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Author: Nathanial E. Watson
Publisher:
ISBN:
Category : Chemometrics
Languages : en
Pages : 117
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Book Description
Three instrumental and computational methods are demonstrated in an endeavor to create novel techniques to extract information from the troves of data generated by multi-dimensional gas chromatography with mass spectrometry. Initially, these methods are considered within the context of targeted and non-targeted experimental design. The tile-based Fisher Ratio with null distribution analysis is first evaluated and validated within the non-targeted realm. The method is shown to be fast and accurate. Forty-six of the fifty-four benchmarked changing metabolites previously discovered were found by the new methodology while consistently excluding all but one of the benchmarked nineteen false positive metabolites previously identified. This was achieved in less than 5% of the time required for the previous method. Later, the three-dimensional gas chromatograph is improved to include mass spectrometric detection. This instrument provides four dimensions (4D) of chemical selectivity and includes significant improvements to total selectivity (mass spectrometric and chromatographic), peak identification, and operational temperature range relative to previous models of the GC3 reported. Useful approaches to visualize the 4D data are presented. The GC3 - TOFMS instrument experimentally achieved total peak capacity, nc,3D, ranging from 5000 to 9600 (x̅= 7000, s = 1700) for 10 representative analytes for 50 min separations with component dimensional peak capacities averaging 406, 3.6, and 4.9 for 1D, 2D, and 3D, respectively. Using this instrument and the well understood Parallel Factor Analysis (PARAFAC) model a new option for targeted analysis is presented. Conceptualizing the GC3 - TOFMS as a one-dimensional gas chromatograph with GC × GC-TOFMS detection the instrument was allowed to create the PARAFAC target window natively. Each first dimension modulation thus created a full GC × GC-TOFMS chromatogram totally amenable to PARAFAC. A simple mixture of 115 compounds and a diesel sample were interrogated through this methodology. All test analyte targets were successfully identified in both mixtures. In addition, mass spectral matching of the PARAFAC loadings to library spectra yielded results greater than 900 in 40 of 42 test analyte cases. Twenty-nine of these cases produced match values greater than 950.
Author: Nathanial E. Watson
Publisher:
ISBN:
Category : Chemometrics
Languages : en
Pages : 117
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Book Description
Three instrumental and computational methods are demonstrated in an endeavor to create novel techniques to extract information from the troves of data generated by multi-dimensional gas chromatography with mass spectrometry. Initially, these methods are considered within the context of targeted and non-targeted experimental design. The tile-based Fisher Ratio with null distribution analysis is first evaluated and validated within the non-targeted realm. The method is shown to be fast and accurate. Forty-six of the fifty-four benchmarked changing metabolites previously discovered were found by the new methodology while consistently excluding all but one of the benchmarked nineteen false positive metabolites previously identified. This was achieved in less than 5% of the time required for the previous method. Later, the three-dimensional gas chromatograph is improved to include mass spectrometric detection. This instrument provides four dimensions (4D) of chemical selectivity and includes significant improvements to total selectivity (mass spectrometric and chromatographic), peak identification, and operational temperature range relative to previous models of the GC3 reported. Useful approaches to visualize the 4D data are presented. The GC3 - TOFMS instrument experimentally achieved total peak capacity, nc,3D, ranging from 5000 to 9600 (x̅= 7000, s = 1700) for 10 representative analytes for 50 min separations with component dimensional peak capacities averaging 406, 3.6, and 4.9 for 1D, 2D, and 3D, respectively. Using this instrument and the well understood Parallel Factor Analysis (PARAFAC) model a new option for targeted analysis is presented. Conceptualizing the GC3 - TOFMS as a one-dimensional gas chromatograph with GC × GC-TOFMS detection the instrument was allowed to create the PARAFAC target window natively. Each first dimension modulation thus created a full GC × GC-TOFMS chromatogram totally amenable to PARAFAC. A simple mixture of 115 compounds and a diesel sample were interrogated through this methodology. All test analyte targets were successfully identified in both mixtures. In addition, mass spectral matching of the PARAFAC loadings to library spectra yielded results greater than 900 in 40 of 42 test analyte cases. Twenty-nine of these cases produced match values greater than 950.
