University of North Florida
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Stuart Chalk, Ph.D.
Department of Chemistry
University of North Florida
Phone: 1-904-620-1938
Fax: 1-904-620-3535
Email: schalk@unf.edu
Website: @unf

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Bernard Lendl

Abbrev:
Lendl, B.
Other Names:
Address:
Institute of Analytical Chemistry, Vienna University of Technology, Getreidemarkt 9/151, A-1060 Vienna, Austria
Phone:
+43-1-58801-15140
Fax:
+43-1-58801-15199

Citations 28

"Raman, IR, And Surface-enhanced Raman Spectroscopy Of Papaverine: An Automated Setup For In Situ Synthesis Of The Silver Substrate And Recording Of The SER Spectra"
Vib. Spectrosc. 2004 Volume 36, Issue 1 Pages 47-55
N. Leopold, J. R. Baena, M. Bolboac, O. Cozar, W. Kiefer and B. Lendl

Abstract: Raman, IR, and surface-enhanced Raman scattering (SERS) were employed for the vibrational characterization of papaverine in the free and adsorbed state. The vibrational assignments were performed by means of density functional theory (DFT) calculations. The adsorption of the molecule was concluded with the isoquinoline and the benzene ring close to the silver surface in tilted orientations. Employing a sequential injection system, an automatized setup was developed for reliable, accurate and rapid recording of FT-SER spectra. The colloidal silver substrate was synthesized in situ in a 2 mL quartz cuvette by reducing silver nitrate with hydroxylamine. The FT-SER spectrum of papaverine obtained with the developed experimental setup exhibits the same characteristics as the SER spectrum obtained with a citrate reduced silver colloid.

"Assessment Of Quantum Cascade Lasers As Mid Infrared Light Sources For Measurement Of Aqueous Samples"
Vib. Spectrosc. 2002 Volume 29, Issue 1-2 Pages 283-289
Malin Kölhed, Michael Haberkorn, Viktor Pustogov, Boris Mizaikoff, Johannes Frank, Bo Karlberg and Bernhard Lendl

Abstract: The potential of Fabry Perot Quantum Cascade (QC) lasers for transmission measurements of aqueous samples was assessed. For this purpose two lasers, one lasing in the region of the bending vibration of water at 1650 cm-1, and the other in the water window at 1080 cm-1, were used in a flow injection system comprising a dedicated fiber-optic flow cell with an adjustable path length. As test analytes a nucleic base (adenine) and a nucleoside (xanthosine) were used. Compound-specific information was obtained as the nucleoside contained a carbohydrate unit in addition to the heteroaromatic ring structure. With the laser emitting at 1650 cm-1 the heteroaromatic ring structure of both compounds gave rise to flow injection peaks, whereas, only the nucleoside, containing the C-O stretching vibrations of the carbohydrate unit, produced peaks also with the laser emitting at 1080 cm-1. The path length limitations were investigated for both lasers. For the 1650 cm-1 laser, path lengths up to 59 mum could be used, despite the strong absorption of water. For xanthosine analysis, the limit of detection (S/N = 3) at this path length was determined to be 0.07 g L-1. Optical path lengths of up to 150 mum could be used for the laser emitting at 1080 cm-1. (C) 2002 Elsevier Science B.V. All rights reserved.

"Flow-through Sensors For Enhancing Sensitivity And Selectivity Of FTIR Spectroscopy In Aqueous Media"
Vib. Spectrosc. 1999 Volume 19, Issue 1 Pages 1-10
B. Lendl and R. Schindler

Abstract: The principle of novel flow-through sensor systems with FTIR spectroscopic detection is presented on the example of the determination of organic acids in aqueous solution. The constructed flow-through sensor system is based on trapping of derivatized porous polymer beads in a conventional IR transmission cell and integration of the flow cell into a sequential injection (SI) manifold. By the SI-manifold sample pre-conditioning, sample-sensor interaction and sensor regeneration were performed in an automated and highly reproducible way. The polymer beads used in this study contained anion exchanger groups so that negatively charged molecules such as organic acids present in the anionic form could selectively interact with the polymer beads. Upon pumping a sample through the sensor cell organic acids were retained on the polymer beads whereas non-ionic matrix molecules passed hence allowing to separate the target analytes form the matrix. Apart from that the organic acids were also concentrated onto the polymer beads so that absolute analyte amounts in the low µg range could easily be detected. Linear calibration curves from 0 to 1 mmol L-1 were recorded for acetic and malic acid using a sample volume of 500 µl (s(x0): 0.032 mmol L-1 acetic acid and 0.031 mmol L-1 malic acid). Mixtures of both acids were analyzed as well and it could be shown that by application of multivariate data evaluation procedures (PLS) simultaneous quantification of both acids could be performed successfully using the developed flow-through sensor system.

