Single-Molecule Pycnometry and Shape Analysis of Ions in the Gas Phase.

The analysis of ions and clusters by mobility-classified mass spectrometry provides information on the mobility of analytes in the drift gas and the analyte mass. Mass equivalent and mobility equivalent diameters of globular analytes, such as ions, poly(ethylene glycol) (PEG), and ionic liquid nanodroplets, can be correlated with good accuracy by the Stokes-Millikan mobility model. A prerequisite to such an analysis is, however, the assumption of a globular analyte shape, which then allows determination of material density for globular ions. We show that the analyte density can be evaluated with high precision, independent of any assumptions on the analyte shape, by careful analysis of analyte-PEG-cluster ions following the concept of classical pycnometry. In particular, the analyte is entrapped in a globular PEG-analyte droplet. Based on the now independently derived mobility diameter and volume equivalent diameter, it is possible to attribute two parameters, size and shape, to the analyte molecule. We demonstrate the approach for lysozyme, cyano-cobalamin (vitamin B12), and glucose, which cover two orders of magnitude in analyte mass (180···14 300 Da). The derived densities for these analytes are highly accurate, i.e., they deviate less than 1% from literature values. Our method can be applied to newly synthesized molecules, supramolecular assemblies, isolated biomolecules, and molecular clusters, where only minor amounts of materials are available. The obtained shape parameters of lysozyme and cyano-cobalamin agree well with the expected molecular shapes. Data evaluation relies only on locations of the species in the mass-mobility plane and is in principle independent of any mobility theory. Our approach is thus robust with respect to experimental uncertainties and produces identical results irrespective of the type of mobility classification and drift gas.

Characterization of Electrospray Drop Size Distributions by Mobility-Classified Mass Spectrometry: Implications for Ion Clustering in Solution and Ion Formation Pathways.

One of the outcomes of electrospray ionization is the size distribution of the droplets, which determines, together with the solvent composition and the source gas temperature, the minimum distance from the sprayer tip to the mass spectrometer inlet and therefore the ion transfer efficiency. Even more importantly, the average number of analyte molecules and, if present, contaminant species per droplet depend on the drop size. Consequently, the drop size distribution is a key parameter in nonspecific ion clustering in solution and ion suppression. The finding that small droplet sizes improve the mass spectral quality led to the development of nanoelectrospray sources, which dispense liquid flow rates below 0.1 µL/min and can generate drops with diameters smaller than 100 nm. However, current discussions on the effect of drop size on ion formation pathways and efficiencies remain qualitative because the exact drop size distributions are unknown. Here, we show that ion mobility-classified mass spectrometry of raffinose cluster ions allows us to determine very precisely the drop size distribution generated by the electrospray source in positive- and negative-ion modes. Based on the derived drop size distributions, we can quantitatively predict nonspecific ion clustering and can extract accurate probabilities for emission of species from parent drops upon Coulomb fission.

Mobility-Classified Mass Spectrometry Reveals a Complete Picture of the Electrospray Outcome.

Electrospray ionization mass spectrometry supported by mobility classification (ESI-DMA-MS) is a strong technique for a comprehensive analysis of organic and inorganic clusters and small nanoparticles. In-depth understanding and optimization of the electrospray process are key for unlocking new fields of application and for extension of the accessible range of data. We show that the combination of mobility-classified mass spectrometry (DMA-MS) with an electrospray operated in a well-defined cone-jet mode is capable of providing a full picture of the outcome of the electrospray process including the evaporation residues of the parent droplets. Based on ESI-DMA-MS measurements of lysozyme, we find that clusters up to almost 1 MDa (around 60 lysozyme molecules per cluster) formed as evaporation residues of the parent droplets can be detected. The escape probability of lysozyme molecules from the parent droplets is polarity-dependent. A quantitative analysis of clusters is possible at analyte concentrations where the likeliness of finding more than a single molecule in the generated droplet is low. At higher concentrations, one can, however, quantify the analyte concentration from the detected size of the droplet evaporation residues. The approach is widely applicable for organic and inorganic nanostructures. The results allow clear statements on the conditions under which mixtures of larger molecules, clusters, and nanoparticles are accessible to quantitative analysis by mass spectrometry.

Ionomer and protein size analysis by analytical ultracentrifugation and electrospray scanning mobility particle sizer.

