Online Monitoring Of Reactions Research Articles

TiO2 Photocatalyzed Oxidation of Drugs Studied by Laser Ablation Electrospray Ionization Mass Spectrometry

In drug discovery, it is important to identify phase I metabolic modifications as early as possible to screen for inactivation of drugs and/or activation of prodrugs. As the major class of reactions in phase I metabolism is oxidation reactions, oxidation of drugs with TiO2 photocatalysis can be used as a simple non-biological method to initially eliminate (pro)drug candidates with an undesired phase I oxidation metabolism. Analysis of reaction products is commonly achieved with mass spectrometry coupled to chromatography. However, sample throughput can be substantially increased by eliminating pretreatment steps and exploiting the potential of ambient ionization mass spectrometry (MS). Furthermore, online monitoring of reactions in a time-resolved way would identify sequential modification steps. Here, we introduce a novel (time-resolved) TiO2-photocatalysis laser ablation electrospray ionization (LAESI) MS method for the analysis of drug candidates. This method was proven to be compatible with both TiO2-coated glass slides as well as solutions containing suspended TiO2 nanoparticles, and the results were in excellent agreement with studies on biological oxidation of verapamil, buspirone, testosterone, andarine, and ostarine. Finally, a time-resolved LAESI MS setup was developed and initial results for verapamil showed excellent analytical stability for online photocatalyzed oxidation reactions within the set-up up to at least 1 h.Graphical

On-line monitoring of photolysis reactions using electrospray ionization mass spectrometry coupled with pressurized photoreactor

A simple method by coupling a spray capillary with pressurized photoreactor for on-line monitoring of photolysis reactions was reported. In this method, only an inert gas is used to transfer the reaction solution, which avoids potential sample loss and contamination. The photolysis reaction is online quenched, and therefore, it has advantages of on-line detection and characterization of photolysis reaction intermediates. The assembly is easy to achieve, low-cost, and robust. Different from off-line methods, the photolysis reaction solution is directly transferred to a capillary tip for ionization, avoiding the delay in the detection of reactive intermediates. With this setup, this technique has been applied for on-line monitoring of degradation of cyclophosphamide (CP) in H2O2/UV for which two new intermediates were intercepted and structurally characterized for the first time by tandem mass spectrometry. In H2O2/UV, the degradation of CP occurred via hydroxylation, dehydrogenation, chlorine substitution reaction, and cleavage of the chloroethyl group. These results indicate that the coupling of electrospray ionization mass spectrometry with pressurized photoreactor can detect and characterize the key photolysis reaction intermediates, which render it a potential tool for elucidating the mechanism of water remediation.

Online monitoring of electrocatalytic reactions of alcohols at platinum and gold electrodes in acidic, neutral and alkaline media by capillary electrophoresis with contactless conductivity detection (EC-CE-C4 D).

An EC-CE-C4 D flow system was applied to the investigation of electrocatalytic processes by monitoring carboxylic acids formed during the electro-oxidation at various potentials of primary alcohols (mixture of 1mmol/L of ethanol, n-propanol, n-butanol and n-pentanol) in acidic, neutral and alkaline media. The electro-oxidation was carried out on gold and platinum disk electrodes (3mm of diameter) in a thin-layer electrochemical flow cell. Products were sampled 50μm apart from the electrode directly into the capillary. All the generated carboxylates were determined in near real time (less than 2min) by CE-C4 D in counter-flow mode, with Tris/HCl buffer solution (pH8.6) as BGE. Long sequences of 5-min experiments were run automatically, exploring the applied potential, electrolysis time and solution composition. Electro-oxidation at 1.5V (versus Ag/AgCl quasi-reference) during 50s in acidic medium was found appropriate for both Pt and Au electrodes when the determination of alcohols after derivatization is intended. A noteworthy selectivity effect was observed on the Au electrode. The signal corresponding to pentanoate is similar on both electrodes while the signal of ethanoate (acetate) is four times larger on gold than on platinum. The carboxylate signals were lower in alkaline medium (below the determination limit on Pt) than in acidic and neutral media. On gold, the formation of carboxylates was anticipated (0.85V in alkaline medium versus 1.40V in neutral medium). The automatic online monitoring of electrochemical processes by EC-CE-C4 D holds great potential to investigate ionic/ionizable intermediates/products of new electrocatalysts and/or alternative fuels.