"Determination Of Enzyme Kinetics And Chemometric Evaluation Of Reaction Products By FTIR Spectroscopy On The Example Of β-fructofuranosidase"
Vib. Spectrosc. 1998 Volume 16, Issue 2 Pages 127-135
R. Schindler, H. Le Thanh, B. Lendl* and R. Kellner

Abstract: FTIR spectroscopy in combination with a sequential injection analysis (SIA) manifold was used for the investigation of the kinetic properties of the quant. anal. of the reaction mixtures of β-fructofuranosidase. Different sucrose solutions were prepared automatically and with the quant. results provided by the chemometric spectra evaluation, the Km was estimated to be 43 mM.
Enzyme, β-fructofuranosidase Spectrophotometry Sequential injection Chemometrics Kinetic

"Containerless Reaction Monitoring In Ionic Liquids By Means Of Raman Microspectroscopy"
Lab on a Chip 2007 Volume 7, Issue 1 Pages 126-132
Mercedes López-Pastor, Ana Domínguez-Vidal, María José Ayora-Cañada, Thomas Laurell, Miguel Valcárcel and Bernhard Lendl

Abstract: 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 analyzed 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. © The Royal Society of Chemistry.

"Association Of Methanol And Water In Ionic Liquids Elucidated By Infrared Spectroscopy Using Two-dimensional Correlation And Multivariate Curve Resolution"
J. Phys. Chem. B 2006 Volume 110, Issue 22 Pages 10896-10902
Mercedes López-Pastor, María José Ayora-Cañada, Miguel Valcárcel, and Bernhard Lendl

Abstract: 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-methyhmidazolium hexafluorophosphate (bmimPF6). Serf-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. © 2006 American Chemical Society.

"Toward The Optical Tongue: Flow-through Sensing Of Tannin-protein Interactions Based On FTIR Spectroscopy"
J. Am. Chem. Soc. 2002 Volume 124, Issue 49 Pages 14741-14747
Andrea Edelmann and Bernhard Lendl

Abstract: The interaction of polyphenols (tannins) with proline-rich proteins (gelatin) has been studied using an automated flow injection system with Fourier transform infrared spectroscopic detection to gain insight into chemical aspects related to astringency. In the perception of astringency, a major taste property in red wines and other beverages such as beer, tea, or fruit juices, an interaction between proline-rich salivary proteins and tannins present in the sample takes place. To study this interaction, agarose beads carrying gelatin (a proline-rich protein) were placed in the IR flow cell in such a way that the beads were probed by the IR beam. Using an automated flow system, we injected samples in a carrier stream and flushed over the proteins in a highly reproducible manner. Simultaneously, any retardation due to tannin-protein interactions taking place inside the flow cell was monitored by infrared spectroscopy. Tannins of different sources (grapes, wooden barrels, formulations used in wine making) were investigated, and their flow-through behavior was characterized. Significant differences in their affinity toward gelatin could be observed. Furthermore, because of small but characteristic differences in the IR spectrum, it is possible to distinguish condensed from hydrolyzable tannins. Nonastringent substances such as alcohols, sugars, and acids did not show retention on gelatin. The selectivity of the flow-through sensor was also demonstrated on the example of red and white wine. In contrast to white wine, where no interaction could be observed, in red wine a major interaction of the red wine tannins was found.

"Flow Through Picoliter Dispenser: A New Approach For Solvent Elimination In FT-IR Spectroscopy"
Appl. Spectrosc. 2002 Volume 56, Issue 7 Pages 902-908
Michael Haberkorn, Johannes Frank, Michael Harasek, Johan Nilsson, Thomas Laurell, and Bernhard Lendl

Abstract: A new interface for FT-IR analysis of liquid samples on the basis of solvent elimination is presented. The approach is based on a piezoactuated flow-through microdispenser, a device built of two microstructured silicon wafers designed for micro-liquid handling. It could be verified during preliminary studies using a sequential injection (SI) system for automated liquid handling that the flow-through microdispenser as a possible interface for flow system-FT-IR analysis has the capability of meeting the demands of hyphenated miniaturized liquid handling systems (e.g., µHPLC, microhigh performance liquid chromatography), as it successfully provides highly stable, reliable and reproducible operating conditions for liquid handling in the picoliter range. Moreover, an increase in sensitivity for FT-IR measurements could be achieved, lowering the mass detection limit of sugars (such as the investigated sucrose) to 53 picograms. As is demonstrated on the example of an HPLC separation of a mixture of glucose and fructose, interfacing LC systems to FT-IR using a piezoactuated flow-through microdispenser is a feasible and promising approach.