By combining analytical ultracentrifugation (AUC) in liquid phase and scanning mobility particle sizer (SMPS) in the gas phase, additional information on the particle size and morphology has been obtained for rigid particles. In this paper, we transfer this concept to soft particles, allowing us to analyze the size and molar mass of the short side chain perfluorosulfonic acid ionomer Aquivion® in a dilute aqueous suspension. The determination of the primary size and exact molar mass of this class of polymers is challenging since they are optically transparent and due to the formation of different aggregate structures depending on the concentration and solvent properties. First, validation of AUC and SMPS measurements was carried out using the well-defined biopolymers bovine serum albumin (BSA) and lysozyme (LYZ) to confirm the reliability of the results of the two unique and independent classifying methods. Then, the ionomer Aquivion® was studied using both techniques. From the mean molar mass of 185 ± 14 kDa obtained by AUC, a mean hydrodynamic diameter of 7.6 ± 0.5 nm was calculated. The particle size obtained from SMPS (7.1 nm) agrees very well with the results from AUC showing that the molecule was transferred into the gas phase without significantly changing its structure. In conclusion, the Aquivion® is molecularly dispersed in the used aqueous buffer solution without any aggregate formation in the investigated concentration range (< 2 g l-1).

A Novel Approach for Analyzing the Dissolution Mechanism of Solid Dispersions.

PURPOSE: To analyze the dissolution mechanism of solid dispersions of poorly water-soluble active pharmaceutical ingredients (APIs), to predict the dissolution profiles of the APIs and to find appropriate ways to improve their dissolution rate. METHODS: The dissolution profiles of indomethacin and naproxen from solid dispersions in PVP K25 were measured in vitro using a rotating-disk system (USP II). A chemical-potential-gradient model combined with the thermodynamic model PC-SAFT was developed to investigate the dissolution mechanism of indomethacin and naproxen from their solid dispersions at different conditions and to predict the dissolution profiles of these APIs. RESULTS: The results show that the dissolution of the investigated solid dispersions is controlled by dissolution of both, API and PVP K25 as they codissolve according to the initial API loading. Moreover, the dissolution of indomethacin and naproxen was improved by decreasing the API loading in polymer (leading to amorphous solid dispersions) and increasing stirring speed, temperature and pH of the dissolution medium. The dissolution of indomethacin and naproxen from their amorphous solid dispersions is mainly controlled by the surface reaction, which implies that indomethacin and naproxen dissolution can be effectively improved by formulation design and by improving their solvation performance. CONCLUSIONS: The chemical-potential-gradient model combined with PC-SAFT can be used to analyze the dissolution mechanism of solid dispersions and to describe and predict the dissolution profiles of API as function of stirring speed, temperature and pH value of the medium. This work helps to find appropriate ways to improve the dissolution rate of poorly-soluble APIs.

On the mechanism of Zn4O-acetate precursors ripening to ZnO: How dimerization is promoted by hydroxide incorporation.

We report on a combined experimental and molecular modelling study on Zn4O ion clusters stabilized by acetate molecules (OAc). In particular, ab initio calculations of acetate substitution by hydroxide ions are compared with mass spectrometry data. Though quantum calculations in the gas phase indicate strong energetic preference, no experimental evidence of stable Zn4O(OAc)6-x(OH)x clusters in ethanolic solutions could be observed. This apparent contradiction is rationalized by identifying the supportive role of hydroxide ions for the association of (OAc(-) → OH(-) substituted) Zn4O(OAc)6 and Zn4O(OAc)5 (+) clusters. Mass spectrometry and quantum calculations hint at the stability of (Zn4O)2(OAc)12-x(OH)x dimers with x = 1, 2. Therein, the hydroxide ions establish salt-bridges that allow for the formation of additional Zn3 motifs with the OH above the triangle center-a structural motif close to that of the ZnO-crystal. The association of Zn4O(OAc)6 clusters is thus suggested to involve OAc(-) → OH(-) substitution as an activation step, quickly followed by dimerization and the subsequent agglomeration of oligomers.

Efficient quenching sheds light on early stages of gold nanoparticle formation.