Online monitoring of chemical reactions by polarization-induced electrospray ionization

Polarization-induced electrospray ionization (PI-ESI) is a simple technique for instant generation of gas-phase ions directly from a microliter-sized droplet for mass spectrometric analysis. A sample droplet was placed over a dielectric substrate and in proximity (2–3 mm) to the inlet of a mass spectrometer. Owing to the polarization effect induced by the high electric field provided by the mass spectrometer, the droplet was polarized and the electrospray was generated from the apex of the droplet. The polarization-induced electrospray could last for tens of seconds, which was sufficiently long to monitor fast reactions occurring within few seconds. Thus, we demonstrated the feasibility of using the droplet-based PI-ESI MS for the online monitoring of fast reactions by simply mixing two droplets (5–10 μL) containing reactants on a dielectric substrate placed in front of a mass spectrometer applied with a high voltage (−4500 V). Schiff base reactions and oxidation reactions that can generate intermediates/products within a few seconds were selected as the model reactions. The ionic reaction species generated from intermediates and products can be simultaneously monitored by PI-ESI MS in real time. We also used this approach to selectively detect acetone from a urine sample, in which acetone was derivatized in situ. In addition, the possibility of using this approach for quantitative analysis of acetone from urine samples was examined.

In-situ nanoelectrospray for high-throughput screening of enzymes and real-time monitoring of reactions

The in-situ and high-throughput evaluation of enzymes and real-time monitoring of enzyme catalyzed reactions in liquid phase is quite significant in the catalysis industry. In-situ nanoelectrospray, the direct sampling and ionization method for mass spectrometry, has been applied for high-throughput evaluation of enzymes, as well as the on-line monitoring of reactions. Simply inserting a capillary into a liquid system with high-voltage applied, analytes in liquid reaction system can be directly ionized at the capillary tip with small volume consumption. With no sample pre-treatment or injection procedure, different analytes such as saccharides, amino acids, alkaloids, peptides and proteins can be rapidly and directly extracted from liquid phase and ionized at the capillary tip. Taking irreversible transesterification reaction of vinyl acetate and ethanol as an example, this technique has been used for the high-throughput evaluation of enzymes, fast optimizations, as well as real-time monitoring of reaction catalyzed by different enzymes. In addition, it is even softer than traditional electrospray ionization. The present method can also be used for the monitoring of other homogenous and heterogeneous reactions in liquid phases, which will show potentials in the catalysis industry.

Use of a Quartz Crystal Microbalance for Studying the Effects of Propyl Gallate on the Interactions of Interleukin-6 and Its Soluble Receptor

Quartz Crystal Microbalance (QCM) is known as a surface sensitive analytical device capable of on-line monitoring of interfacial reactions. To study the interaction between interleukin-6 (IL-6) and its soluble receptor (sIL-6R), as well as the effects of propyl gallate on IL-6/sIL-6R binding based on it, a QCM was employed to monitor the binding responses. In the experiments, sIL-6R was immobilized on the surface of QCM substrate, then different concentrations of IL-6 were added onto the substrate, respectively. The response frequency shift caused by the binding between IL-6 and sIL-6R was recorded. The results showed that sIL-6R was immobilized on the substrate surface with the density of 3.43 ng/mm2, IL-6 then bound to the immobilized sIL-6R specifically. FAST fit program was used to analyse the data obtained of the association of IL-6 and immobilized sIL-6R, fitting it to the biphasic kinetic equations. Kinetic constantsfor IL-6 binding to sIL-6R were determined from the plots of Kon versus the concentration of IL-6, kass=3.41×104 M-1 s-1, kdiss=1.80×10-2 s-1, thus, KD=5.28×10-7 M, KA=1.89×106 M-1. Furthermore, the effects of propyl gallate (PG) on interaction of IL-6 with sIL-6R were studied. The results showed that PG was able to enhance the binding between IL-6 and sIL- 6R. We propose that the interaction between IL-6 and sIL-6R is one of the targets which PG can act on in vivo. The QCM offers an effective way and a new clue to the study on the mechanism of propyl gallate at molecular level.