"Determination Of Free Fatty Acids In Edible Oils By Continuous-flow Analysis With FT-IR Spectroscopic Detection"
Appl. Spectrosc. 2001 Volume 55, Issue 3 Pages 356-360
María Jose Ayora Cañada, Antonio Ruiz Medina, and Bernhard Lendl

Abstract: A flow injection system with Fourier transform infrared spectroscopic detection was developed for the rapid determination of the free fatty acid content in edible oils. The method is based on complete deprotonation of the free fatty acids and evaluation of the intensity of the upsilon (as)-(COO-) band formed at 1570 cm-1 with respect to a baseline point at 1534 cm-1. Upon deprotonation the absorption of the carboxylic acid moieties is shifted toward lower frequency, thus avoiding spectral interferences of the upsilon -C=O from esters, ketones, or aldehydes also present in the sample. Optimum operational conditions were obtained by merging the sample stream first with a carrier stream of 25% (v/v) toluene in 2-propanol followed by merging with a 0.3 M solution of KOH in the same solvent mixture and measurement of the obtained solution after a short (2 s) reaction time, In this way saponification of the oil could be reduced to a minimum, whereas deprotonation was already completed. The obtained calibration curve was linear from 0 to 2.5% oleic acid, and the coefficient of variation of the method was 3.3%. The developed method was applied to the determination of the total amount of free fatty acids in olive, sunflower, and corn oils, delivering results that were in good agreement with those obtained by the official reference method proposed by the European Community. Furthermore, due to its high degree of automation and throughput (40 samples h-1), the developed method is suitable for quality control in routine applications.

"Improved Fiber-detector Coupling For MIR Spectroscopy Employing Shaped Silver Halide Fibers"
Appl. Spectrosc. 2000 Volume 54, Issue 10 Pages 1417-1422
J. Frank, R. Schindler, O. Lendl, and B. Lendl

Abstract: An improved fiber-detector coupling for mid-infrared (MIR) spectroscopy employing silver halide fibers (AgClxBr1-x) and a mercury cadmium telluride (MCT) detector was developed. The key feature was the use of specially machined fiber pieces capable of focusing MIR radiation onto small detector elements. For this purpose new units for the machining and characterization of silver halide fibers were constructed. For fiber machining, a computer-controlled turning lathe was developed, which allowed highly precise fabrication of various fiber tips such as spherically, hyperboloidally, and paraboloidally shaped ones. The intensity distribution in space of MIR radiation emitted by the produced fibers was investigated with a dedicated MCT detector employing a flexible fiber-detector coupling. For this purpose the position of the fiber was kept constant while the light distribution in front of the fiber tip was scanned with the MCT element by moving the detector housing via stepper motors. Different fiber tip geometries were investigated for their influence on the spatial light distribution. With the use of shaped fiber tips, focusing of the IR beam was achieved, allowing the use of smaller detector elements, which in turn improves the signal-to-noise ratio in the case of MCT detection elements. Optimum conditions were found for fibers having a shape radius of 1 mm, being two times the fiber radius, whereas no significant differences between spherically and hyperboloidally as well as paraboloidally shaped fibers were observed. The developed fiber-detector coupling was furthermore combined with a fiber-optic flow-through cell for transmission measurement (optical path: 30 mum) and incorporated in a how injection analysis system. With this experimental setup, reasonable MIR spectra of an aqueous sucrose solution could be recorded by injecting as little as 30 nL of sample containing 0.5 µg of sucrose.

"Flow Analysis-based Surface-enhanced Raman Spectroscopy Employing Exchangeable Microbeads As SERS-active Surfaces"
Appl. Spectrosc. 2000 Volume 54, Issue 7 Pages 1012-1018
Bernhard Lendl, Horst Ehmoser, Johannes Frank, and Rupert Schindler

Abstract: This paper reports on a new fully automated approach for highly sensitive and robust surface-enhanced Raman spectroscopic (SERS) detection using flow systems. An aqueous solution of silver nitrate was reduced by the addition of sodium borohydride and the thus formed silver sols adsorbed on Sephadex anion exchanger microbeads (dry bead size: 50-120 µm) present in the reaction mixture. In this way microbeads carrying a SERS-active layer were generated that could be easily pumped in the how system without facing problems due to Ag-sol adsorption on the Teflon(R) tubing. After online generation of the SERS-active microbeads, the analyte (test analyte: nicotinic acid) was added, and the homogeneous SERS-microbead-analyte suspension pumped into a specially designed dow cell where the beads were retained and the SERS spectra measured with a Fourier transform (FT)-Raman instrument with a laser wavelength of 1064 nm. Afterwards the SERS microbeads were discarded by flow reversal and new, freshly prepared SERS microbeads together with the analyte could be introduced automatically into the flow system. The developed method is characterized by complete computer control of all operation steps, high sensitivity of the SERS measurement, high precision, and long-term stability. The concentration dependence of the SERS signals was investigated over a range from 5.10^-6 M to 0.1 M nicotinic acid. The signal intensities increased until 1 mM, after which no further rise in the SERS intensities was found. Typical precision achieved was 8% throughout the whole calibration range.
Adsorption

