A new flow injection procedure for an assay of Fe(III) by using salicylate obtained from antipyre... more A new flow injection procedure for an assay of Fe(III) by using salicylate obtained from antipyretic powder, which is a cheap and easily available reagent, is proposed. A red complex was continuously monitored by a laboratory-made green LED colorimeter. A linear calibration was obtained in the range of 1-20 mg Fe l −1 with a detection limit of 0.5 mg Fe l −1 and R.S.D.s of 1.4-5.4% (n = 3, for 1-20 mg Fe l −1). The new procedure was applied to assay iron contents in pharmaceutical preparations. The results were in good agreement with those of the USP standard method.
A new method for the separation of denatured alpha-, beta- and kappa- caseins by hydrophobic inte... more A new method for the separation of denatured alpha-, beta- and kappa- caseins by hydrophobic interaction chromatography (HIC) is proposed. The method is based on an easy solubilization of commercial and real samples by 4.0 M guanidine thiocyanate (GdmSCN) and elution on a TSK-Gel(R) Phenyl-5PW column (TosoHaas) in the presence of 8.0 M urea in the mobile phase. The procedure, applied to commercial caseins and to real, raw samples (whole milk powder and fat-free yoghurt) is not expensive, it requires common high performance liquid chromatography (HPLC) instrumentation and allows the separation of caseins also in the presence of whey proteins. Quantitative results on the analysis of alpha-, beta- and kappa-caseins in real samples are also reported.
olites as potential biomarkers. The changes in metabolite concentrations were characterized and c... more olites as potential biomarkers. The changes in metabolite concentrations were characterized and compared between treatment groups. Further comparison determined that 8 metabolites (arachidonic acid, butanoic acid, citric acid, fumaric acid, lactate, malate, propanoic acid, and succinic acid) correlated with early and/or long-term neurodevelopmental outcomes. The combined outcomes of death or cerebral palsy correlated with citric acid, fumaric acid, lactate, and propanoic acid. This change in circulating metabolome after UCO may reflect cellular metabolism and biochemical changes in response to the severity of brain injury and have potential to predict neurodevelopmental outcomes.
Because of the growing number of analysis scenarios involving complex samples, comprehensive two-... more Because of the growing number of analysis scenarios involving complex samples, comprehensive two-dimensional gas chromatography coupled with time-of-flight mass spectrometry (GC×GC–TOF-MS) is now a prominent technique for characterization. However, the limitations on time, expenses, and sample quantities, as well as the need for specialized expertise in comparative analysis, can prevent the discovery of analytes that distinguish multiple samples. This article provides an overview of the development and current status of comparative analysis for GC×GC–TOF-MS data and how key limitations can be overcome with a novel tile-based pairwise analysis method.
Hyphenations of Capillary Chromatography with Mass Spectrometry, 2020
Abstract The challenges of data management and interpretation for purveyors of capillary chromato... more Abstract The challenges of data management and interpretation for purveyors of capillary chromatography instrumentation with mass spectrometric detection are reviewed. Techniques to succeed are detailed within the context of standard analytical targeted, nontargeted, and hybrid targeted/nontargeted work flows. The processes are divided into the common elements of preprocessing, comparative analysis, deconvolution (i.e., mathematical resolution), result analysis, and statistical presentation. Time-honored techniques are discussed throughout with a focus on the added complications created by the data size and density in modern experiments where the samples are analyzed by chromatographic instruments with mass spectrometric detection. Multidimensional and multivariate options are included throughout and the focus constantly returns to harvesting useful information from the windfall of data created by these technologies.
Although comprehensive two-dimensional (2D) gas chromatography (GC × GC) is a powerful technique ... more 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 threedimensional (3D) gas chromatography (GC 3), especially with time-of-flight mass spectrometry detection (GC 3-TOFMS). However, the GC 3-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 GC 3-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 1 D column to the 2 D column, with recently described dynamic pressure gradient modulation (DPGM) as the second modulator from the 2 D column to the 3 D column, which is a total-transfer flow modulation technique. Area measurements of 1 D peaks are compared to the sum of 3 D peak areas to validate the assumption that total-transfer from 1 D to 3 D is accomplished. Additionally, peak heights were amplified by as high as a factor of 177 (x ̅ = 130, s = 47) via comparison of 1 D peak heights to the maximum 3 D 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 (x ̅ = 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.
Abstract Advanced data handling techniques, specifically chemometric data analysis tools, have be... more Abstract Advanced data handling techniques, specifically chemometric data analysis tools, have become an integral part of successful implementation of comprehensive two-dimensional (2D) gas chromatography (GC×GC) for the analysis of complex samples. When GC×GC is coupled with a multichannel detector such as a time-of-flight–mass spectrometer (TOFMS), enormous data sets are produced that require chemometric methods to glean meaningful information. Chemometric methods utilize linear algebra and statistical concepts to reveal underlying chemical relationships in the data that are related to the experimental design. However, the successful application of chemometrics is not without its challenges in order to reap the rewards. Since there are a myriad of chemometric methods, in this chapter we focus on the operation of the most common tools: deconvolution, pattern recognition, discovery-based analysis, and property prediction methods. Considerations of experimental design, preprocessing, data structure, and analysis goals are included. Current applications of these advanced data handling methods are demonstrated to highlight the ability to obtain useful information from complex samples.
