TOPIC V "Comprehensive Separation"

Application of comprehensive two-dimensional gas chromatography for semi volatile organic compounds in particulate matter in aerosol science

The particulate matter of anthropogenic aerosols are suspected and in some cases already convicted to have severe adverse effects for health and environment. The chemical emission profiles from anthropogenic processes are already very complex and could comprise thousands of different chemical species which could condense on such particulate matter. The complexity and possible impact to health may even multiply if aerosols undergo aging due to complex reactions in the atmosphere. Comprehensive two-dimensional gas chromatography time-of-flight mass spectrometry has been proven to be an excellent tool for detailed analyses of the volatile and semi volatile compounds within a very complex matrix. Aerosol particulate matter form emission sources or after artificial or environmental aging are trapped depending on the size of the particles on inert filter material. An automatic desorption procedure is applied and combined with on-line derivatization of the volatile and semi volatile chemical species. The chemical signature is separated and detected by comprehensive two-dimensional gas chromatography time-of-flight mass spectrometry resulting in qualitative and quantitative determinable profiles with thousands of possible target compounds (Schnelle-Kreis, J. et al., 2005). The obtained higher dimensional data sets are further comprehensively analyzed (Welthagen, W. et al., 2003) on a single compound level by applying of special data analysis tools for statistical and multivariate analysis (Vogt, L. et al., 2007; Weggler, B. A. et al., 2014). By this approach highly significance source specific chemical pattern could be identified and further used for interdisciplinary cause and effect studies (Weggler, B. A. et al., 2016; Oeder, S. et al., 2015). A special focused here is the interface between chemistry and biology in HICE.

Selected Ongoing and accomplished studies:

Untargeted Analyses of the Semi-Volatile Organic Fraction in Anthropogenic Particulate Matter. PhD Thesis Dr. Benedikt Weggler, 2016.

Graphical Abstract (Weggler, B. A. et al., 2016). Untargeted Identification of Wood Type-Specific Markers in Particulate Matter from Wood Combustion. Copyright (2016) American Chemical Society.

The awareness of anthropogenic aerosols became more prevalent recently. Due to their influence on human health, such aerosols are of special public interest and discussed therefore highly in media. The present work was carried out within the Helmholtz Virtual Institute of Complex Molecular Systems in Environmental Health (HICE). In HICE the biological response of human lung cells, induced by the uptake of anthropogenic aerosols, is investigated and linked to the chemical and physical properties of the aforesaid aerosols. Within this thesis emissions from residental wood combustion and shipping emissions were investigated. Residential wood combustion emissions and shipping emissions are some major global sources of particulate and gaseous organic pollutants. The particulate matter generated from such combustion processes is composed of a large number of compounds, including condensed semi-volatile organic compounds. The detailed chemical compositions of these emissions are poorly characterized, which prevents the establishment of an accurate effect/cause relationship of the emissions and the adverse health effects. Multidimensional gas chromatography is an appropriate tool for the non-targeted and comprehensive characterisation of complex samples generated from combustion processes. Enhanced with highly sophisticated sample pre-treatment techniques, to reduce the number of potential artefact generating steps, the value of this analysis technique is significantly improved. However, the complex amount of information gained from such comprehensive techniques is associated with difficult and time-consuming data analysis. Because of this obstacle, two- dimensional gas chromatography still receives relatively little use in aerosol science. Untargeted data analyses were performed using the comprehensive measurements. Univariate and multivariate chemometric tools, such as analysis of variance (ANOVA), principal component analysis (PCA), and ANOVA simultaneous component analysis (ASCA), were used to reduce the data to highly significant and specific features. In addition fast and accurate automated compound classification algorithms were developed for initial data screening. This study reveals substances not previously considered in the literature as meaningful markers for aerosol sources and provides valuable input for the establishment of an effect/cause relationship between aerosols and the adverse effects induced in human health.

Development and Evaluation of Multidimensional Gas Chromatographic and Mass Spectrometric Techniques for the Analysis of Highly Complex Chemical Mixtures. PhD Thesis Dr. Werner Welthagen, 2010.

First application of scripting on GC×GC-TOFMS Data for the classification of compound classes found in environmental particulate matter (Welthagen, W. et al., 2003).

