nd dimension (1D/2D) chromatograms are shown. In the bottom trace, the initial 1D part is from the 5th Twister desorption whereas the 2D chromatogram part results from accumulated heart-cuts from all five 1D runs.
Authors

Nobuo Ochiai and
Kikuo Sasamoto,

GERSTEL K.K.,
2-13-18, Nakane, Meguro-ku, Tokyo 152-0031,
Japan

John R. Stuff and
Jacqueline A. Whitecavage,

GERSTEL, Inc.,
701 Digital Dr., Suite J,
Linthicum, MD 21090,
USA

Flavor and Fragrance Analysis

Put on the 1D/2D goggles...

The more complex the sample and the wider the concentration range of analytes, the bigger the challenge for the chromatographer to get a clean separation and sharp peaks. One short-cut is to heart-cut the challenging part(s) of the chromatogram to a 2nd dimension to get a full set of separated peaks. The patented new Selectable 1D/2D-GC/ MS system enables simple and flexible switching between one- and two-dimensional GC/MS analysis on a single GC/MS system.

Determination of flavors and allergens in food, cosmetics and personal care products is certainly not a trivial matter. The matrix is often complex, sometimes requiring extensive sample clean-up and up to several sample preparation steps. Efficient automated sample preparation is a good first step on the way to getting reliable results, but even well prepared samples can produce forests of overlapping peaks making it a case of not being able to see the trees for the forest. When compound peaks overlap, or if a flavor emerges from the Olfactory Detection Port (ODP) without a detectable associated signal from the MS, multi-dimensional GC can be the solution that cuts through the thicket and provides clear, reliable answers where one dimensional GC can not.

Until now, performing multidimensional GC analysis has required the use of a dedicated system with two GCs coupled to each other. Due to the extra cost, and to the often limited utilization in the laboratory, such solutions don’t always provide the best return on investment (ROI). GERSTEL now offers a solution that can be used for routine analysis as well as for special challenges. The patented GERSTEL Selectable 1D/2DGC/MS system is a flexible solution, based on a single standard GC/MS system. It is, in short, a routine analysis system that offers heart-cutting, two-dimensional GC separation, and analyte concentration from multiple injections on demand. The MS detector is used in both dimensions to ensure clear and unequivocal peak identification. Additional detectors, such as a PFPD sulphur-selective detector can be added to the system without modifying the hardware. In simplified terms, the method of operation can be described as follows: When questions arise regarding a section of the standard onedimensional chromatogram, the section in question can be transferred to a 2nd dimension, i.e. a GC column with different polarity installed in a separate module in the same GC. The process of cutting a section of a chromatogram and introducing it to another column is called heart-cutting. The GC system can be used to determine analytes in either the 1st or the 2nd dimension in a flexible manner. Neither the GC run, nor analyte detection is interrupted during the run. Detection of the analytes that were transferred to the 2nd column is performed using the same detector(s) used for the 1st dimension: MSD, ODP, PFPD etc. etc. Should lower detection limits be required for the analyte in question, the system enables heart-cutting from multiple repeat injections combined with cryofocusing on a GERSTEL Cryo Trap System (CTS) of the sections that were cut. The cumulated sections are then transferred to the 2nd dimension column as soon as there is sufficient mass on column to reliably perform the determination. In order to speed up the analysis and eliminate interferences in the 2D chromatogram, the 1D column can be backflushed following the heart-cut(s) in case no further compounds are deemed of interest. A schematic of the system is shown in figure 1. The GC columns used in the system are placed outside the GC oven in Low Thermal Mass (LTM) modules. The LTM technology enables fast heating and cooling for faster analysis as well as independent temperature programming for each column module. The standard GC oven is merely used as a heated chamber for pneumatic connectors and switching devices. Keeping the GC oven at a fixed temperature contributes to the excellent system stability seen in our work with the system so far: Connectors are not subjected to cycles of heating and cooling with the associated material expansion and contraction that can eventually result in system leaks.

The analysis system and the sample preparation used

To check the performance under everyday routine analysis conditions, the selectable 1D/2D-GC/MS-system was used to determine bucchu ketone, which is the main flavor compound in peach flavor and is also found in gin. Typical samples have complex matrices resulting in countless interfering peaks. Because of this, multidimensional separation is the best means of getting information from peaks that otherwise would be hidden when using only 1D separation. With the addition of the ODP, the technique also provides valuable olfactory information from compounds that many times are not detected by the MSD.

The main components of the selectable 1D/2D-GC/MS system are as follows: A GC 6890 equipped with a GERSTEL Cooled Injection System (CIS 4 - PTV-type universal inlet); two Low Thermal Mass (LTM) column modules; 5975C InertXL MSD (both from Agilent Technologies); GERSTEL Thermal Desorption System (TDS) with TDSA autosampler; as well as a GERSTEL CryoTrap System (CTS 2). Alternatively, a GERSTEL Thermal Desorption Unit (TDU) in combination with a MultiPurpose Sampler (MPS) can be used instead of the TDS/TDS-A system. Stir Bar Sorptive Extraction (SBSE) was used to extract flavor compounds from both peach flavor and gin. SBSE is performed using the GERSTEL Twister, a glass encased magnetic stir bar coated with PDMS.