The quality of East African coffee beans has been significantly reduced by a flavor defect known ... more The quality of East African coffee beans has been significantly reduced by a flavor defect known as potato taste defect (PTD) due to the presence of 2-isopropyl-3-methoxypyrazine (IPMP) and 2-isobutyl-3-methoxypyrazine (IBMP). Therefore, the aims of this study were to determine the correlation between these methoxypyrazines and the severity of odor attributed to PTD and discover additional analytes that may be correlated with PTD using Fisher ratio analysis, a supervised discovery-based data analysis method. Specialty ground roasted coffees from East Africa were classified as clean (i.e., no off-odor), mild, medium, or strong PTD. For the samples examined, IPMP was found to discriminate between non-defective and defective samples, while IBMP did not do so. Samples affected by PTD exhibited a wide range of IPMP concentration (1.6-529.9 ng/g). Except for one sample, the IPMP concentration in defective samples was greater than the average IPMP concentration in the non-defective samples (2.0 ng/g). Also, an analysis of variance found that IPMP concentrations were significantly different based on the severity of odor attributed to PTD (p < 0.05). Fisher ratio analysis discovered 21 additional analytes whose concentrations were statistically different based on the severity of PTD odor (p < 0.05). Generally, analytes that were positively correlated with odor severity generally had unpleasant sensory descriptions, while analytes typically associated with desirable aromas were found to be negatively correlated with odor severity. These findings not only show that IPMP concentration can differentiate the severity of PTD but also that changes in the volatile analyte profile of coffee beans induced by PTD can contribute to odor severity.
Dynamic pressure gradient modulation (DPGM) in full modulation mode is optimized for comprehensiv... more Dynamic pressure gradient modulation (DPGM) in full modulation mode is optimized for comprehensive two-dimensional (2D) gas chromatography (GC × GC) with time-of-fight mass spectrometry (TOFMS) detection to obtain high peak capacity separations and demonstrate broad applicability for complex samples. A pulse valve introduces an auxiliary carrier gas flow at a T-union connecting the first dimension (1D) column to the second dimension (2D) column. At a sufficiently high auxiliary pressure (Paux) the 1D flow is temporarily stopped. Then, during each modulation period (PM) the valve is turned off briefly, a period termed the pulse width (pw), allowing the 1D effluent to essentially be reinjected onto the 2D column for the modulated separations. Modifications to the modulator assembly are provided to improve performance. Method optimization is demonstrated for a 116-component test mixture by tuning the Paux and the pw. For a PM = 2 s and 1F of 0.10 ml/min, the optimal pw and initial Paux selected were 200 ms and 330.9 kPa (33 psig), respectively. The 30 min separation of the test mixture provided a 1D peak capacity of 1nc = 330 and a 2D peak capacity of 2nc = 15, hence an ideal 2D peak capacity nc,2D = 1nc × 2nc = 4950. Likewise, the 2D peak capacity corrected for undersampling of the 1D separation was 4500 and corrected for both undersampling and sampling variation via statistical overlap theory was 4090. These results provide a 2-fold improvement in peak capacity relative to the previous DPGM study in full modulation mode for GC × GC-TOFMS. The optimized conditions were applied for a variety of applications: diesel fuel, derivatized cow serum, solid phase microextraction (SPME) of coffee headspace, and SPME of river water headspace. Additionally, the fraction of 2D separation space utilized (fcoverage), as defined by the minimum convex hull method, ranged from 0.60 to 0.85. We observed that any fcoverage correction to 2D peak capacity is highly sample dependent, since all samples, except for the diesel sample, were run with the same separation conditions, and yet the fcoverage ranged from 0.60 to 0.80.