"Mid-IR Spectroscopy For The Quantification Of Metal Ions In Aqueous Solution In The Nanogram Range"
Appl. Spectrosc. 2000 Volume 54, Issue 5 Pages 676-680
Amparo Pérez-Ponce and Bernhard Lendl

Abstract: A novel mid-infrared (IR) dow-through sensor system for the determination of metal ions in aqueous solution was developed. The flow-through sensor comprised weak cation exchanger beads (Sephadex with carboxylic acid moieties) placed in the optical path of a 37 µm IR transmission cell. The combination of the flow-through sensor with a sequential injection analysis (SIA) system allow ed us to carry out the whole analysis procedure including analyte-sensor interaction and sensor regeneration, as well as data acquisition, under computer control. The first step of an analysis sequence was regeneration of the flow-through sensor by rinsing with diluted (0.012 M) hydrochloric acid followed by distilled water, thus protonating the carboxylic acid moieties. Then, after acquisition of a background spectrum and pumping 500 µL of sample followed by 1000 µL of distilled water through the flow-through sensor, the sample spectrum was recorded. Upon passage of the sample, the metal ions were retained on the cation exchanger, giving rise to spectral changes mainly resulting from the exchange of the protons by metal cations. It was found that the spectral changes were characteristic for the metal cations investigated. This observation was exploited to construct a multivariate calibration model for the simultaneous determination of calcium and magnesium ions in water. The developed method covered the range from 0 to 200 nmol for each cation (root mean square error of cross-validation: 9.2, and 7.6 nmol for Ca2+ and Mg2+, respectively) and was successfully applied to the determination of the water hardness of the tap water in our laboratory.

"FTIR Microscopy As Novel Detection System In Micro Flow Injection Analysis"
Anal. Methods Instrum. 1995 Volume 2, Issue 1 Pages 52-54
Kellner, Robert; Lendl, Bernhard

Abstract: A Bruker 113v FTIR spectrometer was coupled with a Bruker IR microscope equipped with a HgCdTe detector. A flow-through cell was developed for this system comprising three parts; the central part of which was constructed of PVC with an inner volume of 1.6 µL and an optical pathlength of 500 µm. The windows consisted of two Si plates (diameter 5.5 mm, thickness 0.75 mm). As the IR beam is focused by the FTIR microscope down to 80 µm it was possible to probe volumes of 10 nl. IBMK was determined in hexane in the ppm range as a model substance. Possible future applications of the system as a µtotal analysis system are discussed.
Methyl isobutyl ketone Organic compound Microscopy Apparatus MIBK

"FTIR Spectroscopy As Detection Principle In Aqueous Flow Analysis"
Anal. Commun. 1999 Volume 36, Issue 4 Pages 123-126
R. Schindler and B. Lendl

Abstract: Whereas FTIR spectroscopic detection is routinely used in gas chromatography (B. Erikson, Anal. Chem., 1998, 70, 801A), its use for liquid chromatography (LC) and flow injection analysis (FIA) is a rather exotic exception. The most prominent reason is the strong IR absorption of most of the common solvents, especially water. Hence FTIR spectroscopy is normally not even considered a valuable detection method. This practice neglects that FTIR spectroscopy offers some unique features which now, using modern instrumentation, can be exploited in an advantageous manner. It is the aim of this Highlight article to demonstrate the wide range of possible applications in LC and FIA. To regard FTIR spectroscopy as too exotic for routine use may be a luxury paid for with the neglect of a simple analytical approach. The term flow analysis (FA) will be used to provide a common cover for both LC and FIA because both rely on the injection of a sample into a flowing stream, passage through a modulator and recording of transient peaks. Although the processes taking place in the modulator are different, being chemical reactions in FIA and separations in LC, the same interfaces can be used for a FTIR spectrometer as a detector. The interfaces used can be divided into two categories, flow through cells where the liquid is probed directly, and solvent removal interfaces where the analyte is separated from the carrier liquid prior to detection. It is necessary to emphasize the complementary nature of these techniques (D. E. Pivonka and K. M. Kirkland, Appl. Spectrosc., 1997, 51, 866) and this Highlight will stress their particular strengths and weaknesses. Special focus is laid on aqueous phase systems because of their high importance in biological systems. As water is certainly the most challenging solvent for IR detection, equal or even better performance of the presented approaches can be expected for other solvents. Additionally three developments will be discussed in detail with respect to their prospects for FA-FTIR instruments: the increasing availability of sophisticated chemometric methods, the miniaturization of analytical instruments and application to combinatorial chemistry.
Combinatorial chemistry

"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"
Anal. Chem. 2003 Volume 75, Issue 9 Pages 2166-2171
Nicolae Leopold, Michael Haberkorn, Thomas Laurell, Johan Nilsson, Josefa R. Baena, Johannes Frank and Bernhard Lendl

Abstract: 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.