The focus of this study was to improve techniques used for the analysis of urban aerosol particulate matter, metabolomic extracts from animal tissues and diesel petrochemical samples. Some of the present techniques in industry, like GC-TOFMS analysis, are already well established and used on a routine basis in many laboratories. GC x GC- TOFMS can be introduced in these fields as a complimentary technique when the current methods fail to reach the detection limits or separation required to identify target compounds. It will also be shown that because of significantly higher spectral purities obtained using GC x GC-TOFMS, due to improved separation compounds can be assigned with improved library matches. In the current work the inherent properties of GC x GC to separate compounds into ordered bands of species with similar chemical or physical properties is investigated, and combined with the unique fragmentation information of compound classes form mass spectrometry detection. This was used to facilitate a new compound classification approach whereby compounds could be grouped into chemical classes through basic classification rules and used in statistical evaluation of samples. These compound class groups were introduced for the statistical evaluation of the organic content of urban aerosol samples as complimentary technique for the ongoing daily monitoring of the organic content of urban aerosol particulate matter (PM2.5) in Augsburg, Germany. GC x GC-TOFMS is also introduced for the first time to the analysis of metabolic extracts from mammalian tissue samples (mice spleen extracts). In a proof of concept study it is shown that tentative biomarker detection is possible using GC x GC-TOFMS. For the biomarker detection new statistical methods were developed and compared to existing methods. When compared to GC-TOFMS which is currently used in the analysis of metabolomic extracts, GC x GC-TOFMS is able to detect more peaks (compounds) in a shorter time frame. This is particularly important where high sample throughput is required for a comprehensive statistical study. A new method for the enhancement of separation is introduced in gas chromatography coupled to selective soft photo ionization time-of-flight mass spectrometry (GC x PI TOFMS). In this approach the existing soft ionization techniques of resonance enhanced mulitphoton ionization (REMPI) and single photon ionization (SPI) were used as selective detectors to detect various different compound class groups within a diesel petroleum sample and present them in a two-dimensional plane of GC separation vs. molecular mass. These two-dimensional representations of GC-separation vs. molecular mass are compared to GC x GC separation. The two-dimensional separation approach with GC x MS is further extended to include an additional GC dimension for a comprehensive three-dimensional separation approach (GC x GC x MS). Finally a VUV lamp is introduced instead of the pulsed laser to produce a continuous source of SPI photons. The continuous source of photons obtained is used to increase the frequency of the detector from 10 Hz to 20 Hz, providing improved coupling to GC x GC. The frequency could be further improved with faster data recording.

Application of comprehensive two-dimensional gas chromatography in the analysis of the metabolome and xenobiotics

The state of a biological system is reflected by its metabolomics profile and the health as well as the environmental and individual conditions may have a severe impact on these profiles. Therefore, a detailed qualitative and quantitative analysis of the metabolome could be used to identify biomarker for diagnostic/forensic reason, to detect early stages of a disease or to investigate the complex mechanism behind metabolic pathways. For a comprehensive analysis endogenous metabolites, exogenous metabolites and xenobiotics in the different biological compartments (cells, tissues, body fluids and gas) and matrices have to be ascertained. Comprehensive two-dimensional gas chromatography time-of-flight mass spectrometry with a sophisticated sampling is able to separate and identify a large part of the vaporizable primary and secondary metabolome for metabolite profiling (Welthagen, W. et al., 2005). Sampling methods for breath gas analysis (Mieth, M. et al., 2010; Gruber, B. et al., 2016) or preparation and extraction methods for different kinds of cells and tissue (Ly-Verdu, S. et al,. 2014) are developed and fitted for target metabolites or general screening. For reproducibility and high throughput, a high degree of automatization is intended. A comparison between different groups (e.g. state of disease or the impurities in a drug (Gröger, T. et al., 2008) is carried out due to a comprehensive statistical and multivariate analysis of the data sets (Ly-Verdu, S. et al., 2015). The aim is always a differentiation of these states base on their chemical profile and an identification of the discriminating metabolites or xenobiotics (Schäffer, M. et al., 2013). A special focus is laid on studies related to Diabetes research.

Selected ongoing and accomplished studies

Development and optimization of non- and minimal-invasive methods for the monitoring of endogenous as well as exogenous volatile or semi-volatile organic compounds in body fluids and breath. PhD Thesis Dr. Beate Gruber, 2016.

Experimental Setup for breath gas and parallel on- and offline sampling. Comparison between online PTR-MS vs offline GC×GC-TOFMS for highly time resolved measurements in breath gas (Gruber, B. et al., 2016). Image: Beate Gruber.

