Biomedical subjects

Peter J Schoenmakers

Publications and source records attributed to Peter J Schoenmakers.

At least 19 recordsLinked to original sources

PubMed2006

Development of an algorithm for peak detection in comprehensive two-dimensional chromatography.

A method for peak detection in two-dimensional chromatography is presented. The algorithm applies first the methods developed for peak detection in one-dimensional chromatography to detect peaks in one dimension. In a second step, a decision tree is applied to decide which one-dimensional peaks are originated from the same compound and have to be 'merged' into one two-dimensional peak. To this end, different features of the peaks (second-dimension peak regions and second-dimension retention times) are compared and different criteria (common peak regions, retention time differences, unimodality in the first dimension) are applied. Different options can be used, depending on the nature of the data. The user controls this decision tree by establishing several options and "switches". The algorithm was tested with GCxGC chromatograms obtained for a commercial air-freshener sample, detecting and merging the modulated peaks belonging to the same compound. Recommendations for the set of options and switches are given. A utility that calculates and sums peak areas from merged peaks is added to facilitate automated quantification. Although the algorithm was developed for GCxGC, its application to comprehensive two-dimensional liquid chromatography (LCxLC) data should at most require minor modifications.

Algorithms↗

PubMed2006

Automatic selection of optimal Savitzky-Golay smoothing.

A method to select the optimal window size of the Savitzky-Golay (SG) algorithm is presented. The approach is based on a comparison of the fitting residuals (i.e., the differences between the input signal and the smoothed signal) with the noise of the instrument. The window size that yields an autocorrelation of the residuals closest to the autocorrelation of the noise of the instrument is considered optimal. The method is applied in two steps. In a first step, the lag-one autocorrelation value of the noise of the instrument is computed through the study of a blank signal. In a second step, the SG algorithm is applied to "smooth" the signal using different window sizes. The method was applied to data from NMR, chromatography, and mass spectrometry and was shown to be robust. It finds the optimal window size for different signal features. This allows the method to be used in an unsupervised way, embedded in a more complex algorithm in which smoothing and/or differentiation of signals is required, provided that the lag-one autocorrelation value of the instrument noise does not change.

Journal Article↗

PubMed2006

Selection of comparison criteria and experimental conditions to evaluate the kinetic performance of monolithic and packed-bed columns.

The present study concerns the problem of finding appropriate experimental conditions and comparison criteria to assess the kinetic performance of LC supports with different sizes or morphologies. A general procedure, based on evaluating each support for its own optimal mobile-phase composition, is proposed. The practical elaboration of the procedure is illustrated using the specific case of a capillary LC separation of a series of polycyclic aromatic test compounds employing silica-monolith capillary columns and capillary columns packed with 6-microm porous particles. To compare the systems for their ability to yield the fastest critical-pair separation, plate-height measurements are transformed into an effective plate number kinetic plot, i.e., a plot of the extrapolated retention time divided by the square of the extrapolated effective plate number (t(R)/N(eff)(2)) versus N(eff). This type of data representation provides a direct and universal basis to compare the kinetic performance of different LC supports and it corrects for differences in retention strength arising from different phase ratios.

Chromatography, Liquid↗

PubMed2006

A graphical method for understanding the kinetics of peak capacity production in gradient elution liquid chromatography.

A novel graphical method for assessing the compromise between conditional peak capacity and separation speed for packed bed columns under gradient conditions has been developed and applied to the separation of peptides. This approach is analogous to and complements the conventional "Poppe plot" used to study plate count in isocratic separations. The use of the new plot can assist the design of appropriate column formats (e.g. particle size and column length) for both dimensions in gradient elution two-dimensional liquid chromatography (2DLC). Particularly for the second dimension of 2DLC, we find that smaller particles provide faster separations even though fast separations based on particles smaller than 2 microm are practically limited by the required miniscule column length. We also find that high temperatures strongly enhance the kinetics of peak capacity production whereas higher pressures help achieve larger absolute peak capacities albeit at the cost of longer analysis time.

Chromatography, Liquid↗

PubMed2006

Molar-mass characterization of cationic polymers for gene delivery by aqueous size-exclusion chromatography.