"Mid-infrared Quantum Cascade Lasers For Flow Injection Analysis"
Anal. Chem. 2000 Volume 72, Issue 7 Pages 1645-1648
B. Lendl, J. Frank, R. Schindler, A. Müller, M. Beck, and J. Faist

Abstract: A Fabry-Perot quantum cascade laser (QCL) was used as a powerful light source for mid infrared (MIR) detection in flow injection analysis. The QCL lased at several wavelengths close to each other within a few wavenumbers (990-1010 cm-1), hence fitting well to the broad absorption bands of molecules in liquid phase. As compared with that of a state-of-the-art Fourier transform spectrometer, the signal-to-noise ratio could be improved by a factor of 50. Additionally, by using a QCL as the light source, optical path lengths of more than 100 m could be used even in aqueous matrixes which reduced the danger of cell clogging. In the example shown here phosphate was determined in Diet Coke samples. The flow injection system used allowed the measurement of the sample at two pH values (5 and 13) at which the analyte was present as H2PO4- and PO43-, respectively. As the analytical readout the difference in IR absorption of H2PO4- and PO43- at the laser wavelengths was taken. The FIA-QCL measurements were corroborated by ion chromatography which was used as a reference method.

"Direct, Reagent-free Determination Of Free Fatty Acid Content In Olive Oil And Olives By Fourier Transform Raman Spectrometry"
Anal. Chim. Acta 2003 Volume 487, Issue 2 Pages 211-220
Barbara Muik, Bernhard Lendl, Antonio Molina-Díaz and María José Ayora-Cañada

Abstract: Fourier transform (FT) Raman spectrometry in combination with partial least squares (PLS) regression was used for direct, reagent-free determination of free fatty acid (FFA) content in olive oils and olives. Oils were directly investigated in a simple flow cell. Milled olives were measured in a dedicated sample cup, which was rotated eccentrically to the horizontal laser beam during spectrum acquisition in order to compensate sample heterogeneity. Both external and internal (leave-one-out) validation were used to assess the predictive ability of the PLS calibration models for FFA content (in terms of oleic acid) in oil and olives in the range 0.20-6.14 and 0.15-3.79%, respectively. The root mean square error of prediction (RMSEP) was 0.29% for oil and 0.28% for olives. The predicted FFA contents were used to classify oils and olives in different categories according to the European Union regulations. Ninety percent of the oil samples and 80% of the olives were correctly classified. These results demonstrate that the proposed procedures can be used for screening of good quality olives before processing, as well as, for the on-line control of the produced oil.

"Mid-infrared Spectroscopy Coupled To Sequential Injection Analysis For The On-line Monitoring Of The Acetone-butanol Fermentation Process"
Anal. Chim. Acta 2001 Volume 438, Issue 1-2 Pages 175-186
Mustafa Kansiz, J. Richard Gapes, Don McNaughton, Bernhard Lendl and K. Christian Schuster

Abstract: Fourier transform infrared (FTIR) spectroscopy, coupled to sequential injection analysis (SIA) was employed fur the on-line monitoring of an acetone-butanol fermentation by simultaneously determining acetone, acetate, ra-butanol, butyrate and glucose from the mid-IR spectra of the samples. The analysis system (SIA-FTIR) developed was completely computer controlled, requiring only minute amounts of NaOH and Na2CO3 as reagents and was capable of performing 30 analyte determinations per hour From a batch fermentation run of 55 h duration, samples were automatically drawn and analyzed at 14 different points of time. The analysis by SIA-FTIR comprised of triplicate injections, from each of which two spectra one with 4 cm-1 the other with 8 cm-1 spectral resolution were obtained. In addition, triplicate reference analysis by gas chromatography and enzymatic test kits were also done at corresponding points of time. Based on the data of the reference analysis a partial least squares (PLS) model was established and validated by a leave-one-out cross validation. Best results were obtained using the second derivatised spectra collected at 8 cm-1 resolution. The regression coefficients and standard errors of prediction (S.E.P.) were as follows: acetone, r = 0.999, S.E.P. = 0.077 g/l; acetate, r = 0.998, S.E.P. = 0.063 g/l; butyrate, r = 0.955 and S.E.P. = 0.058 g/l; il-butanol, r = 0.999, S.E.P. = 0.301 g/l and glucose, r = 0.999, S.E.P. = 0.493 g/l. The high quality of the results obtained, and the observation that the precision of the SIA-FTIR analysis was as good and often better than those of the reference methods, showed that SIA-FTIR is a powerful tool for rapid on-line bioprocess monitoring.

