Total-transfer comprehensive three-dimensional gas chromatography with time-of-flight mass spectrometry.

Although comprehensive two-dimensional (2D) gas chromatography (GC × GC) is a powerful technique for complex samples, component overlap remains likely. An intriguing route to address this challenge is to utilize the additional peak capacity and chemical selectivity provided by comprehensive three-dimensional (3D) gas chromatography (GC3), especially with time-of-flight mass spectrometry detection (GC3-TOFMS). However, the GC3-TOFMS instrumentation reported to date has employed one or both modulators with a duty cycle < 100%, making the potential gain in detection sensitivity over GC × GC modest, or perhaps even worse. Herein, we describe instrumentation for GC3-TOFMS in which both modulators provide total-transfer (100% duty cycle). Specifically, the instrument is based on the facile modification of a commercial thermally modulated comprehensive GC × GC-TOFMS platform for modulation from the 1D column to the 2D column, with recently described dynamic pressure gradient modulation (DPGM) as the second modulator from the 2D column to the 3D column, which is a total-transfer flow modulation technique. Area measurements of 1D peaks are compared to the sum of 3D peak areas to validate the assumption that total-transfer from 1D to 3D is accomplished. Additionally, peak heights were amplified by as high as a factor of 177 (x̅ = 130, s = 47) via comparison of 1D peak heights to the maximum 3D peak heights. Column selection is explored, with emphasis on the resulting peak width-at-base on each dimension and usage of 3D space as evaluation metrics. Using a nonpolar × polar × ionic liquid column combination, an effective peak capacity which considers modulation-induced broadening as high as 32,300 for select analytes was achieved (x̅ = 19,900, s = 10,700). The analytical benefits of employing three selective phases, mass spectrometry detection, and total-transfer modulation are explored with separations of a metabolomics-type sample, i.e., derivatized porcine serum, and a jet fuel spiked with various sulfur-containing compounds.

Dynamic pressure gradient modulation for comprehensive two-dimensional gas chromatography with time-of-flight mass spectrometry detection.

Dynamic pressure gradient modulation (DPGM) is investigated for comprehensive two-dimensional gas chromatography (GC × GC) with time-of-flight mass spectrometry (TOFMS) detection. With DPGM, a commercial pneumatic "pulse" valve is opened to introduce a suitably high auxiliary gas pressure at a T-junction connecting the first dimension (1D) and second dimension (2D) columns during the modulation period (PM), temporarily stopping the 1D flow. The valve is then closed for the duration of a pulse width (pw) to "re-inject" temporally focused 1D eluate onto the 2D column for separation. This flow modulation technique is observed to be compatible with TOFMS detection using a 2D flow rate of 4 ml/min for the separation of a 90-component test mixture. A 25 min separation window using a PM = 1 s and pw = 200 ms for full modulation (and 100% duty cycle) provided an average 1Wb = 4.5 s and 2Wb = 130 ms for a 2D peak capacity of nc,2D = 2700 (100 peaks per min). The detector response enhancement factor (DREF) serves as a metric for the enhanced sensitivity of the modulated relative to the unmodulated 1D peaks, with DREFs ranging between 10 and 20 and about a 5-fold improvement in signal-to-noise ratio (S/N). The bilinear "quality" of the GC × GC data is studied using the chemometric method parallel factor analysis (PARAFAC). Since PARAFAC requires sufficiently trilinear data, the reproducibility of the 2D peak shape for a given analyte is confirmed using lack-of-fit (LOF) and percent variation (R2) metrics. The limit-of-detection (LOD) for the representative analyte hexadecane is determined using PARAFAC, providing an LOD of 0.7 ppb (±0.03 ppb) for three replicates. Seven heavily overlapped analytes are also fully resolved by PARAFAC down to the part-per-million (ppm) concentration level, producing reproducible spectra with a majority of spectral match values (MV) over 800 (RSD ≤ 7.1%). This study provides promising results for DPGM as a flow modulation technique compatible with GC × GC-TOFMS, providing high sensitivity data suitable for chemometric analysis.

Chemometric decomposition of comprehensive two-dimensional gas chromatography time-of-flight mass spectrometry data employing partial modulation in the negative pulse mode.

