Separation of powers | EPFL Graph Search

Separation of powers refers to the division of a state's government into "branches", each with separate, independent powers and responsibilities, so that the powers of one branch are not in conflict with those of the other branches. The typical division into three branches of government, sometimes called the trias politica model, includes a legislature, an executive, and a judiciary. It can be contrasted with the fusion of powers in parliamentary and semi-presidential systems where there can be overlap in membership and functions between different branches, especially the executive and legislative. In most non-authoritarian jurisdictions, however, the judiciary almost never overlaps with the other branches, whether powers in the jurisdiction are separated or fused. The intention behind a system of separated powers is to prevent the concentration of power by providing for checks and balances. The separation of powers model is often imprecisely and metonymically used interchangeably wi

Development of Electrokinetically Based Two-Dimensional Separation Methodologies for Proteomic Studies

Mohammad Reza Pourhaghighi

Proteomic studies require analytical methods capable of separating, identifying and quantifying thousands of proteins. Consequently, developing separation methodologies with high resolving power necessary to separate complex protein samples prior to protein identification via mass spectrometry (MS) has become a major challenge in proteomic studies. It is now demonstrated that a single separation technique is unable to provide a peak capacity sufficient to resolve complex protein mixtures, therefore multidimensional separation methodologies have to be developed. In this context, the highest peak capacity is obtained when the hyphenated methods present separation mechanisms as different as possible. Subsequently, the orthogonality of a two-dimensional (2-D) separation methodology has to be also evaluated as a feature that expresses the efficiency of hyphenation. The objectives of the thesis were mainly focused on the development and application of 2-D separation methodologies in proteomic studies. In the first part of this work, the potential of the combination of Capillary zone electrophoresis (CZE) and matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) is assessed. To further provide a bioanalytical platform with high-sensitivity capabilities, field-enhanced sample injection is integrated as on online preconcentration strategy upstream from the electrokinetic separation. Then, a novel approach to assess the orthogonality of 2-D separation systems based on conditional entropy is introduced. It considers the quantitative distribution of peaks in the entire separation space such that the orthogonality obtained is independent of the number of peaks observed for each separation technique. As another part of this work, the hyphenation of off-gel electrophoresis (OGE) with CZE is revised. While the application of the developed 2-D separation methodology is expanded to protein scale, a significant improvement of the practical peak capacity for peptide separation is also obtained. In this chapter, a computer based model is also developed which is able to digest a specific protein and simulate the 2-D OGE-CZE separation map of resulting peptides. Afterwards, a practical protocol for proteolysis assisted by trypsin loaded NH2-MOSF during OGE separation is developed. Besides savings in experimental effort and enhanced proteolysis efficiency, the straightforward assessment of pI values of peptides after simultaneous digestion and separation with OGE which facilitates peptide identification, is also one valuable aspect of the developed methodology. In the last part of the thesis, the proof of concept of the application of a push-pull microfluidic device for electrotransfer of proteins from a separation gel to a membrane is presented. The membrane can be then subjected to proteolysis and MALDI-MS analysis to visualize with high resolution the protein sample separated.

EPFL2012

Orthogonality of Two-Dimensional Separations Based on Conditional Entropy

Hubert Girault, Mohammad Karzand, Mohammad Reza Pourhaghighi

A new approach to assess the orthogonality of two-dimensional (2-D) separation systems based on conditional entropy is developed. It considers the quantitative distribution of peaks in the entire separation space such that the orthogonality obtained is independent of the number of peaks observed for each separation technique. Therefore, it can be used to compare the orthogonality of different 2-D separation protocols for a given sample. Herein, the developed method has been employed to estimate the orthogonality of peptide separation by off-gel electrophoresis (OGE) hyphenated to capillary zone electrophoresis (CZE).

