Mobile Phase Velocity Research Articles

Separation of Ginsenosides at Elevated Temperature by Ultra High Pressure Liquid Chromatography

Separation of seven ginsenosides at elevated temperature by ultra high pressure liquid chromatography (UHPLC) on reversed phase C18‐ bonded silica stationary phase was studied. All separations were carried out with a binary mobile phase consisting of CH3CN/H2O in isocratic elution mode. It was found that increasing the column temperature resulted in efficient separation and complete resolution of seven ginsenosides. Generally, the separation efficiency increased as a function of temperature. The effect of the mobile phase velocity on the separation efficiency was also investigated.

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.

Comparative study of the performance of columns packed with several new fine silica particles: Would the external roughness of the particles affect column properties?

We measured and compared the characteristics and performance of columns packed with particles of five different C18-bonded silica, 3 and 5μm Luna, 3μm Atlantis, 3.5μm Zorbax, and 2.7μm Halo. The average particle size of each material was derived from the SEM pictures of 200 individual particles. These pictures contrast the irregular morphology of the external surface of the Zorbax and Halo particles and the smooth surface of the Luna and Atlantis particles. In a wide range of mobile phase velocities (from 0.010 to 3mL/min) and at ambient temperature, we measured the first and second central moments of the peaks of naphthalene, insulin, and bovine serum albumin (BSA). These moments were corrected for the contributions of the extra-column volumes to calculate the reduced HETPs. The C-terms of naphthalene and insulin are largest for the Halo and Zorbax materials and the A-term smallest for the Halo-packed column. The Halo column performs the best for the low molecular weight compound naphthalene (minimum reduced HETP, 1.4) but is not as good as the Atlantis or Luna columns for the large molecular weight compound insulin. The Zorbax column is the least efficient column because of its large C-term. The lowest sample diffusivity through these particles, alone, does not account for the results. It is most likely that the roughness of the external surface of the Halo and Zorbax particles limit the performance of these columns at high flow rates generating an unusually high film mass transfer resistance.

Fused-Core Silica Column High-Performance Liquid Chromatography/Tandem Mass Spectrometric Determination of Rimonabant in Mouse Plasma

A high-performance liquid chromatography (HPLC) method using a fused-core silica particle packing was evaluated to allow fast and efficient separation for the analysis of pharmaceutical compounds. Fused-core particles are produced by "fusing" a porous silica layer onto a solid silica particle. The efficiencies of columns packed with 2.7 microm "fused-core" particles (a 0.5 microm porous shell fused to a solid 1.7 microm silica core particle) and 1.7 microm porous particles were compared in reversed-phase HPLC using rimonabant as an analyte. The fused-core silica materials providing the shorter diffusional mass transfer path for solutes are less affected in resolving power by increases in mobile-phase velocity than the sub-2 microm porous silica packings resulting in faster separations and higher sample throughput. This fast HPLC technology is comparable with ultrahigh-pressure liquid chromatography (UHPLC) in terms of chromatographic performance but demands neither expensive ultra-high-pressure instrumentation nor new laboratory protocols. The column effluent was directly connected to the atmospheric pressure chemical ionization (APCI) source prior to tandem mass spectrometric detection. In this work, the described fast HPLC-MS/MS and UHPLC-MS/MS approaches requiring approximately 1.5 min per sample were applied and compared for the determination of the rimonabant in mouse plasma samples at the low nanograms per milliliter region in support of a pharmacodynamic study.

Gradient elution separation and peak capacity of columns packed with porous shell particles

The separation of the tryptic digests of myoglobin and bovine serum albumin were carried out in the gradient elution mode, using water, acetonitrile and TFA as the mobile phase components and columns packed with a new type of shell particles, Halo C 18. These particles give very high efficiencies, characterized with an unusually low eddy diffusion contribution and a small mass transfer contribution. However, because the molecular diffusivities of the peptides in the digest are small, the mobile phase velocity corresponding to the optimum velocity for maximum efficiency is also small, of the order of 0.3 mm/s. The gradient slopes also must be small. Peak capacities of 400 were achieved, with analysis time of the order of an hour.

Pressure-Driven Reverse-Phase Liquid Chromatography Separations in Ordered Nonporous Pillar Array Columns

Building upon the micromachined column idea proposed by the group of Regnier in 1998, we report on the first high-resolution reversed-phase separations in micromachined pillar array columns under pressure-driven LC conditions. A three component mixture could be separated in 3 s using arrays of nonporous silicon pillars with a diameter of approximately 4.3 microm and an external porosity of 55%. Under slightly retained component conditions (retention factor k' = 0.65-1.2), plate heights of about H = 4 microm were obtained at a mobile phase velocity around u = 0.5 mm/s. In reduced terms, such plate heights are as low as hmin = 1. Also, since the flow resistance of the column is much smaller than in a packed column (mainly because of the higher external porosity of the pillar array), the separation impedance of the array was as small as E = 150, i.e., of the same order as the best currently existing monolithic columns. At pH = 3, yielding very low retention factors (k' = 0.13 and 0.23), plate heights as low as H = 2 microm were realized, yielding a separation of the three component mixture with an efficiency of N = 4000-5000 plates over a column length of 1 cm. At higher retention factors, significantly larger plate heights were obtained. More experimental work is needed to investigate this more in depth. The study is completed with a discussion of the performance limits of the pillar array column concept in the frame of the current state-of-the-art in microfabrication precision.