PURPOSE: This study was performed to develop a reliable aqueous size-exclusion chromatography (SEC) method to obtain the absolute molar masses and distributions of various cationic polymers used in gene delivery. METHODS: Water-soluble cationic [2-(dimethylamino) ethyl methacrylate] polymers (PDEs) with different molar masses and low polydispersities were synthesized by living polymerization and these were used to optimize the SEC conditions. Online coupled multiangle light scattering (MALS) detection was applied to obtain the absolute molar masses. Narrow fractions of high molar mass were obtained by semipreparative SEC. RESULTS: It was found that 0.3 M NaAc (pH 4.4) is a suitable eluent in combination with Shodex OHpak SB columns for SEC analysis of PDEs and other cationic polymers, such as poly(L-lysine) and poly(ethylene imine). The absolute molar masses of different PDEs were determined directly using SEC-MALS. A calibration curve was established using narrow PDEs. CONCLUSIONS: A reliable routine method for molar-mass characterization of cationic polymers was established. Because standards of known molar masses with narrow distributions are not commercially available for most polymers used in pharmaceutics and biotechnology, the procedure described in this work can also be applied for molar-mass characterization of other water-soluble polymers.

Cations↗

PubMed2006

Determination of major carotenoids in vegetables by capillary electrochromatography.

A simple and rapid method for the isocratic separation and determination of carotenoids (carotenes and xanthophylls) in vegetables by CEC is described. The capillary column (100 microm ID, 25 cm effective length) was packed with 3 microm Hypersil ODS particles. The optimized mobile phase contained 60% ACN, 35% THF and 5% of a 5 mM Tris aqueous buffer of pH 8. beta-Carotene, lycopene and lutein were separated with efficiencies of 66 000-128 000 plates/m within a short time (less than 12 min for the last peak eluted, 13/13'-cis-beta-carotene). An excellent resolution of the three carotenoids, as well as partial resolution of their geometrical isomers, was achieved. Application to the determination of the analytes in carrot, tomato, spinach and corn was demonstrated.

Carotenoids↗

PubMed2005

A protocol for designing comprehensive two-dimensional liquid chromatography separation systems.

In this paper a protocol is proposed for establishing suitable column dimensions (length and diameters), particle sizes, flow rates, and second-dimension injection volumes (i.e. loop sizes) in comprehensive two-dimensional liquid chromatography (LC x LC). The chromatographer should select the maximum allowable first-dimension retention time, which is approximately equal to the overall analysis time. Also, (s)he should define the maximum allowable pressure in both dimensions and the (minimum) diameter of the first-dimension column. The proposed protocol provides design parameters corresponding to the ideal (theoretically optimal) conditions or to realistic practical conditions. The protocol also allowed us to study the implications of contemporary developments in LC, such as the use of high temperatures (implying reduced viscosities and increased diffusion coefficients), monolithic columns (implying smaller flow-resistance factors), and ultra-high-pressure LC. The combination of (reversed-phase or normal-phase) liquid chromatography with size-exclusion chromatography (LC x SEC) is frequently employed for analysing complex polymers. The proposed protocol is used to design a suitable LC x SEC system under realistic conditions. The results resemble the systems that have been designed and implemented by expert chromatographers, but they also indicate how current practice can be improved.

Algorithms↗

PubMed2005

Performance limits of monolithic and packed capillary columns in high-performance liquid chromatography and capillary electrochromatography.

A method is proposed for the comprehensive characterization and comparison of columns in the high-performance liquid chromatographic (HPLC) and capillary electrochromatographic (CEC) modes. Using this approach, column parameters such as the number of plates, the eddy-diffusion and mass-transfer contributions to peak broadening, the permeability, and the analysis time are incorporated in a single graph and a comparison in terms of efficiency and speed is obtained. The chromatographic performance of silica-based and polymer-based monolithic capillary columns is discussed and a comparison is made with the performance of packed columns. Also, the potential of ultra-high-pressure liquid chromatography is discussed in this context. In the HPLC mode, the best results were obtained with silica monoliths; in the CEC mode, the low-density methacrylate-ester-based monoliths showed the best performance.

Chromatography, High Pressure Liquid↗

PubMed2005

Chemical variance, a useful tool for the interpretation and analysis of two-dimensional chromatograms.

A new method is presented for the analysis of comprehensive two-dimensional data. The approach complements existing chemometric methods for analysing these kinds of data (e.g. multivariate-curve-resolution methods, MCR), and is especially suited for the analysis of families of compounds. The method is based on finding a new pair of axes (replacing the retention time axes) to represent the data. A drop in the matrix rank is observed when one of the axes collects all the variance for a certain family of compounds. This is achieved by introducing retention models (in both chromatographic dimensions) that describe the behaviour of a family of compounds as a function of the property that varies between the members. After this transformation, a single family of compounds gives rise to a single source of variance. This suggests the application of MCR techniques in a second step to separate the contributions of different (overlapped) families of compounds. For the latter application, more research has to be performed. The approach is illustrated using the separation of families of functional poly(methyl-methacrylate) polymers with different numbers of hydroxyl end groups and degrees of polymerisation or, equivalently, with the molecular weight as the key descriptive property within one family.