Author: Christopher E. Freye
Publisher:
ISBN:
Category :
Languages : en
Pages : 320
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Book Description
A combination of four instrumental systems and five chemometric methods are shown to improve the efficiency and resolving power (i.e. peak capacity/ peak capacity production) of multi-dimensional gas chromatography (MDGC) as well as provide straightforward, easily interpretable chemical information. Implementation of a high speed pulse flow valve for two-dimensional gas chromatography (GC × GC) is shown to provide ultra-fast modulation with modulation periods (P[subscript]M) as short as 50 ms. Using a commercially available pulse flow valve, this injection technique performs a combination of vacancy chromatography and frontal analysis, whereby each pulse disturbance in the analyte concentration profile as it exits the first column (1D) results in data that is readily converted into a second separation (2D). A three-step process converts the raw data into a format analogous to a GC × GC separation, incorporating signal differentiation, baseline correction and conversion to a GC × GC chromatogram representation. For a P[subscript]M of 250 ms, the apparent peak width on the 2D, 2W[subscript]b, ranged from 12 to 45 ms producing a 2D peak capacity, 2n[subscript]c, of ~ 10, and the total peak capacity, n[subscript]c,2[subscript]D, was 4300 or a peak capacity production of 650 peaks/min. Next, the use of a high temperature diaphragm valve as a modulator for GC × GC facilitated separation temperatures up to 325 °C. Previous diaphragm valve technology limited use to 175 °C if the valve was mounted in the oven or to 265 °C if the valve was face mounted on the outside of the oven. A 44-component mixture was evaluated and the diaphragm valve created narrow, reproducible peaks on the 2D dimension leading to a peak capacity production of 300 peaks/min and had minimal retention time shifting on the 1D and 2D dimensions. In addition, the high temperature diaphragm valve was shown to increase the detection sensitivity by ~8 times compared to one-dimensional gas chromatography due to zone compression. Furthermore, the high temperature diaphragm valve was proven to be compatible with time-of-flight mass spectrometry (TOFMS). Finally a combination of the high temperature diaphragm valve and pulse flow valve yielded a three-dimensional gas chromatography system (GC3) that had a peak capacity production of 1000 peaks/min which is a ~5 times increase in efficiency compared to other GC3 systems. Investigation of novel chemometric techniques for the analysis of GC × GC is shown to be beneficial in extracting useful chemical information from complicated samples. Kerosene-based rocket fuels were analyzed via a GC × GC – FID system that implemented a high temperature diaphragm valve as a modulator. Using leave-one-out cross validation (LOOCV), the summed GC × GC – FID signal of three compound-class selective 2D regions (alkanes, cycloalkanes, and aromatics) was regressed against previously measured ASTM derived values. Additionally, a more detailed partial least squares (PLS) analysis was performed on compound classes (n-alkanes, iso-alkanes, mono-, di-, and tri-cycloalkanes, and aromatics) as well as the physical properties previously determined by ASTM methods (such as net heat of combustion, hydrogen content, density, kinematic viscosity, sustained boiling temperature and vapor rise temperature). The resulting models had low root mean square errors of cross validation (RMSECV) and had similar outcomes to previously reported results using a GC × GC – TOFMS. Using the information gained from the study, a more extensive study of predicting four physical properties (e.g., viscosity, heat of combustion, hydrogen content, and density) was undertaken using 74 different kerosene-based fuels. Highly reliable PLS models were developed that related chemical composition obtained via GC × GC – TOFMS to fuel properties obtained via ASTM methods. The PLS prediction of the four physical properties (e.g., viscosity, heat of combustion, hydrogen content, and density) had relatively low errors of RMSECV values of 0.0434, 38.1, 0.112, and 0.0037, respectively. Investigation of the linear regression vectors (LRVs) indicate the relationship between the chemical composition and physical properties enabling the chemical compositions of fuels to be altered to meet certain industrial specifications. Using a similar fuel set comprised of 36 kerosene-based fuels, the thermal stability was evaluated using a novel instrument, CRAFTI (Compact Rapid Assessment of Fuel Thermal Integrity). Using a chemometrics-based feature discovery algorithim, the chemical information obtained via GC × GC – TOFMS was correlated to the thermal integrity data. Certain forms of carbon that were deposited within the test article of the CRAFTI instrument were found to strongly correlate with increased backpressure and some of the more prevalent compounds were identified. Next, tile-based Fisher ratio (F-ratio) analysis was applied to tandem ionization time-of-flight mass spectrometry (TI – TOFMS) in order to enhance discovery-based analyses. A hard ionization energy (70 eV) and soft ionization energy (14 eV) were collected concurrently, and the discovery of 12 analytes spiked in diesel fuel was shown to be improved when the two ionization energies were used in tandem resulting in a higher discovery rate while also lowering the number of false positives. Using parallel factor analysis (PARAFAC) the analytes that were “discovered” were deconvoluted in order to obtain their identification via match values. Lastly, the limit of detection (LOD) and limit of quantification (LOQ) were improved by a novel integration method. Signal to noise (S/N) enhancement was theoretically studied using simulations and both the LOD and LOQ can be lowered by a factor of 3. When compared to the two-step, a commonly applied method for quantifying one-dimensional and two-dimensional, the integration method resulted in more accurate and precise measurements at low S/N.
