Development and optimization of multidimensional methods based on online comprehensive microscale liquid chromatography and mass spectrometry

Der technologische Fortschritt zu empfindlichen Detektionstechniken, wie der Massenspektrometrie, zeigt die Komplexität der Proben aus den analytischen Bereichen Proteomik, Genomik, Umweltschutz und Lebensmittelindustrie. Eindimensionale Flüssigkeitschromatographie gekoppelt mit massenspektrometrischer Detektion bietet ein leistungsstarkes Werkzeug zur Analyse solcher komplexer Proben, hat jedoch Limitierungen im Bereich der Peakkapazität. Eine alternative Trenntechnik mit höherer Peakkapazität ist die umfassende zweidimensionale Flüssigkeitschromatographie. Diese Dissertation befasst sich mit der Entwicklung und Optimierung multidimensionaler Methoden basierend auf miniaturisierter umfassender online Flüssigkeitschromatographie gekoppelt mit der Massenspektrometrie. Die Leistungsfähigkeit des miniaturisierten zweidimensionalen Systems gegenüber konventionellen eindimensionalen Systemen wurde mit einer neu entwickelten Methode zum Vergleich solcher Systeme erfolgreich evaluiert. Der Vergleich erfolgte anhand von Multireferenzstandards und Realproben mit komplexer Matrix, hier beispielhaft Kläranlagenzuläufe. Zur automatischen Visualisierung von zwei- bzw. dreidimensionalen Daten wurde ein Workflow auf Basis von Excel Makro-Programmierung entwickelt und angewandt. Der Vergleich des Referenzstandards, der matrixarme Proben repräsentiert, zeigte, dass der klassische eindimensionale Ansatz in der Lage ist, mehr Komponenten zu detektieren. Bei den komplexen Matrixproben wurden doppelt so viele Analyten mit dem miniaturisierten zweidimensionalen System detektiert und eindeutig identifiziert als mit dem eindimensionalen Ansatz. Der Vergleich zeigte nicht nur das enorme Potential des zweidimensionalen Systems, sondern auch deutliche technisch-methodische Schwächen auf, zu denen systembedingte Verdünnung, die Auswahl der stationären Phasen und Datenevaluierung/Datenvisualisierung gehören. Diese Aspekte konnten im Einzelnen adressiert, mögliche Lösungen vorgestellt und diese im Detail diskutiert werden. Um systembedingten Verdünnungen entgegenwirken zu können, wurde in der ersten Dimension zu Beginn eine 50 mm lange stationäre Phase basierend auf graphitisiertem Kohlenstoff eingesetzt. Diese ermöglichte großvolumige Injektionen wässriger Proben, was sich positiv auf die Sensitivität auswirkte, jedoch deutliche Nachteile bezüglich der Signalform der unpolaren Analyten zeigte. In dieser Arbeit wurde daher ein Vorsäulen-Konzept erarbeitet, das auf einer seriellen Kopplung einer 1 cm kurzen Vorsäule mit graphitisiertem Kohlenstoff und einer zusätzlichen Hauptsäule basiert. Die Evaluierung zeigte, dass es möglich ist, ohne signifikante Peakverbreiterung unpolarer Analyten, große Volumina wässriger Proben zu injizieren und zu fokussieren. Um das Vorsäulenkonzept auf der ersten Dimension des miniaturisierten zweidimensionalen Systems bewerten zu können, wurde eine neue Methode zur Bewertung der Orthogonalität/Selektivität auf Basis der Analytverteilung über das zweidimensionale Chromatogramm entwickelt und im Vergleich zu drei etablierten Methoden evaluiert. Die Orthogonalitätsstudie dieser Arbeit zeigte, dass die zusätzliche Selektivität der seriellen Kopplung der Vor- und Hauptsäule auf der ersten Dimension zur besseren Orthogonalität des zweidimensionalen Systems führt. Darüber hinaus steht nun ein einfaches und automatisierbares Tool zur Methodenentwicklung für zweidimensionale Trennsysteme zur Verfügung. Die beste Säulenkombination kann anhand weniger Basismessungen und einem repräsentativen Referenzstandard ermittelt werden. Die weiteren technischen Entwicklungen im Bereich der miniaturisierten zweidimensionalen Flüssigkeitschromatographie liegen vor allem in der Kopplung mit der Ionenmobilitätsspektrometrie