Volatolomics, the study of volatile organic compounds produced by a biological system, is of growing interest for the screening and diagnosis of the human health state. The analysis of volatile organic compounds in body fluids by minimal- and non-invasive methods, such as the analysis of exhaled breath that reflects the volatile composition of the blood stream, has several advantages such as less painful sampling over traditional methods. Volatolomics is very promising for monitoring the condition of the human metabolism, but requires complex and standardized methods. The work presented here focused on the development of new gas chromatographic methods for the monitoring of volatile organic compounds in body fluids and breath during fast proceeding metabolic processes. Breath gas analysis was carried out during a glucose challenge by the simultaneous application of real-time proton-transfer-reaction quadrupole mass spectrometry and off-line needle trap micro extraction combined with comprehensive two-dimensional gas chromatography coupled to time-of-flight mass spectrometry. To gain extensive information on the volatile composition, complementary vacuum ultraviolet absorption spectroscopy was coupled to comprehensive two-dimensional gas chromatography the first time and evaluated for highly polar volatile organic compounds. Glucose challenge responsive breath metabolites could be monitored and identified as small n-alkanols and short chain fatty acids, which might be related to an abnormal glucose metabolism. The results from time-of-flight mass spectrometry studies show good agreement with vacuum ultraviolet absorption spectroscopy measurements. Besides, a highly sensitive and less invasive method for phenanthrene analysis in 1 µL capillary blood was established using thermal extraction in combination with gas chromatography mass spectrometry. The method was successfully applied to the monitoring of phenanthrene blood levels. Thus, this work represents pharmacokinetics of unmetabolized polyaromatic hydrocarbons in human for the first time. In summary, the used pre-concentration techniques allowed a fast, time-resolved sampling as well as extraction of analytes out of the matrix. The combination with highly sophisticated gas chromatographic approaches created powerful tools for the monitoring of the human volatolome.

Chemometric Data Analysis for Untargeted Metabolomic Profiling of Biological Samples by Two – dimensional Gas – Chromatography Mass-Spectrometry. PhD Thesis Dr. Saray Ly-Verdu, 2017

Time-course design for a study to investigate the progression the influence of dietary for the progression of diabetes based on mouse models. The experimental design recreates the typical insulin resistance progression over time sketched in the upper graph. Data from samples measured with GC×GC–ToF–MS were processed, and ASCA analysis was performed to discriminate artificial and experimental induced variations vs biological relevant features (Ly-Verdu, S. et al., 2015).

Metabolomics has established a strong foothold in the field of system biology, as it proved to be the ideal candidate for closing the loop of functional genomic, formed by proteomics and transcriptomics. Genes and proteins are epigenetically regulated, and suffer post-translational modifications. Metabolites instead, represent the downstream biochemical end products that are closer to the phenotype, since the hierarchy of the transcriptome and the proteome amplifies changes in the physiology of cells resulting from gene deletion. Accordingly, it is easier to correlate metabolomic profiles with phenotype. Several institutes participated in a multidisciplinary collaboration connecting targeted and non-targeted proteomics, non-targeted metabolomics, and comprehensive phenotyping in mouse models. In this way, experimental protocols for mice liver perfusion have been established, and sample material from these studies was used in turn to establish protocols for liver tissue sample preparation. All the expertise the metabolomic insight can provide will remain concealed if it was not for the utilisation of a suitable analytical technique. The capacity of comprehensive two-dimensional gas chromatography coupled to time-of-flight mass spectrometry (GC×GC-ToFMS) to meet the requirements for an adequate tool for this purpose is examined in this work. The applicability for the analysis of highly complex matrices is tested and the results obtained confirm the advantages implied in its utilisation. On the one hand, improved separation increases the resolution and allows the identification of many more compounds than one-dimesional GC. On the other hand, TOF mass spectrometry possesses the capacity to acquire data across the full mass range, permitting the rapid identification of trace-level analytes even in the most challenging of samples, such as biological tissue or aerosol samples, GC×GC-ToFMS allows accurate chemical profile characterisation. No subsequent drawing of conclusions will be accomplished if not for the data processing robustness of multivariate statistics methods. Conventional exploratory methods are combined in this thesis and in addition a relatively novel statistical tool is tested, namely Anova Simultaneous Component Analysis (ASCA), for metabolomic study of liver mice samples and aerosols from wood combustions. The versatility of this chemometric tool has proved to be of benefit to extract important information in terms of sample discrimination, but also to excerpt the weight of the source of this information, thereby improving the exactness of the disclosed inferences.This work aims to be a small contribution toward the establishment of how extensive cooperation could face contemporary challenges in the progress of analytical biochemistry. To this end, it is focused toward embracing proficiency of metabolomic comprehension, biological awareness, high-throughput analytical techniques and innovative chemometry.

