High Mobile Phase Velocities Research Articles

Deductive inference on the plate height equation of ultra performance liquid chromatography

In recent years, separation and analysis workers have made considerable progress on ultra performance liquid chromatography (UPLC). A lot of separation works have been completed which have never been done before. But the theory about chromatographic dynamics of UPLC from the beginning up to now, is remaining in 1950's. Some people explain the principle of UPLC by the van Deemter equation, but this is not right. In this paper, the dynamic process of UPLC has been studied. According to the chromatographic dynamics principle and starting from the heat conduction equation, the plate height equation of UPLC including the influence of mobile phase friction heat production has been deduced as follows: [Formula: see text]. The last item on the right represents the contribution of mobile phase friction heat. When the linear velocity of mobile phase is lower, the contribution of mobile phase friction heat tending to become zero, the plate height equation is reduced to the Horvath and Lin equation. When the linear velocity of mobile phase is high, the temperature difference between the column axial center and edge is directly proportional to the mobile phase linear speed square, and the contribution of thermal effect will largely increased. In this paper, the author clearly pointed out that the column efficiency of UPLC has a direct bearing on the column diameter. Using fine diameter column is helpful to implement column efficiency. Too high mobile phase velocity will lead to the column efficiency collapsed.

Practical observations on the performance of bare silica in hydrophilic interaction compared with C18 reversed-phase liquid chromatography

The kinetic performance of a bare silica and C18 phase prepared from the same sub-2μm and 3.5μm base materials were compared in the HILIC and RP mode using both charged and neutral solutes. The HILIC column was characterised using the neutral solute 5-hydroxymethyluridine, the weak base cytosine, and the strong base nortriptyline, the latter having sufficient retention also in the RP mode to allow comparison of performance. Naphthalene was also used as a simple neutral substance to evaluate the RP column alone. The retention factors of all substances were adjusted to give similar values (k′∼5.5) at their respective optimum linear velocities. Reduced van Deemter b-coefficients (determined by curve fitting and by the peak parking method, using a novel procedure involving switching to a dummy column) were significantly lower in HILIC for all substances compared with those found under RP conditions. Against expectation, c-coefficients were always lower in RP when compared with HILIC using sub-2μm particles. While measurement of these coefficients is complicated by retention shifts caused by the influence of high pressure and by frictional heating effects, broadly similar results were obtained on larger particle (3.5μm) phases. The mechanism of the separations was further investigated by examining the effect of buffer concentration on retention. It was concluded that HILIC can sometimes show somewhat inferior performance to RP for fast analysis at high mobile phase velocity, but clearly shows advantages when high column efficiencies, using longer columns at low flow velocity, are employed. The latter result is attributable to the lower viscosity of the mobile phase in HILIC and the reduced pressure requirement as well as the lower b-coefficients.

Performance characteristics of new superficially porous particles

Superficially porous particles (also called Fused-Core, core shell or porous shell particles) show distinct advantages over comparable totally porous particles for separating small molecules. Columns of Fused-Core particles exhibit very high efficiency because of superior eddy dispersion properties (smaller van Deemter A term). The efficiency for columns of 2.7μm Fused-Core particles actually rivals that for sub-2μm totally porous particles with only about one-half the back pressure. These Fused-Core particles show special advantages with larger molecules for fast separations at high mobile phase velocities because of superior mass transfer (kinetic) properties (smaller van Deemter C term). This report describes the effect of different particle size and porous shell thicknesses on chromatographic performance for Fused-Core particles. Particle characteristics can significantly affect factors of separation importance. For example, the reduced plate height of packed columns is affected by particle diameter. Interestingly, larger Fused-Core particles show smaller reduced plate heights than smaller Fused-Core particles. Also, porous shell thickness has a strong effect on solute retention as well as separation efficiency, and particle surface area has a direct influence on sample loading characteristics. Fused-Core particles with a wide range of physical characteristics have been developed that allows the preparation of stable, efficient packed columns.

