A survey among Flemish gastroenterologists about endoscopic sedation practices in colorectal cancer screening.

Background & study aims: The sedation levels and methods used for colonoscopy in colorectal cancer screening programs vary from country to country and from continent to continent. Little is known in the literature about how frequently the different sedation levels are used in colorectal cancer screening colonoscopies. We made a survey among all Flemish gastroenterologists (GI) to determine how frequently they use the different sedation modalities in this target population and to determine the motives of the GI to opt for one or another sedation modality. Patient and methods: An online survey was sent to all 329 Flemish GI by e-mail. A reminder e-mail was sent one month later. Participants could indicate how frequently (by percentage) they used the different sedation methods (no sedation, minimal sedation, conscious sedation, deep sedation) and which sedative medication they administered. In addition, they were asked to indicate their main motives for choosing a specific sedation method. Descriptive statistics were used. Results: 112 out of 329 GI answered the questionnaire (response rate 34%). Anesthesia monitored care is the most frequently used sedation modality, followed by conscious sedation. Patient preference is the main motive for most GI to use each sedation modality. Conclusions: Anesthesia monitored care is currently the most frequently used sedation regimen to perform a colonoscopy in the FIT positive population or in the colorectal cancer screening program in Flanders. The motives given by the GI for choosing one or another sedation modality are not always congruent with current scientific evidence or guidelines.

Considerations for the use of ultra-high pressures in liquid chromatography for 2.1mm inner diameter columns.

The current contribution investigates the effects of viscous heat dissipation in chromatographic columns (with an emphasis on so-called narrow bore columns with an inner diameter of 2.1mm) using numerical simulations of the temperature and velocity profiles and the resulting band broadening, for the first time at operating pressures up to 2000bar. When operating columns under well-thermostatted conditions to maintain a constant temperature of the mobile phase, a dramatic increase in plate heights can be observed that voids any advantage one could expect from the possibility to use smaller particles offered by the increased pressure limit. It is also clearly demonstrated that, even when the column is not temperature controlled, the backflow of heat along the wall can causes a significant loss in performance under standard operating conditions in a still air oven. It is found that for operating pressure above 1250bar, a significant (relative to the typical column performance) contribution to the observed plate height will be caused by viscous heating effects, which increases with increasing temperature dependency of the retention factor. In addition, unprecedented experimental measurements of the temperature effects at an operating pressure up to 2600bar were performed on a 10cm long, 2.1mm ID column showing a dramatic temperature increase up to 60°C relative to the inlet temperature when using methanol as a mobile phase.

Optimization and evaluation of radially interconnected versus bifurcating flow distributors using computational fluid dynamics modelling.

Two main groups of flow distributors, viz. "bifurcating distributors" (BF) and "radially interconnected distributors" (RI), as well as some hybrid distributors were investigated. Computational fluid dynamics was used to evaluate the performance of the distributors and to establish the design yielding the most uniform velocity field and the smallest variance for the bands emerging from the distributor. A minimum channel width of 100 µm was considered to allow the use of micro-milling techniques for chip fabrication. The main factors that influenced the values of band variances were identified. The performance of the distributors was found to correlate most strongly with the volume of the flow distributors. The separation bed should be positioned immediately after, but not against the flow distributor. It was concluded that BF distributors perform best in terms of band variance. The values of band variances for the BF distributor decreased with increasing angle between bifurcation branches and the lowest value of about 0.01 mm(2) was found for α=175°. Both BF and RI flow distributors were found to perform reasonably well when imperfections were present in the structure. However, severe blockages (exceeding 75% of the cross-sectional area and length) of channels in, especially, BF flow distributors may jeopardize their performance.

Quantification aspects of constant pressure (ultra) high pressure liquid chromatography using mass-sensitive detectors with a nebulizing interface.