Prior to the application of non-targeted chemometric techniques (i.e., Fisher ratio analysis), pr... more Prior to the application of non-targeted chemometric techniques (i.e., Fisher ratio analysis), pre-processing steps must be carried out on the data collected using liquid chromatography-quadrupole time of flight (LC-QTOF) mass spectrometry. Data compression is the most important pre-processing step due to the vast number of high-resolution mass channels (m/z; 0.0001 Da) collected. Our first approach was to bin the mass spectral dimension to 0.01 Da; however, this approach was computationally expensive and did not preserve the highresolution data. Therefore, we started using a data compression strategy outlined by Tauler et al., which defines "regions of interest" (ROI) in the LC-QTOF data (Fig. 1). Since high resolution MS data provides an irregular number of measured m/z and signal intensity pairs at each retention time, this method searches for ROIs in the raw data that has signals higher than a set threshold and are present in high densities. Those ROIs are then reorganized into a data matrix, which contains the signal intensities for the ROI m/z at every time point. These first two steps are repeated for each individual chromatogram in the analysis. Next, to analyze multiple samples simultaneously, the individual ROI data matrices must be augmented. It is important to note that individual ROI matrices can have different ROI m/z values; therefore, the augmentation step
Tile-based Fisher ratio (F-ratio) analysis has recently been developed and validated for discover... more Tile-based Fisher ratio (F-ratio) analysis has recently been developed and validated for discovery-based studies of highly complex data collected using comprehensive two-dimensional gas chromatography coupled with time-of-flight mass spectrometry (GC ×GC-TOFMS). In previous studies, interpretation and utilization of F-ratio hit lists has relied upon manual decomposition and quantification performed by chemometric methods such as parallel factor analysis (PARAFAC), or via manual translation of the F-ratio hit list information to peak table quantitative information provided by the instrument software (ChromaTOF). Both of these quantification approaches are bottlenecks in the overall workflow. In order to address this issue, a more automatable approach to provide accurate relative quantification for F-ratio analyses was investigated, based upon the mass spectral selectivity provided via the F-ratio spectral output. Diesel fuel spiked with 15 analytes at four concentration levels (80, 40, 20, and 10 ppm) produced three sets of two class comparisons that were submitted to tile-based F-ratio analysis to obtain three hit lists, with an F-ratio spectrum for each hit. A novel algorithm which calculates the signal ratio (S-ratio) between two classes (eg., 80 ppm versus 40 ppm) was applied to all mass channels (m/z) in the F-ratio spectrum for each hit. A lack of fit (LOF) metric was utilized as a measure of peak purity and combined with Fratio and p-values to study the relationship of each of these metrics with m/z purity. Application of a LOF threshold coupled with a p-value threshold yielded a subset of the most pure m/z for each of the 15 spiked analytes, evident by the low deviations (< 5%) in S-ratio relative to the true concentration ratio. A key outcome of this study was to demonstrate the isolation of pure m/z without the need for higher level signal decomposition algorithms.
We investigate the extent to which comprehensive three-dimensional gas chromatography (GC3) provi... more We investigate the extent to which comprehensive three-dimensional gas chromatography (GC3) provides a signal enhancement (SE) and a signal-to-noise ratio enhancement (S/NRel) relative to one-dimensional (1D)-GC. Specifically, the SE is defined as the ratio of the tallest 3D peak height from the GC3 separation to the 1D peak height from the unmodulated 1D-GC separation. A model is proposed which allows the analyst to predict the theoretically attainable SE (SET) based upon the peak width and sampling density inputs. The model is validated via comparison of the SET to the experimentally measured SE (SEM) obtained using total-transfer GC3 (100% duty cycle for both modulators) with time-of-flight mass spectrometry detection. Two experimental conditions were studied using the same GC3 column set, differing principally in the modulation period from the 1D to 2D columns: 4 s versus 8 s. Under the first set of conditions, the average SEM was 97 (±22), in excellent agreement with the SET of 97 (±18). The second set of conditions improved the average SEM to 181 (±27), also in agreement with the average SET of 176 (±26). The average S/NRel following correction for the mass spectrum acquisition frequency was 38.8 (±11.2) and 59.0 (±27.2) for the two sets of conditions. The enhancement in S/N is largely attributed to moving the signal to a higher frequency domain where the impact of "low frequency" noise is less detrimental. The findings here provide strong evidence that GC3 separations can provide enhanced detectability relative to 1D-GC and comprehensive two-dimensional gas chromatography (GC×GC) separations.
Basic principles are introduced for implementing discovery-based analysis with automated quantifi... more Basic principles are introduced for implementing discovery-based analysis with automated quantification of data obtained using comprehensive three-dimensional gas chromatography with flame ionization detection (GC 3-FID). The GC 3-FID instrument employs dynamic pressure gradient modulation, providing full modulation (100% duty cycle) with a fast modulation period (P M) of 100 ms. Specifically, tile-based Fisher-ratio analysis, previously developed for comprehensive two-dimensional gas chromatography with time-of-flight mass spectrometry (GC ×GC-TOFMS), is adapted and applied for GC 3-FID where the third chromatographic dimension (3 D) is treated as the "spectral" dimension. To evaluate the instrumental platform and software implementation, ten "non-native" compounds were spiked into a ninety-component base mixture to create two classes with a concentration ratio of two for the spiked analyte compounds. The Fisher ratio software identified 95 locations of potential interest (i.e., hits), with all ten spiked analytes discovered within the top fourteen hits. All 95 hits were quantified by a novel signal ratio (Sratio) algorithm portion of the F-ratio software, which determines the time-dependent S-ratio of the 3 D chromatograms from one class to another, thus providing relative quantification. The average S-ratio for spiked analytes was 1.94 ± 0.14 mean absolute error (close to the nominal concentration ratio of two), and 1.06 ± 0.16 mean absolute error for unspiked (i.e., matrix) components. The appearance of the S-ratio as a function of 3 D retention time in the GC 3 dataset, referred to as an S-ratiogram, provides indication of peak purity for each hit. The unique shape of the S-ratiogram for hit 1, α-pinene, suggested likely 3 D overlap. Parallel factor analysis (PARAFAC) decomposition of the hit location confirmed that overlap was occurring and successfully decomposed α-pinene from a highly overlapped (3 R s = 0.1) matrix interferent.
