Biomedical subjects

Gert Desmet

Publications and source records attributed to Gert Desmet.

At least 19 recordsLinked to original sources

PubMed2006

Domain size-induced heterogeneity as performance limitation of small-domain monolithic columns and other LC support types.

We have computed the band broadening and the flow resistance in a series of apparently self-similar porous LC support structures, all having the same mean geometric ratios and external porosity, but with a decreasing scale and disturbed by a scale-independent variance on the size and position of the porous solid zone elements. The study shows in a general and qualitative way that each type of LC support that is produced using a manufacturing process displaying a fixed (i.e., domain-size independent) variance on the size and position of the produced solid zone elements will eventually encounter a limit beyond which a further reduction of the domain size can no longer be expected to yield a significant gain in separation speed. This is currently observed in practice for silica monoliths and could also compromise the performance of photolithographically etched columns.

Journal Article↗

PubMed2006

An automated injection system for sub-micron sized channels used in shear-driven-chromatography.

This paper describes a method to automatically and reproducibly inject sharply delimited sample plugs in the shallow (i.e., sub-micron) channels typically used in shear driven chromatography. The formation of asymmetric plugs, which typically occurs during loading of the sample in wide channels, is circumvented by etching a slit in the middle of the channel that is connected to a micro-well and a vacuum system with syringes for the supply of both the analyte and the mobile phase. The design of the injection slit was supported by a series of CFD simulations to optimize its shape and that of the corresponding injection well. The system was intensively tested experimentally and showed good reproducibility, both for the width and the area of the injected peaks (relative standard deviations are max. 4 and 6%, respectively). The concentration of the injected plug was found to be approximately 80% of the original sample concentration. It was also observed that with the current setup the lower limit of the peak width was about 120 microm. This is a consequence of the fact that the peak width originating from the convection filling step becomes negligible to the contribution of diffusion during the filling and flushing time. Being fully automated and perfectly closed, the presently proposed injection system also paves the way to integrate other functionalities in shear driven chromatography, i.e. gradient elution and parallelization.

Journal Article↗

PubMed2006

Future of high pressure liquid chromatography: do we need porosity or do we need pressure?

Making a theoretical study supported by experiments of the kinetic advantages of increased inlet pressures versus increased external porosity using impedance plots of analysis time versus required plate number, it is found that both approaches more or less have the same effect on the kinetic performance. The need to change a given system to one with an increased inlet pressure or with an increased external porosity can best be assessed from the optimal plate number (N(opt)) of the system. When the pursued application requires a plate number that is larger than N(opt), any increase in inlet pressure and external porosity is beneficial. When the required plate number is smaller than N(opt), any increase in inlet pressure and external porosity should preferentially be accompanied by an overall reduction of the feature sizes of the support. The degree to which this feature size reduction can be realized in practice will to a large extent determine which of the two approaches will be the dominant system of the future.

Chromatography, High Pressure Liquid↗

PubMed2006

Detection enhancement in nano-channels using micro-machined silicon groove.

The present paper reports on an experimental study of the possibility to use a micro-machined detection groove to enhance the detection sensitivity in flat-rectangular nano-channels for ultra-rapid liquid chromatography separations. Transversally running detection grooves with three different axial widths (respectively, 2, 4 and 6 microm) and one depth (4.75 microm) were tested in glass and silicon channels for the whole range of detectable fluorescein isothiocyanate isomer I, FITC, concentrations. The groove with the most square-like cross-section (i.e., 4 microm wide and 4.75 microm deep) yielded the best combination of detection gain and minimal additional band broadening. In a 1cm long channel, the effective plate loss caused by the 4 microm wide groove would only be of the order of 20%, while the gain in S/N-ratio was of the order of a factor of 5. The detection groove concept yields larger gains in silicon channel substrates than in glass channel substrates, due to the larger stray light losses occurring in the latter.

Chromatography, Liquid↗

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

Theoretical calculation of the retention enthalpy effect on the viscous heat dissipation band broadening in high performance liquid chromatography columns with a fixed wall temperature.

