Performance evaluation of evaporative light scattering detection and charged aerosol detection in reversed phase liquid chromatography.

In pharmaceutical industry ultraviolet (UV) detection is often used as the preferred detection technique in HPLC analysis since most pharmaceutical compounds possess a UV-absorbing chromophore. However, in case the active pharmaceutical ingredient (API) does not have a UV-absorbing chromophore, or if some of the impurities present lack a chromophore, they will not be detected in routine HPLC analysis employing only a UV detector and alternative detection schemes have to be used. Refractive index detection or mass spectroscopy (MS) can be used but these detectors have their intrinsic weaknesses, such as lack of sensitivity or high cost. With the appearance of semi-universal techniques such as evaporative light scattering detection (ELSD), and more recent, charged aerosol detection (CAD), detection of non-UV-absorbing compounds became feasible without having to resort to such complex or costly detection methods. This paper evaluates the different performance characteristics such as sensitivity, linearity, accuracy and precision of both the ELSD and CAD detector coupled to HPLC. One disadvantage of this type of detector is the non-linear response behaviour which makes direct linear regression for making calibration curves inaccurate.

On the optimisation of the bed porosity and the particle shape of ordered chromatographic separation media.

We report on a theoretical study wherein we considered a large number of ordered two-dimensional porous pillar arrays with different pillar shapes and widely varying external porosity and calculated the flow resistance and the band broadening (under retentive conditions) over the complete range of practical velocities using a commercial computational fluid dynamics software package. It is found that the performance of the small porosity systems is very sensitive to the exact pillar shape, whereas this difference gradually disappears with increasing porosity. The obtained separation impedances are very small in comparison to packed bed and monolithic columns and decrease with increasing porosity. If accounting for the current micromachining limitations, a proper selection of the exact shape and porosity even becomes more critical, and different design rules are obtained depending on whether porous or non-porous pillars are considered.

Influence of the packing heterogeneity on the performance of liquid chromatography supports.

We report on a series of plate height and flow resistance data obtained via computational fluid dynamics simulations in a simplified two-dimensional (2D) mimic of real packed bed and monolithic columns. By varying the external porosity (0.4 < epsilon < 0.8) and the degree of packing randomness, a good qualitative insight in the relationship between the packing porosity and heterogeneity and the general chromatographic performance parameters is obtained, unbiased by any differences in phase retention factor k', mobile phase diffusivity or viscosity or intra-skeleton porosity. The results provide a quantitative support for the use of domain size reduced plate heights as a means to compare the performance of chromatographic beds with a different porosity, as it was found that packings with a similar degree of packing heterogeneity yield very similar domain size reduced h(min)-values, nearly completely independent of the porosity. The study also clearly shows that the presence of preferential flow paths (inevitably accompanied by the presence of more clustered regions) leads to a decrease of the flow resistance, but also leads to a strong increase of the band broadening if supports with the same porosity epsilon and the same radial width are compared. For the presently considered 2D system, the flow resistance reduction is too small to overcome the corresponding strong increase in band broadening, such that the presence of preferential flow paths always leads to an overall increase of the separation impedance.

Influence of the pillar shape on the band broadening and the separation impedance of perfectly ordered 2-D porous chromatographic media.

We report on a computational study assessing the effect of the pillar shape in perfectly ordered porous chromatographic media. Using computational fluid dynamics to compare the band broadening and flow resistance characteristics of a large number of different pillar shapes, it is found that the most axially elongated shapes yield the best chromatographic performance and that diamonds are to be preferred over ellipsoids. The former pack into a more uniform pore space and display a smaller C(s) value, whereas the latter pack into a locally constricted pore space and therefore generate a considerably larger flow resistance. For the presently considered case of a densely packed array (epsilon = 0.4), changing the pillar shape from a cylinder to a more elongated diamond, for example, reduces the minimal plate heights from h(min) = 0.84 to h(min) = 0.72, the C factor from C = 0.062 to C = 0.050, and the separation impedance from E(min) = 330 to E(min) = 220, without affecting the number of interchannel coupling points.

Computational study of the band broadening in two-dimensional etched packed bed columns for on-chip high-performance liquid chromatography.