Partial modulation via a pulse flow valve operated in the negative pulse mode is developed for hi... more Partial modulation via a pulse flow valve operated in the negative pulse mode is developed for high-speed onedimensional gas chromatography (1D-GC), comprehensive two-dimensional (2D) gas chromatography (GC×GC), and comprehensive three-dimensional gas chromatography (GC 3). The pulse flow valve readily provides very short modulation periods, P M , 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 pseudo-steady state analyte stream is modulated, and 8 analytes are baseline resolved (R s ≥ 1.5) in a 200 ms window, providing a peak capacity, n c , of 14 at unit resolution (R s = 1.0). Additionally, the pulse width, p w , of the pulse flow valve "injection" relationship to peak width-at-base, w b , 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, 2 D, with 2 w b ranging from 7 to 12 ms, providing an exceptional 2 D peak capacity, 2 n c of ~12 using a P M of 100 ms. Next, a 12 min separation of a diesel sample using a P M of 300 ms is presented. The 1 w b is ~4 s, resulting in a 1 n c of ~180, and 2 w b is ~18 ms, resulting in a 2 n c of ~17, thus achieving a n c,2D of ~3,000 in this rapid GC×GC diesel separation. Finally, GC 3 with time-of-flight mass spectrometry detection (TOFMS) using a P M of 100 ms applied between the 2 D and 3 D columns is reported. Narrow third dimension, 3 D, peaks 3 w b ~ 15 ms were obtained, resulting in a GC 3 peak capacity, n c,3D , of ~35,000, in a 45 min separation.
differences were also observed between non-rapid progressors and non-progressors. In terms of hap... more differences were also observed between non-rapid progressors and non-progressors. In terms of haplogroups, compared with the most common haplogroup H, the haplogroup J showed a lower frequency in the rapid progressors group in relation to non-progressors, although this difference did not reach the statistical significance (OR¼0,526; 95% CI: 0,251-1,101; p¼0,088). When the analysis included the major H subtypes, the most frequent sub-haplogroup H1 appeared over-represented in the rapid progressors group in relation to both non-progressors (OR¼1,832; 95%CI: 1,102-3,044) and non-rapid progressors (OR¼1,587; 95%CI: 0,876-2,877), being this difference statistically significant in the case of non-progressors (p¼0,020). After re-categorizing the dependent variable into rapid-progressors and non-rapid progressors, in addition to the significant association of the classical risk factors such as gender (female), age, BMI, contralateral OA, previous injury of target knee and total WOMAC at baseline, the sub-haplogroup H1 appeared significantly associated as a risk factor for rapid progression too (OR¼ 1,781; 95%CI: 1,087-2,919; p¼0,022) (Table 1). Conclusions: The mtDNA haplogroups, more specifically the major subhaplogroup H1, increase the risk of rapidly progressive OA of the knee. The assignment of this mitochondrial genetic sub-haplogroup could be useful as complementary genetic biomarker for the early identification of this OA phenotype.
... Size-Exclusion Chromatography with Dynamic Surface Tension Detection: Analysis of Polymers an... more ... Size-Exclusion Chromatography with Dynamic Surface Tension Detection: Analysis of Polymers and Proteins Robert E. Synovec1, Bethany A. Staggemeier1, Emilia Bramanti2 , Wes WC Quigley1, and Bryan J. Prazen1 ... E.; Nabi, Α.; Skogerboe, KJ; Synovec, RE Anal. Bioanal. ...
A new flow injection procedure for an assay of Fe(III) by using salicylate obtained from antipyre... more A new flow injection procedure for an assay of Fe(III) by using salicylate obtained from antipyretic powder, which is a cheap and easily available reagent, is proposed. A red complex was continuously monitored by a laboratory-made green LED colorimeter. A linear calibration was obtained in the range of 1-20 mg Fe l −1 with a detection limit of 0.5 mg Fe l −1 and R.S.D.s of 1.4-5.4% (n = 3, for 1-20 mg Fe l −1). The new procedure was applied to assay iron contents in pharmaceutical preparations. The results were in good agreement with those of the USP standard method.