The problem of the additional band broadening arising from the trans-column temperature profile under conditions of significant viscous heat dissipation has been extended by including the retention enthalpy effect. This effect can significantly aggravate the trans-column migration velocity profile of retained species as compared to the already known zero-retention enthalpy case. An approximate analytical solution for the accompanying additional band broadening could be established. The solution is valid in nearly all practically relevant cases and shows how the additional band broadening varies as a function of the generated heat, the retention coefficient and the retention enthalpy. Surveying all possible HPLC operating conditions, it was found that the inclusion of the retention enthalpy effect leads to significantly larger viscous heating plate height contributions than those predicted by the zero-retention enthalpy solution (up to a factor of 10 or more).

Chromatography, High Pressure Liquid↗

PubMed2006

Practical constraints in the kinetic plot representation of chromatographic performance data: theory and application to experimental data.

It is demonstrated that the kinetic plot representation of experimental plate height data can also account for practical constraints on the column length, the peak width, the viscous heating, and the mobile-phase velocity without needing any iterative solution routine. This implies that the best possible kinetic performance to be expected from a given tested support under any possible set of practical optimization constraints can always be found using a directly responding calculation spreadsheet template. To show how the resulting constrained kinetic plots can be used as a powerful design and selection tool, the method has been applied to a series of plate height measurements performed on a number of different commercial columns for the same component (butyl-parabene) and mobile-phase composition. The method, for example, allows one to account for the fact that the advantageous solutions displayed by the silica monolith and 5 microm particle columns in the large plate number range of the free kinetic plot are no longer accessible if applying a maximal column length constraint of Lmax = 30 cm. In the plate number range that remains accessible, the investigated sub-2 mum particle columns in any case perform (at least for the presently considered parabene separation) better than the 3.5 mum particle columns or silica monolith, especially if considering the use of system pressures exceeding 400 bar. The constrained kinetic plot method can also be used to select the best-suited column length from an available product gamma to perform a separation with a preset number of plates. One of the optimization results that is obtained in this case is that sometimes a significant gain in analysis time can be obtained by selecting a longer column, yielding the desired plate number at a larger velocity than that for a shorter column.

Algorithms↗

PubMed2006

A dimensionless number analysis of the hybridization process in diffusion- and convection-driven DNA microarray systems.

The present theoretical analysis aims at providing a general understanding of the combined effect the many different process variables have on the hybridization rate in diffusion- and convection-driven DNA microarray systems. It is shown that all process variables can be grouped into only four different dimensionless numbers (the Damkohler number Da, the dimensionless association constant kappa(A), the dimensionless initial concentration C'(0) and a geometrical ratio alpha). These four numbers have a straightforward physical meaning and only contain easily measurable parameters. Reducing the solution space from 7D to 4D, the dimensionless number representation greatly facilitates the insight in the conditions leading to the occurrence of diffusion-limited hybridization rates in both diffusion- and convection-driven DNA microarray systems. This in turn simplifies their design and the interpretation of the experimental results that are obtained with these systems.

Diffusion↗

PubMed2006

A discussion of the possible ways to improve the performance of silica monoliths using a kinetic plot analysis of experimental and computational plate height data.

Using kinetic plots to analyse the performance of some of the best silica monoliths found in the literature shows that the current generation of silica monoliths outperform particulate beds only in the high plate-number region (roughly N > 40000). The plots also reveal the existence of a so-called 'forbidden region' wherein no existing chromatographic support seems to be able to operate. To investigate several possible approaches to intrude this forbidden region, computational fluid dynamics simulations of the flow field and band-broadening characteristics of a simplified structural mimic of real silica monoliths were made for five different porosities (epsilon = 0.38, 0.49, 0.60, 0.72, 0.86). It was found that entering the forbidden region will necessitate new synthesis methods, yielding either a strong improvement of the structural homogeneity (if assuming constant domain size conditions) or a decrease of the domain size (if assuming constant homogeneity conditions).

Computer Simulation↗

PubMed2006

Diffusion limitation: a possible source for the occurrence of doughnut patterns on DNA microarrays.

