Diffusion coefficients of an extensive set of pharmaceutical compounds in supercritical fluid chromatography over a wide range of mobile phase compositions.

Diffusion data are essential for adequate analysis of the kinetic separation performance of any chromatographic system. Unfortunately, for Supercritical Fluid Chromatography (SFC), very little data is available of the diffusion coefficients in mobile phases typically used in contemporary methods, i.e. with a non-negligible amount of polar modifier such as methanol. In this work, a relative simple method which only requires minor modifications to a standard commercially available SFC instrument is used to determine the diffusion coefficient of an extensive set of pharmaceutical compounds in the range of 10-50 vol% of modifier (methanol) in CO2. By using a traditional SFC column, the solute is first separated from the sample solvent plug, before entering a long capillary, where the band broadening can be linked to its diffusion coefficient using the Taylor-Aris equation. By using two UV-detectors, before and after the capillary, the effect of the dispersion in the column can be eliminated and the true volumetric flow rate determined. It was found that in the investigated range of conditions, the change in mobile phase viscosity in a first approximation allows to predict the variation in diffusion coefficient. Chemical structure and more particularly functional groups can however have a significant effect on the diffusion coefficient.

Detailed numerical study of the peak shapes of neutral analytes injected at high solvent strength in short reversed-phase liquid chromatography columns and comparison with experimental observations.

We report on a numerical investigation of the different steps in the development of the spatial concentration profiles developing along the axis of a liquid chromatography column when injecting large relative volumes (>10 to 20% of column volume) of analytes dissolved in a high solvent strength solvent band as can be encountered in the second dimension (2D) column of a two-dimensional liquid chromatography (2D-LC) system. More specifically, we made a detailed study of the different retention and the axial band broadening effects leading to the double-headed peak shapes or strongly fronting peaks that can be experimentally observed under certain conditions in 2D-LC. The establishment of these intricate peak profiles is discussed in all its fine, mechanistic details. The effect of the volume of the column, the volume and the shape of the sample band, the retention properties of the analyte and the band broadening experienced by the analytes and the sample solvent are investigated. A good agreement between the simulations and the experimental observations with caffeine and methylparaben injected in acetonitrile/water (ACN/H2O) mobile phase with different injection volumes is obtained. Save the difference in dwell volume, key features of experimental and simulated chromatograms agree within a few %. The simulations are also validated against a number of simple mathematical rules of thumb that can be established to predict the occurrence of a breakthrough fraction and estimate the amount of breakthrough.

Column-in-valve designs to minimize extra-column volumes.

We report on the design and performance of in-house built column cartridges that can be directly screwed into the ports of a commercial rotor-stator valve to minimize extra-column band broadening and pressure-drop losses when pursuing ultra-fast separations such as those needed in 2D and 3D-LC separations. Two basic designs were evaluated and were compared with the results obtained with a commercial screw-in column cartridge. The system produces an extra-column band broadening as low as 0.05 to 0.1 µL2 for the employed UV-detector set-up. Despite these very low values, the obtained separation efficiency of the in-house fabricated cartridge columns was very low, corresponding to a reduced minimal plate height around h=7 at the very best, which, for the 1.7 µm particle and 26.4 mm long columns corresponds to a number of theoretical plates of N=2200 under isocratic conditions. A similar poor performance was obtained with a commercial column cartridge with similar dimensions using the same set-up. One possible explanation of the observed performance could be found in the inner diameter of the column cartridges (i.d. =0.75 mm and 1 mm) which, for the employed sub 2-µm particles, falls into a region of column diameters that, according to literature models, is most likely to suffer from inherent packing problems.

Peak sharpening limits of solvent-assisted post-column refocusing to enhance detection limits in liquid chromatography.

We report on a numerical and experimental study of the limits of peak refocusing and concentration enhancement that can be obtained with solvent front-assisted peak remobilization in a trap column receiving peaks eluting from a preceding analytical column. It is shown that the upper limit of peak refocusing can best be pursued by injecting a sufficiently large volume in a sufficiently narrow capillary and elute it with a sufficiently steep (ballistic) gradient. Corresponding equations offering a quantitative description have been derived and verified experimentally. For the latter purpose, peak volumes of the order of 0.5 to 2.0 µL were refocused in a dedicated set-up capable of trapping µL-sized peaks in a 75 µm i.d. capillary and remobilized using a nano-LC pump propelling an acetonitrile/isopropyl alcohol mixture with a viscosity matching that of the trapping solvent. Injecting 2.0 µL peaks, a peak refocusing factor of 17.3 could be achieved.

Optimal mixing rate in reverse phase liquid chromatography. Experimental evaluations.

The topic of this report is experimental verification of previously published theoretical predictions. The mixing rate (Rϕ) is the temporal rate of increasing a fraction of stronger solvent in the mobile phase of gradient LC. The optimal Rϕ (Rϕ,Opt) is the one at which a required peak capacity (n) of gradient LC analysis is obtained in the shortest time. The key factors affecting Rϕ,Opt are the sample molecular weight (M), the void time (tM), and the column pressure condition - Rϕ,Opt in a column operating below the instrumental pressure limit is 2-3 times higher than Rϕ,Opt in a column operating at the instrumental pressure limit. Using previously proposed speed optimization criteria, t/si, where t, s and i are the analysis time, the separation capacity (a metric proportions to n), and the order (i=2 or i=4 for analyses operating below or at the instrumental pressure limit, respectively), parameter Rϕ,Opt for analyses of small-molecule samples has been experimentally found. For both orders, the agreement with the theoretical prediction can be considered as very good. Base on the experimental results, we continue to recommend the earlier proposed theoretically based mixing rate of 5%/tM as a default for all analyses of small-molecule samples (100