A new flow injection procedure for an assay of Fe(III) by using salicylate obtained from antipyre... more A new flow injection procedure for an assay of Fe(III) by using salicylate obtained from antipyretic powder, which is a cheap and easily available reagent, is proposed. A red complex was continuously monitored by a laboratory-made green LED colorimeter. A linear calibration was obtained in the range of 1-20 mg Fe l −1 with a detection limit of 0.5 mg Fe l −1 and R.S.D.s of 1.4-5.4% (n = 3, for 1-20 mg Fe l −1). The new procedure was applied to assay iron contents in pharmaceutical preparations. The results were in good agreement with those of the USP standard method.
A new method for the separation of denatured alpha-, beta- and kappa- caseins by hydrophobic inte... more A new method for the separation of denatured alpha-, beta- and kappa- caseins by hydrophobic interaction chromatography (HIC) is proposed. The method is based on an easy solubilization of commercial and real samples by 4.0 M guanidine thiocyanate (GdmSCN) and elution on a TSK-Gel(R) Phenyl-5PW column (TosoHaas) in the presence of 8.0 M urea in the mobile phase. The procedure, applied to commercial caseins and to real, raw samples (whole milk powder and fat-free yoghurt) is not expensive, it requires common high performance liquid chromatography (HPLC) instrumentation and allows the separation of caseins also in the presence of whey proteins. Quantitative results on the analysis of alpha-, beta- and kappa-caseins in real samples are also reported.
olites as potential biomarkers. The changes in metabolite concentrations were characterized and c... more olites as potential biomarkers. The changes in metabolite concentrations were characterized and compared between treatment groups. Further comparison determined that 8 metabolites (arachidonic acid, butanoic acid, citric acid, fumaric acid, lactate, malate, propanoic acid, and succinic acid) correlated with early and/or long-term neurodevelopmental outcomes. The combined outcomes of death or cerebral palsy correlated with citric acid, fumaric acid, lactate, and propanoic acid. This change in circulating metabolome after UCO may reflect cellular metabolism and biochemical changes in response to the severity of brain injury and have potential to predict neurodevelopmental outcomes.
Because of the growing number of analysis scenarios involving complex samples, comprehensive two-... more Because of the growing number of analysis scenarios involving complex samples, comprehensive two-dimensional gas chromatography coupled with time-of-flight mass spectrometry (GC×GC–TOF-MS) is now a prominent technique for characterization. However, the limitations on time, expenses, and sample quantities, as well as the need for specialized expertise in comparative analysis, can prevent the discovery of analytes that distinguish multiple samples. This article provides an overview of the development and current status of comparative analysis for GC×GC–TOF-MS data and how key limitations can be overcome with a novel tile-based pairwise analysis method.
Hyphenations of Capillary Chromatography with Mass Spectrometry, 2020
Abstract The challenges of data management and interpretation for purveyors of capillary chromato... more Abstract The challenges of data management and interpretation for purveyors of capillary chromatography instrumentation with mass spectrometric detection are reviewed. Techniques to succeed are detailed within the context of standard analytical targeted, nontargeted, and hybrid targeted/nontargeted work flows. The processes are divided into the common elements of preprocessing, comparative analysis, deconvolution (i.e., mathematical resolution), result analysis, and statistical presentation. Time-honored techniques are discussed throughout with a focus on the added complications created by the data size and density in modern experiments where the samples are analyzed by chromatographic instruments with mass spectrometric detection. Multidimensional and multivariate options are included throughout and the focus constantly returns to harvesting useful information from the windfall of data created by these technologies.
Although comprehensive two-dimensional (2D) gas chromatography (GC × GC) is a powerful technique ... more 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 threedimensional (3D) gas chromatography (GC 3), especially with time-of-flight mass spectrometry detection (GC 3-TOFMS). However, the GC 3-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 GC 3-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 1 D column to the 2 D column, with recently described dynamic pressure gradient modulation (DPGM) as the second modulator from the 2 D column to the 3 D column, which is a total-transfer flow modulation technique. Area measurements of 1 D peaks are compared to the sum of 3 D peak areas to validate the assumption that total-transfer from 1 D to 3 D is accomplished. Additionally, peak heights were amplified by as high as a factor of 177 (x ̅ = 130, s = 47) via comparison of 1 D peak heights to the maximum 3 D 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 (x ̅ = 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.
Abstract Advanced data handling techniques, specifically chemometric data analysis tools, have be... more Abstract Advanced data handling techniques, specifically chemometric data analysis tools, have become an integral part of successful implementation of comprehensive two-dimensional (2D) gas chromatography (GC×GC) for the analysis of complex samples. When GC×GC is coupled with a multichannel detector such as a time-of-flight–mass spectrometer (TOFMS), enormous data sets are produced that require chemometric methods to glean meaningful information. Chemometric methods utilize linear algebra and statistical concepts to reveal underlying chemical relationships in the data that are related to the experimental design. However, the successful application of chemometrics is not without its challenges in order to reap the rewards. Since there are a myriad of chemometric methods, in this chapter we focus on the operation of the most common tools: deconvolution, pattern recognition, discovery-based analysis, and property prediction methods. Considerations of experimental design, preprocessing, data structure, and analysis goals are included. Current applications of these advanced data handling methods are demonstrated to highlight the ability to obtain useful information from complex samples.
