Bead injection for surface enhanced Raman spectroscopy: automated on-line monitoring of substrate generation and application in quantitative analysis

The technique of bead injection has been adapted for surface enhanced Raman scattering (SERS) to substantially improve precision, long term stability and sensitivity of SERS detection in analytical chemistry. For this purpose a fully automated flow system comprising a dedicated flow-cell has been developed and tested. With the developed flow-cell, which contains two inlet and two outlet channels, it is possible to retain, perfuse and discharge minute amounts of polymer beads while monitoring all steps by Raman spectroscopy. First, beads carrying cation exchanger moieties were retained in the flow-cell and subsequently perfused with a silver nitrate and a hydroxylamine solution using one inlet of the flow cell. By this sequence homogeneous SERS active silver layers were formed on the beads. The uniformity of the achieved silver layer was studied by secondary electron microscopy. For measurement, the analyte was then introduced from the second inlet channel such that the interaction between the activated SERS beads and analyte occurred in close proximity and within the focus of the laser excitation beam. Due to the complete computer control of all experimental steps, including bead entrapment, SERS layer generation, sample introduction and final bead removal, highly reproducible conditions for SERS were achieved. The method was developed using 9-aminoacridine as a test molecule. Quantitative studies were carried out for 9-aminoacridine and acridine showing linear calibrations from 1-100 nmol l(-1) and 50-1,000 nmol l(-1), respectively, using a sample volume of 200 microl each. Typical relative standard deviations were 4.7% for 9-aminoacrine and 5.8% for acridine.     

Matroid bases with cardinality constraints on the intersection

Mathematical Programming - Given two matroids $$\mathcal {M}_{1} = (E, \mathcal {B}_{1})$$ and $$\mathcal {M}_{2} = (E, \mathcal {B}_{2})$$ on a common ground set E with base sets $$\mathcal...     

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.     

On-line infrared detection in aqueous micro-volume systems

Mid infrared spectroscopy is a non-destructive technique that can provide detailed information on important, molecule-specific features such as the conformation and functional groups of a large range of compounds. Infrared spectroscopy is now an established and frequently used technique for qualitative analysis, i.e. the identification of chemical constituents in a sample. In addition, its use for quantitative purposes has grown dramatically in recent years. It is important to realise that the analytical problem defines the mode of operation and implementation of the FTIR technique. This Highlight article focuses on the advantages and scope of on-line FTIR detection strategies. However, in common with all techniques, on-line FTIR detection has a number of potential shortcomings, which are also discussed.     

Identification of lipophilic bioproduct portfolio from bioreactor samples of extreme halophilic archaea with HPLC-MS/MS

Extreme halophilic archaea are a yet unexploited source of natural carotenoids. At elevated salinities, however, material corrosivity issues occur and the performance of analytical methods is strongly affected. The goal of this study was to develop a method for identification and downstream processing of potentially valuable bioproducts produced by archaea. To circumvent extreme salinities during analysis, a direct sample preparation method was established to selectively extract both the polar and the nonpolar lipid contents of extreme halophiles with hexane, acetone and the mixture of MeOH/MTBE/water, respectively. Halogenated solvents, as used in conventional extraction methods, were omitted because of environmental considerations and potential process scale-up. The HPLC-MS/MS method using atmospheric pressure chemical ionization was developed and tuned with three commercially available C₄₀ carotenoid standards, covering the wide polarity range of natural carotenoids, containing different number of OH-groups. The chromatographic separation was achieved on a C₃₀ RP-HPLC column with a MeOH/MTBE/water gradient. Polar lipids, the geometric isomers of the C₅₀ carotenoid bacterioruberin, and vitamin MK-8 were the most valuable products found in bioreactor samples. In contrast to literature on shake flask cultivations, no anhydrous analogues of bacterioruberin, as by-products of the carotenoid biosynthesis, were detected in bioreactor samples. This study demonstrates the importance of sample preparation and the applicability of HPLC-MS/MS methods on real samples from extreme halophilic strains. Furthermore, from a biotechnological point-of-view, this study would like to reveal the relevance of using controlled and defined bioreactor cultivations instead of shake flask cultures in the early stage of potential bioproduct profiling.     

Determination of yeast assimilable nitrogen content in wine fermentations by sequential injection analysis with spectrophotometric detetection

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 nm 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%.     

Quantum cascade laser modulation for correction of matrix-induced background changes in aqueous samples

A robust method for the determination of carbon dioxide in sugar containing solutions using a single distributed feedback quantum cascade laser (QCL) has been developed. By switching the driving current between two selected values, 1.6 and 1 A, emission wavelengths at 2341.4 and 2341.6 cm^-1 could be achieved. The method is based on absorbance measurements in transmission and the calculation of the absorbance differences between both wavenumbers. This allows the elimination of indirect matrix interference produced on carbon dioxide measurements with increasing sugar concentrations. A flow injection setup was employed to produce carbon dioxide standards from a series of bicarbonate solutions (0–3 g/l) by adjusting the pH with a sodium hydroxide/citric acid buffer solution to pH 3.13. Different concentrations (0–90 g/l) of sugar were also mixed on line with the analyte to study their influence on carbon dioxide measurement. As the difference in the two evaluated wavelengths is small compared to the absorption peak of CO₂, the analytical readout of the QCL modulation can be seen as a proportional parameter to the first derivative of FTIR spectra in this spectral region. PACS 42.55.Px; 42.62.Fi     

Analytical potential of mid-infrared detection in capillary electrophoresis and liquid chromatography: a review

Literature published in the last decade concerning the use of mid-infrared spectrometry as a detection system in separation techniques employing a liquid mobile phase is reviewed. In addition to the continued use of isocratic liquid chromatographic (LC) techniques, advances in chemometric data evaluation techniques now allow the use of gradient techniques on a routine basis, thus significantly broadening the range of possible applications of LC-IR. The general trend towards miniaturized separation systems was also followed for mid-IR detection where two key developments are of special importance. Firstly, concerning on-line detection the advent of micro-fabricated flow-cells with inner volumes of only a few nL for transmission as well as attenuated total reflection measurements enabled on-line mid-IR detection in capillary LC and opened the path for the first successful realization of on-line mid-IR detection in capillary zone electrophoresis as well as micellar electrokinetic chromatography. Secondly, concerning off-line detection the use of micro-flow through dispensers now enables to concentrate eluting analytes on dried spots sized a few tens of micrometers, thus matching the dimensions for sensitive detection by mid-IR microscopy. Finally in an attempt to increase detection sensitivity of on-line mid-IR detection, mid-IR quantum cascade lasers have been used. Applications cover the field of food analysis, environmental analysis and the characterization of explosives among others. Best detection sensitivities for on-line and off-line detection have been achieved in miniaturized systems and are in the order of 50 ng and 2 ng on column, respectively.