zur Erhöhung der Informationsdichte von unbekannten Proben. Diesbezüglich darf die Evaluierung neuer Algorithmen zur automatischen Datenprozessierung nicht außer Acht gelassen werden.The technological progress to more sensitive detection techniques such as mass spectrometry reveals the complexity of samples from fields as diverse as proteomics, genomics, environmental protection and food science. One dimensional liquid chromatography coupled to mass spectrometric detection is a powerful tool for the analysis of such complex samples, but it has limitations in terms of peak capacity. An alternative separation technique with higher peak capacity is comprehensive two dimensional liquid chromatography. This thesis deals with the development and optimization of multidimensional methods based on microscale comprehensive online liquid chromatography coupled with mass spectrometry. The performance of the microscale two-dimensional system was evaluated by a comparison to a conventional one-dimensional system. For the comparison multi-reference standards as well as samples with a complex matrix (influent of a wastewater treatment plant) were used. An automatic workflow based on Excel macro programming was developed and applied for the visualization of the two- or three-dimensional data sets. The comparison of the reference standards showed that it is possible to detect more compounds with the conventional one-dimensional approach. In contrast, for the complex sample it was possible to detect and identify two times more compounds with the microscale two-dimensional approach than with the conventional one-dimensional approach. The comparison showed not only the enormous potential of the two-dimensional system, but also significant technical and methodological weaknesses, including dilution caused by the system, the selection of stationary phases and data evaluation/data visualization. These aspects were addressed in this thesis and possible solutions are presented and discussed in detail. To counteract dilution caused by the system, a 50 mm long stationary phase based on porous graphitic carbon was used in the first dimension of the microscale system at the beginning. This allowed the injection of large volumes of aqueous samples, which resulted in a better sensitivity. However, significant disadvantages relating to the peak shape of non-polar analytes could be observed. Therefore, a pre-column concept was developed in this work that is based on a serial coupling of a 1 cm short pre-column filled with porous graphitic carbon and an additional main column. The concept evaluation revealed that it is possible to inject large volumes of aqueous samples without significant peak broadening for non-polar analytes. To evaluate the pre-column concept in the first dimension of the microscale two-dimensional system, a new method for the calculation of the orthogonality and selectivity based on the compound distribution across the separation space was developed. Additionally, the new method was compared with three established methods reported in literature. The orthogonality study of this work showed that the additional selectivity of the serially coupled columns in the first dimension results in a higher orthogonality of the two-dimensional system. In addition, there is now a simple and automatable tool available for method development in the field of two-dimensional separation techniques. The best column combination can be determined based on a few basic measurements and a representative reference standard. Further technical developments in the field of microscale two-dimensional liquid chromatography could focus on the coupling with ion mobility spectrometry to further increase the analytical information gained from measurement of unknown samples. In that regard, the evaluation of new algorithms for automatic data processing should also be pursued

