Enhanced Fluidity Liquid Chromatography for Biological Applications

Journal of Chromatography A, 2004

The type of the stationary phase for reversed-phase liquid chromatography significantly affects the sample polarity range that can be covered using gradients of organic solvents in water. The polarity range available for gradient separations of samples containing compounds differing in the lipophilic parts of the molecules can be characterized by "gradient lipophilic capacity", Pl, based on the retention of standard compounds with a repeat lipophilic structural unit, such as a methylene group. The gradient lipophilic capacity is also suitable to characterize the separation possibilities of the columns in non-aqueous reversed-phase gradient elution of strongly non-polar compounds, such as triacylglycerols. In the same way, the suitability of various columns for reversed-phase gradient separations of oligomers can be characterized by "gradient oligomer capacity", as demonstrated in the example of oligo(ethylene glycols). To enable a comparison of the properties of stationary phases independent of column efficiency and dimensions, the gradient lipophilic capacity or the gradient oligomer capacity should be normalized for a "standard" column plate number, gradient range and volume (in column holdup volume units). The gradient lipophilic capacity or the gradient oligomer capacity and the number of compounds that can be resolved during a gradient run decrease as the initial concentration of the strong solvent in the mobile phase increases and (or) the gradient time decreases. These quantities can be used to select a suitable column and to adjust the optimum gradient profile (the initial composition of the mobile phase and the gradient steepness) with respect to the time of analysis and the number of oligomers or other compounds with regular repeat structural groups that can be resolved during the gradient run.

Journal of Chromatography A, 2009

Advances in modern high-performance liquid chromatography (HPLC) have led to increased interest in the comparison of the ultimate performance limits of methodologies aimed at increasing the resolving power per unit time. Kinetic plot-based methods have proven invaluable in facilitating such evaluations. However, in bridging the gap between fundamental comparisons and the eventual practical applicability of kinetic performance data, the effect of analyte properties have thus far largely been neglected. Using pharmaceutical compounds as representative real-life analytes, it is demonstrated that noteworthy differences in the optimal kinetic performance of a chromatographic system are observed compared to data for common test compounds. For a given stationary phase particle size, higher optimal-and maximum plate numbers, corresponding to increased analysis times, are measured for pharmaceutical compounds. Moreover, it is found that the optimal particle size/maximum pressure combination depends on the analyte under investigation, with the beneficial range of efficiencies for small particles shifted towards higher plate numbers for drug molecules. It is further demonstrated that the pH of the mobile phase plays a crucial role in determining the kinetic performance of pharmaceutical compounds. These data clearly indicate that data for test compounds do not reflect the performance attainable for pharmaceutical compounds and highlights the importance of using real-life samples to perform kinetic evaluations.

Journal of Chromatography A, 1978

By measuring the activity coefficients of so!utes and moderator in the mobile phase and the capacity ratios, it was possible to study separately the dependence of the competition term and the solvent interaction term on the composition of the mobile phase. This was done for mobile phases consistin g of 2-propanol and ethyl acetate as moderators in n-heptane, in combination with silica with a high surface area. It was found that both the competition and solvent interaction effects contribute significantly to the change in retention with changing composition of the mobile phase. For a considerable concentration range of the moderator, the stationary phase effects can be understood as a competition for the surface between the moderator and the solutes, while assuming an adsorbed layer of fixed size consisting of moderator and solute. For higher moderator concentrations the competion effect diminishes, and it seems that in this instance solutes can enter the layer without displacing the moderator. The measurement of activity coefficients was also applied to the mobile phases acetonitrile-water and methanol-water used in reversed-phase adsorption chromatography. Comparisons are made with retention data obtained with octyl-modified silica. Preliminary results suggest that the assumption of constant activity of solutes in the stationary phase is not tenable.

Chromatographia, 1995

Using the concept of an effective concentration a method is proposed for predicting retention in liquid chromatography with multicomponent mobile phases based on experiments with corresponding binary ones. The method is verified for normal and reversed-phase systems with ternary mobile phases and agrees closely with experimental data.

Journal of Chromatography A, 2005

Correlations between the separation selectivity in aqueous and non-aqueous reversed-phase systems and in normal-phase LC systems were investigated for samples containing different numbers of two repeat structural elements. Such samples are best separated in "orthogonal" two-dimensional chromatographic systems, showing selectivity for one type of the repeat structural element only in the first dimension and for the other structural element only in the second dimension. The number of resolved compounds improves as the degree of orthogonality of the separation systems increases with decreasing correlation between the selectivities for the sample structural distribution in the two dimensions. Orthogonal systems with non-correlated selectivities for each repeat structural element provide the highest number of separated peaks and regular arrangement of the peaks over the two-dimensional retention space according to the individual structural element distribution and the best use of the available peak capacity. Fully orthogonal systems are difficult to find in practice. Partially orthogonal system with correlated selectivities for one structural type distribution, but with one system non-distinguishing the distribution for the other structural element are still useful for the two-dimensional separations. The correlations between the selectivities for repeat regular structural increments were employed to evaluate the suitability of phase systems for two-dimensional HPLC separations. The selectivity correlation in various reversed-phase and normal-phase systems was evaluated for two sample types: (1) Various RP columns show significantly inversely correlated selectivities for acyl lengths and numbers of double bonds distribution, but the differences in the double bond selectivity can be used for practical separations of triacylglycerols with the same equivalent carbon numbers. (2) Synthetic EO-PO block (co)oligomers with two-dimensional distribution of oxyethylene and oxypropylene monomer units were separated according to the two distribution types using on-line two-dimensional reversed-phase-normal-phase LC with a C18 column in the first dimension and an aminopropyl silica column in the second dimension.

Analytica Chimica Acta, 2006

Computational methods have been used to study the effect of the molecular diffusivity of analytes on the band broadening and more particular the A-term band broadening, in liquid chromatography. The study was conducted on a two-dimensional mimic of a packed bed with nonporous particles (k = 0) and on a hypothetical perfectly ordered packed bed. The advantage of this computational approach is that the molecular diffusion coefficient of the solute in the mobile phase can be varied freely without affecting other parameters. The considered systems hence also formed a perfect test case for Giddings reduced plate height data representation method. The fact that the plate height values for the different D mol-cases were found to fall exactly on the same single reduced plate height curve hence simultaneously validates Giddings (undisputed) theory and the presently proposed method of chromatographic data simulation. The obtained results are also ideally suited for plate height model discrimination, because they yield highly accurate band broadening data (devoid of any experimental scatter) over a very broad range of reduced velocities (ν up to 9000). The best agreement between simulation data and theory is found for the Knox model in which the Knox coefficient was lowered from the widely used n = 1/3 to a value of n = 0.23. This value agrees well with recent data obtained by Popovici et al. in experimental plate height studies of size exclusion chromatography (SEC) of polymers in a range of reduced velocities amounting up to nearly ν = 10 5 .

Analytical Chemistry, 2006

Journal of Chemical Education, 2009