Partial modulation in the negative pulse mode was optimized for comprehensive two-dimensional gas chromatography (GC × GC) coupled to time-of-flight mass spectrometry (TOFMS). With partial modulation in the negative pulse mode, a flow of auxiliary carrier gas is applied through a pulse valve to a T-junction joining the first (1D) and second (2D) dimension columns for nearly all of the modulation period (PM). This results in dilution of the 1D eluate, followed by briefly turning off the pulse valve for a specified pulse width (pw), effectively "injecting" undiluted 1D eluate onto the 2D column. The raw data has the appearance of 2D separations superimposed on top of the 1D separation. While high peak capacity GC × GC data are produced, there are challenges that needed to be addressed regarding the use of chemometrics for analyte decomposition, identification, and quantification. Herein, these data analysis challenges are addressed using multivariate curve resolution - alternating least squares (MCR-ALS). An isothermal separation of a 15-component mixture of similar compounds is obtained in 20 s using a PM = 250 ms and a pw = 6 ms. Various peak overlap situations were purposely produced to facilitate the chemometric method demonstration. The MCR-ALS chromatographic loadings (peak profiles) were used to determine retention times (tR) and width-at-base (Wb) in both separation dimensions. MCR-ALS readily decomposed 1D × 2D regions for analytes with severe 1D overlap if they were fully resolved on 2D. Decomposition was more challenging for analytes severely overlapped on both GC × GC dimensions when their spectra were similar, though ultimately all 15 compounds were successfully decomposed. Additionally, the 2D concentration peak profiles obtained via MCR-ALS are demonstrated to be reliable for identification and quantifiable. The predicted versus prepared concentration values are in good agreement for two representative analytes, with percent deviation values of -5.6% (±2.2%) for 1-hexene, and 1.8% (±3.4%) for 2-pentanone.

Dynamic pressure gradient modulation for comprehensive two-dimensional gas chromatography.

We report the discovery, preliminary investigation, and demonstration of a novel form of differential flow modulation for comprehensive two-dimensional (2D) gas chromatography (GC×GC). Commercially available components are used to apply a flow of carrier gas with a suitable applied auxiliary gas pressure (Paux) to a T-junction joining the first (1D) and second (2D) dimension columns. The 1D eluate is confined at the T-junction, and introduced for 2D separation with a cyclic rhythm, dependent upon the relationship of the modulation period (PM) to the pulse width (pw), where pw is defined as the time interval when the auxiliary gas flow at the T-junction is off. We refer to this flow modulation technique as "dynamic pressure gradient modulation" (DPGM) since a pressure gradient oscillates with the PM along the 1D and 2D column ensemble providing temporary stop-flow conditions and fast 2D flow rates, resulting in 100% duty cycle and full modulation. A 90-component test mixture was used to evaluate the technique with a pw of 60 ms and a PM of 750 ms. The resulting peaks were narrow, with 2Wb ranging from about 20-180 ms. With an average 1Wb of 3 s and a 2nc of 10, a 2D peak capacity, nc,2D, for the 25 min separation was 5000. The detector response enhancement factor (DREF) is reported, defined as the peak height of the highest modulated 2D peak divided by the unmodulated 1D peak height (DREF = 2h/1h). The DREF ranged from about 7-87, depending on the 1Wb and 2Wb for a given analyte. A diesel sample was analyzed to demonstrate performance with a complex sample. Based upon the average 1Wb of 5 s and an average 2Wb of 168 ms, a nc,2D of 8640 was obtained for the 60 min diesel separation. Finally, the modulation principle was investigated as a function of PM, pw, and the volumetric flow rates, 1F and 2F. The measured 2Wb correlate well with the theoretical 2D injected width, given by 2Winj = (1F/2F) ·PM. However, the relevant 1F appears to be dictated by the 1D flow rate when no pressure is applied (during the pw interval), instead of 1F being the average flow rate on 1D (defined by the 1D dead time). The findings provide strong evidence for a differential flow modulation mechanism.

Impact of data bin size on the classification of diesel fuels using comprehensive two-dimensional gas chromatography with principal component analysis.