The present contribution investigates the quantitation aspects of mass-sensitive detectors with nebulizing interface (ESI-MSD, ELSD, CAD) in the constant pressure gradient elution mode. In this operation mode, the pressure is controlled and maintained at a set value and the liquid flow rate will vary according to the inverse mobile phase viscosity. As the pressure is continuously kept at the allowable maximum during the entire gradient run, the average liquid flow rate is higher compared to that in the conventional constant flow rate operation mode, thus shortening the analysis time. The following three mass-sensitive detectors were investigated: mass spectrometry detector (MS), evaporative light scattering detector (ELSD) and charged aerosol detector (CAD) and a wide variety of samples (phenones, polyaromatic hydrocarbons, wine, cocoa butter) has been considered. It was found that the nebulizing efficiency of the LC-interfaces of the three detectors under consideration changes with the increasing liquid flow rate. For the MS, the increasing flow rate leads to a lower peak area whereas for the ELSD the peak area increases compared to the constant flow rate mode. The peak area obtained with a CAD is rather insensitive to the liquid flow rate. The reproducibility of the peak area remains similar in both modes, although variation in system permeability compromises the 'long-term' reproducibility. This problem can however be overcome by running a flow rate program with an optimized flow rate and composition profile obtained from the constant pressure mode. In this case, the quantification remains reproducibile, despite any occuring variations of the system permeability. Furthermore, the same fragmentation pattern (MS) has been found in the constant pressure mode compared to the customary constant flow rate mode.

Kinetic performance comparison of fully and superficially porous particles with sizes ranging between 2.7 µm and 5 µm: Intrinsic evaluation and application to a pharmaceutical test compound.

The reintroduction of superficially porous particles has resulted in a leap forward for the separation performance in liquid chromatography. The underlying reasons for the higher efficiency of columns packed with these particles are discussed. The performance of the newly introduced 5 µm superficially porous particles is evaluated and compared to 2.7 µm superficially porous and 3.5 and 5 µm fully porous columns using typical test compounds (alkylphenones) and a relevant pharmaceutical compound (impurity of amoxicillin). The 5 µm superficially porous particles provide a superior kinetic performance compared to both the 3.5 and 5 µm fully porous particles over the entire relevant range of separation conditions. The performance of the superficially porous particles, however, appears to depend strongly on retention and analyte properties, emphasizing the importance of comparing different columns under realistic conditions (high enough k) and using the compound of interest.

Comparison of the quantitative performance of constant pressure versus constant flow rate gradient elution separations using concentration-sensitive detectors.

This contribution discusses the difference in chromatographic performance when switching from the customary employed constant flow rate gradient elution mode to the recently re-introduced constant pressure gradient elution mode. In this mode, the inlet pressure is maintained at a set value even when the mobile phase viscosity becomes lower than the maximum mobile phase viscosity encountered during the gradient program. This leads to a higher average flow rate compared to the constant flow rate mode and results in a shorter analysis time. When both modes carry out the same mobile phase gradient program in volumetric units, normally identical selectivities are obtained. However, small deviations in selectivity are found due to the differences in pressure and viscous heating effects. These selectivity differences are of the same type as those observed when switching from HPLC to UHPLC and are inevitable when speeding up the analysis by applying a higher pressure. It was also found that, when using concentration-sensitive detectors, the constant pressure elution mode leads to identical peak areas as the constant flow rate mode. Also the linearity is maintained. In addition, the repeatability of the peak area and retention time remains the same when switching between both elution modes.

Kinetic performance limits of constant pressure versus constant flow rate gradient elution separations. Part I: theory.

We report on a general theoretical assessment of the potential kinetic advantages of running LC gradient elution separations in the constant-pressure mode instead of in the customarily used constant-flow rate mode. Analytical calculations as well as numerical simulation results are presented. It is shown that, provided both modes are run with the same volume-based gradient program, the constant-pressure mode can potentially offer an identical separation selectivity (except from some small differences induced by the difference in pressure and viscous heating trajectory), but in a significantly shorter time. For a gradient running between 5 and 95% of organic modifier, the decrease in analysis time can be expected to be of the order of some 20% for both water-methanol and water-acetonitrile gradients, and only weakly depending on the value of V(G)/V0 (or equivalently t(G)/t0). Obviously, the gain will be smaller when the start and end composition lie closer to the viscosity maximum of the considered water-organic modifier system. The assumptions underlying the obtained results (no effects of pressure and temperature on the viscosity or retention coefficient) are critically reviewed, and can be inferred to only have a small effect on the general conclusions. It is also shown that, under the adopted assumptions, the kinetic plot theory also holds for operations where the flow rate varies with the time, as is the case for constant-pressure operation. Comparing both operation modes in a kinetic plot representing the maximal peak capacity versus time, it is theoretically predicted here that both modes can be expected to perform equally well in the fully C-term dominated regime (where H varies linearly with the flow rate), while the constant pressure mode is advantageous for all lower flow rates. Near the optimal flow rate, and for linear gradients running from 5 to 95% organic modifier, time gains of the order of some 20% can be expected (or 25-30% when accounting for the fact that the constant pressure mode can be run without having to leave a pressure safety margin of 5-10% as is needed in the constant flow rate mode).