Author: Fulton G. Kitson
Publisher: Academic Press
ISBN: 0080532322
Category : Science
Languages : en
Pages : 395
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Book Description
This guide provides, under one cover, a wealth of practical information designed to facilitate the effectiveness of the GC/MS user. Separation conditions for numerous compound types are provided along with derivatized and underivatized compounds. A section on how to interpret mass spectral data, an extensive correlation of ion masses and neutral losses with possible structures, and examples of mass spectra are provided to further aid structure determination. Also included are basic information on instrumentation, ionization methods, quantitation, tips on the operation of mass spectrometers, the best derivatization procedures for a variety of compound types, troubleshooting techniques, and a variety of other information found to be useful to the practicing user of GC/MS instrumentation. This guide would be immediately valuable to the novice as well as the experienced GC/MS user who may not have the breadth of experience covered in this book.Key Features* Condenses and organizes recent and essential information for new and experienced GC/MS users* Comprehensively indexed and referenced* Includes practical methods of analysis* Serves as a text reference for short practical courses on the subject
Author:
Publisher:
ISBN:
Category : Chemistry
Languages : en
Pages : 716
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Book Description
Reports NIST research and development in the physical and engineering sciences in which the Institute is active. These include physics, chemistry, engineering, mathematics, and computer sciences. Emphasis on measurement methodology and the basic technology underlying standardization.
Author: Daniel Bahaghighat
Publisher:
ISBN:
Category :
Languages : en
Pages : 155
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Book Description
A combination of four instrumental systems and one chemometric method are presented that improves the efficiency, resolving power (i.e. peak capacity/ peak capacity production), and lessens the typical time of multi-dimensional gas chromatography (MDGC) separation in a straightforward, easily interpretable manner. Application of partial modulation via a commercially available high speed pulse flow valve for two-dimensional gas chromatography (GC×GC) is shown to provide ultra-fast modulation with modulation periods (P[subscript]M) as short as 50 ms. This technique performs a combination of vacancy chromatography and frontal analysis by an injection of carrier gas at the union of the first column (1D) and second column (2D). Each pulse disturbance in the analyte concentration profile as it exits the first column (1D) results in vacancy like data that is readily converted into a second separation (2D). A three-step process converts the raw data into a format equivalent to a traditional GC×GC separation chromatogram: 1. signal differentiation, 2. inversion of data, 3. baseline correction. The first instrumental system (GC×GC-Flame Ionization Detector (FID) with a P[subscript]M of 500 ms, separating a 115-component mixture composed of a wide range of boiling points (36–372 °C) compounds with apparent peak widths on the 2D, 2W[subscript]b, ranged from 10 to 40 ms, producing a 2D peak capacity, 2n[subsciprt]c, of ~ 20, and the total peak capacity, n[subscript]c,2[subscript]D, was 7200 or a peak capacity production of 1200 peaks/min. For a P[subscript]M of 75 ms, separating a low boiling point 15-component mixture isothermally, apparent peak widths on the 2D, 2W[subscript]b, averaged 10 ms producing a 2D peak capacity, 2n[subscript]c, of ~ 7.5, with a peak capacity production of 950 peaks/min. The second system incorporated a high temperature diaphragm valve modulator and a pulse valve flow modulator to create a three-dimensional gas chromatography system (GC3) with a peak capacity production of 1000 peaks/min which is a ~5 times increase in efficiency compared to other GC3 systems. The third instrumental design established capability with a time-of-flight mass spectrometer (TOF), a method was developed for GC×GC-TOF separation in which a concentration study was conducted with an 18-component mixture and a P[subscript]M of 50 ms. The subsequent data was deconvoluted with multivariate curve resolution-alternating least squares (MCR-ALS) in order to obtain their identification via match values. The resulting MCR-ALS data was converted in a similar manner as before into GC×GC chromatograms. Lastly, the pulse valve flow modulator was demonstrated to conduct continuous gas sampling of a system via one dimensional (1D) chromatography. The method applies the partial modulation technique to create frontal analysis peaks that are then transformed into a 1D chromatogram of analytes from a dynamic system that present a novel method of continuous sampling.