Comprehensive Analysis of petrochemical matrices

Petroleum and petrochemical Matrices from downstream processes manifest a great complexity in their chemical nature. Although containing a limited number of chemical classes, these matrices consist of a plethora of individual compounds within these classes. The vast development and improvement of multi-dimensional gas chromatograph methods (i.e. GC×GC) over the past years, has allowed for a more than suffice characterization for end products such as middle distillates and make comprehensive two-dimensional gas chromatography time-of-flight mass spectrometry to the method of choice for a comprehensive qualitative and quantitative analysis. The qualitative analysis will be supported by a linking of a very ordered elution pattern of the different compound classes with their uniform MS fragmentation pattern. While a qualitative analysis of the semi volatile boiling range has now become standard, a comprehensive quantitative analysis (Jennerwein, M. K. et al., 2014) and the analysis of higher boiling cuts or residuals is more challenging and focus of our work (Jennerwein, M. et al., 2017 (accepted)). Next to GC×GC with nominal mass detection also other thermal sample introduction methods like thermo gravimety (Geissler, R. et al., 2009) and direct insertion probe will be applied. For a better identification and visualization of compound classes high resolution time-of-flight mass spectrometry and spectrometric detection methods are applied.

Ongoing and accomplished studies

Investigation of Bitumen, heavy petroleum cuts and residues by mass spectrometry and thermal introduction methods. PhD Thesis Uwe Käfer, ongoing

The combination of different approaches for the characterization of high boiling matrices derived from petroleum. Image: Uwe Käfer.

The research is focused on the chemical Analysis of petroleum vacuum residues and bitumen. The work is part of an AiF research project for bitumen characterization, which is carried out together with cooperation partner ASG. Because of the complex nature of heavy petroleum samples, multiple techniques are combined for a comprehensive analysis. LC-fractionation and High resolution Mass spectrometry take key roles in the studies. Different ionization methods and sample inlet systems for High resolution mass spectrometry are combined to investigate important mechanisms in the chemistry of heavy petroleum compounds.

Application of comprehensive two-dimensional Gas Chromatography Time-of-Flight Mass Spectrometry and Visual Basic Script for detailed Analysis of fossil and biogenic Fuels. PhD Thesis Dr. Maximilian Jennerwein, 2017

Comprehensive qualitative and quantitative classification of petroleum derived fuels by GC×GC-TOFMS (Jennerwein, M. K. et al., 2014). Copyright (2014) American Chemical Society.

Comprehensive two-dimensional gas chromatography (GC×GC) hyphenated with time-of-flight mass spectrometry (TOFMS) was applied for a quantification of fossil and biogenic fuels. The focus here was on middle distillates and crude oils. The development of the analysis method was possible due to the inherent features of GC×GC so separate complex samples based on their physical and chemical properties into compound groups. Furthermore, the classification of contained compound groups was possible based on the characteristic mass fragmentation produced by TOFMS. A discussion of the advantages and disadvantages of the basically different column combinations for GC×GC –TOFMS for the analysis of petrochemical samples was also in the scope of the method development. Common middle distillates can contain more than 10.000 different compounds depending on the produced distillation cut. These compounds can be classified to homologues series of different compound classes. For the development of a complete group-type quantification method, a sophisticated calibration method had to be developed, taking into account the decreasing detector response along with increasing carbon number. By this approach, in was possible to perform a quantification of common middle distillates to a degree of more than 99 %(m/m) (Jennerwein, M. K. et al., 2014). The classification of the different substance classes was achieved by the application of Visual Basic Script (VBS) programming, based on the characteristic mass fragmentation. The quantitative results were compared to standard quantification methods. In this regard some shortcoming of the quantification method of aromatic compound using HPLC and refractive index detection (RID) could be found. Advantages of the quantification using GC×GC –TOFMS could be shown especially in the case of modern premium diesel fuels. Furthermore, the approach could be applied for renewable resources in form of different kinds of wood. Contrary to the analysis of fossil fuels, the focus was on emissions of the combustion. An expansion of the method to high temperature applications enabled the analysis of crude oils and high boiling distillation products. The data evaluation using VBS classifications was extended to polyaromatic and heteroaromatic compounds. In addition, atmospheric and vacuum distillation processes were applied for the production of numerous distillation cuts. These cuts were used for the generation of virtual distillation cuts within the 2D separation space. By this purpose, the concept for a two-dimensional simulated distillation was developed.