Practical comparison of 2.7 μm fused-core silica particles and porous sub-2 μm particles for fast separations in pharmaceutical process development

Fused-core silica stationary phases represent a key technological advancement in the arena of fast HPLC separations. These phases are made by fusing a 0.5 μm porous silica layer onto 1.7 μm nonporous silica cores. The reduced intra-particle flow path of the fused particles provides superior mass transfer kinetics and better performance at high mobile phase velocities, while the fused-core particles provide lower pressure than sub-2 μm particles. In this work, chromatographic performance of the fused-core particles (Ascentis Express) was investigated and compared to that of sub-2 μm porous particles (1.8 μm Zorbax Eclipse Plus C18 and 1.7 μm Acquity BEH C18). Specifically, retention, selectivity, and loading capacity were systematically compared for these two types of columns. Other chromatographic parameters such as efficiency and pressure drop were also studied. Although the fused-core column was found to provide better analyte shape selectivity, both columns had similar hydrophobic, hydrogen bonding, total ion-exchange, and acidic ion-exchange selectivities. As expected, the retention factors and sample loading capacity on the fused-core particle column were slightly lower than those for the sub-2 μm particle column. However, the most dramatic observation was that similar efficiency separations to the sub-2 μm particles could be achieved using the fused-core particles, without the expense of high column back pressure. The low pressure of the fused-core column allows fast separations to be performed routinely on a conventional LC system without significant loss in efficiency or resolution. Applications to the HPLC impurity profiling of drug substance candidates were performed using both types of columns to validate this last point.

Modeling of thermal processes in high pressure liquid chromatography: II. Thermal heterogeneity at very high pressures

Advanced instruments for liquid chromatography enables the operation of columns packed with sub-2 μm particles at the very high inlet pressures, up to 1000 bar, that are necessary to achieve the high column efficiency and the short analysis times that can be provided by the use of these columns. However, operating rather short columns at high mobile phase velocities, under high pressure gradients causes the production of a large amount of heat due to the viscous friction of the eluent percolating through the column bed. The evacuation of this heat causes the formation of significant axial and radial temperature gradients. Due to these thermal gradients, the retention factors of analytes and the mobile phase velocity are no longer constant throughout the column. The consequence of this heat production is a loss of column efficiency. We previously developed a model combining the heat and mass balance of the column, the equations of flow through porous media, and a linear isotherm model of the analyte. This model was solved and validated for conventional columns operated under moderate pressures. We report here on the results obtained when this model is applied to columns packed with very fine particles, operated under very high pressures. These results prove that our model accounts well for all the experimental results. The same column that elutes symmetrical, nearly Gaussian peaks at low flow rates, under relatively low pressure drops, provides strongly deformed, unsymmetrical peaks when operated at high flow rates, under high pressures, and under different thermal environments. The loss in column efficiency is particularly important when the column wall is kept at constant temperature, by immersing the column in a water bath.

Density gradients in packed columns: II. Effects of density gradients on efficiency in supercritical fluid separations

To investigate how fluid compressibility affects efficiency in supercritical fluid separations, band dispersion along a packed capillary column was measured from on-column elution rate profiles obtained under solvating gas chromatography (SGC) conditions; this allowed efficiency to be determined with respect to position along the column. Theoretical efficiency was also modeled. The model indicates that the primary cause of band broadening in SGC is high mobile phase velocity near the column outlet. However, the experimental results show that significant band broadening also occurs near the column inlet in a region that corresponds to high elution rates of the analyte. On-column detection also revealed spatial focusing of the analyte as it moves down the column density gradient.

