Biomedical subjects

Bernhard Lendl

Publications and source records attributed to Bernhard Lendl.

At least 19 recordsLinked to original sources

PubMed2006

Determination of peroxide-based explosives using liquid chromatography with on-line infrared detection.

A nondestructive analytical method for peroxide-based explosives determination in solid samples is described. Reversed-phase high-performance liquid chromatography in combination with on-line Fourier transform infrared (FT-IR) detection is used for the analysis of triacetonetriperoxide (TATP) and hexamethylenetriperoxide diamine (HMTD). In contrast to other liquid chromatographic methods with optical detection, no derivatization or decomposition of the peroxides is required. The peroxides are identified and quantified via their characteristic absorption spectra in the mid-infrared range of the electromagnetic spectrum. The detection limit of 0.5 mmol L-1 for HMTD and 1 mmol L-1 for TATP allows the identification of the explosives in complex matrixes.

Journal Article↗

PubMed2006

On-line monitoring of pH junctions in capillary electrophoresis using Fourier transform infrared spectrometry.

Fourier transform mid-infrared spectroscopic detection is proposed as an on-line detection technique for the study of on-line preconcentration processes in capillary electrophoresis (CE). The molecule-specific information contained in mid-IR spectra can be used to directly determine the chemical compositions of individual zones and their boundaries. This paper reports on pH junctions employed in myoglobin analysis. On-line mid-IR detection allowed the shape of the sample peak to be monitored as well as the chemical compositions of the surrounding zones. From this information it was possible to obtain detailed insights into the actual chemical compositions of the individual zones governing the efficiency of the preconcentration technique applied. The principle of measurement outlined here can therefore also be regarded as a promising one for investigating other on-line preconcentration techniques, like stacking, sweeping, and pH junction-sweeping among others.

Journal Article↗

PubMed2006

Containerless reaction monitoring in ionic liquids by means of Raman microspectroscopy.

Reaction monitoring by Raman microspectroscopy in levitated room temperature ionic liquid (RTIL) droplets is reported. Due to their non-volatility, RTIL droplets are well-suited to act as wall-less microreactors. The droplets were produced by a piezoelectric flow-through microdispenser connected to an automated flow injection system and were levitated by an acoustic trap. Taking advantage of the flow system versatility, the sequence of reagents was easily changed to study a model organic reaction: the Knoevenagel condensation. The reaction was followed by Raman microspectrometry and the obtained spectra were analysed using multivariate curve resolution to retrieve the concentration profiles and pure spectra of reactants, intermediates and products involved in the reaction. In addition, information about solvation interactions was obtained by monitoring the desolvation process taking place when a volatile co-solvent evaporated from the droplet.

Journal Article↗

PubMed2006

Association of methanol and water in ionic liquids elucidated by infrared spectroscopy using two-dimensional correlation and multivariate curve resolution.

Water and methanol associations in ionic liquids (ILs) have been studied by means of FTIR spectroscopy. Spectra at different concentrations of water or methanol in ILs were obtained by means of on-line dilution using a flow injection analysis system. Spectral features in the OH stretching region revealed that most of the water and methanol molecules tended to be isolated from each other and to interact with the anion of the IL via H bonding. By means of two-dimensional correlation spectroscopy, the formation of methanol and water dimers was also detected. Multivariate curve resolution was used to recover pure spectra and concentration profiles of the different species. Methanol dimers form at concentrations higher than 0.8% (w/w) in the three studied ILs, 1-ethyl-3-methylimidazolium tetrafluoroborate (emimBF4), 1-butyl-3-methylimidazolium tetrafluoroborate (bmimBF4), and 1-butyl-3-methylimidazolium hexafluorophosphate (bmimPF6). Self-association of water molecules takes place in emimBF4 and bmimBF4 at a molar ratio similar to that of methanol molecules; however, water dimers cannot be detected in bmimPF6, the most hydrophobic IL studied. No evidence was found that bigger water clusters are formed in these ILs at the studied cosolvent concentrations.

Journal Article↗

PubMed2006

Detection of albumin unfolding preceding proteolysis using Fourier transform infrared spectroscopy and chemometric data analysis.

The hydrolysis of bovine serum albumin with protease K at 60 degrees C has been studied by means of infrared spectroscopy. Two-dimensional correlation spectroscopy (2DCoS) has been used to study spectral changes in the reaction. The use of the multivariate curve resolution-alternating least-squares method applied to infrared measurements allowed the recovery of pure infrared spectra and concentration profiles of the different species involved in the reaction. Special attention was paid to the careful inspection of residuals again using 2DCoS. In this way, a heat-induced unfolding step previous to protein hydrolysis was identified. The infrared spectra of the intermediate species showed a more disordered structure than native albumin, the decrease in alpha-helix conformation being especially noticeable. The formation of beta-sheet aggregates due to heating was detected too.