Doughnut shaped hybridization patterns on DNA microarrays are mainly allocated to spotting or drying artifacts. The present study reports on results obtained from four different approaches that when combined generate a better view on the occurrence of these patterns. This study points out that doughnuts are not only formed during the spotting and drying process, but the hybridization process itself can be considered as an important cause. A combination of computer simulations, theoretical, optical, and experimental techniques shows how ring-shaped hybridization patterns occur when diffusion-limited conditions are present during the hybridization process. The theoretical assumptions as well as the simulations indicate that, for the basic geometry of a microarray hybridization experiment, a large amount of binding molecules reach the spot from the sides (and not from above the spot), leading to a preferential binding on the rims of the spot. These patterns seem to occur especially during hybridization with short oligonucleotides that have a very high binding probability and fast hybridization kinetics. Longer target DNA molecules lead to a more evenly distributed intensity signal. Furthermore, the diffusion-limited conditions also lead to pronounced hybridization intensity patterns on the scale of a whole spot block, where larger intensities are obtained on the edges of the block compared with the spots laying in the center of the block.

Animals↗

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

Ultra-rapid separation of an angiotensin mixture in nanochannels using shear-driven chromatography.

The present paper reports on the separation of a mixture of fluorescein isothiocyanate-labeled angiotensin I and II peptides in a shear-driven nanochannel with a C18-coating and using an eluent consisting of 5% acetonitrile in 0.02 M aqueous phosphate buffer at pH 6.5. The flat-rectangular nanochannel in fused silica consisted of an etched structure in combination with a flat moving wall. The very fast separation kinetics that can be achieved in a nanochannel allowed to separate the angiotensin peptides in less then 0.2 s in a distance of only 1.8 mm. Plate heights as small as 0.4 microm were calculated after substraction of the injection effect.

Angiotensin I↗

PubMed2005

Measurements of diffusion coefficients in 1-D micro- and nanochannels using shear-driven flows.

The present paper describes a method for measuring the molecular diffusion coefficient of fluorescent molecules in microfluidic systems. The proposed static shear-driven flow method allows one to perform diffusion measurements in a fast and accurate manner. The method also allows one to work in very thin (i.e. submicron) channels, hence allowing the investigation of diffusion in highly confined spaces. In the deepest investigated channels, the obtained results were comparable to the existing literature values, but when the channel size dropped below the micrometer range, a significant decrease (more than 30%) in molecular diffusivity was observed. The reduction of the diffusivity was most significant for the largest considered molecules (ssDNA oligomers with a size ranging between 25 to 100 bases), but the decrease was also observed for smaller tracer molecules (FITC). This decrease can be attributed to the interactions of the analyte molecules with the channel walls, which can no longer be neglected when the depth of the channel reaches a critical value. The change in diffusivity seems to become more explicit as the molecular weight of the analytes increases.

DNA, Single-Stranded↗

PubMed2005

DNA microarray enhancement using a continuously and discontinuously rotating microchamber.

It is demonstrated that the most efficient way to enhance DNA microarray analysis consists of a maximal reduction of the total device volume (to keep the concentration of the available DNA as high as possible), combined with the creation of a strong lateral convective transport of the sample. In the present study, DNA microarray hybridizations are performed in a set of rotating, circular microchambers covering exactly the spotted area of the microarray and with a depth varying between 70 and 1.6 microm. Rotating the microchamber substrate while keeping the microarray stationary, the rotating microchamber bottom wall literally drags the sample past the microarray spots with a velocity which is independent of the fluid layer thickness. Interestingly, it was found that transporting the sample in a discontinuous mode (with stop periods of several minutes) not only yields a more stable and reproducible operation, it also yields significantly larger hybridization intensities (typically a factor of 2-3 larger) than a continuous rotation. This seems to be due to the fact that the velocity field disturbs the binding process at the binding site level. Working under limiting DNA sample mass conditions, the system yielded in a short, 30-min experiment already a 5-fold increase of the hybridization intensity, as compared to a conventional microscope slide/coverslip system operated overnight under diffusion-driven conditions. Compared to a commercial pump-around hybridization system, the gain was even more impressive, precisely due to the fact that the pump-around system requires larger volumes, which with a fixed amount of available genetic material leads to the application of more diluted samples.

DNA, Complementary↗

PubMed2005

Geometry-independent plate height representation methods for the direct comparison of the kinetic performance of LC supports with a different size or morphology.