On-line Monitoring of Continuous Flow Chemical Synthesis Using a Portable, Small Footprint Mass Spectrometer

For on-line monitoring of chemical reactions (batch or continuous flow), mass spectrometry (MS) can provide data to (1) determine the fate of starting materials and reagents, (2) confirm the presence of the desired product, (3) identify intermediates and impurities, (4) determine steady state conditions and point of completion, and (5) speed up process optimization. Recent developments in small footprint atmospheric pressure ionization portable mass spectrometers further enable this coupling, as the mass spectrometer can be easily positioned with the reaction system to be studied. A major issue for this combination is the transfer of a sample that is representative of the reaction and also compatible with the mass spectrometer. This is particularly challenging as high concentrations of reagents and products can be encountered in organic synthesis. The application of a portable mass spectrometer for on-line characterization of flow chemical synthesis has been evaluated by coupling a Microsaic 4000 MiD to the Future Chemistry Flow Start EVO chemistry system. Specifically, the Hofmann rearrangement has been studied using the on-line mass spectrometry approach. Sample transfer from the flow reactor is achieved using a mass rate attenuator (MRA) and a sampling make-up flow from a high pressure pump. This enables the appropriate sample dilution, transfer, and preparation for electrospray ionization. The capability of this approach to provide process understanding is described using an industrial pharmaceutical process that is currently under development. The effect of a number of key experimental parameters, such as the composition of the sampling make-up flow and the dilution factor on the mass spectrometry data, is also discussed.

On-line monitoring of chemical reactions by using bench-top nuclear magnetic resonance spectroscopy.

Real-time nuclear magnetic resonance (NMR) spectroscopy measurements carried out with a bench-top system installed next to the reactor inside the fume hood of the chemistry laboratory are presented. To test the system for on-line monitoring, a transfer hydrogenation reaction was studied by continuously pumping the reaction mixture from the reactor to the magnet and back in a closed loop. In addition to improving the time resolution provided by standard sampling methods, the use of such a flow setup eliminates the need for sample preparation. Owing to the progress in terms of field homogeneity and sensitivity now available with compact NMR spectrometers, small molecules dissolved at concentrations on the order of 1 mmol L(-1) can be characterized in single-scan measurements with 1 Hz resolution. Owing to the reduced field strength of compact low-field systems compared to that of conventional high-field magnets, the overlap in the spectrum of different NMR signals is a typical situation. The data processing required to obtain concentrations in the presence of signal overlap are discussed in detail, methods such as plain integration and line-fitting approaches are compared, and the accuracy of each method is determined. The kinetic rates measured for different catalytic concentrations show good agreement with those obtained with gas chromatography as a reference analytical method. Finally, as the measurements are performed under continuous flow conditions, the experimental setup and the flow parameters are optimized to maximize time resolution and signal-to-noise ratio.

Online monitoring of chemical reactions by contactless atmospheric pressure ionization mass spectrometry

Journal of Mass SpectrometryVolume 47, Issue 5 p. 586-590 JMS Letters Online monitoring of chemical reactions by contactless atmospheric pressure ionization mass spectrometry Cheng-Huan Hsieh, Cheng-Huan Hsieh Department of Applied Chemistry, National Chiao Tung University, Hsinchu, 300 TaiwanSearch for more papers by this authorChin-Sheng Chao, Chin-Sheng Chao Department of Applied Chemistry, National Chiao Tung University, Hsinchu, 300 TaiwanSearch for more papers by this authorKwok-Kong Tony Mong, Kwok-Kong Tony Mong Department of Applied Chemistry, National Chiao Tung University, Hsinchu, 300 TaiwanSearch for more papers by this authorYu-Chie Chen, Corresponding Author Yu-Chie Chen yuchie@mail.nctu.edu.tw +886-3-5131527 | Fax: +886-3-5723764 Department of Applied Chemistry, National Chiao Tung University, Hsinchu, 300 TaiwanYu-Chie Chen, Department of Applied Chemistry, National Chiao Tung University, Hsinchu 300, Taiwan. E-mail: yuchie@mail.nctu.edu.twSearch for more papers by this author Cheng-Huan Hsieh, Cheng-Huan Hsieh Department of Applied Chemistry, National Chiao Tung University, Hsinchu, 300 TaiwanSearch for more papers by this authorChin-Sheng Chao, Chin-Sheng Chao Department of Applied Chemistry, National Chiao Tung University, Hsinchu, 300 TaiwanSearch for more papers by this authorKwok-Kong Tony Mong, Kwok-Kong Tony Mong Department of Applied Chemistry, National Chiao Tung University, Hsinchu, 300 TaiwanSearch for more papers by this authorYu-Chie Chen, Corresponding Author Yu-Chie Chen yuchie@mail.nctu.edu.tw +886-3-5131527 | Fax: +886-3-5723764 Department of Applied Chemistry, National Chiao Tung University, Hsinchu, 300 TaiwanYu-Chie Chen, Department of Applied Chemistry, National Chiao Tung University, Hsinchu 300, Taiwan. E-mail: yuchie@mail.nctu.edu.twSearch for more papers by this author First published: 10 May 2012 https://doi.org/10.1002/jms.2983Citations: 27Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Citing Literature Volume47, Issue5May 2012Pages 586-590 RelatedInformation