Animals↗

PubMed2006

Automated sample preparation and analysis using a sequential-injection-capillary electrophoresis (SI-CE) interface.

A fully automated sequential-injection-capillary electrophoresis (SI-CE) system was developed using commercially available components as the syringe pump, the selection and injection valves and the high voltage power supply. The interface connecting the SI with the CE unit consisted of two T-pieces, where the capillary was inserted in one T-piece and a Pt electrode in the other (grounded) T-piece. By pressurising the whole system using a syringe pump, hydrodynamic injection was feasible. For characterisation, the system was applied to a mixture of adenosine and adenosine monophosphate at different concentrations. The calibration curve obtained gave a detection limit of 0.5 microg g(-1) (correlation coefficient of 0.997). The reproducibility of the injection was also assessed, resulting in a RSD value (5 injections) of 5.4%. The total time of analysis, from injection, conditioning and separation to cleaning the capillary again was 15 minutes. In another application, employing the full power of the automated SIA-CE system, myoglobin was mixed directly using the flow system with different concentrations of sodium dodecyl sulfate (SDS), a known denaturing agent. The different conformations obtained in this way were analysed with the CE system and a distinct shift in migration time and decreasing of the native peak of myoglobin (Mb) could be observed. The protein samples prepared were also analysed with off-line infrared spectroscopy (IR), confirming these results.

Journal Article↗

PubMed2006

On-line reaction monitoring in the liquid phase using two mid-infrared quantum cascade lasers simultaneously.

On-line monitoring of a model reaction was performed by employing two pulsed mid-infrared Fabry-Pérot quantum cascade lasers (QCL). The emission maxima of the QCLs were located at 1393 and 1080 cm(-1). An optical system of parabolic mirrors and a ZnSe beam splitter combined the two laser beams and allowed a transmission cell to be probed with both QCLs simultaneously. The reaction mixture was pumped continuously through a cell that had an optical path of 48 microm. This dual QCL system allowed fast absorption measurements of the reaction mixture at two distinct wavenumbers. The reaction under study was the oxidation of sulfite to sulfate with hydrogen peroxide acting as oxidant. On-line measurements of the chemical reaction allowed direct, real-time monitoring of sulfate formation and hydrogen peroxide depletion.

Journal Article↗

PubMed2005

On-line hyphenation of quantum cascade laser and capillary electrophoresis.

We report the first successful hyphenation of a Fabry Pérot quantum cascade (QC) laser to a capillary electrophoresis system. This involved use of a dedicated IR-transparent flow cell, made of CaF2, constructed by means of SU-8 based lithography and low temperature wafer bonding techniques. Adenosine, guanosine, xanthosine and adenosine-5'-monophosphate were separated in a borate-containing separation electrolyte (10 mM, pH 9.3). Functional group (carbohydrate) detection was accomplished by use of the 1080 cm(-1) emission line of the available QC-laser. The assessable optical path length could be increased, from the normally available 10-15 microm in CE-FTIR analyses, to 60 microm using this powerful mid-infrared laser and aqueous solutions.

Adenosine↗

PubMed2005

Flow-through microdispenser for interfacing micro-HPLC to Raman and mid-IR spectroscopic detection.

A flow-through microdispenser has been coupled to a micro HPLC separation system and used as a solvent elimination interface for Fourier transform infrared (FTIR) and Raman spectroscopic detection of the separated compounds. Using the microdispenser picoliter sized droplets can be generated and deposited on an appropriate target placed on a computerized x, y-stage. Evaporation of volatile solvent and buffer is rapid and allows analysis of the obtained dry deposits by various techniques. Due to the destruction free character of Raman and FTIR spectroscopy they can be applied sequentially to interrogate the same deposit. In the reported application five phenolic acids typically present in wine have been separated on a C-18 column technique using a mixture of water, methanol and acetic acid as mobile phase. For spectrum acquisition infrared and Raman microscopes have been used. The spectra recorded from the dried deposits of the separated compounds agreed well with the reference spectra of corresponding components.

Chromatography, High Pressure Liquid↗

PubMed2005

Direct monitoring of lipid oxidation in edible oils by Fourier transform Raman spectroscopy.