Author: Derrick V. Gough
Publisher:
ISBN:
Category :
Languages : en
Pages : 118
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Book Description
The potential efficiency and applicability of gas chromatography is advanced through the continued development and use of the pulse valve modulator. New advancements in modulation techniques are reported with the pulse valve modulator concurrently with the use of commercial chemometric algorithms and new data visualization techniques. First, the application of partial modulation in the negative pulse mode (NPM) is demonstrated. The NPM is developed for high-speed, one-dimensional gas chromatography (1D-GC), comprehensive two-dimensional (2D) gas chromatography (GCxGC), and comprehensive three-dimensional gas chromatography (GC3). This modulation technique is shown to be more beneficial than previous work with partial modulation in the positive pulse mode (PPM). The NPM produces comparable analyte peak widths-at-base (w[subscript]b) as the PPM but does so with greater S/N and with less data processing required. In the 1D-GC mode, 8 analytes are baseline resolved (Rs ≥ 1.5) in a 200 ms window, providing a peak capacity, nc, of 14 at unit resolution (Rs = 1.0). Demonstrating this higher efficiency to a GCxGC, a 20-component test mixture is separated. Analytes were separated on the second-dimension column, 2D, with 2wb ranging from 7 to 12 ms, providing an exceptional 2D peak capacity, 2nc of ~12 using a P[subscript]M of 100 ms. Next, the NPM is applied to a GC3 with time-of-flight mass spectrometry detection (TOFMS). Narrow third dimension, 3D, peaks 3wb ~ 15 ms were obtained, resulting in a GC3 peak capacity, n[subscript]c,3D, of ~35,000, in a 45 min separation. While high peak capacity has been produced, there are challenges that needed to be addressed regarding the use of commercially available data analysis techniques to ensure method translation can occur. The NPM data has the appearance of 2D separations superimposed on top of the 1D separation, which is unique. Multivariate curve resolution – alternating least squares (MCR-ALS) is used to demonstrate the potential for method translation with the NPM technique. A mixture of 15 similar analytes was isothermally separated to purposefully create several scenarios of peak overlap within 20 s. Despite the high degree of overlap, the NPM data is shown to be amenable to MCR-ALS, with all 15 analytes able to be decomposed, separated, and identified. The potential for quantification is demonstrated for two representative analytes, with percent deviation values of -5.6% (± 2.2%) for 1-hexene, and 1.8% (± 3.4%) for 2-pentanone. Next, the pulse valve modulator was used in the full modulation mode to demonstrate further advancements and quantifiable gains in S/N with GC3 instruments. The detector response enhancement factor (DREF) is a quantifiable measure of the increase in S/N ratio of analyte signal as the number of dimensions is increased. We calculated DREF values of ~ 37 for dodecene and ~ 21 for tridecane. Additionally, the 2Dx3D peak capacity was demonstrated at 30 peaks per 1.5 s. Last, a method to visualize the 2Dx3D chromatogram is presented, in which four successive modulations are lined up and one can “see” analytes arriving at and eluting from the TOFMS detector.
Author: Fahad Saeed
Publisher: Springer Nature
ISBN: 3031019601
Category : Science
Languages : en
Pages : 146
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Book Description
To date, processing of high-throughput Mass Spectrometry (MS) data is accomplished using serial algorithms. Developing new methods to process MS data is an active area of research but there is no single strategy that focuses on scalability of MS based methods. Mass spectrometry is a diverse and versatile technology for high-throughput functional characterization of proteins, small molecules and metabolites in complex biological mixtures. In the recent years the technology has rapidly evolved and is now capable of generating increasingly large (multiple tera-bytes per experiment) and complex (multiple species/microbiome/high-dimensional) data sets. This rapid advance in MS instrumentation must be matched by equally fast and rapid evolution of scalable methods developed for analysis of these complex data sets. Ideally, the new methods should leverage the rich heterogeneous computational resources available in a ubiquitous fashion in the form of multicore, manycore, CPU-GPU, CPU-FPGA, and IntelPhi architectures. The absence of these high-performance computing algorithms now hinders scientific advancements for mass spectrometry research. In this book we illustrate the need for high-performance computing algorithms for MS based proteomics, and proteogenomics and showcase our progress in developing these high-performance algorithms.
