Fast, high peak capacity separations in gas chromatography-time-of-flight mass spectrometry.

Peak capacity production (i.e., peak capacity per separation run time) is substantially improved for gas chromatography-time-of-flight mass spectrometry (GC-TOFMS) and applied to the fast separation of complex samples. The increase in peak capacity production is achieved by selecting appropriate experimental conditions based on theoretical modeling of on-column band broadening, and by reducing the injection pulse width. Modeling to estimate the on-column band broadening from experimental parameters provided insight for the potential of achieving GC separations in the absence of off-column band broadening, i.e., the additional band broadening not due to the on-column separation process. To optimize GC-TOFMS separations collected with a commercial instrumental platform, off-column band broadening from injection and detection needed to be significantly reduced. Specifically for injection, a commercially available thermal modulator is adapted and applied (referred to herein as thermal injection) to provide a narrow injection pulse, while the TOFMS provided a data collection rate of 500 Hz, initially averaged to 100 Hz for data storage. The use of long, relatively narrow open tubular capillary columns and a 30 °C/min programming rate were explored for GC-TOFMS, specifically a 20 m, 100 µm inner diameter (i.d.) capillary column with a 0.4 µm film thickness to benefit column capacity, operated slightly below the optimal average linear gas velocity (at ~2 mL/min, due to the flow rate constraint of the TOFMS). Standard autoinjection with a 1:100 split resulted in an average peak width of ~1.2 s, hence a peak capacity production of 50 peaks/min. Metabolites in the headspace of urine were sampled by solid-phase microextraction (SPME), followed by thermal injection and a ~7 min GC separation (with a ~6 min separation time window), producing ~660 ms peak widths on average, resulting in a total peak capacity of ~550 peaks (at unit resolution) and a peak capacity production of ~90 peaks/min (~2-fold improvement relative to standard autoinjection with the 1:100 split). This total peak capacity production achieved is equivalent to, or greater than, that currently utilized in metabolomics studies using GC/MS, but with much slower separations, on the order of 40 to 60 min, corresponding to a 5-fold or greater GC/MS analysis throughput rate.

High throughput analysis of atmospheric volatile organic compounds by thermal injection--isothermal gas chromatography--time-of-flight mass spectrometry.

Sixty one volatile organic compounds (VOCs) from a standard gas mixture were separated via isothermal gas chromatography coupled with time-of-flight mass spectrometry (GC-TOFMS) in a ≈ 35s separation time window (≈ 45 s separation). The VOCs in the standard gas mixture were selected based on the EPA TO-15 methodology. The high throughput separation was achieved with a relatively high total peak capacity (n(c) ≈ 114), by simultaneously minimizing both on-column and off-column peak width broadening. The on-column contributions to peak width broadening were minimized by taking into account and applying GC separation theory for the selection of column dimensions and carrier gas flow rate conditions. Both fast cryogenic focusing and re-injection of compounds (implemented via a commercially available thermal modulator and referred to herein as thermal injection (TI) and fast TOFMS detection (100 scans/s)) were applied to reduce off-column sources of peak width band broadening (sometimes referred to as off-column band broadening). Cryogenic focusing during TI and minimal band broadening-based dilution during separation resulted in preconcentration factors for the detected peaks ranging from 78 (1,4-dichlorobenzene) to 420 (propylene). Since the injected volume for preconcentration was 500 µl, and based on the detected noise levels at selected m/z for each analyte compound, the concentration limit of detection (LOD) ranged from 67 ppbv (parts per billion by volume) for propylene, to 4 ppbv for freon-12. While application of standard VOC analysis conditions leads to separation times typically ranging from ≈ 30 to 50 min, the isothermal GC-TOFMS method reported herein represents a 40-fold improvement in analysis time while maintaining peak capacity and detection sensitivity that is comparable to traditional GC-MS VOC analysis.

High-speed cryo-focusing injection for gas chromatography: reduction of injection band broadening with concentration enrichment.