Kinetic performance limits of constant pressure versus constant flow rate gradient elution separations. Part II: experimental.

We report on a first series of experiments comparing the selectivity and the kinetic performance of constant flow rate and constant pressure mode gradient elution separations. Both water-methanol and water-acetonitrile mobile phase mixtures have been considered, as well as different samples and gradient programs. Instrument pressures up to 1200 bar have been used. Neglecting some small possible deviations caused by viscous heating effects, the experiments could confirm the theoretical expectation that both operation modes should lead to identical separation selectivities provided the same mobile phase gradient program is run in reduced volumetric coordinates. Also in agreement with the theoretical expectations, the cP-mode led to a gain in analysis time amounting up to some 17% for linear gradients running from 5 to 95% of organic modifier at ultra-high pressures. Gains of over 25% were obtained for segmented gradients, at least when the flat portions of the gradient program were situated in regions where the gradient composition was the least viscous. Detailed plate height measurements showed that the single difference between the constant flow rate and the constant pressure mode is a (small) difference in efficiency caused by the difference in average flow rate, in turn leading to a different intrinsic band broadening. Separating a phenone sample with a 20-95% water-acetonitrile gradient, the cP-mode leads to gradient plate heights that are some 20-40% smaller than in the cF-mode in the B-term dominated regime, while they are some 5-10% larger in the C-term dominated regime. Considering a separation with sub 2-µm particles on a 350 mm long coupled column, switching to the constant pressure mode allowed to finish the run in 29 instead of in 35 min, while also a larger peak capacity is obtained (going from 334 in the cF-mode to 339 in the cP-mode) and the mutual selectivity between the different peaks is fully retained.

Kinetic performance of reversed-phase C18 high-performance liquid chromatography columns compared by means of the Kinetic Plot Method in pharmaceutically relevant applications.

Four fully porous C18 columns (Hypersil Gold, ACE3, Xbridge and Gemini NX), widely employed in the pharmaceutical industry, were compared in terms of efficiency and analysis speed with the Kinetic Plot Method. Weakly basic, medium-sized, N-containing pharmaceutical compounds from GlaxoSmithKline Research and Development were used as test molecules. Isocratic elution was carried out at pH 4.5 and pH 8.0 with acetonitrile as organic modifier. The columns under evaluation included highly pure silica supports (Hypersil Gold, ACE3) and hybrid polymer-silica supports (XBridge, Gemini NX). Both types of columns claim for nearly absent secondary interactions with ionized silanol groups and are therefore applicable in a wide pH range. This is an important feature for method development purposes in pharmaceutical industries. The Kinetic Plot Method was used to compare the support characteristics and assess the kinetic performance of the columns in different experimental conditions. Although the evaluated columns have roughly identical particle diameters (from 3.0 to 3.5µm) according to their manufacturers, large differences in kinetic performance were observed at pH 4.5 that can be accounted for by different flow resistances, porosities and average particle diameters, experimentally determined from scanning electron microscopy and laser light scattering experiments on loose stationary phase material. The ACE3 column was the best performing support among the evaluated columns, due to its excellent efficiency and average flow resistance. The better performance of the ACE3 column was due to its better packing quality, as could be derived from its impedance plot. Kinetic plots of resolution of a critical pair versus analysis time and column length were established at pH 8.0. These plots can be used as a method development tool to tailor the separation conditions to the required resolution of a given critical pair, combining efficiency and selectivity features of the column.

Integrated fluidic system for bio-molecule separation.

An integrated fluidic system has been fabricated, capable of separating a mixture of different bio-molecules into its components. It is composed of a filter and an actuator; the pressure generated by the actuator sustains the flow of the mixture through the filter. The actuator is made by stacking several layers of conductive polymer. Actuator strain in excess of 10% has been obtained, which corresponds to a fluid flow of 3 microL/min in the fabricated system. The filter consists of an ordered array of Si micro-pillars. A mixture composed of DNA fragments of different length (300 and 400 base-pair) has been effectively separated by using the fabricated filter and chromatographic techniques.

Relationship between the particle size distribution of commercial fully porous and superficially porous high-performance liquid chromatography column packings and their chromatographic performance.