Influence of Viscous Friction Heating on the Efficiency of Columns Operated under Very High Pressures

When columns packed with very fine particles are operated at high mobile phase velocities, the friction of the mobile phase percolating through the column bed generates heat. This heat dissipates along and across the column and axial and radial temperature gradients appear. The wall region of the column tends to be cooler than its center, and due to the influence of temperature on the mobile phase viscosity and on the equilibrium constant of analytes, the band velocity is not constant across the column. This radial heterogeneity of the temperature distribution across the column contributes to band broadening. This phenomenon was investigated assuming a cylindrically symmetrical column and using the general dispersion theory of Aris, which relates the height equivalent to the theoretical plate (HETP) contribution due to a radial heterogeneity of the column to the radial distribution of the linear velocities of a compound peak and to the radial distribution of its apparent dispersion coefficients in the column bed. The former is known from the temperature gradient across the column, the temperature dependencies of the mobile phase viscosity, and the retention factor of the compound. The latter is derived from the known expression of the transverse reduced HETP equation for the column. The values of the HETP calculated with the Aris model and a classical HETP equation were compared to those measured on a 2.1 x 50 mm Acquity BEH-C(18) column, run at flow rates of 0.6, 0.95, 1.30, and 1.65 mL/min, with pure acetonitrile as the mobile phase and naphtho[2,3-a]pyrene as the retained compound. These two sets of data are in generally good agreement, although the experimental values of the HETP tend to increase faster with increasing mobile phase velocity than the calculated values.

Practical assessment of frictional heating effects and thermostat design on the performance of conventional (3 μm and 5 μm) columns in reversed-phase high-performance liquid chromatography

A practical investigation of frictional heating effects in conventional C18 columns was undertaken, to investigate whether problems found for sub-2 μm columns were also present for those of particle size 3 μm and 5 μm and different internal diameter. The influence of a water bath, a still air heater, and a forced air heater on performance was investigated. Heating effects were substantial, with a decrease in k of almost 15% for toluene over the flow rate range ∼0.4–2.3 mL/min with a 15 cm × 0.46 cm ID column packed with 3 μm particles. Heating effects on retention increased with increasing solute k, with increase in the column ID, with decrease in the column particle size, and with decrease in the set column oven temperature. While the water bath minimised axial temperature gradients and thus its effect on k, radial temperature gradients were potentially serious with this system, especially at high mobile phase velocity, even with columns containing 5 μm particles. In contrast to the effects of axial temperature gradients in 4.6 mm columns, very little difference in Van Deemter plots was noted between the three different thermostats with 2 mm ID columns, even when 3 μm particles were used. However, the efficiency of 2 mm columns for peaks of low or moderate k ( k < 4) can be compromised by the extra dead volume introduced by the heating systems, even with conventional HPLC systems with otherwise minimised extra column volume.

Consequences of the radial heterogeneity of the column temperature at high mobile phase velocity

When a high velocity stream of mobile phase percolates through a chromatographic column, the bed cannot remain isothermal. Due to the mobile phase decompression, heat is generated along the column. Longitudinal and radial temperature gradients take place along and across its bed. The various consequences of this thermal heterogeneity are calculated and their effects on the column efficiency investigated for a 0.46cm×25cm stainless steel column packed with 5μm particles. The maximum pressure drop applied was varied from 0.1 to 2kbar. The amplitude of the longitudinal temperature gradient can be estimated on the basis of the integral heat balance equation applied to the whole column and of measurements of the eluent temperature at the column exit. Assuming that the radial gradient is parabolic and the longitudinal gradient linear, the amplitude of the radial gradient can be determined on the basis of the energy balance across the column and of direct measurements of the radial gradient at high inlet pressures. A radial temperature gradient causes a radial distribution of the eluent viscosity, hence of its local velocity. The result is that bands move faster in their center than along the wall, become warped, hence a radial concentration gradient, similar in origin to the one observed in open cylindrical tubes. Diffusion relaxes this gradient. If there is only a longitudinal temperature gradient, the column efficiency would be 30% smaller for a 2kbar pressure drop than if there is no longitudinal temperature gradient. However, when both a longitudinal and a radial temperature gradient coexist, there is a large loss of efficiency. If the influence of the diffusive relaxation of the radial concentration gradient is neglected, the peak shape would be broad and exhibit a marked shoulder.