In order to maximize peak capacity and detection sensitivity of fast gas chromatography (GC) separations, it is necessary to minimize band broadening, and in particular due to injection since this is often a major contributor. A high-speed cryo-focusing injection (HSCFI) system was constructed to first cryogenically focus analyte compounds in a 6 cm long section of metal MXT column, and second, reinject the focused analytes by rapidly resistively heating the metal column via an in-house built electronic circuit. Since the cryogenically cooled section of column is small (∼750 nl) and the direct resistive heating is fast (∼6000 °C/s), HSCFI is demonstrated to produce an analyte peak with a 6.3 ms width at half height, w(1/2). This was achieved using a 1m long column with a 180 µm inner diameter (i.d.) operated at an absolute head pressure of 55 psi and an oven temperature of 60 °C, with a 10 V pulse applied to the metal column for 50 ms. HSCFI was also used to demonstrate the head space sampling and fast GC analysis of an aqueous solution containing six test analytes (acetone, methanol, ethanol, toluene, chlorobenzene, pentanol). Using Henry's law constants for each of the analytes, injected mass limits of detection (LODs) were typically in the low pg levels (e.g., 1.2 pg for acetone) for the high speed separation. Finally, to demonstrate the use of HSCFI with a complex sample, a gasoline was separated using a 20 m × 100 µm i.d. column and the stock GC oven for temperature programming, which provided a separation time of 200 s and an average peak width at the base of 440 ms resulting in a total peak capacity of 460 peaks (at unit resolution).

Fast, high peak capacity separations in comprehensive two-dimensional gas chromatography with time-of-flight mass spectrometry.

Peak capacity production is substantially improved for two-dimensional gas chromatography coupled with time-of-flight mass spectrometry (GC×GC-TOFMS) and applied to the fast separation of a 28 component liquid test mixture, and two complex vapor samples (a 65 component volatile organic compound test mixture, and the headspace of warm ground coffee beans). A high peak capacity is achieved in a short separation time by selecting appropriate experimental conditions based on theoretical modeling of on-column band broadening, and by reducing the off-column band broadening by applying a narrow, concentrated injection pulse onto the primary column using high-speed cryo-focusing injection (HSCFI), referred to as thermal injection. A long, relatively narrow open tubular capillary column (20 m, 100 µm inner diameter (i.d.) with a 0.4 µm film thickness to benefit column capacity) was used as the primary column. The initial flow rate was 2 ml/min (60 cm/s average linear flow velocity) which is slightly below the optimal average linear gas velocity of 83 cm/s, due to the flow rate constraint of the TOFMS vacuum system. The oven temperature programming rate was 30°C/min. The secondary column (1.8m, 100 µm i.d. with a 0.1 µm film thickness) provided a relatively high peak capacity separation, concurrent with a significantly shorter modulation period, P(M), than commonly applied with the commercial instrument. With this GC×GC-TOFMS instrumental platform, compounds in the 28 component liquid test mixture provided a ∼7 min separation (with a ∼6.5 min separation time window), producing average peak widths of ∼600 ms full width half maximum (FWHM), resulting in a peak capacity on the primary column of ∼400 peaks (at unit resolution). Using a secondary column with a 500 ms P(M), average peak widths of ∼20 ms FWHM were achieved, thus providing a peak capacity of 15 peaks on the second dimension. Overall, an ideal orthogonal GC×GC peak capacity of ∼6000 peaks (at unit resolution) was achieved (or a ß-corrected orthogonal peak capacity of ∼4400, at an average modulation ratio, M(R), of ∼2). This corresponds to an ideal orthogonal peak capacity production of ∼1000 peaks/min (or ∼700 peaks/min, ß-corrected). For comparison, standard split/split-less injection techniques with a 1:100 split, when combined with standard GC×GC conditions typically provide a peak capacity production of ∼100 peaks/min, hence the instrumental platform we report provides a ∼7-fold to 10-fold improvement.

Study of the interdependency of the data sampling ratio with retention time alignment and principal component analysis for gas chromatography.