The quality of East African coffee beans has been significantly reduced by a flavor defect known ... more The quality of East African coffee beans has been significantly reduced by a flavor defect known as potato taste defect (PTD) due to the presence of 2-isopropyl-3-methoxypyrazine (IPMP) and 2-isobutyl-3-methoxypyrazine (IBMP). Therefore, the aims of this study were to determine the correlation between these methoxypyrazines and the severity of odor attributed to PTD and discover additional analytes that may be correlated with PTD using Fisher ratio analysis, a supervised discovery-based data analysis method. Specialty ground roasted coffees from East Africa were classified as clean (i.e., no off-odor), mild, medium, or strong PTD. For the samples examined, IPMP was found to discriminate between non-defective and defective samples, while IBMP did not do so. Samples affected by PTD exhibited a wide range of IPMP concentration (1.6-529.9 ng/g). Except for one sample, the IPMP concentration in defective samples was greater than the average IPMP concentration in the non-defective samples (2.0 ng/g). Also, an analysis of variance found that IPMP concentrations were significantly different based on the severity of odor attributed to PTD (p < 0.05). Fisher ratio analysis discovered 21 additional analytes whose concentrations were statistically different based on the severity of PTD odor (p < 0.05). Generally, analytes that were positively correlated with odor severity generally had unpleasant sensory descriptions, while analytes typically associated with desirable aromas were found to be negatively correlated with odor severity. These findings not only show that IPMP concentration can differentiate the severity of PTD but also that changes in the volatile analyte profile of coffee beans induced by PTD can contribute to odor severity.
Dynamic pressure gradient modulation (DPGM) in full modulation mode is optimized for comprehensiv... more Dynamic pressure gradient modulation (DPGM) in full modulation mode is optimized for comprehensive two-dimensional (2D) gas chromatography (GC × GC) with time-of-fight mass spectrometry (TOFMS) detection to obtain high peak capacity separations and demonstrate broad applicability for complex samples. A pulse valve introduces an auxiliary carrier gas flow at a T-union connecting the first dimension (1D) column to the second dimension (2D) column. At a sufficiently high auxiliary pressure (Paux) the 1D flow is temporarily stopped. Then, during each modulation period (PM) the valve is turned off briefly, a period termed the pulse width (pw), allowing the 1D effluent to essentially be reinjected onto the 2D column for the modulated separations. Modifications to the modulator assembly are provided to improve performance. Method optimization is demonstrated for a 116-component test mixture by tuning the Paux and the pw. For a PM = 2 s and 1F of 0.10 ml/min, the optimal pw and initial Paux selected were 200 ms and 330.9 kPa (33 psig), respectively. The 30 min separation of the test mixture provided a 1D peak capacity of 1nc = 330 and a 2D peak capacity of 2nc = 15, hence an ideal 2D peak capacity nc,2D = 1nc × 2nc = 4950. Likewise, the 2D peak capacity corrected for undersampling of the 1D separation was 4500 and corrected for both undersampling and sampling variation via statistical overlap theory was 4090. These results provide a 2-fold improvement in peak capacity relative to the previous DPGM study in full modulation mode for GC × GC-TOFMS. The optimized conditions were applied for a variety of applications: diesel fuel, derivatized cow serum, solid phase microextraction (SPME) of coffee headspace, and SPME of river water headspace. Additionally, the fraction of 2D separation space utilized (fcoverage), as defined by the minimum convex hull method, ranged from 0.60 to 0.85. We observed that any fcoverage correction to 2D peak capacity is highly sample dependent, since all samples, except for the diesel sample, were run with the same separation conditions, and yet the fcoverage ranged from 0.60 to 0.80.