Monitoring the nitrification and identifying the endpoint of ammonium oxidation by using a novel system of titrimetry

Based on the structure of the hybrid respirometer previously developed in our group, a novel implementation for titrimetry was developed, in which two pH electrodes were installed at the inlet and outlet of the measuring cell. The software capable of digital filtering and titration time delay correction was developed in LabVIEW. The hardware and software of the titrimeter and the respirometer were integrated to construct a novel system of respirometry-titrimetry. The system was applied to monitor a batch nitrification process. The obtained profiles of oxygen uptake rate (OUR) and hydrogen ion production rate (HPR) are consistent with each other and agree with the principle of the biological nitrification reaction. According to the OUR and HPR measurements, the oxidized ammonium concentrations were estimated accurately. Furthermore, the endpoint of ammonium oxidation was identified with much higher sensitivity by the HPR measurement. The system could be potentially used for on-line monitoring of biochemical reactions occurring in any kind of bioreactors because its measuring cell is completely independent of the bioreactor.

Fluorescence spectroscopy as a novel method for on-line analysis of biocatalytic C–C bond formations

On-line analysis of bioprocesses is of increasing interest avoiding the time delay for off-line sample preparation and the following analyses via chromatographic methods. Moreover, continuous monitoring of the reaction components during chemo- or biocatalytic transformations provides a direct control of the process. Since productivity of the processes can be controlled simultaneously, on-line monitoring of the processes is attractive for industrial applications. The reliable in situ monitoring of biocatalyzed reactions has been a challenge where reactions run in aqueous solutions. Limited work has been published on the use of spectroscopic methods for on-line analysis of biocatalytic reactions up to now. However, in this communication two dimensional (2D)-fluorescence spectroscopy has been proved to be an effective tool for on-line monitoring of the carboligation reactions catalyzed by wild type benzoylformate decarboxylase (BFD) from Pseudomonas putida . BFD is a thiamine diphosphate (ThDP)-dependent enzyme that catalyzes the asymmetric C–C bond formation to ( S )-2-hydroxypropiophenone (( S )-2-HPP) starting from benzaldehyde and acetaldehyde. The analysis of the fluorescence spectra was achieved by chemometric modeling performing principle component analysis (PCA) and partial least square (PLS) regression. The derived chemometric models were used for the validation of concentrations of yielded 2-HPP and the substrate benzaldehyde with low root mean square error of calibration (RMSEC).

SOCIETE FRANCAISE DE NEUROLOGIE (SFN)

Unstable biopolymer solutions inevitably lead to mis-characterization of macromolecular properties and irreproducible results. Even under stable or quasi-stable conditions, persistent aggregates can hamper reliable characterization, especially using light scattering methods. Various pitfalls in characterizing biopolymers were worked through, including determination of solution stability zones, dissolution kinetics, estimation of fraction of aggregate populations, and the relationship between batch and fractionation methods. Chitosans, polyampholytic biopolymers with isoelectric point around pH = 6.0, with varying degrees of carboxymethylation were studied. Instability was determined vs. pH and ionic strength using a high throughput screening method, simultaneous multiple sample light scattering (SMSLS). With stable solution conditions determined, equilibrium batch and multi-detector GPC characterization of molecular weight, intrinsic viscosity, and polyelectrolyte properties was made. Finally, a first attempt at continuous online monitoring of the modification reaction itself was made and compared to FTIR analysis of carboxymethylation on discrete aliquots. Given the range of possible characterization problems, multiple approaches with independent instruments may be required for reliable natural product characterization. Online monitoring of modification reactions may lead to rapid advances in understanding and preparation of natural products.

Fluorescence anisotropy assay for pharmacological characterization of ligand binding dynamics to melanocortin 4 receptors

Fluorescence anisotropy assay was implemented for characterization of ligand binding dynamics to melanocortin 4 (MC(4)) receptors. This approach enables on-line monitoring of reactions that is essential for estimation of more correct binding parameters, understanding of ligand binding and its regulation mechanisms, and design of new drugs with desirable properties. Two different red-shifted fluorophore-labeled peptide ligands, Cy3B-NDP-alpha-MSH and TAMRA-NDP-alpha-MSH, were used and compared in assays that monitored their binding to MC(4) receptors in membranes of Sf9 insect cells. The Cy3B dye-labeled ligand exhibited improved performance in assays when compared with the TAMRA-labeled ligand, having higher photostability, insensitivity to buffer properties, and better signal/noise ratio. The binding of both ligands to membranes of Sf9 cells expressing MC(4) receptors was saturable and with high affinity. All studied MC(4) receptor-specific nonlabeled ligands displaced fluoroligands' binding in a concentration-dependent manner with potencies in agreement with their pharmacological activities. On-line monitoring of the reactions revealed that equilibrium of peptide binding was not reached even after 3h. Real-time monitoring of ligand binding dynamics enabled us to find optimal experimental conditions for each particular ligand and an improved estimate of their binding parameters.