The separation efficiency and kinetics of several commercial HPLC particle types (both fully porous and superficially porous) have been investigated using a pharmaceutical weakly basic N-containing compound as a test molecule. A strong trend between the particle size distribution (PSD) of the particles and the typically employed "goodness of packing"-parameters was observed. The relative standard deviation of the PSD of the tested particles ranged between 0.05 and 0.2, and in this range, a near linear relationship between the A-term constant, the h(min)-value and the minimal separation impedance was found. The experimental findings hence confirm the recent observations regarding the relationship between the narrow PSD of the recently commercialized porous-shell particles and their superior efficiency and kinetic performance. The outcome also suggests that the performance of the current generation of fully porous particle columns could be significantly improved if the PSD of these particles could be reduced.

The kinetic plot method applied to gradient chromatography: theoretical framework and experimental validation.

The kinetic plot method, originally developed for isocratic separations, was extended to the practically much more relevant case of gradient elution separations. A set of explicit as well as implicit data transformation expressions has been established. These expressions can readily be implemented in any calculation spread-sheet program, and allow to directly turn any experimental data set representing the relation between the separation efficiency and the flow rate measured on a single column into the kinetic performance limit curve of the tested separation medium. Since the kinetic performance limit curve is based on an extrapolation to columns with a different length, it should be realized that the curve is only valid under the assumption that the gradient time and the delay time (if any) are adapted such that the analytes are subjected to the same relative mobile phase history when the column length is changed. Both experimental and numerical data are presented to corroborate the fact that the kinetic performance limit curves that are obtained using the proposed expressions are indeed independent of the column length the experimental data were collected in. Deviations might arise if excessive viscous heating occurs in columns with a pronounced non-adiabatic thermal behaviour.

Towards a solution for viscous heating in ultra-high pressure liquid chromatography using intermediate cooling.

A generic solution is proposed for the deleterious viscous heating effects in adiabatic or near-adiabatic systems that can be expected when trying to push the column operating pressures above the currently available range of ultra-high pressures (i.e., 1200 bar). A set of proof-of-principle experiments, mainly using existing commercial equipment, is presented. The solution is based on splitting up a column with given length L into n segments with length L/n, and providing an active cooling to the capillaries connecting the segments. In this way, the viscous heat is removed at a location where the radial heat removal does not lead to an efficiency loss (i.e., in the thin connection capillaries), while the column segments can be operated under near-adiabatic conditions without suffering from an unacceptable rise of the mobile phase temperature. Experimental results indicate that the column segmentation does not lead to a significant efficiency loss (comparing the performance of a 10 cm column with a 2 cm x 5 cm column system), whereas, as expected, the system displays a much improved temperature stability, both in time (because of the shortened temperature transient times) and in space (reduction of the average axial temperature rise by a factor n). The method also prevents a large backflow of heat along the column wall that would lead to large efficiency losses if one would attempt to operate columns at pressures of 1500 bar or more. A real-world pharmaceutical example is given where this improved temperature robustness could help in moderating the changes in selectivity during method transfer from a low to a high pressure operation, although the complex non-linear behavior of the viscous heating and high pressure effects result in lower than expected improvement.

Numerical and analytical solutions for the column length-dependent band broadening originating from axisymmetrical trans-column velocity gradients.

Trans-column velocity gradients arising from radial variations in packing density or mobile phase temperature lead to a plate height contribution that, in the case of for example a 4.6mm column, may increase over several tens of centimeters before it reaches a constant value. Considering a wide variety of different trans-column velocity profiles, including Giddings' general polynomial expression and several simplified partially flat profiles, and performing a set of analytical calculations (to establish an expression for the long time-limit constant value H(infinity)) and numerical simulations (to calculate the band broadening in the transient regime), it was found that the column length-dependent variation of this plate height contribution can be very closely approximated by a simple exponential-law expression. The availability of the latter will greatly simplify the experimental analysis of radial column heterogeneity effects, especially considering that this expression is independent of the radial dispersion, the column diameter, and the average velocity and maximum velocity difference. Surprisingly, the exponential-law expression is to a first approximation also independent of the shape of the velocity profile, provided the velocity profile does not become flat over a substantially large part of the cross-section. In the latter case, the transient curve obeys a more complex law, but can nevertheless still be approximated by an exponential-law expression, though with a different (larger) decay constant.

Characterization of porous silicon integrated in liquid chromatography chips.