Author: Xu Zeng
Publisher:
ISBN:
Category :
Languages : en
Pages : 380
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Book Description
The determination and accurate identification of fatty acids (FAs) in biological samples remain challenging due to the complexity of these compounds and confounding sample matrix effects. This thesis highlights a range of method developments and applications using advanced multidimensional gas chromatography (MDGC) and comprehensive two dimensional gas chromatography (GCxGC) techniques, coupled with mass spectrometry (MS) for FA profiling and structural identification.An investigation of appropriate combinations of stationary phases was studied prior to the subsequent multidimensional analytical method development. Recently introduced ionic liquid (IL) capillary GC stationary phases covering a wide polarity range have been evaluated for fatty acid methyl esters (FAMEs) analysis in terms of their elution patterns and retention indices (e,g. ECL) on the IL columns.The performance of a newly proposed integrated GCxGC / heart-cut (H/C) MDGC-FID method for isomeric PUFA separation was then evaluated. As a practical application, abundant long chain UFA isomers with carbon numbers ranging from 18 to 22 in marine oil, as well as a dairy food product, can almost be fully separated by using GCxGC. Additionally, more than 7 other FA compounds were found in the same region by switching the system to H/C MDGC-FID under a modified GC condition. Both these applications employed fast analysis conditions.Next, GCxGC was coupled to the quadrupole-accurate mass time of flight mass spectrometry (QTOFMS). This was done to achieve accurate detection and structural confirmation of individual known or unknown branched FA obtained through predicted empirical formula and accurate mass information. The approach was applied to phospholipid fatty acids (PLFAs) from complex forest soil samples in order to investigate the microbial community. Several high abundance branched hydroxyl- (OH-), iso-/anteiso- and cyclopropyl- (cy-) FAME were clearly determined. Tentative identities of trace level OH-FAME and unusual epoxidised FAME were found according to their predicted empirical formulae and elemental compositions. Epoxidised FA were previously found in the metabolic pathway of a Gram-positive soil bacterium Bacillus megaterium. The findings indicate that this approach has great potential towards fingerprinting and FA biomarker discovery in environmental research. Lastly, comprehensive fast GCxMS with a positive chemical ionisation mode (PCI) was proposed for FA analysis in medical studies. Method performance was evaluated by application to different types of FAs including PUFAMEs and bacterial FAMEs (e.g. branched FAMEs) as well as to real biological samples (e.g. wild type and transgenic mouse brains associated with Alzheimer's disease). Methanol was selected as the most appropriate PCI chemical reagent for total FA profiling. A comparison of both rapid comprehensive GCxPCI-QMS and GCxGC approaches, for fast screening capabilities and the rapid quantitative determination of long chain PUFA isomers in mice brain associated with Alzheimer's disease, was conducted in this study. An evaluation of both methods was based on the comparison of their 2D presentations, calibration linearity and minimum levels of detection. Fast GCxMS data demonstrated the good quantification potential of this approach towards biological FA analysis, especially in medical studies.
Author: Ahmed Mohamed Mostafa
Publisher:
ISBN:
Category :
Languages : en
Pages : 282
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Book Description
Comprehensive two-dimensional gas chromatography (GCxGC) is among the most powerful methods used to separate complex samples. Two columns of different selectivities are coupled in series through a special interface (modulator). The main role of the modulator is to trap and/or sample the primary column effluent and inject it into the secondary column. This results in an enhanced sensitivity, increased peak capacity and structured chromatograms. Practically all thermal modulators in use today are equipped with two trapping stages to prevent problems related to analyte breakthrough, which makes their design more complicated. In this work, The sensitivity of GCxGC coupled to two different detectors, time-of-flight mass spectrometer (GCxGC-TOFMS) and flame ionization detector (GCxGC-FID) was compared to the sensitivity of conventional one-dimensional gas chromatography (GC-TOFMS and GC-FID) by determining the limits of detection (LOD) for a series of different compounds such as n-alkanes and alcohols using both approaches. Different modulation periods were used for GCxGC ranging from 2 to 8 seconds. In addition, different types of inlet ferrules were used to study their effect on both systems. In general, the LODs in GCxGC were lower by at least an order of magnitude. A new liquid nitrogen-based single-stage cryogenic modulator was developed and characterized. In addition, a new liquid nitrogen delivery system was developed. Band breakthrough was prevented using changes in the carrier gas viscosity with temperature to reduce the carrier gas flow during desorption. Injection band widths for n-alkanes of 30-40 ms at half height were obtained. Most importantly, even the solvent peak could be perfectly modulated, which is impossible with any commercially available thermal modulator. Moreover, the newly developed liquid nitrogen supply system reduced liquid nitrogen consumption to ~30 L per day versus 50-100 L per day for commercially available modulators. Evaluation of the newly developed system for the GCxGC separation of some real samples such as regular gasoline and diesel fuel showed that the analytical performance of this single-stage modulator rivals that of the more complicated dual-stage designs. The technique was tested in various applications. Headspace solid phase microextraction in combination with GCxGC coupled to time-of-flight mass spectrometry (HS-SPME-GCxGC-TOFMS) were used for the detailed investigation of the impact of malolactic fermentation (MLF) using three commercial Oenococcus oeni strains on the volatile composition of Pinotage wines. The technique was also applied for the characterization of Pinotage wine volatiles and blue honeysuckle berries volatiles.