Ultrasonication-assisted spray ionization mass spectrometry for on-line monitoring of organic reactions

A straightforward on-line monitoring of organic reactions by ultrasonication-assisted spray ionization mass spectrometry (UASI MS) is demonstrated in this work.

Characterization of stability, aggregation, and equilibrium properties of modified natural products; The case of carboxymethylated chitosans

Unstable biopolymer solutions inevitably lead to mis-characterization of macromolecular properties and irreproducible results. Even under stable or quasi-stable conditions, persistent aggregates can hamper reliable characterization, especially using light scattering methods. Various pitfalls in characterizing biopolymers were worked through, including determination of solution stability zones, dissolution kinetics, estimation of fraction of aggregate populations, and the relationship between batch and fractionation methods. Chitosans, polyampholytic biopolymers with isoelectric point around pH = 6.0, with varying degrees of carboxymethylation were studied. Instability was determined vs. pH and ionic strength using a high throughput screening method, simultaneous multiple sample light scattering (SMSLS). With stable solution conditions determined, equilibrium batch and multi-detector GPC characterization of molecular weight, intrinsic viscosity, and polyelectrolyte properties was made. Finally, a first attempt at continuous online monitoring of the modification reaction itself was made and compared to FTIR analysis of carboxymethylation on discrete aliquots. Given the range of possible characterization problems, multiple approaches with independent instruments may be required for reliable natural product characterization. Online monitoring of modification reactions may lead to rapid advances in understanding and preparation of natural products.

Direct determination of ephedrine intermediate in a biotransformation reaction using infrared spectroscopy and PLS

Fourier transform mid-infrared spectroscopy (FT-MIR) coupled with a homemade attenuated total reflectance (ATR) flow-cell was used for on-line monitoring of a biotransformation reaction. The reaction was also monitored off-line by gas chromatography–mass spectrometry (GC–MS) enabling to establish a multivariate model for the infrared data based on partial least squares (PLS) regression. The method developed allowed the simultaneous determination of the substrate, two intermediates and the final product involved in the reduction reaction of 1-phenyl 1,2-propanedione at an initial concentration of 0.5% (v/v). The reaction was accomplished with a whole-cell suspension of Saccharomyces cerevisiae in a phosphate buffer of pH 3.0 at 32 ± 1 °C. The ATR infrared monitoring was performed directly on the suspension cell without any separation process or extraction over 3 h, totaling 188 spectra. The data were split into two subsets, with 158 times for calibration and 30 times for validation. The results showed that the proposed method may be used for on-line monitoring of the biotransformation reactions when the initial concentration is very low.

Applicability of Fiber-Optic-Based Raman Probes for On-Line Reaction Monitoring of High-Pressure Catalytic Hydrogenation Reactions

This study evaluates the applicability of fiber-optic-based Raman probes for on-line reaction monitoring of high-pressure catalytic hydrogenation reactions in batch autoclaves. First, based on trends in the strong intensity of the 945 cm(-1) C-O-C vibration of 1,3-dioxolane, the effect of various experimental parameters on sensitivity was evaluated and can be summarized as follows: (1) above 500 rpm a linear increase in stirring speed induces a linear decrease in Raman intensity; (2) a linear increase in hydrogen pressure also leads to a linear decrease of the Raman signal; (3) linear temperature elevation exponentially decreases the Raman intensity; and (4) increasing the catalyst particle concentration results in a steep nonlinear decrease of the Raman signal. Light scattering by gas bubbles, or combined scattering and absorption by (black) catalyst particles, reducing the amount of light collected by the optical fiber probe, explain the observed experimental trends. Second, the sensitivity of Raman spectroscopy was directly compared with attenuated total reflection-Fourier transform infrared (ATR-FT-IR) spectroscopy in the analysis of three different hydrogenation reactions over a Cu-ZnO catalyst. From the applied target molecules, diethyl maleate hydrogenation could be very well analyzed by Raman spectroscopy due to the high Raman scattering efficiency of the C=C bond, while for analysis of the hydrogenation of gamma-butyrolactone or 1-butanal, ATR-FT-IR is the technique of choice.