The formation mechanism of plasmonic gold nanoparticles (Au NPs) by fast NaBH4 induced reduction of the precursors is still under debate. In this work we introduce a simple method to access intermediate species of Au NPs by quenching the solid formation process at desired time periods. In this way, we take advantage of the covalent binding of glutathione on Au NPs to stop their growth. By applying a plethora of precise particle characterization techniques, we shed new light on the early stages of particle formation. The results of in situ UV/vis measurements, ex situ sedimentation coefficient analysis by analytical ultracentrifugation, size exclusion high performance liquid chromatography, electrospray ionization mass spectrometry supported by mobility classification and scanning transmission electron microscopy suggest an initial rapid formation of small non-plasmonic Au clusters with Au10 as the main species followed by their growth to plasmonic Au NPs by agglomeration. The fast reduction of gold salts by NaBH4 depends on mixing which is hard to control during the scale-up of batch processes. Thus, we transferred the Au NP synthesis to a continuous flow process with improved mixing. We observed that the mean volume particle sizes and the width of the particle size distribution decrease with increasing flow rate and thus higher energy input. Mixing- and reaction-controlled regimes are identified.

Structure and adsorption behavior of high hydrostatic pressure-treated ß-lactoglobulin.

HYPOTHESIS: High hydrostatic pressure treatment causes structural changes in interfacial-active ß-lactoglobulin (ß-lg). We hypothesized that the pressure-induced structural changes affect the intra- and intermolecular interactions which determine the interfacial activity of ß-lg. The conducted experimental and numerical investigations could contribute to the mechanistic understanding of the adsorption behavior of proteins in food-related emulsions. EXPERIMENTS: We treated ß-lg in water at pH 7 with high hydrostatic pressures up to 600 MPa for 10 min at 20 °C. The secondary structure was characterized with Fourier-transform infrared spectroscopy (FTIR) and circular dichroism (CD), the surface hydrophobicity and charge with fluorescence-spectroscopy and ζ-potential, and the quaternary structure with membrane-osmometry, analytical ultracentrifugation (AUC) and mass spectrometry (MS). Experimental analyses were supported through molecular dynamic (MD) simulations. The adsorption behavior was investigated with pendant drop analysis. FINDINGS: MD simulation revealed a pressure-induced molten globule state of ß-lg, confirmed by an unfolding of ß-sheets with FTIR, a stabilization of α-helices with CD and loss in tertiary structure induced by an increase in surface hydrophobicity. Membrane-osmometry, AUC and MS indicated the formation of non-covalently linked dimers that migrated slower through the water phase, adsorbed more quickly due to hydrophobic interactions with the oil, and lowered the interfacial tension more strongly than reference ß-lg.

How to avoid interfering electrochemical reactions in ESI-MS analysis.

The presence of electrochemical reactions occurring in an electrospray processes at the point where the current enters the liquid is discussed since the early 1990's. This current transfer to the liquid results in oxidation or reduction of either electrolyte species in the liquid sprayed or of the electrode material in contact with the liquid. As a result, new chemical species are generated. These products of the electrochemical reaction might be detected as altered species in mass spectra; they might be volatile and not recognized at all or accumulate on the electrode surface and cause cross contamination later on. In other cases, it might happen that the products of the electrochemical reactions are the only detectable species formed from an otherwise nondetectable analyte. An electrospray setup in which electrochemical reactions do not interfere with the analyte under investigation excludes the electrochemical reaction as source of sample contamination and sample altering and may serve as reference setup for experiments focused on the electrochemical reaction itself. We present a simple and inexpensive current coupling approach and specify operation conditions for which any impact of the electrochemical reaction on the sample under investigation is inherently excluded. On the basis of a practical example, we show the impact of the electrochemical reaction on sample composition and demonstrate the benefit of using the proposed current coupling method. Because of the obvious benefit of this method and its simple realization, it has the potential to be employed as standard feeding approach, especially for electrosprays operated at small flow rates.

Analysis of Tribo-Charging during Powder Spreading in Selective Laser Sintering: Assessment of Polyamide 12 Powder Ageing Effects on Charging Behavior.

Powder flowability is key to achieving high process stability and part quality by application of smooth and dense layers in selective laser sintering (SLS). This study sheds light on the rarely investigated effect of tribo-electric charge build-up during powder delivery in the SLS process. This is achieved by a novel approach to quantify electrostatic potentials during doctor blading. The presented model setup is used in combination with charge spectrometry and impedance spectroscopy to investigate the alterations in tribo-electric charging behavior for the most commonly used laser sintering material polyamide 12 in its virgin and aged, c.f. reused, states. We show that the electrostatic charge build-up is significantly enhanced for aged polymer powder material, likely contributing to altered performance in SLS processing.