Christian W. Klampfl

Simple test system for single molecule recognition force microscopy

We have established an easy-to-use test system for detecting receptor–ligand interactions on the single molecule level using atomic force microscopy (AFM). For this, avidin–biotin, probably the best characterized receptor–ligand pair, was chosen. AFM sensors were prepared containing tethered biotin molecules at sufficiently low surface concentrations appropriate for single molecule studies. A biotin tether, consisting of a 6 nm poly(ethylene glycol) (PEG) chain and a functional succinimide group at the other end, was newly synthesized and covalently coupled to amine-functionalized AFM tips. In particular, PEG800 diamine was glutarylated, the mono-adduct NH2–PEG–COOH was isolated by ion exchange chromatography and reacted with biotin succinimidylester to give biotin–PEG–COOH which was then activated as N-hydroxysuccinimide (NHS) ester to give the biotin–PEG–NHS conjugate which was coupled to the aminofunctionalized AFM tip. The motional freedom provided by PEG allows for free rotation of the biotin molecule on the AFM sensor and for specific binding to avidin which had been adsorbed to mica surfaces via electrostatic interactions. Specific avidin–biotin recognition events were discriminated from nonspecific tip–mica adhesion by their typical unbinding force (∼40 pN at 1.4 nN/s loading rate), unbinding length (<13 nm), the characteristic nonlinear force–distance relation of the PEG linker, and by specific block with excess of free d-biotin. The convenience of the test system allowed to evaluate, and compare, different methods and conditions of tip aminofunctionalization with respect to specific binding and nonspecific adhesion. It is concluded that this system is well suited as calibration or start-up kit for single molecule recognition force microscopy.

CE with MS detection: a rapidly developing hyphenated technique.

Thirty years of Electrophoresis – 22 years of CE‐MS; what has happened to this hyphenated technique within the last decades? Was it able to develop from a scientifically interesting and promising but exotic technique, available exclusively to a restricted group of users with pronounced technical skills, to an almost routine technique (at least in some fields of application) performed on commercially available equipment? What might be the future of CE‐MS with respect to technical developments or new fields of application? The present review paper will try to provide answers to some of these questions. Current progress as well as possible future directions in CE‐MS will be displayed using the example of selected publications from several areas.

Analysis of melamine in milk powder by CZE using UV detection and hyphenation with ESI quadrupole/TOF MS detection

An easy to employ method for the determination of melamine in milk powder samples using CZE with either UV or MS detection is presented. The separation capabilities of CZE allowed keeping sample preparation rather simple. Before analysis, milk powder samples were dissolved in water, de‐fatted with dichloromethane and diluted with ACN to precipitate the protein, filtrated and injected without further pretreatment. The developed CZE method allowed the determination of melamine in milk powder down to a level of 0.50 mg/kg using UV detection and 0.25 mg/kg when ESI quadrupole/TOF MS was employed for detection.

Determination of organic acids in food samples by capillary zone electrophoresis

A comprehensive survey of the use of capillary zone electrophoresis for the determination of organic acids in food and beverage samples is presented. The analytes discussed in this paper include low-molecular-mass organic acids, amino acids, vitamin related compounds and free fatty acids.

Investigations on the Coupling of High-Performance Liquid Chromatography to Direct Analysis in Real Time Mass Spectrometry

A novel hyphenated technique namely the combination of HPLC with direct analysis in real time (DART) mass spectrometry (MS) is presented. The coupling of HPLC to DART-MS was achieved by a simple interface design with a capillary end piece transferring the HPLC effluent to the ionization region of the DART. Flow rates (0.3-1.6 mL min(-1)) and inner diameters of the capillary (50-150 microm) were harmonized to provide a stable liquid jet. Ionization-related parameters optimized included positioning of the capillary end piece with respect to He outlet of the ionization source and the MS inlet, He heater temperature and He flow rate. Among DART-MS voltages, the grid electrode voltage proved to have the most pronounced effect on signal intensities. A major benefit of this setup is the possibility to employ (commonly not MS-compatible) HPLC eluents such as phosphate buffers up to a phosphate concentration of 120 mM even at typical HPLC flow rates such as 1 mL min(-1) and above without negative side effects like contamination of the ion source or ion suppression. Experiments evaluating the correlation of signal intensity to mass-flow and concentration revealed that DART-MS can be seen as a mass flow sensitive detector. The usability of this hyphenated technique has been tested on the example of four parabenes (measured in the negative ion mode) as well as a set of pyrazine derivatives (measured in the positive ion mode). For the parabenes limits of detection (LOD) in the range of 20-55 microg L(-1) and linear ranges from at least 200-10000 microg L(-1) with correlation coefficients better than 0.997 were obtained.

Direct ionization methods in mass spectrometry: An overview.