Potential of silica monolithic columns in peptide separations

The objective of the work described here was to evaluate the efficacy of silica monolith supports in high-speed reversed-phase liquid chromatography (RPLC) of peptides. This was done using a commercial Chromolith column with an octadecylsilane stationary phase and a tryptic digest of cytochrome c. Columns (100mm×4.6mm) were operated at mobile phase velocities ranging from 1ml/min (2.0mm/s) to 10ml/min (25mm/s). There was little noticeable change over this flow rate range in either resolution, peak elution volume, or analyte concentration in collected fractions. It was concluded that capillary columns in this silica monolith format would be particularly valuable in peptide separations for proteomics. There was, however, a small, but perceptible contamination of peaks at high mobile phase velocity with earlier eluting analytes. Based on the fact that peak shape did not change at high mobile phase velocity, it is suggested that this phenomena might be due to the presence of peptide conformers in structural equilibrium on the sorbent surface. When elution rate exceeds the rate of conformer interchange, conformers could elute as broadened or even separate peaks.

Preparative Monolithic Silica Sorbents (PrepRODs™) and Their Use in Preparative Liquid Chromatography

The use of monolithic silica sorbents for the isolation of substances by preparative liquid chromatography is demonstrated. Preparative liquid chromatography is recognized as a valuable technique for the isolation and purification of substances in the pharmaceutical and fine chemicals industry. The system technology has meanwhile reached a high standard, and the greatest future improvements are expected to arise from new and improved adsorbents. Monolithic silica sorbents offer some unique features for preparative liquid chromatography. They exhibit high efficiencies even at high flow rates due to their fast convective mass transfer and can therefore be used at very high mobile phase velocities, leading to high productivity and hence to maximum process economy. The benefits of this new type of adsorbent are illustrated for an example in batch-chromatographic mode and an example using the continuous simulated moving bed (SMB) technology.

Characterization of protein adsorption by composite silica-polyacrylamide gel anion exchangers II. Mass transfer in packed columns and predictability of breakthrough behavior

Rates of uptake of proteins by a composite silica-polyacrylamide gel ion exchanger known as Q-HyperD are obtained for hydrodynamic conditions identical to those found in a chromatographic column operated at a high mobile phase velocity by means of a shallow bed technique. Under conditions of intraparticle mass transfer control, mass transfer rates in the shallow bed were the same as those measured when the sorbent particles were suspended in an agitated vessel, indicating that the intraparticle mass transfer rate is independent of the hydrodynamic conditions outside the particle and consistent with a purely diffusional transport mechanism. The predictability of breakthrough curves based on kinetic parameters obtained from batch experiments is assessed by comparing experimental results for different proteins with predictions of a model which incorporates the external film resistance and intraparticle diffusion. This model is in excellent agreement with the experimental results even at extremely high mobile phase velocities. Very large dynamic capacities are obtained with columns packed with Q-HyperD media as a result of their high static capacity, rapid uptake kinetics and mechanical strength that prevents compression of the particles even at elevated flow-rates.

High velocity reversed-phase chromatography of proteins and peptides: use of conventional C18, 300 Å, 15 μm particles