Principal component analysis (PCA) is a widely applied chemometric tool for classifying samples using comprehensive two-dimensional (2D) gas chromatography (GC × GC) separation data. Classification via PCA can be improved by 2D binning of the data. A "standard operating procedure (SOP) bin size" is often applied to improve the S/N and to mitigate potential retention time misalignment issues. The SOP bin size is generally selected to be slightly larger than the typical 2D peak dimensions. In this study we examine to what extent a single SOP bin size is optimal for all of the class comparisons that can be made in a single PCA scores plot. For this purpose, a GC × GC-FID dataset comprised of 5 different diesel fuels (i.e., 5 sample classes), each run with 4 replicates using a reverse column configuration (polar 1D column and non-polar 2D column) was utilized. The dataset was collected within about one day, which minimized retention time misalignment in order to allow the study to focus on S/N enhancement concurrent with maintaining the chemical selectivity provided by the GC × GC separations. A total of 110 bin sizes were evaluated. Degree-of-class separation (DCS) was utilized as a quantitative metric to assess the impact of binning in improving separation in the scores plot. The DCS was calculated pair-wise between nearest neighbor sample classes for each of the 5 sample classes in the scores plot (5 sample class pairs). Results indicated the SOP bin size did not provide the highest DCS for any of the 5 fuel pairs. Each fuel pair is found to have its own optimal bin size, suggesting the binning finds the balance between S/N optimization concurrent with leveraging the chemical selectivity information differences in the samples as manifested in their GC × GC separation "patterns". Robustness of the findings in this study were supported by leaving out one fuel at a time and re-running the PCA models.

Development of Ultrafast Separations Using Negative Pulse Partial Modulation To Enable New Directions in Gas Chromatography.

Partial modulation via a pulse flow valve operated in the negative pulse mode is developed for high-speed one-dimensional gas chromatography (1D-GC), comprehensive two-dimensional (2D) gas chromatography (GC × GC), and comprehensive three-dimensional gas chromatography (GC3). The pulse flow valve readily provides very short modulation periods, PM, demonstrated herein at 100, 200, and 300 ms, and holds significant promise to increase the scope and applicability of GC instrumentation. The negative pulse mode creates an extremely narrow, local analyte concentration pulse. The reproducibility of the negative pulse mode is validated in a 1D-GC mode, where a pseudosteady-state analyte stream is modulated, and 8 analytes are baseline resolved (resolution, Rs ≥ 1.5) in a 200 ms window, providing a peak capacity, nc, of 14 at unit resolution ( Rs = 1.0). Additionally, the pulse width, pw, of the pulse flow valve "injection" relationship to peak width-at-base, wb, resolution between peaks and detection sensitivity are studied. To demonstrate the applicability to GC × GC, a high-speed separation of a 20-component test mixture of similar, volatile analytes is shown. Analytes were separated on the second-dimension column, 2D, with 2 wb ranging from 7 to 12 ms, providing an exceptional 2D peak capacity, 2 nc, of ∼12 using a modulation period ( PM) of 100 ms. Next, a 12 min separation of a diesel sample using a PM of 300 ms is presented. The 1 wb is ∼4 s, resulting in a 1 nc of ∼180, and 2 wb is ∼18 ms, resulting in a 2 nc of ∼17, thus achieving a nc,2D of ∼3000 in this rapid GC × GC diesel separation. Finally, GC3 with time-of-flight mass spectrometry (TOFMS) detection using a PM of 100 ms applied between the 2D and 3D columns is reported. Narrow third dimension, 3D, peaks with 3 wb of ∼15 ms were obtained, resulting in a GC3 peak capacity, nc,3D, of ∼35 000 in a 45 min separation.

Column selection approach to achieve a high peak capacity in comprehensive three-dimensional gas chromatography.

The separation power of comprehensive three-dimensional gas chromatography (GC3) is substantially enhanced through proper selection of the phase volume ratio, ß, of each column relative to each other on successive dimensions. Consideration and application of the ratio of phase volume ratios, ßr, or ß ratio, between successive dimensions has been a relatively un-studied approach to maximize separating power in comprehensive multidimensional GC instrument design. Herein, proper selection of ßr in multidimensional GC is shown to control the elution temperature, Te, of analytes throughout a 40 min primary (1D) column separation, and thus better control width-at-base, W, on all three dimensions. Specifically, between the 1D and secondary (2D) columns, a ßr of 0.45 was applied, and between the 2D and tertiary (3D) columns a ßr of 1.0 was applied. A total ideal peak capacity of 30,600, or a peak capacity production of ~770 peaks/min, was accomplished with the GC3 instrument with the reconfigured parameters. Additionally, due to the complex nature of this three-dimensional data, a novel approach to "slicing" the chromatographic run into user-defined time intervals is shown. This novel way to view the data still elicits a traditional GC×GC chromatograms, but with the focus on 2D × 3D separations. Moreover, due to proper ßr selection, every 2 s window (i.e. every 1D modulation period) is shown to have a peak capacity of ~50-100 for each 2D × 3D separation. This high overall peak capacity (30,600) and peak capacity per 1D modulation (~50-100), courtesy of proper column selection, is demonstrated to hold great promise to physically separate truly complex mixtures.