Properties of porous silicon which are relevant for use of the material as a stationary phase in liquid chromatography chips, like porosity, pore size and specific surface area, were determined with high-resolution SEM and N(2) adsorption-desorption isotherms. For the anodization conditions investigated, porosity is between 20 and 60%, pore sizes between 2 and 5 nm and specific surface area between 130 and 410 m(2)/cm(3). It was established that under identical anodization conditions, porous layer formation is 10-15% slower on micromachined pillars than on flat substrates, and depends on geometrical parameters like pillar diameter and height and interpillar spacing. In microchannels containing pillars with a porous silicon shell, chromatographic experiments on a coumarin dye mixture were performed, which in comparison with non-porous pillars showed a significant increase of the retention factors, resulting from the large internal surface of the porous pillars. The increased relative retention of one of the coumarin dyes, C480, could be correlated quantitatively with the measured internal surface of the porous layer. Due to the small pore size, these porous shell columns are particularly suitable for analytical or preparative separation of low-molecular weight molecules, with applications in metabolomics, food quality control, or medical diagnostics.

Errors involved in the existing B-term expressions for the longitudinal diffusion in fully porous chromatographic media Part II: experimental data in packed columns and surface diffusion measurements.

Peak parking experiments have been performed on three RP-HPLC different columns, using two different components and a variable mobile phase composition. The aim of the study was to investigate whether the B-term diffusion expressions currently used in the literature (which are all Knox-type models) should be replaced by the effective diffusion expressions that have been developed in the frame of the effective medium theory (EMT). Although the EMT-expressions are not fully accurate either (the mathematics of the complex interactions between different diffusion zones that are in close contact are too demanding to catch them in an exact analytical expression), they at least are physically sound and do not violate Maxwell's basic law of diffusion. Further they also provide a much better approximation of the numerically calculated effective diffusivity in the theoretical test situation considered in part I. The present study shows that the values of the surface or stationary phase diffusion coefficient that are derived from peak parking models can depend heavily on the employed B-term model. The EMT-based B-term expressions lead to values of the surface diffusion coefficient that vary much less strongly with the phase retention factor than if one of the Knox-type models is used to analyze the data. This implies that, since all peak parking experiments that have been performed in the past have all been interpreted with a Knox-type model, the conclusions that have been drawn from these studies should all be moderated or at least revisited.

Errors involved in the existing B-term expressions for the longitudinal diffusion in fully porous chromatographic media Part I: computational data in ordered pillar arrays and effective medium theory.

Numerically solving the effective diffusion in a simplified representation of a chromatographic bed, it was found that the B-term expressions that have up to now been used in the literature, and which can all be reduced to either Deff=(gamma mDm+k'gamma sDs)/(1+k') or Deff=(gamma meDm+k''Dpart)/(1+k''), can no longer be considered to be unconditionally valid. This could be demonstrated by showing that the simulated diffusion data are in agreement with the mathematically sound effective medium theory (EMT), whereas the B-term expressions used up to now in literature are in conflict with the EMT, a theory that is widely accepted in all other fields of science. It is also shown that the use of the existing B-term expressions can lead to very large measurement errors (up to a 100% and more) for the determination of the stationary phase diffusion coefficient gamma sDs from peak parking experiments. The representation of the B-term diffusion should in the future hence be based on the Deff expressions that can be derived from the EMT. These are physically sound and are also more accurate than the classical B-term expressions used up to now.

Understanding and design of existing and future chromatographic support formats.

The present contribution reviews the use of alternative support formats as a means to surpass the chromatographic performance of the packed bed of spheres. First, a number of idealized structures are considered to obtain a general insight in how the performance of a chromatographic support depends on its shape and size, using the isocratic peak-capacity generation speed as the main performance indicator. Using this criterion, it is found that the packing density or, equivalently, the external porosity, is the most important of all geometrical shape factors. Depending on whether the sample consists of weakly or strongly retained components, the optimal external porosity can be expected to vary between 60% and a value near 100%. The optimal exploitation of a high external porosity, however, also requires overall shrinkage of the domain size, towards and into the sub-micron range. With the current fabrication technologies, this requirement seems difficult to achieve. In the presence of a lower limit on the characteristic support size, each range of desired plate numbers or peak capacities has its own optimal external porosity, ranging from a very low value (high packing density) for high speed, small peak capacity applications, to very high external porosities (low packing density) for applications requiring a very large peak capacity. Subsequently, the obtained theoretical insights are used to review and discuss the past and current research on alternative support formats. Finally, a number of emerging micro- and nano-fabrication technologies are introduced and their potential for the future production of supports with improved shape and homogeneity is discussed.