Experiments were conducted to study the effects of mobile phase velocity on the reversed-phase chromatography of peptides ans proteins using a mono-modal pore size (300 Å) C18 spherical silica packing. This material was packed into several 5 × 0.46 cm columns for gradient elution studies using ribonuclease A, insulin, lysozyme and myoglobin. Baseline separation of these proteins was achieved within 90 seconds. Using a two-minute linear gradient from 15 to 65% acetonitrile (in 0.1% trifluoroacetic acid), resolution improved with velocity. Enhanced performance was attributed to the concurrent increase in gradient volume with higher mobile phase velocity. The frontal adsorption capacity of lysozyme was 25 and 23 mg/ml at 220 and 3600 Å packing material. These values are equivalent (within an experimental error of ±2 mg/ml) clearly demonstrating that lysozyme ( M r 14 300) fully permeates the 300 Å pores during operation at high mobile phase velocity. Comparison of protein diffusion velocity with the distances involved in pore penetration substantiates the feasibility of this observation. Loading studies were conducted at both 360 and 3600 cm/h using the protein test mixture. The resulting chromatograms were very similar indicating that, under certain circumstances, separations can be run on conventional particles at velocities 5 to 10 times greater than currently practised. The preparative implications are discussed.

Model for retention and efficiency in open-tubular supercritical fluid chromatography

A model for open-tubular supercritical fluid chromatography at constant mass flow-rate, which takes into account the effects of temperature, density and pressure drop on solute retention and dispersion, is developed and applied to the elution of normal alkanes (C 8-C 40) on a polysiloxane stationary phase using CO 2 mobile phase using CO 2 mobile phase at 40 to 120°C. An exact expression for resolution is derived in terms of observed chromatographic parameters, and the effect of swelling on solute diffusion coefficients in the stationary phase is considered. Plots of apparent plate height vs. temporal average velocity show the onset of gradient-induced band spreading for a variety of conditions. Swelling of the stationary phase is shown to be critical to the achievement of acceptable efficiency for heavy solutes at low temperatures, and the prediction of a second maximum in resolution at high mobile phase velocities suggests the possibility of extremely fast separations by supercritical fluid chromatography.

Use of chromatographic data to determine the molecular weight of a solute eluted from a liquid chromatographic column

The diffusivities of 69 different compounds in a n-hexane—ethyl acetate solvent mixture have been determined and a precise relationship between solute diffusivity, molecular weight and molar volume established. The band dispersion for each of the same solutes has also been measured employing a liquid chromatographic column designed to emphasize the resistance-to-mass transfer factor and minimize thermal effects resulting from the use of high pressures and high mobile phase velocities. The effect of the capacity ratio of a solute on the resistance-to-mass transfer factor is determined and the relationship between the bandwidth of a solute, its diffusivity and its molecular weight established. A procedure is outlined for the determination of the molecular weight of a solute from the measurement of its bandwidth, when eluted from a liquid chromatographic column, within an error of 13% for 90% of the solutes examined providing the density of the solute lies between 0.85 and 1.25 g/ml.

Use of microbore columns for rapid liquid chromatographic separations

The use of microbore columns for high-speed chromatographic separation is investigated, and apparatus is described that can be usd with such columns. Specific details of detector cell construction are given, and photosensor specifications are suggested to provide the necessary fast detector response time. Specifications are also provided for appropriate data acquisition equipment. The performance of microbore columns operated at high mobile phase velocities is examined, and the effect of mobile phase velocity on column efficiency investigated. The precisions of quantitative analysis by peak height and peak area measurements are compared for a microbore columns operated at high linear mobile phase velocity. An example of the separation of a seven-component mixture in less than 30 sec is included.

Use of oxiranes in the preparation of bonded phase supports

A variety of stationary phases were bonded to silaceous supports through an intermediate silane coupling agent, γ-glycidoxypropylsilane. Consequently, a series of ion-exchange, hydrophobic, and hydrophilic supports were prepared that are stable, withstand high mobile phase velocities, and have sufficient porosity to allow the partitioning of biopolymers. Liquid-liquid partition chromatography of proteins was achieved with phosphate buffers while the separation of a series of aromatic compounds was accomplished on the same columns with hexane. Anion- and cation-exchange supports were found to have hemoglobin ion- exchange capacities similar to classical cross-linked dextrans while allowing separation speeds 10–20 times those of carbohydrate gel supports.