Prior to the application of non-targeted chemometric techniques (i.e., Fisher ratio analysis), pr... more Prior to the application of non-targeted chemometric techniques (i.e., Fisher ratio analysis), pre-processing steps must be carried out on the data collected using liquid chromatography-quadrupole time of flight (LC-QTOF) mass spectrometry. Data compression is the most important pre-processing step due to the vast number of high-resolution mass channels (m/z; 0.0001 Da) collected. Our first approach was to bin the mass spectral dimension to 0.01 Da; however, this approach was computationally expensive and did not preserve the highresolution data. Therefore, we started using a data compression strategy outlined by Tauler et al., which defines "regions of interest" (ROI) in the LC-QTOF data (Fig. 1). Since high resolution MS data provides an irregular number of measured m/z and signal intensity pairs at each retention time, this method searches for ROIs in the raw data that has signals higher than a set threshold and are present in high densities. Those ROIs are then reorganized into a data matrix, which contains the signal intensities for the ROI m/z at every time point. These first two steps are repeated for each individual chromatogram in the analysis. Next, to analyze multiple samples simultaneously, the individual ROI data matrices must be augmented. It is important to note that individual ROI matrices can have different ROI m/z values; therefore, the augmentation step
Tile-based Fisher ratio (F-ratio) analysis has recently been developed and validated for discover... more Tile-based Fisher ratio (F-ratio) analysis has recently been developed and validated for discovery-based studies of highly complex data collected using comprehensive two-dimensional gas chromatography coupled with time-of-flight mass spectrometry (GC ×GC-TOFMS). In previous studies, interpretation and utilization of F-ratio hit lists has relied upon manual decomposition and quantification performed by chemometric methods such as parallel factor analysis (PARAFAC), or via manual translation of the F-ratio hit list information to peak table quantitative information provided by the instrument software (ChromaTOF). Both of these quantification approaches are bottlenecks in the overall workflow. In order to address this issue, a more automatable approach to provide accurate relative quantification for F-ratio analyses was investigated, based upon the mass spectral selectivity provided via the F-ratio spectral output. Diesel fuel spiked with 15 analytes at four concentration levels (80, 40, 20, and 10 ppm) produced three sets of two class comparisons that were submitted to tile-based F-ratio analysis to obtain three hit lists, with an F-ratio spectrum for each hit. A novel algorithm which calculates the signal ratio (S-ratio) between two classes (eg., 80 ppm versus 40 ppm) was applied to all mass channels (m/z) in the F-ratio spectrum for each hit. A lack of fit (LOF) metric was utilized as a measure of peak purity and combined with Fratio and p-values to study the relationship of each of these metrics with m/z purity. Application of a LOF threshold coupled with a p-value threshold yielded a subset of the most pure m/z for each of the 15 spiked analytes, evident by the low deviations (< 5%) in S-ratio relative to the true concentration ratio. A key outcome of this study was to demonstrate the isolation of pure m/z without the need for higher level signal decomposition algorithms.
We investigate the extent to which comprehensive three-dimensional gas chromatography (GC3) provi... more We investigate the extent to which comprehensive three-dimensional gas chromatography (GC3) provides a signal enhancement (SE) and a signal-to-noise ratio enhancement (S/NRel) relative to one-dimensional (1D)-GC. Specifically, the SE is defined as the ratio of the tallest 3D peak height from the GC3 separation to the 1D peak height from the unmodulated 1D-GC separation. A model is proposed which allows the analyst to predict the theoretically attainable SE (SET) based upon the peak width and sampling density inputs. The model is validated via comparison of the SET to the experimentally measured SE (SEM) obtained using total-transfer GC3 (100% duty cycle for both modulators) with time-of-flight mass spectrometry detection. Two experimental conditions were studied using the same GC3 column set, differing principally in the modulation period from the 1D to 2D columns: 4 s versus 8 s. Under the first set of conditions, the average SEM was 97 (±22), in excellent agreement with the SET of 97 (±18). The second set of conditions improved the average SEM to 181 (±27), also in agreement with the average SET of 176 (±26). The average S/NRel following correction for the mass spectrum acquisition frequency was 38.8 (±11.2) and 59.0 (±27.2) for the two sets of conditions. The enhancement in S/N is largely attributed to moving the signal to a higher frequency domain where the impact of "low frequency" noise is less detrimental. The findings here provide strong evidence that GC3 separations can provide enhanced detectability relative to 1D-GC and comprehensive two-dimensional gas chromatography (GC×GC) separations.
Basic principles are introduced for implementing discovery-based analysis with automated quantifi... more Basic principles are introduced for implementing discovery-based analysis with automated quantification of data obtained using comprehensive three-dimensional gas chromatography with flame ionization detection (GC 3-FID). The GC 3-FID instrument employs dynamic pressure gradient modulation, providing full modulation (100% duty cycle) with a fast modulation period (P M) of 100 ms. Specifically, tile-based Fisher-ratio analysis, previously developed for comprehensive two-dimensional gas chromatography with time-of-flight mass spectrometry (GC ×GC-TOFMS), is adapted and applied for GC 3-FID where the third chromatographic dimension (3 D) is treated as the "spectral" dimension. To evaluate the instrumental platform and software implementation, ten "non-native" compounds were spiked into a ninety-component base mixture to create two classes with a concentration ratio of two for the spiked analyte compounds. The Fisher ratio software identified 95 locations of potential interest (i.e., hits), with all ten spiked analytes discovered within the top fourteen hits. All 95 hits were quantified by a novel signal ratio (Sratio) algorithm portion of the F-ratio software, which determines the time-dependent S-ratio of the 3 D chromatograms from one class to another, thus providing relative quantification. The average S-ratio for spiked analytes was 1.94 ± 0.14 mean absolute error (close to the nominal concentration ratio of two), and 1.06 ± 0.16 mean absolute error for unspiked (i.e., matrix) components. The appearance of the S-ratio as a function of 3 D retention time in the GC 3 dataset, referred to as an S-ratiogram, provides indication of peak purity for each hit. The unique shape of the S-ratiogram for hit 1, α-pinene, suggested likely 3 D overlap. Parallel factor analysis (PARAFAC) decomposition of the hit location confirmed that overlap was occurring and successfully decomposed α-pinene from a highly overlapped (3 R s = 0.1) matrix interferent.