"Sequential Injection Fourier Transform Infrared Spectroscopy For The Simultaneous Determination Of Organic Acids And Sugars In Soft Drinks Employing Automated Solid Phase Extraction"
Anal. Chim. Acta 2000 Volume 422, Issue 1 Pages 63-69
Hai LeThanh and Bernhard Lendl

Abstract: A fully automated method for the rapid determination of organic acids (citric-, malic- and tartaric acid) and sugars (glucose, fructose, and sucrose) in soft drinks by sequential injection Fourier transform infrared (FTIR) spectroscopy is presented. A convective interaction media (CIM) disc carrying quaternary amino moieties was added as a solid phase extraction column to the flow system. Upon injection of a sample the organic acids were completely retained on the CIM disc whereas sugars passed to the flow cell. The organic acids were subsequently eluted by injection of an alkaline (pH 8.5) 1 M sodium chloride solution and recorded in their fully deprotonated form as a second flow injection peak. In both cases, the FTIR spectra corresponding to the peak maxima were selected for data evaluation. Two partial least squares models, one for sugars and the other for organic acids, were constructed based on the analysis of standards containing all six analytes. The developed method was applied to natural samples yielding results which were in good agreement with those obtained by an external reference method (enzymatic test kits). Deviations in the results were 3.4. and 4.1% for citric and malic acid and ranged from 4.7-5.1% for the sugars. The developed method is characterized by its short analysis time, experimental simplicity and its potential applications in routine analysis and process control.
Citric acid l-Malic acid Tartaric acid Glucose Fructose Sucrose Soft drink Spectrophotometry Sequential injection Preconcentration Method comparison Solid phase extraction

"Sheath-flow Fourier Transform Infrared Spectrometry For The Simultaneous Determination Of Citric, Malic And Tartaric Acids In Soft Drinks"
Anal. Chim. Acta 2000 Volume 417, Issue 1 Pages 41-50
María José Ayora-Cañada and Bernhard Lendl

Abstract: A new approach for the determination of organic acids in soft drinks based on pH modulation and Fourier transform infrared spectroscopic detection is presented. The analytical readout was taken from the spectral changes induced by the pH change of the sample. The pH modulation was carried out in a novel sheath-flow cell which was connected to a sequential injection analysis system. The sheath-flow cell comprised three stream lines flowing adjacent to each other in a strongly laminar fashion, The sample (pH similar to 9) was introduced in the central channel whereas reagent (hydrochloric acid) was introduced in the outer channels. As a consequence of the laminar flow profile, hardly any mixing between the stream lines was observed while maintaining the flow, hence allowing the measurement of the sample spectrum at alkaline pH (similar to 9). Upon stopping the flow, diffusion of protons from the outer stream lines into the central line occurred resulting in a complete protonation of the analyte (pH similar to 2). The spectral changes were calculated and the region between 1400 and 1180 cm-1 used to set-up a partial least squares (PLS) calibration model. For the PLS model only standards containing the analytes but no matrix molecules were used. By evaluation of the spectral changes induced by the pH modulation the PLS model could successfully be applied to test samples containing sugars as well as to natural soft drinks.
Citric acid l-Malic acid Tartaric acid Soft drink Spectrophotometry Sequential injection Sheath flow Partial least squares Stopped-flow

"Simultaneous Determination Of Enzyme Activities By FTIR-spectroscopy In An One-step Assay"
Anal. Chim. Acta 1999 Volume 391, Issue 1 Pages 19-28
R. Schindler and B. Lendl

Abstract: The simultaneous quantification of β-fructofuranosidase (invertase, EC 3.2.1.26) and glucan 1,4-α-glucosidase amyloglucosidase, EC 3.2.1.3) activities in an one-step assay is reported. The approach is based on chemometric evaluation of the mid-IR spectra of the reaction mixture, measured with a sequential injection (SI) system with FTIR spectroscopic detection. Two partial least squares (PLS) calibration models were established automatically. The first model was calculated from reaction mixtures after the action of standard mixtures of both enzymes. The other was built up with mixtures of the substrates (maltose, sucrose) and products (fructose, glucose) without any enzymatic reaction. The establishment of the second model was more rapid as no time for an enzymatic reaction is necessary. Both models gave comparable results for the prediction of mixtures containing 0-2.0 U/ml of each enzyme, with mean errors between 0.08 and 0.05 U/ml.
Multivariate calibration Sequential injection