Further conceptual and instrumental development for chromatography based comprehensive analysis and data analysis

The concept and practical realization for comprehensive two-dimensional gas chromatography has been established already in 1984 by Giddings and the concept has been successfully translated to other chromatographically techniques. After a while of instrumental development the technique has now matured. Our group further expands the concept of a higher dimensionally separation with a special focus on the detection methods as well as alternating sample introduction methods. For a further comprehensive analysis high resolution time-of-flight mass spectrometry is applied and/or mass spectrometry is upgraded by single photon ionization to integrate mass defect or molecular ion information into a higher dimensional separation space (Welthagen, W. et al., 2007; Eschner, M. et al., 2010). Alternatively also other new selective spectroscopic detection methods like vacuum ultraviolet absorption spectroscopy are hyphenated to extend the separation space by a new dimension (Gröger, T. et al., 2016). To overcome the thermal limitation of gas chromatography alternative thermal sample introduction methods like thermal gravimetry and simultaneous thermal analysis are applied and extended to a higher dimensional separation (Saraji-Bozorgzad, M. R. et al., 2010).

GC-SPI: Combined electron ionization / single photon ionization source for time-of-flight mass spectrometry (Gröger, T. et al., 2016)

Next to instrumental development our group is also interested in the development and application of data analysis concept for higher dimensional data sets. Beside peak based approaches we also applying pixel or image based approaches (Groeger, T. et al., 2008) to compare such data sets (Gröger, T. and R. Zimmermann, 2011).

The left picture shows 3D Data set obtained by GC × GC–TOFMS analysis of rats’ plasma. Figure (B) and (C) indicate discriminating features after different times of exposure (Gröger, T. and R. Zimmermann, 2011).

Ongoing and accomplished studies

A Vacuum Ultraviolet Absorption Array Spectrometer as a Selective Detector for Comprehensive Two-Dimensional Gas Chromatography: Concept and First Results. Thomas Gröger, 2016. 

Concept VUV Absorption Spectroscopy hyphenated to GC×GC (Groeger, T. et al., 2008). Copyright (2016) American Chemical Society.

Fast and selective detectors are very interesting for comprehensive two-dimensional gas chromatography (GC × GC). This is particularly true if the detector system can provide additional spectroscopic information on the compound structure and/or functionality. Other than mass spectrometry (MS), only optical spectroscopic detectors are able to provide selective spectral information. However, until present the application of optical spectroscopy technologies as universal detectors for GC × GC has been restricted mainly due to physical limitations such as insufficient acquisition speed or high detection limits. A recently developed simultaneous-detection spectrometer working in the vacuum ultraviolet (VUV) region of 125−240 nm overcomes these limitations and meets all the criteria of a universal detector for GC × GC. Peak shape and chromatographic resolution is preserved and unique spectral information, complementary to mass spectrometry data, is gained. The power of this detector is quickly recognized as it has the ability to discriminate between isomeric compounds or difficult to separate structurally related isobaric species; thus, it provides additional selectivity. A further promising feature of this detector is the data analysis concept of spectral filtering, which is accomplished by targeting special electronic transitions that allows for a fast screening of GC × GC chromatograms for designated compound classes.

Coupling of Gas Chromatographic Methods with Single Photon Ionization Mass Spectrometry for Multidimensional Characterization of Complex Samples. PhD-Thesis Dr. Markus Escher, 2011.

Effect of benzene substitution on absolute photoionization cross sections. Substituents with alkyl substitution or rather permethylation. Ionization of these molecules is almost exclusively achieved from the highest occupied molecular orbital (HOMO) with a central excitation energy of 9.8 eV.

This work describes the realization of novel concepts in coupling of gas chromatography (GC) to mass spectrometry (MS) by utilization of soft single photon ionization (SPI) on the basis of the electron-beam pumped rare gas excimer light source (EBEL). The innovative EBEL-technology enables generation of intense and brilliant vacuum ultraviolet radiation. By means of SPI-MS molecules can be detected mostly fragmentation-free. Thus, molecules with different molecular ion masses can be separated within the same mass spectrum resembling a boiling point separation in GC. Furthermore, by combining GC and SPI-MS a comprehensive two-dimensional characterization (GC×MS) of complex samples can be obtained which is in its results similar to comprehensive two-dimensional gas chromatography (GC×GC). By application of a fast time-of-flight mass spectrometer with SPI capability and coupling to GC×GC even a three-dimensional separation was realized. Moreover, a method for rapid switching between classical electron ionization and EBEL-based SPI was developed and successfully applied to gas chromatographic analysis of diesel fuel. At last, after determination of photoionization cross sections of hazardous compounds present in cigarette smoke a puff-resolved quantification of these molecules was enabled by on-line coupling of GC×MS to a smoking machine.

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