Fourier transform Raman spectroscopy has been used to investigate the chemical changes taking place during lipid oxidation in several edible oils. Oxidative degradation of six vegetable oils was accelerated by heating at 160 degrees C. Formation of aldehydes was detected, and saturated as well as alpha,beta-unsaturated aldehydes could be identified with the help of pure component spectra. The formation of conjugated double bond systems and the isomerisation of cis to trans double bonds was observed in the C=C stretching region and found to follow a distinct pattern for the different oils. It was possible to associate these differences to the fatty acid composition. The time-dependent intensity changes in certain Raman bands were compared to conventional parameters used to determine the extent of oxidation in oils, such as anisidine value and K(270), and showed good correlation.

Fatty Acids↗

PubMed2004

On-line determination of the intracellular poly(beta-hydroxybutyric acid) content in transformed Escherichia coli and glucose during PHB production using stopped-flow attenuated total reflection FT-IR spectrometry.

An automated approach for rapid on-line monitoring of the solid and liquid phases present in bioprocesses based on mid-IR Fourier transform spectrometry is introduced. The principles of this new approach are presented using the example of the quantitative and qualitative analysis of poly(beta-hydroxybutyric acid) (PHB) accumulated in living bacterial cells as well as dissolved glucose during two 27-h fermentation processes. FT-IR spectra were recorded on-line using a diamond-attenuated total reflection (ATR) cell connected to the fermentation broth by means of a computer-controlled flow system. For calibration of the FT-IR method, standard reference analysis procedures for PHB (gas chromatography) and glucose (HPLC) were used. While pumping the fermentation broth through the flow cell, the recorded spectra corresponded to the fermentation solution, which allowed the determination of glucose in a range from 3.8 to -10.3 g/L. Upon stopping the flow, the cells settled on the ATR surface, and the thus recorded spectra enabled the determination of the intracellular PHB content of Escherichia coli in a range from 0.005 to 0.766 g/L. Errors of cross-validation of 0.264 g/L for glucose and 0.037 g/L for PHB were obtained. Application of one PLS calibration model to another fermentation was possible with prediction errors of 0.493 g/L for glucose and 0.105 g/L for PHB. Furthermore, from the position and shape of the PHB carbonyl band, it could be concluded that the PHB granules inside the E. coli are predominantly amorphous.

3-Hydroxybutyric Acid↗

PubMed2004

Discrimination of olives according to fruit quality using Fourier transform Raman spectroscopy and pattern recognition techniques.

Fourier transform Raman spectroscopy combined with pattern recognition has been used to discriminate olives of different qualities. They included samples of sound olives, olives with frostbite, olives that have been collected from the ground, fermented olives, and olive samples with diseases. Milled olives were measured in a dedicated sample cup, which was rotated during spectrum acquisition. A preliminary study of the data set structure was performed using hierarchical cluster analysis and principal component analysis. Two supervised pattern recognition techniques, K-nearest neighbors and soft independent modeling of class analogy (SIMCA), were tested using a "leave-a-fourth-out" cross-validation procedure. SIMCA provided the best results, with prediction abilities of 95% for sound, 93% for frostbite, 96% for ground, and 92% for fermented olives. The olive samples with diseases (too few to define a class) were included in the validation and recognized as not belonging to any class. None of the damaged olive samples was wrongly predicted to the class of sound olives. With this approach a selection of sound olives for the production of high-quality virgin olive oil can be achieved.

Fourier Analysis↗

PubMed2004

Determination of oil and water content in olive pomace using near infrared and Raman spectrometry. A comparative study.

Near infrared (NIR) reflectance and Raman spectrometry were compared for determination of the oil and water content of olive pomace, a by-product in olive oil production. To enable comparison of the spectral techniques the same sample sets were used for calibration (1.74-3.93% oil, 48.3-67.0% water) and for validation (1.77-3.74% oil, 50.0-64.5% water). Several partial least squares (PLS) regression models were optimized by cross-validation with cancellation groups, including different spectral pretreatments for each technique. Best models were achieved with first-derivative spectra for both oil and water content. Prediction results for an independent validation set were similar for both techniques. The values of root mean square error of prediction (RMSEP) were 0.19 and 0.20-0.21 for oil content and 2.0 and 1.8 for water content, using Raman and NIR, respectively. The possibility of improving these results by combining the information of both techniques was also tested. The best models constructed using the appended spectra resulted in slightly better performance for oil content (RMSEP 0.17) but no improvement for water content.

Least-Squares Analysis↗

PubMed2004

Raman spectroscopy in chemical bioanalysis.