"Simultaneous Determination Of α-amylase And Amyloglucosidase Activities Using Flow Injection Analysis With Fourier Transform Infrared Spectroscopic Detection And Partial Least-squares Data Treatment"
Anal. Chim. Acta 1998 Volume 366, Issue 1-3 Pages 35-43
R. Schindler, B. Lendl* and R. Kellner

Abstract: A flow injection analysis (FIA) system with fourier transform IR (FTIR) spectroscopic detection for the simultaneous determination of amyloglucosidase (exo-glucan 1,4-α-glucohydrolase, EC 3.2.1.3) and α-amylase (endo-glucan 1,4-α-glucanohydrolase, EC 3.2.1.1) activities in aqueous solution is proposed. Starch hydrolysis catalyzed by both enzymes was monitored in the mid-IR range by recording IR-spectra of the assay solutions before and after hydrolysis. The intensities of the resulting difference spectra were directly related to the enzyme activities. Furthermore, the different reaction pathways of the two enzymes under investigation produced distinct spectral changes resulting in difference spectra characteristic for each enzyme. Using partial least squares (PLS) regression it was possible to simultaneously determine both enzyme activities covering a range of 38-150 U/l (630-2500 nkat/l) for amyloglucosidase and a range of 500-3750 U/l (8300-62000 nkat/l) for α-amylase. Absorption due to the sample matrix was successfully eliminated by calculation of the difference spectra. The developed method was also successfully applied to the anal. of spiked fermentation broth samples yielding mean deviations of 2.6% and 10.8% for amyloglucosidase and α-amylase, respectively.
α-Amylase Amyloglucosidase Fermentation broth Spectrophotometry Simultaneous analysis Catalysis Interferences Partial least squares

"Automated Sample Preparation And Analysis Using A Sequential-injection- Capillary Electrophoresis (SI-CE) Interface"
Analyst 2006 Volume 131, Issue 6 Pages 739-744
Stephan Kulka, Guillermo Quintás and Bernhard Lendl

Abstract: 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 ?g 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 analyzed 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 analyzed with off-line infrared spectroscopy (IR), confirming these results. © The Royal Society of Chemistry 2006.

"A Mid-IR Flow-through Sensor For Direct Monitoring Of Enzyme Catalysed Reactions. Case Study: Measurement Of Carbohydrates In Beer"
Analyst 2002 Volume 127, Issue 1 Pages 109-113
M. Haberkorn, P. Hinsmann and B. Lendl

Abstract: A novel mid-IR flow-through sensor for in situ monitoring of the enzymatic reaction of amyloglucosidase with carbohydrates was developed. Amyloglucosidase was immobilized on agarose beads with N-ethyl-N-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and directly placed in a conventional IR flow-through cell. The carbohydrate content of various beer samples was then determined by following the enzymatic hydrolytic cleavage of carbohydrates to glucose with Fourier-transform infrared (FTIR) spectroscopy. The whole procedure was done in an automated way operating in the stopped flow mode by incorporating the flow-through sensor in a sequential injection (SI) manifold. As the immobilized enzyme was directly probed by the IR beam, an in situ study of the enzymatic reaction was possible enabling determination of the Michaelis-Menten constant of the immobilized enzyme. A linear calibration curve was recorded using maltose standards in the range between 0.86 and 7.13 g L-1. The proposed method was successfully applied to the determination of the carbohydrate content of four different beer samples by the standard addition method. Moreover experiments showed the possibility of monitoring in situ the immobilization of the enzyme as well as a small organic acid (malic acid) on the agarose beads using EDC.

"A Rapid Method For Peroxide Value Determination In Edible Oils Based On Flow Analysis With Fourier Transform Infrared Spectroscopic Detection"
Analyst 2001 Volume 126, Issue 2 Pages 242-246
Antonio Ruíz, María Jose Ayora Cañada and Bernhard Lendl

Abstract: The development of an automated, rapid and highly precise method for determination of the peroxide value in edible oils based on a continuous flow system and Fourier transform infrared (FTIR) spectroscopic detection is described. The sample stream was mixed with a solvent mixture consisting of 25% (v/v) toluene in hexanol which contained triphenylphosphine (TPP). The hydroperoxides present in the sample reacted stoichiometrically with TPP to give triphenylphosphine oxide (TPPO) which has a characteristic and intense absorption band at 542 cm-1. A 10% (m/v) TPP solution in the solvent mixture and a 100 cm reaction coil were necessary for complete reaction. FTIR transmission spectra were recorded using a flow cell equipped with CsI windows having an optical pathlength of 100 mum. By using tert-butyl hydroperoxide spiked oil standards and evaluation of the band formed at 542 cm-1 a linear calibration graph covering the range 1-100 PV (peroxide value; meq. O-2 kg-1 oil) was obtained. The relative standard deviation was 0.23% (n = 11) and the throughput 24 samples h-1. The developed system was also applied to the determination of PV in olive, sunflower and corn oils, showing good agreement with the official reference method of the European Community which is based on titration using organic solvents. The results obtained clearly show that the developed method is superior to the standard wet chemical method, hence suggesting its application in routine analysis and quality control.