Author: Sarah Elizabeth Prebihalo
Publisher:
ISBN:
Category :
Languages : en
Pages : 151
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Book Description
Comprehensive two-dimensional gas chromatography (GCxGC) coupled with time-of-flight mass spectrometry (TOFMS) is a powerful analytical technique capable of separating complex mixtures, providing valuable information about the chemical composition of samples. However, the inherent data density associated with three-dimensional data provides a unique challenge to analytical chemists. As a result, significant effort has been invested in utilizing advanced chemometrics to glean meaningful information about samples from large and complex data sets. Herein, this dissertation introduces several investigations conducted on optimizing separation conditions to be amenable to chemometric deconvolution algorithms as well as the development, study, and application of advanced chemometric techniques applied to GCxGC-TOFMS data. To begin, the metric trilinear deviation ratio (TDR) is utilized to study the impact of experimental parameters such as column selection and modulation period, PM, on the quantitative accuracy of parallel factor analysis (PARAFAC) deconvolution. TDR scales with increasing change in second dimension retention time, delta2tR, associated with pseudo-isothermal conditions on the second dimension, 2D, and quantitative accuracy decreases as TDR increases. Two column sets were utilized with varying film thickness on the first column, 1D, and each column set was studied using two PM for a total of 4 experiments. It was reported that using 1D columns with larger film thicknesses allows the analyst to employ a shorter PM, in turn lowering the delta2tR, leading to higher quantitative accuracy. Many GCxGC-TOFMS studies relate to identifying class distinguishing analytes and can be tedious when performed manually. Fortunately, the use of discovery-based chemometric tools such as principal component analysis (PCA) and Fisher ratio (F-ratio) analysis has increased in popularity as less time-intensive and automated techniques for untargeted analyses. To begin, this dissertation will investigate mass channel purity obtained via the tile-based F-ratio algorithm using diesel fuel spiked with non-native analytes using GCxGC-TOFMS. The F-ratio algorithm, considered a supervised discovery technique because class membership is known a priori, was first used to "discover" the spiked non-native analytes. Then, using a novel signal ratio (S-ratio) algorithm, the mass channel selectivity information output by the F-ratio method was studied using three statistical metrics: null distribution analysis, p-value, and lack-of-fit (LOF). The result of this investigation revealed that a mass channel has a high likelihood of being pure when its p-value and LOF are sufficiently low. Finally, F-ratio analysis was applied to a dataset including patients with an anterior cruciate ligament (ACL) injury to discover potential biomarkers of post-traumatic osteoarthritis (PTOA) post-injury. Standard F-ratios are calculated by the between class variance divided by the sum of the within-class variance, scaling up as the between class variance increases and the within-class variance remains sufficiently small. However, many biological studies involve significant biological variance (~30%) that may not be associated with disease state or injury severity, etc. Herein, the standard tile-based F-ratio algorithm was modified to use only the within-class variance associated with control samples. It was expected that the control class contained less within-class variance relative to the patient class, due to the expectation that some patient samples would be associated with increased severity of injury or the presence of coexisting conditions. Hit lists (metabolites discovered via F-ratio) from standard F-ratio and control-normalized F-ratio were studied and directly compared to establish a comprehensive metabolome of potential biomarkers for PTOA development post ACL injury. Reported in this dissertation is a discussion on the complementary nature of standard and control-normalized F-ratio, followed by demonstration of class distinguishing metabolites via PCA.
