Kinetic performance optimisation for liquid chromatography: Principles and practice (original) (raw)
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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.
Journal of Chromatography A, 2008
The review highlights the fundamentals and the most prominent achievements in the field of high-performance liquid chromatography (HPLC) column development over a period of nearly 50 years. After a short introduction on the structure and function of HPLC columns, the first part treats the major steps and processes in the manufacture of a particle packed column: synthesis and control of particle morphology, sizing and size analysis, packing procedures and performance characterization. The next section is devoted to three subjects, which reflect the recent development and the main future directions of packed columns: minimum particle size of packing, totally porous vs. core/shell particles and column miniaturization. In the last section an analysis is given on an alternative to packed columns-monolithic columns, which have gained considerable attraction. The challenges are: improved packing design based on modeling and simulation for targeted applications, and enhanced robustness and reproducibility of monolithic columns. In the field of miniaturization, particularly in chip-based nano-LC systems, monoliths offer a great potential for the separation of complex mixtures e.g. in life science.
Journal of Chromatography A, 2012
The kinetic performance of 0.5 mm × 50 mm columns packed with 2.7 m Halo-C 18 core-shell particles and 3 m EP-120-C 18 fully porous particles fitted on an Eksigent LC-Express Ultra HPLC system were measured. The instrument contribution to band broadening was obtained by directly connecting the injection valve and the detector cell with a short, narrow PEEKSIL tube. The connections between the column and the connecting tubes, the column endfittings and its frits contribute to band spreading and are responsible for a significant rear peak tailing, even for retained compounds, resulting in a significant loss of efficiency. Our results show that the HPLC system could outperform the current VHPLC systems using 2.1 mm I.D. columns packed with 1.7 m particles if it were using 0.5 mm I.D. columns packed with 1 m particles, if it could operate at a few kbar pressure drop, and if the sum of the contributions of the instrument, column endfittings and the column frits to band dispersion were three times smaller than it is at present.
Use of the kinetic plot method to analyze commercial high-temperature liquid chromatography systems
Journal of Chromatography A, 2007
Using a set of experimentally determined plate height data obtained on three commercial high-temperature HPLC supports, and evaluating their isocratic separation speed potential under the application of a set of instrumental constraints, a qualitative map of the practically achievable critical pair separation speed potential of high-temperature HPLC has been established. The obtained data show that the gain in separation speed is more strongly affected by the instrumental limitations in the high-temperature range than it is for the low temperatures. For the presently considered case of alkylbenzene separations, the potential gain in analysis time that can be obtained by going from T = 30 to 120 • C in the presence of a typical set of instrumental limitations nevertheless remains of the order of a factor of 2-4. The study also shows that improvements on the instrumentation side (increased detector frequency, pumping flow rate, smaller extra-column volumes,. . .) are indispensable to fully benefit from the high temperature advantages for all separations requiring less than 10,000 effective theoretical plates.
Effect of Pressure, Particle Size, and Time on Optimizing Performance in Liquid Chromatography
Analytical Chemistry, 2009
Although the principles of optimization of HPLC have a long history starting with the work of Giddings in the 1960s, and continuing with work by Knox and Guiochon extending into the 1990s we continue to see statements that flatly contradict theory. A prominent example is the notion that optimum 'performance', as measured by plate count, is always obtained by operating conventional length columns (e.g., 5 to 15 cm) at eluent velocities corresponding to the minimum plate height in the van Deemter curve. In the last decade the introduction of 'Poppe plots' by Poppe and 'kinetic plots' by Desmet and others has simplified the selection of 'optimum' conditions, but it is evident that many workers are not entirely comfortable with this framework. Here we derive a set of simple, yet accurate equations that allow rapid calculation of the column length and eluent velocity that will give either the maximum plate count in a given time, or a given plate count in the shortest time. Equations are developed for the optimum column length, eluent velocity and thus plate count for both the case when particle size is pre-selected, and when particle size is optimized along with eluent velocity and column length. Although both of these situations have been previously considered the implications of the resulting equations have not been previously made explicit. Lack of full understanding of the consequences of the differences between these two cases is very important and responsible for many erroneous conclusions. The simple closed-form equations that result from this work complement the graphical, iterative approaches of Poppe and Desmet; the resulting compact framework allows practitioners to rapidly and effectively find the operating parameters needed to achieve a specific separation goal in the shortest time, and to compare emerging technologies (e.g., high pressure, high temperature, and different particle types) in terms of their impact on achievable plate counts and speeds in HPLC. A web-based calculator based on the equations presented here is now available (http://homepages.gac.edu/\~dstoll/calculators/optimize.html). "The fastest possible analysis with any column design will be achieved with a column operated at the maximum possible pressure drop and having a length as to give the plate number necessary to perform the desired separation." 1
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, 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.
Chromatography, 2015
Traditionally, column performance in liquid chromatography has been studied using information from the elution of probe compounds at different flow rates through van Deemter plots, which relate the column plate height to the linear mobile phase velocity. A more recent approach to characterize columns is the representation of the peak widths (or the right and left peak half-widths) for a set of compounds versus their retention times, which, for isocratic elution, give rise to almost linear plots. In previous work, these plots have been shown to facilitate the prediction of peak profiles (width and asymmetry) with optimization purposes. In this work, a detailed study on the dependence of the peak widths (or half-widths) on the flow rate is reported. A new approach to quantify the deterioration of column performance for slow and fast flow rates and to characterize chromatographic columns is proposed. The approach makes use of the width (or half-widths) for a set of compounds with similar interaction kinetics and does not require knowledge of the extra-column contributions to the total variance. The chromatographic data of two sets of compounds of different natures (sulfonamides and β-blockers), eluted from Spherisorb and Chromolith columns with acetonitrile-water mixtures, are used to illustrate the approach.
Types of High-Performance Liquid Chromatography (HPLC) Columns: A Review
FoodTech: Jurnal Teknologi Pangan
Some chemical analysts, especially those working in the food sector, often find it difficult to choose the HPLC columns for their research. Every so often they tend to modify their method to fit whatever HPLC column available in their laboratory instead of looking for the column type best suited to their experiments. Other than that, HPLC column types are often very limited discussed in the class. This is particularly disadvantageous for those who have sufficient access to select the HPLC column they need for the best result. The lack of insight into the types of HPLC columns available in the market also influenced their decision to select the right column in their analysis to a large extent. This article briefly reviews the differences between the commonly used Particle-Packed Columns with the newer yet less frequently used Monolithic Columns. The types of HPLC columns based on polarity, molecular size, and the electrical charge will be described further, along with the working pri...