The advantages of representing experimental plate height data as a plot of Kv/u0(2) or H2/Kv versus Kv/(Hu0) instead of as H versus u0 are discussed (Kv=column permeability). Multiplying the values on both axes by the ratio of a reference pressure drop and mobile-phase viscosity, the obtained plots directly yield the kinetic performance limits of the tested support structure, without any need for further numerical optimization. Directly showing the range of plate numbers or analysis times wherein the tested support geometry can yield faster separations or produce more plates than another support type, such kinetic plots are ideally suited to compare the performance of differently shaped or sized LC supports. The approach hence obviates the need for a common reference length, which is a clear problem if it is attempted to compare differently shaped supports on the basis of their flow resistance phi and reduced plate height h. It is also shown how an MS Excel template file, only requiring the user to paste the column permeability Kv and a series of experimental (u0, H) data, can be used to automatically establish a series of so-called kinetic performance (KP) numbers, which can be used to completely describe the performance characteristics of the considered support. The advantages of the proposed data representation methods are demonstrated by applying them to several recent literature plate height data sets, showing that the obtained kinetic plots directly visualize the range of plate numbers where new approaches such as ultra-high-pressure HPLC or the use of open-porous silica monoliths can be expected to provide a substantial gain and where not. The data analysis also showed that the most generally relevant KP numbers are N(opt) (the plate number for which the support achieves its best analysis time/pressure cost ratio), t(opt) (the time needed to obtain N(opt) plates), and t(1K) (the time needed to generate 1000 or 1 kilo of theoretical plates). These KP numbers are much more informative than the H(min), u(0,opt), and Kv data traditionally employed to quantify the performance of LC supports.

Journal Article↗

PubMed2005

Experimental validation of the tetrahedral skeleton model pressure drop correlation for silica monoliths and the influence of column heterogeneity.

This paper describes the use of computational fluid dynamics for the calculation of the flow resistance through computer-generated models resembling silica monoliths. This study was undertaken to determine the effect of skeleton heterogeneity on the flow resistance and, more precisely, to test the hypothesis that increased skeleton heterogeneity decreases the flow resistance. To evaluate the proposed model, 24 real silica monoliths have been prepared using the same method, covering a wide range of skeleton sizes (2.2 microm < d(s) < 8 microm) and porosities (0.47 < epsilon < 0.66). The permeability of these monoliths was determined by pressure drop measurements, and structural information was obtained by image analysis of laser scanning confocal microscopy-generated 3D images of the skeleton structure. The results indicate that the presence of preferential flow paths due to an increased heterogeneity of the flow through pore space reduces the flow resistance of monolithic media. It is also shown that the pore size is hence a much better suited scaling dimension than the skeleton size to reduce the permeability of monolithic columns.

Journal Article↗

PubMed2005

A first principles explanation for the experimentally observed increase in A-term band broadening in small domain silica monoliths and other chromatographic supports.

The present computational study illustrates how the existence of a residual lower limit on the variance of the skeleton and through-pore size of monolithic columns can be expected to severely compromise the possibility to prepare well-performing small domain monolithic columns. Adopting rather conservative estimates for the minimal standard deviation on the pore and the skeleton size (0.2 and 0.04 microm, respectively), the presented calculations show that, if such a fixed lower limit on the size variance exists, it will be impossible to decrease the A-term band broadening below a given critical value, no matter how small the domain size is made. From a given critical domain size value on, any attempt to further decrease the domain size without being able to co-reduce the size variance can be expected to be counterproductive and leads to an increase instead of to a further decrease of the plate heights.

Chromatography, Liquid↗

PubMed2005

Comparison of a pump-around, a diffusion-driven, and a shear-driven system for the hybridization of mouse lung and testis total RNA on microarrays.

In the present study, we demonstrate the benefits of a shear-driven rotating microchamber system for the enhancement of microarray hybridizations, by comparing the system with two commonly used hybridization techniques: purely diffusion-driven hybridization under coverslip and hybridization using a fully automated hybridization station, in which the sample is pumped in an oscillating manner. Starting from the same amount of DNA for the three different methods, a series of hybridization experiments using mouse lung and testis DNA is presented to demonstrate these benefits. The gain observed using the rotating microchamber is large: both in terms of analysis speed (up to tenfold increase) and in final spot intensity (up to sixfold increase). The gain is due to the combined effect of the hybridization chamber miniaturization (leading to a sample concentration increase if comparing iso-mass conditions) and the transport enhancement originating from the rotational shear-driven flow induced by the rotation of the chamber bottom wall.

Animals↗