Comprehensive two-dimensional gas chromatography and time-of-flight mass spectrometry detection with a 50 ms modulation period.

An ultrafast flow modulation period, PM of 50 ms, for comprehensive two-dimensional (2D) gas chromatography (GC × GC) with time-of-flight mass spectrometry (TOFMS) detection is demonstrated, producing narrow peak widths, 2W (4σ width-at-base on the 2D dimension), demonstrating the potential for ultrafast (2D) separations with high peak capacity. The modulator is a pulse flow valve that injects a narrow pulse of carrier gas at a user defined PM, at the union between the 1D and 2D columns. The raw data produced combines the properties of vacancy chromatography and frontal analysis. Deconvolution of the raw data using unconstrained multivariate curve resolution alternating least squares (MCR-ALS) analysis facilitates identification and quantification for overlapped analyte peaks. The peak profile loadings obtained from MCR-ALS are converted into traditional appearing GC × GC data through a process commonly used with frontal analysis. An 18-component test mixture at seven different injected mass levels was studied. The 2D peaks generated ranged from an 2W of 16 to 36 ms with an average of 26 ms. At an on-column injected mass of 14 ng per analyte, an average mass spectral match value, MV, of 822 was achieved using in-house collected spectra for comparison, with an average match value RSD of 7.1%. Calibration of overlapped test analytes was evaluated using the areas of the MCR-ALS loadings, with excellent quantification demonstrated. The advancement demonstrated in modulation performance for GC × GC represents a significant decrease in PM as most commercial modulators have a minimum PM of 1 s, while maintaining the benefits of a duty cycle of essentially 1.0, which promises to enable new chemical analyzer designs, compatible with the vacuum requirements of the TOFMS detector.

Ultrafast separations via pulse flow valve modulation to enable high peak capacity multidimensional gas chromatography.

Ultrafast modulation with a modulation period PM ≥ 50ms via a pulse flow valve is demonstrated for comprehensive two-dimensional gas chromatography (GC×GC) and comprehensive three-dimensional (3D) gas chromatography (GC3). Significant increases in peak capacity and peak capacity production are achieved for GC×GC and GC3 relative to previous studies due to using pulse flow valve modulation. Due to the nature of the "partial" modulation process, the separation dimension following pulse flow valve modulation is not a traditional chromatogram, rather requires data processing to convert the data to expose the encoded chromatographic information, producing "apparent" chromatographic peaks. In the GC×GC mode, a 115-component test mixture was evaluated using a PM of 500ms, creating an apparent 2D peak width-at-base 2W with an average of 25ms, producing a 2nc of 20. Based on the average 1W of 1.0s for the 6min first dimension 1D separation, an ideal peak capacity nc,2D of 7200 is achieved (1,200/min peak production). For a high-speed GC×GC separation (30s run), a PM of 75ms produced apparent 2W of 8ms, ideal for the third dimension of a GC3 instrument. Using the knowledge gained from this high-speed GC×GC experiment, the pulse flow valve was implemented as the second modulator in GC3. Three samples were evaluated in the GC3 mode: a simple mixture containing 18 compounds (to illustrate basic concepts), the 115-component test mixture (to determine peak capacity figures-of-merit), and a diesel spiked with 8 polar compounds (to illustrate chemical selectivity benefits of GC3). For the 115-component test mixture with a 1PM of 1.2s and a 2PM of 60ms, average 1W of 3.2s, 2W of 130ms, and apparent 3W of 13ms were produced, resulting in a 1nc of 210, 2nc of 9.2, and 3nc of 5, respectively. Hence, an ideal peak capacity, nc,3D of ∼10,000 for GC3 was achieved for the 11min 1D separation window of the 115-component test mixture.