While the Twister stirs the sample, analytes are efficiently absorbed in the PDMS phase. Different Twisters are available with different phase volumes. Depending on the phase volume the analyte, and the sample volume SBSE can provide up to 1000 times better sensitivity than SPME.

Successful implementation

Samples were prepared for SBSE as follows: Peach flavor sample: The sample was spiked to a concentration of 1 μg/mL bucchu ketone. 200 μL aliquots were pipetted into 10 mL screw cap headspace vials containing 9.8 mL bottled water to achieve a concentration of 0.02 μg/mL bucchu ketone in 10 mL of solution. Gin sample: 0.5 mL aliquots of gin were pipetted into 10 ml screw cap headspace vials containing 4.5 ml bottled water.

A conditioned Twister was added to each vial. The vials were screw capped, and the samples stirred at room temperature for 1hr. Twisters were rinsed with water, dabbed dry, and placed into separate conditioned TDS tubes. The TDS tubes were finally placed in the TDS-A autosampler for analysis. All further steps were performed automatically. As a first step, the Twisters that had been used to extract the standard solutions were analyzed. Bucchu ketone was found in the retention time window between 10 and 11 minutes in the 1st dimension (1D) chromatogram. Performing a heart-cut of this time window resulted in a 2nd dimension (2D) chromatogram with a number of peaks that could not have been separated in the 1st dimension (figure 2).

The same approach was used for the peach flavor sample: A heart cut was performed of the retention time window from 10 to 11 minutes. The co-eluting compounds that were transferred to the 2nd dimension were separated on the 2D column and identified. The 2nd dimension separation can follow either immediately after the heart-cut period or when the complete 1D separation has been finalized. The chromatograms from the two separations are acquired sequentially using the same MSD and combined into one GC/MS chromatogram. To ensure that late eluting analytes from the 1D column do not interfere with the 2D column chromatogram, the 1D column can be backflushed. In the case of the peach flavor sample, 1D column backflush was not necessary, since no compounds from the 1D column co-eluted with the compounds separated on the 2D column (figure 3). In case the 2D column separation doesn‘t yield satisfactory answers due to lack of sensitivity it is possible to perform heart-cuts from multiple injections and to concentrate the transferred compounds by cryofocusing before releasing the combined fractions to the 2D column performing a single separation and compound determination. To investigate the effectiveness of this approach, we cryofocused and then transferred five heart-cut fractions to the 2D column and compared the resulting chromatogram with one obtained from a single heart-cut fraction. The resulting chromatograms are shown in figure 4. Sharp peaks and excellent separation are obtained even after extended multi-step cryofocusing and the bucchu ketone signal is increased by a factor of 5.6 as can be seen in table 1. This result clearly shows the efficiency of the selectable 1D/2D-GC/MS system.

   Peak Area
 1 Twister  1,535,403
 5 Twisters  8,587,702
Table 1: Peak area for bucchu ketone as a function of
number of twisters

The analysis of gin using the Selectable 1D/2D-GC/MS system provided highly satisfactory results, similar to the results for peach flavor mentioned above. To determine the bucchu ketone level in a gin sample, a heart-cut was taken from the 1D chromatogram (Figure 5) between 9.36 and 10.35 minutes and the fraction transferred to the 2D column. Transferring heart-cut fractions from 5 separate injections and cryofocusing these for a single 2D run resulted in a significant increase in sensitivity as can be seen in figure 7.

Conclusion

Selectable 1D/2D-GC/MS has been used successfully in flavor and fragrance analysis, both for food, beverage, body care, and cosmetic products. In addition to flavors, offflavors are an important application area for the 1D/2D technology. Off-flavors often need to be tracked down and determined both in the products themselves and in the packaging used and often the concentrations involved are at ultra-trace levels. The 1D/2D system presented here provides the analyst with a powerful and comprehensive tool consisting of different polarity columns for multi-dimensional separation of a wide range of compounds even in complex matrices. Further the added capability to concentrate flavor compounds from multiple injections is extremely helpful when tracking down off-flavors with low odor thresholds. Since the complete solution is built into a single standard GC/MS system, investment costs remain very reasonable. Further, the heart-cut fraction can be analyzed on the 2nd dimension column using the same MSD. Additional detection possibilities such as a PFPD or an Olfactory Detection Port (ODP) can be integrated and used without adding complexity to the operation of the system. Unlike standard multi-dimensional GC/MS systems, the 1D/2D system uses the MSD as monitoring detector for the 1D separation as well, ensuring that heart-cut sections are correctly chosen for the compounds in question and providing much additional information on the sample in general. Our experience after approximately two years is that the 1D/2D system is a rugged and reliable system for routine 1D analysis and that it is easily switched to 2D mode when additional separation power is needed. The GERSTEL MAESTRO software provides easy and convenient set-up by mouse-click of the entire system from one integrated method and one integrated sequence table.