An in-depth study is presented to better understand how data reduction via averaging impacts retention alignment and the subsequent chemometric analysis of data obtained using gas chromatography (GC). We specifically study the use of signal averaging to reduce GC data, retention time alignment to correct run-to-run retention shifting, and principal component analysis (PCA) to classify chromatographic separations of diesel samples by sample class. Diesel samples were selected because they provide sufficient complexity to study the impact of data reduction on the data analysis strategies. The data reduction process reduces the data sampling ratio, S(R), which is defined as the number of data points across a given chromatographic peak width (i.e., the four standard deviation peak width). Ultimately, sufficient data reduction causes the chromatographic resolution to decrease, however with minimal loss of chemical information via the PCA. Using PCA, the degree of class separation (DCS) is used as a quantitative metric. Three "Paths" of analysis (denoted A-C) are compared to each other in the context of a "benchmark" method to study the impact of the data sampling ratio on preserving chemical information, which is defined by the DCS quantitative metric. The benchmark method is simply aligning data and applying PCA, without data reduction. Path A applies data alignment to collected data, then data reduction, and finally PCA. Path B applies data reduction to collected data, and then data alignment, and finally PCA. The optimized path, namely Path C, is created from Paths A and B, whereby collected data are initially reduced to fewer data points (smaller S(R)), then aligned, and then further reduced to even fewer points and finally analyzed with PCA to provide the DCS metric. Overall, following Path C, one can successfully and efficiently classify chromatographic data by reducing to a S(R) of ∼15 before alignment, and then reducing down to S(R) of ∼2 before performing PCA. Indeed, following Path C, results from an average of 15 different column length-with-temperature ramp rate combinations spanning a broad range of separation conditions resulted in only a ∼15% loss in classification capability (via PCA) when the loss in chromatographic resolution was ∼36%.

Utilizing a constant peak width transform for isothermal gas chromatography.

A computational approach to partially address the general elution problem (GEP), and better visualize, isothermal gas chromatograms is reported. The theoretical computational approach is developed and applied experimentally. We report a high speed temporally increasing boxcar summation (TIBS) transform that, when applied to the raw isothermal GC data, converts the chromatographic data from the initial time domain (in which the peak widths in isothermal GC increase as a function of their retention factors, k), to a data point based domain in which all peaks have the same peak width in terms of number of points in the final data vector, which aides in preprocessing and data analysis, while minimizing data storage size. By applying the TIBS transform, the resulting GC chromatogram (initially collected isothermally), appears with an x-axis point scale as if it were instrumentally collected using a suitable temperature program. A high speed GC isothermal separation with a test mixture containing 10 compounds had a run time of ∼25 s. The peak at a retention factor k ∼0.7 had a peak width of ∼55 ms, while the last eluting peak at k ∼89 (i.e., retention time of ∼22 s) had a peak width of ∼2000 ms. Application of the TIBS transform increased the peak height of the last eluting peak 45-fold, and S/N ∼20-fold. All peaks in the transformed test mixture chromatogram had the width of an unretained peak, in terms of number of data points. A simulated chromatogram at unit resolution, studied using the TIBS transform, provided additional insight into the benefits of the algorithm.

Achieving high peak capacity production for gas chromatography and comprehensive two-dimensional gas chromatography by minimizing off-column peak broadening.

By taking into consideration band broadening theory and using those results to select experimental conditions, and also by reducing the injection pulse width, peak capacity production (i.e., peak capacity per separation time) is substantially improved for one dimensional (1D-GC) and comprehensive two dimensional (GC×GC) gas chromatography. A theoretical framework for determining the optimal linear gas velocity (the linear gas velocity producing the minimum H), from experimental parameters provides an in-depth understanding of the potential for GC separations in the absence of extra-column band broadening. The extra-column band broadening is referred to herein as off-column band broadening since it is additional band broadening not due to the on-column separation processes. The theory provides the basis to experimentally evaluate and improve temperature programmed 1D-GC separations, but in order to do so with a commercial 1D-GC instrument platform, off-column band broadening from injection and detection needed to be significantly reduced. Specifically for injection, a resistively heated transfer line is coupled to a high-speed diaphragm valve to provide a suitable injection pulse width (referred to herein as modified injection). Additionally, flame ionization detection (FID) was modified to provide a data collection rate of 5kHz. The use of long, relatively narrow open tubular capillary columns and a 40°C/min programming rate were explored for 1D-GC, specifically a 40m, 180µm i.d. capillary column operated at or above the optimal average linear gas velocity. Injection using standard auto-injection with a 1:400 split resulted in an average peak width of ∼1.5s, hence a peak capacity production of 40peaks/min. In contrast, use of modified injection produced ∼500ms peak widths for 1D-GC, i.e., a peak capacity production of 120peaks/min (a 3-fold improvement over standard auto-injection). Implementation of modified injection resulted in retention time, peak width, peak height, and peak area average RSD%'s of 0.006, 0.8, 3.4, and 4.0%, respectively. Modified injection onto the first column of a GC×GC coupled with another high-speed valve injection onto the second column produced an instrument with high peak capacity production (500-800peaks/min), ∼5-fold to 8-fold higher than typically reported for GC×GC.