Prediction of Peak Shape and Characterization of Column Performance in Liquid Chromatography as a Function of Flow Rate (original) (raw)
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Analytical Chemistry, 2006
It is demonstrated 4 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 µm 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 L max) 30 cm. In the plate number range that remains accessible, the investigated sub-2 µm particle columns in any case perform (at least for the presently considered parabene separation) better than the 3.5 µm 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.
Effect of analyte properties on the kinetic performance of liquid chromatographic separations
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, 2007
A kinetic plot based method has been used to experimentally predict the optimal particle size yielding the maximal isocratic peak capacity in a given analysis time. Applying the method to columns of three different manufacturers and characterizing them by separating a 4-component paraben mixture at 30 • C, it was consistently found that the classical 3 and 3.5 m particles provide the highest peak capacity for typical isocratic separation run times between 30 and 60 min when operating the columns at a conventional pressure of 400 bar. Even at 1000 bar, the sub-2 m particles only have a distinct advantage for runs lasting 30 min or less, while for runs lasting 45 min or longer the 3 and 3.5 m again are to be preferred. This finding points at the advantage for high-resolution separations that could be obtained by producing 3 and 3.5 m particle columns that can be operated at elevated pressures.
Journal of Chromatography A, 2007
Theoretical aspects of temperature in liquid chromatography (LC) have mostly been studied to elucidate changes in retention behavior of small and large molecules in various solvents. That temperature also plays a significant role in chromatographic performance is less known. Kinetic plots are an established tool to predict chromatographic performance in terms of speed and efficiency that can be obtained with a certain particle size at the maximum attainable column pressure. In this paper, temperature effects on mobile phase viscosity and analyte diffusion are incorporated in these plots to prove that superior performances are within experimental reach for conventional LC columns and equipment. Verification of the modified kinetic plots with experimental data points is presented.
Enhanced Fluidity Liquid Chromatography for Biological Applications
The following work describes the successful application of enhanced fluidity liquid (EFL) mobile phases to improving isocratic chromatographic separation of biological molecules in hydrophilic interaction liquid chromatography (HILIC) mode. The mobile phase was buffered methanol/water with carbon dioxide added to create the enhanced fluidity liquid. Nucleosides (adenosine, uridine, cytidine, guanosine) were employed as the test sample for this method comparison. Using UV detection at 262 nm, the separation of the sample molecules was studied under each mobile phase condition. Increases in peak resolution between all four were observed as a function of increasing additions of carbon dioxide to create the enhanced fluidity liquid.
Column packings for high performance liquid chromatography: Present state and future development
Journal of Radioanalytical and Nuclear Chemistry Articles, 1994
A short overview of HPLC column packings is presented. The properties of chromatographic carriers and the possibilities to combine the solid matrices with organic polymeric stationary phases are elucidated in detail. The latest achievements and anticipated future developments in the area are outlined. Column packings belong to the most important and the most rapidly developing constituents of liquid chromatographs. The properties of the column packings determine several important parameters of the chromatographic processes, namely their selectivity, efficiency and sample capacity thus setting the limits of performance, throughput and speed of separation. High performance liquid chromatography (HPLC) column packings consist of a chromatographic carrier and of a stationary phase. The stationary phase is chemically or physically immobilized on the carrier and its most important part represent(s) the chromatographic function(s). The chromatographic function is a ligand either electroneutral or ionic, attached to the carrier either directly or via a spacer. Alternatively, the chromatographic functions can be carried by macromolecules. Sometimes the molecules provided with the chromatographic functions are attached to the carrier only temporarily, being in dynamic equilibrium with the same molecules added to the mobile phase. ~ Some LC column packings are chemically uniform, i.e., the chemical compositions of the chromatographic carrier and of the stationary phase including chromatographic functions are identical. More often, however, the composition of the carrier matrix differs from the composition of the stationary phase. The stationary phases in general and chromatographic function in particular are responsible for the adsorption, ion interactions, associations including chelation and other kinds of complexation, affinity interactions, partition between stationary and mobile phase, etc., of the separated molecules. The differences in the above interactions
Journal of Chromatography A, 2009
In this paper a comparison of two column characterisation systems is reported: the method based on the hydrophobic-subtraction model of Dolan and Snyder (HS method) versus the method developed at the Katholieke Universiteit Leuven (KUL method). Comparison was done for seven different pharmaceutical separations (fluoxetine, gemcitabine, erythromycin, tetracycline, tetracaine, amlodipine and bisacodyl), using a set of 59 columns. A ranking was built based on an F value (KUL) or F s value (HS) versus a (virtual) reference column. Both methods showed similar probabilities of ranking patterns. Correlation of the respective test parameters of both approaches was poor. Both methods are not perfect and do not match well, but anyhow yield results which allow, with a relatively high certainty, the selection of similar or dissimilar columns as compared to a reference column. An analyst that uses either of the two methods will end up with a similar probability to choose an adequate column. From a practical point of view, it must be noted that the KUL method is easier to use.
Kinetic performance optimisation for liquid chromatography: Principles and practice
Journal of Separation Science, 2011
This HPLC tutorial focuses on the preparation and use of kinetic plots to characterise the performance in isocratic and gradient LC. This graphical approach allows the selection of columns (i.e. optimum particle size and column length) and LC conditions (operating pressure and temperature) to generate a specific number of plates or peak capacity in the shortest possible analysis time. Instrument aspects including the influence of extracolumn effects (maximum allowable system volume) and thermal operating conditions (oven type) on performance are discussed. In addition, the performance characteristics of porous-shell particle-packed columns and monolithic stationary phases are presented and the potential of future column designs is discussed.
Journal of Chromatography A, 1980
This paper reports the influences of detector time constant variations od plate count calculations and peak retention times in the standardisation of high-performance liquid chromatographic columns. A recommendation is made here that column efficiencies should be quoted at zero time constant in order to remove the variability in plate count introduced by varying time constant. For detectors which do not have variable time constant controls, it is recommended that the calculation of plate numbers should be made on solutes having capacity factors in the region 5-6. Further recommendations made are that the peak symmetry correlation ratio should be used to evaluate system performance only when calculated at zero time constant and that the detector time constant should not exceed one hundredth of the peak width for peaks used in the calculation of plate numbers.
Journal of Chromatography A, 1997
The packing behavior of a typical 10 Ixm C~ stationary phase was studied in terms of the resultant column efficiency and capacity factor. The column-to-column reproducibility of these parameters under identical packing procedures is assessed. Correlation of these parameters and the column void volume to the column packing density is reported. Two regimes were studied; that of poor-and well-packed columns. For poorly packed columns, the column-to-column variability is high, but a concomitantly poor same-column reproducibility of measurement suggests that little statistically significant difference exists between different columns packed with the same procedure. There is also no statistically significant correlation between the column parameters and the packing densities, however, the poorest columns showed a degradation of performance after the drying procedures used to obtain the column masses. Well-packed columns showed much less degradation upon drying. For the well-packed columns, statistically significant column-to-column differences were observable, mainly due to a high same-column precision of measurement. Analysis of the results suggests that even well-packed columns are not optimally packed and that regions of high and low density coexist along the column. The results are compared to those achieved with semi-preparative columns packed with the same slurry procedure and preparative columns packed under dynamic axial compression. Poor day-to-day, same-column reproducibility (degradation) under ambient conditions was observed in conjunction with a high column-to-column variability for the semi-preparative columns.