Searching for a Stochastic Background of Gravitational Waves with LIGO

The Laser Interferometer Gravitational-wave Observatory (LIGO) has performed the fourth science run, S4, with significantly improved interferometer sensitivities with respect to previous runs. Using data acquired during this science run, we place a limit on the amplitude of a stochastic background of gravitational waves. For a frequency independent spectrum, the new limit is $\Omega_{\rm GW} < 6.5 \times 10^{-5}$. This is currently the most sensitive result in the frequency range 51-150 Hz, with a factor of 13 improvement over the previous LIGO result. We discuss complementarity of the new result with other constraints on a stochastic background of gravitational waves, and we investigate implications of the new result for different models of this background.Comment: 37 pages, 16 figure

Online and Splitless NanoLC × CapillaryLC with Quadrupole/Time-of-Flight Mass Spectrometric Detection for Comprehensive Screening Analysis of Complex Samples

A novel multidimensional separation system based on online comprehensive two-dimensional liquid chromatography and hybrid high-resolution mass spectrometry has been developed for the qualitative screening analysis and characterization of complex samples. The core of the system is a consistently miniaturized two-dimensional liquid chromatography that makes the rapid second dimension compatible with mass spectrometry without the need for any flow split. Elevated temperature, ultrahigh pressure, and a superficially porous sub-3-μm stationary phase provide a fast second dimension separation and a sufficient sampling frequency without a first dimension flow stop. A highly loadable porous graphitic carbon stationary phase is employed in the first dimension to implement large volume injections that help countervailing dilution caused by the sampling process between the two dimensions. Exemplarily, separations of a 99-component standard mixture and a complex wastewater sample were used to demonstrate the performance of the dual-gradient system. In the second dimension, 30 s gradients at a cycle time of 1 min were employed. One multidimensional separation took 80–90 min (∼120 min including extended hold and re-equilibration in the first dimension). This approach represents a cost-efficient alternative to online LC × LC strategies working with conventionally sized columns in the rapid second dimension, as solvent consumption is drastically decreased and analytes still are detectable at environmentally relevant concentrations

Search of S3 LIGO data for gravitational wave signals from spinning black hole and neutron star binary inspirals

We report on the methods and results of the first dedicated search for gravitational waves emitted during the inspiral of compact binaries with spinning component bodies. We analyze 788 hours of data collected during the third science run (S3) of the LIGO detectors. We searched for binary systems using a detection template family designed specially to capture the effects of the spin-induced precession of the orbital plane. We present details of the techniques developed to enable this search for spin-modulated gravitational waves, highlighting the differences between this and other recent searches for binaries with non-spinning components. The template bank we employed was found to yield high matches with our spin-modulated target waveform for binaries with masses in the asymmetric range 1.0 Msol < m1 < 3.0 Msol and 12.0 Msol < m2 < 20.0 Msol which is where we would expect the spin of the binary's components to have significant effect. We find that our search of S3 LIGO data had good sensitivity to binaries in the Milky Way and to a small fraction of binaries in M31 and M33 with masses in the range 1.0 Msol < m1, m2 < 20.0 Msol. No gravitational wave signals were identified during this search. Assuming a binary population with spinning components and Gaussian distribution of masses representing a prototypical neutron star - black hole system with m1 ~ 1.35 Msol and m2 ~ 5 Msol, we calculate the 90%-confidence upper limit on the rate of coalescence of these systems to be 15.9 yr^-1 L_10^-1, where L_10 is 10^10 times the blue light luminosity of the Sun.Comment: 18 pages, 8 figure

Upper limits on gravitational wave emission from 78 radio pulsars

We present upper limits on the gravitational wave emission from 78 radio pulsars based on data from the third and fourth science runs of the LIGO and GEO 600 gravitational wave detectors. The data from both runs have been combined coherently to maximize sensitivity. For the first time, pulsars within binary (or multiple) systems have been included in the search by taking into account the signal modulation due to their orbits. Our upper limits are therefore the first measured for 56 of these pulsars. For the remaining 22, our results improve on previous upper limits by up to a factor of 10. For example, our tightest upper limit on the gravitational strain is 2.6×10-25 for PSR J1603-7202, and the equatorial ellipticity of PSR J2124–3358 is less than 10-6. Furthermore, our strain upper limit for the Crab pulsar is only 2.2 times greater than the fiducial spin-down limit

Publisher’s Note: First cross-correlation analysis of interferometric and resonant-bar gravitational-wave data for stochastic backgrounds [Phys. Rev. DPRVDAQ0556-2821 76, 022001 (2007)]

This paper was published online on 9 July 2007 with incorrect affiliation numbering in the author list. The affiliations have been corrected as of 23 July 2007. The text is correct in the printed version of the journal