Wim Th. Kok

Influence of temperature on vibrational spectra and consequences for the predictive ability of multivariate models.

Temperature, pressure, viscosity, and other process variables fluctuate during an industrial process. When vibrational spectra are measured on- or in-line for process analytical and control purposes, the fluctuations influence the shape of the spectra in a nonlinear manner. The influence of these temperature-induced spectral variations on the predictive ability of multivariate calibration model is assessed. Short-wave NIR spectra of ethanol/water/2-propanol mixtures are taken at different temperatures, and different local and global partial least-squares calibration strategies are applied. The resulting prediction errors and sensitivity vectors of a test set are compared. For data with no temperature variation, the local models perform best with high sensitivity but the knowledge of the temperature for prediction measurements cannot aid in the improvement of local model predictions when temperature variation is introduced. The prediction errors of global models are considerably lower when temperature variation is present in the data set but at the expense of sensitivity. To be able to build temperature-stable calibration models with high sensitivity, a way of explicitly modeling the temperature should be found.

Linear techniques to correct for temperature-induced spectral variation in multivariate calibration

Abstract The influence of external physical variation such as temperature fluctuations on near-infrared (NIR) spectra and their effect on the predictive power of calibration models such as PLS have been studied. Different methods to correct for the temperature effect by explicitly including the temperature in a calibration model have been tested. The results are compared to the implicit inclusion, which takes the temperature into account only through the calibration design. Two data sets are used, one well-designed data set measured in the laboratory and one industrial data set consisting of measurements for process samples. For both data sets, the explicit inclusion of the temperature in the calibration models did not result in an improvement of the prediction accuracy compared to implicit inclusion.

Capillary electrochromatography with macroporous particles

The performance of macro-porous particles in capillary electrochromatography is studied. Three reversed-phase stationary phases with pore diameters between 500 A and 4000 A have been tested for separation efficiency and mobile phase velocity. With these stationary phases, a large portion of the total flow appears to be through the pores of particles, thereby increasing the separation efficiency through a further decrease of the flow inhomogeneity and through enhancement of the mass transfer kinetics. The effects of pore size and mobile phase composition on the plate height and mobile phase velocity have been studied. With increasing buffer concentrations and larger pore diameters, higher mobile phase velocities and higher separation efficiencies have been obtained. Columns packed with 7 microns particles containing pores with a diameter of 4000 A generated up to 430,000 theoretical plates/m for retained compounds. Reduced plate heights as low as 0.34 have been observed, clearly demonstrating that a significant portion of the flow is through the pores. For the particles containing 4000 A pores no minimum was observed in the H-u plot up to linear velocities of 3.3 mm/s, suggesting that the separation efficiency is dominated by axial diffusion. On relatively long (72 cm) columns, efficiencies of up to 230,000 theoretical plates/column have been obtained under non-optimal running conditions. On short (8.3 cm) columns fast separations could be performed with approximately 15,000 theoretical plates generated in less than 30 s.

Conductive carbon cement as electrode matrix for cobalt phthalocyanine modified electrodes for detection in flowing solutions

Abstract A comparison has been made of the performance of electrodes modified with cobalt phthalocyanine catalyst prepared in different ways for use in flowing solutions. Because of leaching direct adsorption of cobalt phthalocyanine (CoPC) on a glassy carbon electrode surface had little analytical utility. Use of a polystyrene film prevents leaching of CoPC from the electrode surface to some extent, albeit at the expense of sensitivity. A new composite electrode with conductive carbon cement as electrode matrix was found to be comparable to a carbon paste electrode in terms of the electrocatalytic properties of CoPC towards thiol compounds (N-acetylcysteine, cysteine, glutathione and dithiothreitol) both in quiescent and flowing solutions, but is obviously superior in terms of long-term stability in flowing solutions. An advantage of the use of conductive carbon cement is that it can be repacked, so that the normal electrode body for carbon paste can be utilized. The modified electrode has been applied in liquid chromatography for the determination of cysteine in urine samples. The decrease of the electrode sensitivity over 5 h was about 5% at a flow-rate of 1.8 ml min−1 and a fixed potential of 0.7 V vs. Ag/AgCl.

Post-column reaction system for fluorescence detection in capillary electrophoresis

Abstract A system for post-column derivatization in capillary electrophoresis has been developed and evaluated. In this system, reagent is added through a porous tube connecting the separation capillary with a reactor capillary. The reagent solution is driven by a regulated air pressure applied on the separation capillary inlet and the reagent vessel simultaneously. The system has been applied for the fluorescence detection of amino acids using o-phthalaldehyde as reagent. The reactor capillary geometry and flow-rates were optimized with respect to sensitivity and separation efficiency on the basis of measured reaction rate constants. A ten-fold increase in sensitivity could be obtained by using “bubble-cell” capillaries instead of normal fused-silica capillaries for detection. Amino acids were separated in a pH 9.7 borate buffer. Plate numbers in the order of 100 000 to 150 000 were obtained. Detection limits were typically between 2·10−6 and 4·10−6 mol l−1, or 0.05 to 0.1 pmol injected. The method was applied to the determination of free amino acids in urine samples.