Algorithms↗

PubMed2005

Tailoring the morphology of methacrylate ester-based monoliths for optimum efficiency in liquid chromatography.

Methacrylate ester-based monolithic stationary phases were prepared in situ in fused-silica capillaries and simultaneously in vials. The influence of the composition of the polymerization mixture on the morphology was studied with mercury intrusion porosimetry, scanning electron microscopy, and nitrogen adsorption measurements. A high-density porous polymeric material with a unimodal pore-size distribution was prepared with 40 wt % monomers and 60 wt % solvent in the mixture. A low-density material, prepared with a 20:80 ratio of monomers versus pore-forming solvent, showed a bimodal pore-size distribution and a much finer structure than the high-density monolith. The characteristic pore size could be controlled by changing the ratio of pore-forming solvents. With increasing solvent polarity, both the pore size and the dimension of the globules increased. The best efficiency in the CEC mode was obtained with an average pore size of 600 nm. Low-density monoliths exhibited lower A- and C-terms than high-density monoliths. With the optimal monolithic material, a minimum plate height of 5 mum could be obtained. The low-density monolith also performed better in the HPLC mode, giving a minimum plate height of 15 mum and a much higher flow permeability than that of the high-density material.

Journal Article↗

PubMed2005

Practical aspects of using methacrylate-ester-based monolithic columns in capillary electrochromatography.

Methacrylate-ester-based monoliths containing quaternary ammonium groups were prepared in situ in capillary columns and in simultaneous experiments in vials, employing thermal initiation. The chromatographic properties of the monoliths were determined with capillary electrochromatography (CEC), and their morphology was studied with mercury-intrusion porosimetry on the bulk materials. Materials with different, well repeatable pore-size distributions could be prepared. A satisfactory column-to-column and run-to-run repeatability was obtained for the electro-osmotic mobility, the retention characteristics (k-values) and the efficiency on the columns prepared and tested in the CEC mode. A relatively high electro-osmotic flow was observed in the direction of the positive electrode. The electro-osmotic mobility was found to be influenced only marginally by mobile-phase parameters such as the pH, ionic strength, and acetonitrile content. The retention behavior of the monolithic columns was similar to that of columns packed with C18-modified silica particles. Columns could be prepared with optimum plate heights ranging from 6 microm for unretained compounds to 20 microm for well retained (k=2.5) polyaromatic hydrocarbons. However, for specific analytes a - still unexplained - lower chromatographic column efficiency was observed.

Chromatography, Micellar Electrokinetic Capillary↗

PubMed2005

Fast size-exclusion chromatography--theoretical and practical considerations.

Fast SEC is a very interesting modification of conventional SEC. The need for it emerges from combinatorial chemistry and high-throughput experimentation, where high-speed analyses are required. The different approaches to change the speed of analysis are extensively described in this paper. Special attention is paid to the trade-off between analysis time and resolution and to the selection of optimal column lengths and flow rates. Simulations are used to design and to understand experiments. Integrity plots are constructed to judge the quality of various SEC systems. Fast separations in size-exclusion chromatography are found to be more favorable than suggested by conventional theory. The results are based on experimental data obtained for polystyrene using THF as mobile phase.

Chromatography, Gel↗

PubMed2005

Comprehensive two-dimensional liquid chromatography for the characterization of functional acrylate polymers.

Comprehensive two-dimensional liquid chromatography-size-exclusion chromatography (LC x SEC) was investigated as a tool for the characterization of functional poly(methyl methacrylate) (PMMA) polymers. Ultraviolet-absorbance and evaporative light-scattering detection (ELSD) were used. A simple method to quantify ELSD data is presented. Each data point from the ELSD chromatogram can be converted into a mass concentration using experimental calibration curves. The qualitative and quantitative information obtained on two representative samples is used to demonstrate the applicability of LC x SEC for determining the mutually dependent molar-mass distributions (MMD) and functionality-type distributions (FTD) of functional polymers. The influence of the molar mass on the retention behavior in LC was investigated using LC x SEC for hydroxyl-functional PMMA polymers. The critical conditions, at which retention is--by definition--independent of molar mass, were not exactly the same for PMMA series with different end-groups. Our observations are in close agreement with theoretical curves reported in the literature. However, for practical applications of LC x SEC it is not strictly necessary to work at the exact critical solvent composition. Near-critical conditions are often sufficient to determine the mutually dependent distributions (MMD and FTD) of functional polymers.

Calibration↗

PubMed2005

Stochastic theory of size exclusion chromatography: peak shape analysis on single columns.