Advances in instrumentation are making Raman spectroscopy the tool of choice for an increasing number of (bio)chemical applications. Raman is an interesting option for several reasons, including the sensitivity to small structural changes, non-invasive sampling capability, minimal sample preparation, and high spatial resolution in the case of Raman micro-spectroscopy. Herein we discuss the most recent technical approaches employed, from the well-known surface enhanced resonance Raman spectroscopy to non-linear Raman techniques such as coherent anti-stokes Raman spectroscopy (CARS) and related techniques. Relevant applications of Raman spectroscopy in the fields of clinical pathology, in vivo and ex vivo imaging, classification and detection of microorganisms and chemical analysis in the past three years are also included.

Biosensing Techniques↗

PubMed2004

On-line fermentation monitoring by mid-infrared spectroscopy.

A new method for on-line monitoring of fermentations using mid-infrared (MIR) spectroscopy has been developed. The method has been used to predict the concentrations of glucose and ethanol during a baker's yeast fermentations. A completely automated flow system was employed as an interface between the bioprocess under study and the Fourier transform infrared (FT-IR) spectrometer, which was equipped with a flow cell housing a diamond attenuated total reflection (ATR) element. By using the automated flow system, experimental problems related to adherence of CO(2) bubbles to the ATR surface, as well as formation of biofilms on the ATR surface, could be efficiently eliminated. Gas bubbles were removed during sampling, and by using rinsing steps any biofilm could be removed from the ATR surface. In this way, constant measuring conditions could be guaranteed throughout prolonged fermentation times (approximately 8 h). As a reference method, high-performance liquid chromatography (HPLC) with refractive index detection was used. The recorded data from different fermentations were modeled by partial least-squares (PLS) regression comparing two different strategies for the calibration. On the one hand, calibration sets were constructed from spectra recorded from either synthetic standards or from samples drawn during fermentation. On the other hand, spectra from fermentation samples and synthetic standards were combined to form a calibration set. Differences in the kinetics of the studied fermentation processes used for calibration and prediction, as well as the precision of the HPLC reference method, were identified as the main chemometric sources of error. The optimal PLS regression method was obtained using the mixed calibration set of samples from fermentations and synthetic standards. The root mean square errors of prediction in this case were 0.267 and 0.336 g/L for glucose and ethanol concentration, respectively.

Biofilms↗

PubMed2004

Separation and on-line distinction of enantiomers: a non-aqueous capillary electrophoresis Fourier transform infrared spectroscopy study.

We report on the separation and on-line distinction of (R,S)-3,5-dinitrobenzoyl leucine (DNB-Leu) enantiomers with non-aqueous capillary electrophoresis (CE) and Fourier transform infrared (FT-IR) spectroscopic detection using O-(tert-butyl carbamoyl) quinine (tBuCQN) as the chiral selector (CS). Due to stereoselective intermolecular interactions--particularly ionic interactions, hydrogen bonding, and pi-pi-interactions--the enantiomers undergo enantioselective complex and ion-pair formation, respectively, with the CS enabling CE separation and direct identification with FT-IR detection. Especially the (S)-enantiomer of the analyte shows significant changes in the mid-infrared region upon complexation, allowing for a clear spectral distinction between both enantiomers. In this way FT-IR spectroscopy represents a novel and attractive detection method for CE enantiomeric separations providing qualitative stereochemical information on the interactions between the chiral selector and the enantiomers, which is hardly accessible by other CE detection methods.

Dinitrobenzenes↗

PubMed2003

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.

Journal Article↗

PubMed2003

Time-resolved Fourier transform infrared spectrometry using a microfabricated continuous flow mixer: application to protein conformation study using the example of ubiquitin.

We report on the use of time-resolved Fourier transform infrared spectroscopy (FT-IR) to study chemically induced conformational changes of proteins using the example of ubiquitin. For this purpose a micromachined mixer is coupled to a conventional IR transmission cell with a pathlength of 25 microm and operated in both the continuous and the stopped-flow mode. This experimental set-up allows the elucidation of reaction pathways in the time frame of about 500 milliseconds to seconds with little reagent consumption and low pressure. For continuous flow measurements employed in the time frame from 0.5 to 1.4 s the reaction time is determined by the flow rate used as the connection between the point of confluence in the micromixer and the flow cell was kept constant in all experiments. For stopped-flow experiments (>1.4 s) the time is determined by data acquisition of the rapid scanning infrared spectrometer. Ubiquitin, a small well-known protein with 76 amino acid residues, changes its conformation from native to A-state with the addition of methanol under low pH conditions. We investigated the conformational change in the time frame from 0.5 to 10 s by mixing ubiquitin (20% methanol-d(4)) with an 80% methanol-d(4) solution at pD 2 by evaluating the time dependent changes in the amide I band of the protein.

Amides↗