"A Novel Flow Injection Procedure For Determination Of Phosphate In Industrial Raw Phosphoric Acid"
Analyst 2000 Volume 125, Issue 6 Pages 1211-1213
Houyem Abderrazak, Mohamed Dachraoui and Bernhard Lendl

Abstract: A new method for the rapid determination of the phosphate content of highly concentrated industrial raw phosphoric acid, as obtained in the Tunisian mining industry, is reported. The method is based on the injection of the phosphate-containing sample in to a stream of FeCl3 and subsequent measurement of the decrease in absorption at 334 nm due to the formation of an iron(III) phosphate complex. With the developed method, a linear calibration curve from 0.1 to 20 g L-1 H3PO4 was obtained. For the analysis of samples drawn from a phosphoric acid production site, dilution by a factor of 100 was necessary in order to fit the linear range of the calibration curve. With the developed procedure a sampling frequency of 30 samples per hour and a standard deviation of 1.4% was achieved. The developed method was applied to the analysis of natural samples and the results obtained were in excellent agreement with those obtained by gravimetry.

"Surface Enhanced Raman Spectroscopy As A Molecular Specific Detection System In Aqueous Flow-through Systems"
Analyst 1998 Volume 123, Issue 5 Pages 1057-1060
N. Weißenbacher, B. Lendl, J. Frank, H. D. Wanzenböck and R. Kellner

Abstract: Surface enhanced Raman scattering (SERS) is proposed as a mol. specific technique for direct measurements of organic molecules in aqueous solutions An FT-Raman spectrometer was interfaced with a flow injection manifold operated in the stopped-flow mode enabling reproducible collection of SERS spectra due to the automation of the anal. procedure. For SERS a solid state substrate placed in a newly developed flow-cell was used. Multiple measurements on one single SERS substrate were achieved by rinsing the substrate with reagents such as 3 M KCl or 0.1 M NaOH solutions prior to the next measurements to remove retained analytes from the surface of the SERS substrate. This procedure allowed for improved precision as compared with a conventional batch approach. Quant. aspects were studied by establishing a calibration curve for nicotinic acid which was used as a model analyte. A linear dependence of the recorded SERS intensities from the logarithm of the analyte concentration. was obtained throughout the whole studied concentration. range (0.001-0.1 M, correlation coefficient r2 = 0.97). The standard deviation of the method sx0 is 0.122 mM and detection limit 1.7 mM, respectively. The results demonstrate the potential of SERS spectroscopy to be used as a molecular specific detector in aqueous flow systems such as flow injection anal.
Nicotinic acid Raman Raman Stopped-flow Flowcell Apparatus Method comparison

"Determination Of Yeast Assimilable Nitrogen Content In Wine Fermentations By Sequential Injection Analysis With Spectrophotometric Detetection"
Anal. Bioanal. Chem. 2002 Volume 374, Issue 1 Pages 167-172
Barbara Muik, Andrea Edelmann, Bernhard Lendl, María José Ayora-Cañada

Abstract: An automated method for measuring the primary amino acid concentration in wine fermentations by sequential injection analysis with spectrophotometric detection was developed. Isoindole-derivatives from the primary amino acid were formed by reaction with o-phthaldialdehyde and N-acetyl-L-cysteine and measured at 334 mn with respect to a baseline point at 700 nm to compensate the observed Schlieren effect. As the reaction kinetic was strongly matrix dependent the analytical readout at the final reaction equilibrium has been evaluated. Therefore four parallel reaction coils were included in the flow system to be capable of processing four samples simultaneously. Using isoleucine as the representative primary amino acid in wine fermentations a linear calibration curve from 2 to 10 mM isoleucine, corresponding to 28 to 140 mg nitrogen/L (N/L) was obtained. The coefficient of variation of the method was 1.5% at a throughput of 12 samples per hour. The developed method was successfully used to monitor two wine fermentations during alcoholic fermentation. The results were in agreement with an external reference method based on high performance liquid chromatography. A mean-t-test showed no significant differences between the two methods at a confidence level of 95%.