The stochastic theory of size exclusion chromatography (SEC) was applied to analyze the peak shape of chromatograms obtained with a wide range of polystyrene standards on various columns. The columns were packed with stationary phases of different pore sizes. The stochastic-dispersive model of SEC results in a peak shape model that fits well the symmetrical and asymmetrical peaks observed in SEC. From the peak shape parameters obtained after nonlinear parameter estimation, information can be gained regarding the fundamental characteristics of the size exclusion process. When a series of polymer standards are analyzed on one column-in a manner similar to other methods of inverse chromatography-the stationary phase can be characterized. The dependence of the ingress and the egress processes on the relative size of the macromolecule to the pore size was determined. We found that for small molecules the selectivity in SEC arises from the ingress process, while when the size of the macromolecule is comparable to that of the pore-i.e., close to the exclusion limit-the egress process will also strongly affect the selectivity.

Journal Article↗

PubMed2005

Poppe plots for size-exclusion chromatography.

Poppe plots provide a clear and unambiguous way to discuss the performance limits of separation systems. The effects of particle size, pressure drop and column permeability can be illustrated using such plots. The performance limits of size-exclusion chromatography are of interest, due to developments in combinatorial chemistry and high-throughput experimentation. In these fields, fast separations of high-molecular-weight analytes are required. In this paper, Poppe plots will be presented for size-exclusion chromatography. Because of the very high-reduced velocities encountered, the Poppe plots are found to be significantly different from those commonly observed in HPLC. Fast separations in size-exclusion chromatography are not as unfavourable as suggested by conventional theory. The results are based on experimental data obtained for a wide range of polystyrenes (1.7-3.25 kDa) using THF as mobile phase, but may be equally valid in other cases.

Chromatography, Gel↗

PubMed2005

Novel system for classifying chromatographic applications, exemplified by comprehensive two-dimensional gas chromatography and multivariate analysis.

For practical chromatographers it is extremely difficult to judge the merits and limitations of new technological developments. On the other hand, it is nearly impossible for those at the forefront of technology to judge the implications of their efforts for all specific applications of chromatography. Both chromatographers and researchers can be aided by a classification of the numerous specific applications into a few well-defined categories. In this paper, we propose such a classification of all chemical analysis by chromatography into three generic types of applications, viz. target-compound analysis, group-type separation, and fingerprinting. The requirements for each type are discussed in general terms. The classification scheme is applied to assess the benefits and limitations of comprehensive two-dimensional gas chromatography (GC x GC) and the possible additional benefits of using multivariate-analysis (MVA) techniques for each type of application. The conclusions pertaining to the generic types of applications are indicative for the implications of new developments for specific chemical analysis by chromatography.

Chromatography, Gas↗

PubMed2005

Contactless conductivity detection of synthetic polymers in non-aqueous size-exclusion electrokinetic chromatography.

A capacitively coupled contactless conductivity detection (CCD) system has been applied for the detection of neutral synthetic polymers in capillary size-exclusion electrokinetic chromatography (SEEC). Polystyrene standards, that were used as a model compounds, were separated on a capillary column packed with porous 10 microm silica particles with an electrokinetically driven mobile phase, and detected by CCD and UV detection simultaneously. Mass-calibration curves for polystyrene were constructed. Satisfactory results were obtained for the linearity, the run-to-run repeatability (<0.2% for the relative retention and <4% for the peak area) and the robustness of the detector. One of the major issues in this preliminary study was to investigate the origin of the peaks observed for the polystyrene standards. The effect of the molar mass of the polystyrenes on the sensitivity was small. Therefore, the signals obtained could not be explained as the result of an increased viscosity and a decreased solution conductivity of the solute zone. An alternative hypothesis is suggested, and recommendations for further research are given.

Calibration↗

PubMed2004

Band broadening in size-exclusion chromatography of polydisperse samples.

Understanding and controlling the band broadening is essential to obtain accurate molar-mass distributions by size-exclusion chromatography (SEC). In this paper, band broadening in SEC is reviewed from a contemporary perspective. The observed band broadening is due to dispersion inside and outside the chromatographic column (undesirable band broadening) and to the polydispersity of the sample (desirable SEC selectivity). The various contributors to band broadening are discussed. Integrity plots are introduced as a tool to evaluate the performance of specific SEC columns at given experimental conditions. For narrow polymer standards on single SEC columns the observed peak width is dominated by the chromatographic dispersion. MALDI-ToF-MS is demonstrated as an alternative to determine the PDI of narrowly distributed samples. The plate heights encountered at very high reduced velocities are found to be lower than expected. This is advantageous for fast separations by SEC.

Chromatography, Gel↗