American Chemical Society2011

New Analytical Methods for Size Fractionated, Quantitative, and Element Specific Analysis of Metallic Engineered Nanoparticles in Aerosols and Dispersions

Harald Hagendorfer

The application of engineered nanoparticles (ENP) increased almost exponentially during the last decade. Next to carbon based ENP, metal based ENP are the next most commonly employed. Utilization of such particles for medical applications, remediation of toxic substances, consumer products, new materials, and numerous other fields has been reported already. Despite their extraordinary success and implementation, concerns have been raised about possible negative effects in humans and the environment. Once released, ENP are easily distributed via the atmosphere and the aquatic environment where an exposure to organisms can occur. To understand ENP release, life cycle (fate), and exposure as well as to evaluate their toxicological mechanisms, appropriate, media-specific (aerosols and dispersions) analytical methods are required. First, the development of a new set-up to determine ENP in aerosols is presented. For this purpose online (SMPS) and offline (electrostatic sampling on TEM grids) aerosol particle measurement techniques were combined. The aim was to study the release and fate of metallic ENP from commercial available spray products. Method development and verification was performed employing a well characterized aqueous Ag-NP spray product. The investigations have shown that the method offers the possibility to determine size, and element -specific nanoparticle release. Furthermore information about particle morphology and quantity could be obtained. It was revealed, that the release of nanoparticles strongly depends on the type of spray. Pump sprays did not show to produce airborne ENP in the aerosol, whereas propellant gas sprays released nanoparticles in substantial quantities. The release of nanoparticles was correlated with the droplet size distributions generated from the different spray types. Following method development, the setup was applied to determine ENP release of commercial available sprays, indicated to contain ENP. The obtained data was useful to model the consumer exposure of ENP from such products. Second, a set-up to determine ENP in dispersions was developed. For this purpose an asymmetric flow field flow fractionation (A4F) apparatus was coupled to an UV/Vis, light scattering- and inductively coupled plasma mass spectrometric (ICPMS) detector, simultaneously. In the beginning, method development and systematic investigations of prospects and limitations was performed employing well-characterized Au nanoparticles (Au-NP). The investigation revealed that A4F is a versatile separation technique for metallic ENP. In contrast, size determination with the A4F system, derived from the retention times of nanoparticles in the chromatogram, was erroneous. Unspecific interaction of ENP with the A4F channel membrane led to retention time shifts resulting in biased size information. For this reason, online dynamic light scattering (DLS) was employed and it offered a reliable determination of nanoparticle dimensions. Determination of nanoparticle concentration also proved to be to be problematic. Unspecific loss of nanoparticles in the A4F separation channel led to insufficient recovery prevented direct quantification of nanoparticles. To overcome this problem an alternative method was employed. With ICPMS after ultracentrifugation, quantification of the ionic and particulate -fraction of metallic ENP dispersions was possible. Validation of the set-up was performed with certified Au-NP reference materials obtained from NIST and results showed excellent agreement. Combining both of these new methods provided a comprehensive understanding of the metallic ENP systems. After method development with well defined nanoparticle systems, the set-up was tested for its applicability to "real world" samples. First, the characterization of commercial available, polydisperse, commercial Ag nanoparticle (Ag-NP) products was performed. The new developed set-up allowed obtaining an element specific, mass, and number size distribution. Furthermore, Ag-NP concentrations as well as the toxicological relevant ionic Ag concentration could be determined. Figures of merit compared to transmission electron microscopy (TEM) and batch-DLS results were in good agreement and the new method was advantageous in terms of analysis time and reliability. Furthermore the possibility to characterize SiO2 coated Au nanoparticles (Au@SiO2) was investigated. These particles are employed for Shell-Isolated Nanoparticle Enhanced Raman Spectroscopy (SHINERS). It was shown that in terms of particle size characterization, the method provided excellent data. However, determination of Si concentrations to calculate the shell thickness of such particles was impeded by the poor A4F peak shape as well as the high Si background in ICPMS. In conclusion, both setups show great potential for the characterization of metallic ENP either in aerosols or dispersions, respectively.

EPFL2011