Within this paper a sub-group of ambient ionization mass spectrometry namely direct ionization mass spectrometry techniques are reviewed. They are characterized by the generation of an electrospray directly from the sample investigated. Prominent representatives include paper spray mass spectrometry, tissue spray mass spectrometry, probe electrospray ionization or thin-layer chromatography mass spectrometry. Applications of all major direct ionization techniques within different fields such as biomedical analysis, analysis of natural products, analysis of technical products and food analysis, just to name a few, are discussed and relevant parameters are listed in five Tables.

Determination of carbohydrates by capillary electrophoresis with electrospray‐mass spectrometric detection

A method for the determination of underivatized carbohydrates using capillary electrophoresis (CE) with detection by electrospray ionization – mass spectrometry (ESI‐MS) presented. Highly alkaline carrier electrolytes based on volatile organic bases like is diethylamine (DEA) combined with MS detection in the negativ‐ion mode proved to be the optimum solution for the separation and detection of these analytes. Optimization of the carrier electrolyte composition has been performed with respect to its pH, ionic strength as well as the addition of an organic modifier. The influence of the DEA concentration in the sheath liquid on parameters like peak shapes or signal‐to‐noise (S/N) ratios was also investigated. Limits of detection (LOD) were in the range of 0.5–3.0 mgL–1 and calibration was linear over an order of magnitude for almost all solutes investigated. Finally, the applicability of this method for the analysis of real samples was demonstrated with wine samples.

Determination of underivatized amino acids in beverage samples by capillary electrophoresis

A method for the analysis of free amino acids in beverage samples by capillary electrophoresis and direct UV detection at 185 nm is presented. Separations were performed in a strongly acidic carrier electrolyte containing an alkanesulfonic acid and varying amounts of acetonitrile. The organic modifier permitted a manipulation of the separation selectivity for a number of analytes investigated in this study, thereby allowing design of appropriate carrier electrolyte compositions for a given analytical problem. Orange juices as well as beer samples could be analyzed with respect to their content of free amino acids. In this way, different types of beer could be distinguished by their amino acid patterns.

Determination of low-molecular-mass anionic compounds in beverage samples using capillary zone electrophoresis with simultaneous indirect ultraviolet and conductivity detection

The determination of low-molecular-mass anionic components in beer samples using capillary zone electrophoresis with simultaneous direct conductivity and indirect UV detection is described. Optimization of the carrier electrolyte composition has been performed taking into account parameters such as pH as well as those that were found to be crucial for its compatibility with both detection principles employed in this paper. The latter are UV absorptivity, electrophoretic mobility of the buffer co-ion and the buffer co-ion concentration, which strongly influence the quality (signal-to-noise ratio, chromatographic resolution of adjacent peaks) of the electropherogram. Best results could be achieved with a carrier electrolyte consisting of 4-aminobenzoic acid, tetradecyltrimethylammonium bromide electroosmotic flow reversal and a pH of 5.75, adjusted using histidine. Using this carrier electrolyte, limits of detection ranging from 0.218 mg l−1 for pyroglutamate down to 0.018 mg l−1 for chloride could be obtained.

Separation of hydrophobic polymer additives by microemulsion electrokinetic chromatography.

Microemulsion electrokinetic chromatography (MEEKC) has been applied to the separation of some phenolic antioxidants [Irganox 1024, Irganox 1035, Irganox 1076, Irganox 1010, Irganox 1330, Irgafos 138, Irganox 168 and 2,6-di-tert.-butyl-4-methylphenol (BHT)]. Due to the extremely hydrophobic nature of these analytes, they could not be separated using standard MEEKC conditions and two alternative approaches were investigated. Using an acidic buffer (phosphate, pH 2.5) to effectively suppress the electroosmotic flow, the addition of 2-propanol to the aqueous phase of the microemulsion buffer to improve partitioning of the analytes, and a negative separation voltage, separation of five of the analytes in under 10 min was possible. The second approach, using a basic buffer (borate, pH 9.2) and a positive separation voltage resulted in complete resolution of all eight analytes. A mixed surfactant system comprising the anionic sodium dodecyl sulfate (SDS) and neutral Brij 35 was used to reduce the overall charge and with it the mobility of the droplets, and hence the separation time. Using an optimised MEEKC buffer consisting of 2.25% (w/w) SDS, 0.75% (w/w) Brij 35, 0.8% (w/w) n-octane, 6.6% (w/w) 1-butanol, 25% (w/w) 2-propanol and 64.6% (w/w) 10 mM borate buffer (pH 9.2) the eight target analytes were baseline separated in under 25 min. For these analytes, MEEKC was found to be superior to micellar electrokinetic chromatography in every respect. Specifically, the solubility of the analytes was better, the selectivity was more favourable, the analysis time was shorter and the separation efficiency was up to 72% higher when using the MEEKC method. Detection limits from 5.4 to 26 microg/ml were obtained and the calibration plot was linear over more than one order of magnitude. The optimised method could be applied to the determination of Irganox 1330 and Irganox 1010 in polypropylene.