The chromatographic performance of several straightforward two-dimensional etched packed bed column lay-outs (equilaterally staggered arrays of, respectively, circular, hexagonal, and diamond-like pillars) has been compared using commercial computational fluid dynamics software. In all cases, the bed porosity was kept at epsilon = 0.4 and a retained component with zone capacity ratio k" = 2 was considered. Exploring the use of six different possible characteristic dimensions to bring the Van Deemter plots of the three different considered particle shapes into agreement, none of them yielded a perfect agreement. Using the pillar volume-based equivalent cylinder diameter (deq) as the characteristic dimension, the diamond-like pillars yielded a significantly smaller h(min) value than the cylinders and the hexagons (h(min) approximately equal to 0.74 for the former versus h(min) approximately equal to 0.83 for the two latter). Including the flow resistance into the analysis, it was found that the "hydrodynamic" shape of the particles has an important influence on the separation impedance E. The more axially elongated diamond pillars yielded an Emin number as small Emin = 180 (for a retained component with k" = 2), i.e. about 40% smaller than the cylinders and the hexagons (Emin = 300-330). The obtained h(min) and Emin values are also significantly smaller than the values often cited for the best possible packed bed HPLC columns. We believe this is a consequence of the assumed perfect homogeneity of the etched structures, and hence hints at the potential benefits of perfectly ordered chromatographic columns, as was already inferred by Knox [J. Chromatogr. A 831 (1999) 3; 960 (2002) 7] and He et al. [Anal. Chem. 70 (1998) 3790].

Advantages of perfectly ordered 2-D porous pillar arrays over packed bed columns for LC separations: a theoretical analysis.

A series of theoretical calculations is presented to quantify the gain in LC separation efficiency that can be expected if the traditionally used packed bed columns were replaced by columns with a perfectly ordered flow-through pore network. It is shown that a perfectly ordered 2-D array of porous cylindrical pillars could yield reduced plate heights as small as h = 0.65 (for k' ' = 0.75) to h = 0.85 (for k' ' = 2) and separation impedances as small as E = 200 (for k' ' = 0.75) to E = 300 (for k' ' = 2) without having to compromise on the porosity (epsilon = 0.4) and the retention capacity of the packed bed of spheres. Fitting the calculated van Deemter plots with Knox's equation especially shows a strong decrease of the A-term contribution, hence confirming that the improved column performance indeed stems from the increased homogeneity of the packing. The presented results, hence, provide a clear quantitative support for Knox's recent argumentation that the use of more uniform beds could greatly enhance the efficiency of pressure-driven LC.

Sub-second liquid chromatographic separations by means of shear-driven chromatography.

Utilizing the concept of shear-driven chromatography, we have been able to realize reversed-phase LC separations in flat rectangular nano-channels coated with a C8 monolayer and being as thin as 100 nm. At this scale, the separation kinetics are strongly enhanced, as is witnessed by the extremely short time (< 0.1 s) needed to separate a mixture of coumarin dyes. The observed plate numbers are still relatively small, because the experiments were conducted in ultra-short columns (< or = 1 mm) and under injection band width-limiting conditions.

Shear-flow-based chromatographic separations as an alternative to pressure-driven liquid chromatography.

It is only by developing specially designed injection and detection systems that shear-driven chromatography can become a viable alternative to HPLC. In the present paper, a dedicated zero dead-volume injection procedure is presented with which sample volumes can be injected reproducibly in the required picoliter range. In addition, a transversal detection groove system is designed which should allow to perform on-line UV-VIS absorption measurements with path lengths in the millimeter range, with an acceptable theoretical plate loss (only 20% in a 5 cm long channel) and acting as a nearly perfect wave guide.

Experimental demonstration of the possibility to perform shear-driven chromatographic separations in micro-channels.

The possibility to perform shear-driven chromatographic separations in micro-channels is demonstrated, using a novel laser-jet printed microfluidic channel system. The obtained theoretical plate numbers are in fair agreement with the theoretical calculations. Theoretical extrapolations of the separation speeds and detection limits which can be achieved when further miniaturizing the current system are presented as well.