Spectroscopic on-line monitoring of reactions in dispersed medium: Chemometric challenges

Emulsion and suspension polymerizations are important industrial processes for polymer production. The end-user properties of polymers depend strongly on how the polymerization reactions proceed in time ( i.e. a batch or semicontinuous, rate of reagents feeding, etc.). In other words, these reactions are process dependent, which makes the successful process control a key point to ensure high-quality products. In several process control strategies the on-line monitoring of reaction performance is required. Due to the multiphase nature of the emulsion and suspension processes, there is a lack of sensors to perform successful on-line monitoring. Near infrared and Raman spectroscopies have been pointed out as useful approaches for monitoring emulsion and suspension polymerizations and several applications have been described. In such instance, the chemometric approach on relating near infrared and Raman spectra to polymer properties is widely used and has proven to be useful. Nevertheless, the multiphase nature of emulsion and suspension polymerizations also represents a challenge for the chemometric approach based on multivariate calibration models and demands the development of new methods. In this work, a set novel results is presented from the monitoring of 15 batch emulsion reactions that show the chemometric challenge to be faced on development of new methods for successful monitoring of processes taken under dispersed medium. In order to discuss these results, several chemometric approaches were revised. It is shown that Raman and NIR spectroscopic techniques are suitable for on-line monitoring of monomer concentration and polymer content during the polymerizations, as well as medium heterogeneity properties, i.e. average particle size. It is also shown that Hotteling and Q statistics, widely used in chemometrics, might fail in monitoring these reactions, while an approach based on principal curves is able to overcome such restriction.

On-line monitoring of airborne chemistry in levitated nanodroplets: in situ synthesis and application of SERS-active Ag-Sols for trace analysis by FT-Raman spectroscopy.

We report a new strategy for on-line monitoring of chemical reactions in ultrasonically levitated, nanoliter-sized droplets by Raman spectroscopy. A flow-through microdispenser connected to an automated flow injection system was used to dose picoliter droplets into the node of an ultrasonic trap. Taking advantage of the flow-through characteristics of the microdispenser and the versatility of the automated flow system, a well-defined sequence of reagents could be injected via the microdispenser into the levitated droplet placed in the focus of the collection optics of the Fourier transform Raman spectrometer. In that way, chemical reactions could be carried out and monitored on-line. The developed system was used for fast, reproducible, in situ synthesis of a highly active surface enhanced Raman scattering (SERS) sol resulting from the reduction of silver nitrate with hydroxylamine hydrochloride in basic conditions. With this chemical system, SERS substrate preparation could be achieved at room temperature and in short time. The in situ prepared silver sol was used for trace analysis of several organic test molecules that were injected into the levitated SERS-active droplet again using the microdispenser. The concentration dependence of the SERS spectra was studied using 9-aminoacridine, revealing that down to the femtogram region high-quality SERS spectra could be obtained. Additionally, SERS spectra of 6-mercaptopurine, thiamine, and acridine were recorded in the levitated drop as well.

Pre-steady-state kinetics of enzymatic reactions studied by electrospray mass spectrometry with on-line rapid-mixing techniques

This chapter focuses on recent developments that allow the direct on-line monitoring of enzymecatalyzed reactions by electrospray ionization (ESI) mass spectrometry (MS). In these experiments, an enzyme/substrate mixture is injected directly into the ESI source of the mass spectrometer, while the reaction proceeds in solution. With this approach it is possible to overcome many limitations of traditional kinetic methods. The new ESI MS-based techniques have the potential to become standard tools for studying enzymatic reactions in the pre-steady-state regime. These techniques are useful for kinetic studies in other fields of chemistry and biochemistry. The current time resolution of these techniques (tens of milliseconds) is not as good as that of conventional quench-flow and stopped-flow experiments. Certain restrictions may be encountered when choosing solvent systems that are compatible with on-line monitoring of the reaction mixture by ESI MS. Salts, detergents, and pH buffers may interfere with the electrospray process, leading to low signal intensities and the formation of adduct species. The electrospray process is compatible with pH buffers that are based on volatile components, such as acetic acid, formic acid, ammonia, or piperidine. It might be feasible to develop microdialysis systems for kinetic experiments that allow the rapid desalting of the reaction mixture immediately before it is analyzed by ESI MS.