Optimal gradient operation in comprehensive liquid chromatography × liquid chromatography systems with limited orthogonality

A novel strategy is described for designing optimal second dimension (2D) gradient conditions for a comprehensive two-dimensional liquid chromatography system where the two dimensions are not fully orthogonal. Using the approach developed here, the initial and final organic modifier content values resulting in the highest coverage of separation space can be derived for each 2D gradient run. Theory indicates that these values can be determined by adapting 2D gradient operation to the degree of orthogonality. The new method is tested on a comprehensive two-dimensional liquid chromatography system that uses reversed phase (RP) columns showing different selectivities in the two dimensions. A comparison between analyses carried out using normal and optimized 2D gradients showed that the latter allow a more efficient use of analysis time. This can result either in an improved peak capacity or in decreasing total analysis time, depending on the final goal of the experiment. In the latter scenario, the number of separated peaks is comparable to that obtained using gradients spanning a wide range of organic modifier but, now, in half the time. As test samples complex mixtures of peptides were analyzed.

Determination of sugars by capillary electrophoresis with electrochemical detection using cuprous oxide modified electrodes

The separation by capillary electrophoresis and off-column amperometric detection of underivatized sugars has been studied. A palladium-metal union was used as grounded cathode to decouple the high separation voltage before detection. With a second piece of capillary the field decoupler was connected to a home-made T-shaped detection cell. Cuprous oxide modified microelectrodes were prepared and evaluated. For the detection of sugars they were operated at a constant potential of + 0.60 V. The peak-height reproducibility with these electrodes over a period of 18 h (25 injections) was within 7% absolutely and within 4% when one of the analytes was regarded as internal standard. To preserve the flat electroosmotic flow profile in the separation capillary, pressure compensation was applied. The contribution of various sources to the system zone variance was studied. Under optimized conditions plate numbers of over 100 000 were obtained. For the baseline separation of a mixture of sugars 0.10 mol I-’ sodium hydroxide solutions had IO be used as background electrolyte. Detection limits of l-2 pmol I-’ were found for various carbohydrates.

Application of flow field-flow fractionation for the characterization of macromolecules of biological interest: a review

An overview is given of the recent literature on (bio) analytical applications of flow field-flow fractionation (FlFFF). FlFFF is a liquid-phase separation technique that can separate macromolecules and particles according to size. The technique is increasingly used on a routine basis in a variety of application fields. In food analysis, FlFFF is applied to determine the molecular size distribution of starches and modified celluloses, or to study protein aggregation during food processing. In industrial analysis, it is applied for the characterization of polysaccharides that are used as thickeners and dispersing agents. In pharmaceutical and biomedical laboratories, FlFFF is used to monitor the refolding of recombinant proteins, to detect aggregates of antibodies, or to determine the size distribution of drug carrier particles. In environmental studies, FlFFF is used to characterize natural colloids in water streams, and especially to study trace metal distributions over colloidal particles. In this review, first a short discussion of the state of the art in instrumentation is given. Developments in the coupling of FlFFF to various detection modes are then highlighted. Finally, application studies are discussed and ordered according to the type of (bio) macromolecules or bioparticles that are fractionated.

Post-column derivatization for fluorescence and chemiluminescence detection in capillary electrophoresis

Instrumental developments and applications of post-column derivatization for fluorescence and chemiluminescence detection in capillary electrophoresis (CE) are reviewed. Various systems to merge the reagent solution with the separation medium have been developed, including coaxial capillary reactors, gap reactors and free solution or end-column systems. For all reactor types the geometry of the system, as well as the method to propel the reaction mixture (by pressure or by voltage) appeared to be critical to preserve the separation efficiency. Plate numbers of over 100,000 could be realised with different reactors. The strict requirements on the rate of post-column derivatization reactions to be applied in CE limit the number of different reagents that have been used. For fluorescence detection, with laser or lamps as the excitation source, so far mainly o-phthalaldehyde and its naphthalene analogue have been used as reagent. Derivatization systems that are based on complexation reactions also showed good promise for application in CE. Detection limits could be obtained that were comparable to those obtained after pre-column derivatization. Various reagents for chemiluminescence detection (e.g. the luminol and peroxyoxalate systems) have been studied. The often complicated chemistry involved made application of these reagents in CE even more difficult. Results obtained so far, in terms of sensitivity, have not been up to expectation, with detection limits usually in the order of micromol l(-1).

Hollow-fibre flow field-flow fractionation of polystyrene sulphonates

Abstract Hollow-fibre flow field-flow fractionation has been used for the characterization and separation of sodium polystyrene sulphonates in aqueous solutions. The elution behaviour of the polymers was found to follow theoretical predictions with a fair accuracy. Diffusion coefficients of standards of different molecular mass could be calculated from elution data. A method was developed to determine the polydispersity of narrow standards by measuring peak widths under different flow regimes. Flow programming has been used to fractionate samples with a wide molecular mass range.