Partial modulation via a pulse flow valve operated in the negative pulse mode is developed for hi... more Partial modulation via a pulse flow valve operated in the negative pulse mode is developed for high-speed onedimensional gas chromatography (1D-GC), comprehensive two-dimensional (2D) gas chromatography (GC×GC), and comprehensive three-dimensional gas chromatography (GC 3). The pulse flow valve readily provides very short modulation periods, P M , 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 pseudo-steady state analyte stream is modulated, and 8 analytes are baseline resolved (R s ≥ 1.5) in a 200 ms window, providing a peak capacity, n c , of 14 at unit resolution (R s = 1.0). Additionally, the pulse width, p w , of the pulse flow valve "injection" relationship to peak width-at-base, w b , 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, 2 D, with 2 w b ranging from 7 to 12 ms, providing an exceptional 2 D peak capacity, 2 n c of ~12 using a P M of 100 ms. Next, a 12 min separation of a diesel sample using a P M of 300 ms is presented. The 1 w b is ~4 s, resulting in a 1 n c of ~180, and 2 w b is ~18 ms, resulting in a 2 n c of ~17, thus achieving a n c,2D of ~3,000 in this rapid GC×GC diesel separation. Finally, GC 3 with time-of-flight mass spectrometry detection (TOFMS) using a P M of 100 ms applied between the 2 D and 3 D columns is reported. Narrow third dimension, 3 D, peaks 3 w b ~ 15 ms were obtained, resulting in a GC 3 peak capacity, n c,3D , of ~35,000, in a 45 min separation.
differences were also observed between non-rapid progressors and non-progressors. In terms of hap... more differences were also observed between non-rapid progressors and non-progressors. In terms of haplogroups, compared with the most common haplogroup H, the haplogroup J showed a lower frequency in the rapid progressors group in relation to non-progressors, although this difference did not reach the statistical significance (OR¼0,526; 95% CI: 0,251-1,101; p¼0,088). When the analysis included the major H subtypes, the most frequent sub-haplogroup H1 appeared over-represented in the rapid progressors group in relation to both non-progressors (OR¼1,832; 95%CI: 1,102-3,044) and non-rapid progressors (OR¼1,587; 95%CI: 0,876-2,877), being this difference statistically significant in the case of non-progressors (p¼0,020). After re-categorizing the dependent variable into rapid-progressors and non-rapid progressors, in addition to the significant association of the classical risk factors such as gender (female), age, BMI, contralateral OA, previous injury of target knee and total WOMAC at baseline, the sub-haplogroup H1 appeared significantly associated as a risk factor for rapid progression too (OR¼ 1,781; 95%CI: 1,087-2,919; p¼0,022) (Table 1). Conclusions: The mtDNA haplogroups, more specifically the major subhaplogroup H1, increase the risk of rapidly progressive OA of the knee. The assignment of this mitochondrial genetic sub-haplogroup could be useful as complementary genetic biomarker for the early identification of this OA phenotype.
... Size-Exclusion Chromatography with Dynamic Surface Tension Detection: Analysis of Polymers an... more ... Size-Exclusion Chromatography with Dynamic Surface Tension Detection: Analysis of Polymers and Proteins Robert E. Synovec1, Bethany A. Staggemeier1, Emilia Bramanti2 , Wes WC Quigley1, and Bryan J. Prazen1 ... E.; Nabi, Α.; Skogerboe, KJ; Synovec, RE Anal. Bioanal. ...
A new flow injection procedure for an assay of Fe(III) by using salicylate obtained from antipyre... more A new flow injection procedure for an assay of Fe(III) by using salicylate obtained from antipyretic powder, which is a cheap and easily available reagent, is proposed. A red complex was continuously monitored by a laboratory-made green LED colorimeter. A linear calibration was obtained in the range of 1-20 mg Fe l −1 with a detection limit of 0.5 mg Fe l −1 and R.S.D.s of 1.4-5.4% (n = 3, for 1-20 mg Fe l −1). The new procedure was applied to assay iron contents in pharmaceutical preparations. The results were in good agreement with those of the USP standard method.
Uploads
Papers by Robert Synovec