Application of a chromatography model with linear gradient elution experimental data to the rapid scale-up in ion-exchange process chromatography of proteins

We applied the model described in our previous paper to the rapid scale-up in the ion exchange chromatography of proteins, in which linear flow velocity, column length and gradient slope were changed. We carried out linear gradient elution experiments, and obtained data for the peak salt concentration and peak width. From these data, the plate height (HETP) was calculated as a function of the mobile phase velocity and iso-resolution curve (the separation time and elution volume relationship for the same resolution) was calculated. The scale-up chromatography conditions were determined by the iso-resolution curve. The scale-up of the linear gradient elution from 5 to 100 mL and 2.5 L column sizes was performed both by the separation of β-lactoglobulin A and β-lactoglobulin B with anion-exchange chromatography and by the purification of a recombinant protein with cation-exchange chromatography. Resolution, recovery and purity were examined in order to verify the proposed method.

The influence of grafted polymer architecture and fluid hydrodynamics on protein separation by entropic interaction chromatography

Entropic interaction chromatography (EIC) provides efficient size-based separation of protein mixtures through the entropy change associated with solute partitioning into a layer of hydrophilic homopolymer that has been end-grafted within the pores of a macroporous chromatography support. In this work, surface-initiated atom-transfer radical polymerization (ATRP) is used to prepare a library of EIC stationary phases covering a wide range of grafted-chain densities and molecular weights. Exhaustive chain cleavage and analysis by saponification and GPC-MALLS, respectively, show that the new ATRP synthesis procedure allows for excellent control over graft molecular weight and polydispersity. The method is used to prepare high-density grafts (up to 0.164 +/- 0.005 chains/nm(2)) that extend the range of EIC applications to include efficient buffer-exchange and desalting of protein preparations. Reducing the graft density allows for greater partitioning of high molecular weight solutes, extending the linear range of the selectivity curve. Increasing graft molecular weight also alters selectivity, but more directly affects column capacity by increasing the volume of the grafted layer. Protein partitioning in high-density EIC columns is found to decrease with mobile-phase velocity (u). Although solute mass transfer resistances leading to an increase in plate height can explain this effect, pressure drop data across the column are indicative of weak convective flow through at least a fraction of the grafted architecture. Modeling of the grafted brush properties in the presence of solvent flow by subjecting a self-consistent-field theory representation of the brush to a viscous shear force predicts that the grafted chains will tilt and elongate in the direction of flow. The shear force may therefore act to reduce the number of conformations available to chains, increasing their rigidity without significantly altering the thickness of the grafted layer. A reduction in protein partitioning is then predicted when the dependence on u of the solute entropy loss is stronger than that of the grafted polymer, a condition met at high graft densities.

Performance of octadecylsilylated monolithic silica capillary columns of 530 μm inner diameter in HPLC

Monolithic silica capillary columns were successfully prepared in a fused silica capillary of 530 microm inner diameter and evaluated in HPLC after octadecylsilylation (ODS). Their efficiency and permeability were compared with those of columns pakked with 5-microm and 3-microm ODS-silica particles. The monolithic silica columns having different domain sizes (combined size of through-pore and skeleton) showed 2.5-4.0-times higher permeability (K= 5.2-8.4 x 10(-14) m2) than capillary columns packed with 3-mm particles, while giving similar column efficiency. The monolithic silica capillary columns gave a plate height of about 11-13 microm, or 11 200-13 400 theoretical plates/150 mm column length, in 80% methanol at a linear mobile phase velocity of 1.0 mm/s. The monolithic column having a smaller domain size showed higher column efficiency and higher pressure drop, although the monolithic column with a larger domain size showed better overall column performance, or smaller separation impedance (E value). The larger-diameter (530 microm id) monolithic silica capillary column afforded a good peak shape in gradient elution of proteins at a flow rate of up to 100 microL/min and an injection volume of up to 10 microL.

Generation of Ultrahigh Peak Capacity LC Separations via Elevated Temperatures and High Linear Mobile-Phase Velocities

The use of a combination of ultraperformance liquid chromatography at approximately 11,000 psi on sub 2-microm particles combined with reversed-phase gradient chromatography at a temperature of 90 degrees C is described as applied to the analysis of endogenous and drug metabolites in human and animal urine. By using elevated temperatures, back pressures can be reduced while maintaining high flow rates and chromatographic efficiency, with peaks 1-3 s wide at the base. Application to urine samples provided a peak capacity of approximately 700 for a 10-min analysis and greater than approximately 1000 in 1 h. Despite the narrow nature of the peaks, good quality mass spectra were also obtained, allowing the identification of typical drug and endogenous metabolites. These ultra-high-resolution chromatograms should be ideal for the analysis of complex samples in, for example, metabolite identification, impurity identification, and metabonomic/metabolomic studies. Applications in natural product analysis and proteomics can also be envisaged.

Retention properties of hydrophobically end-capped poly(ethylene glycol)s on a β-cyclodextrin support

High-performance liquid chromatography (HPLC) was used to examine the retention behavior of monomethoxypoly(ethylene glycol)s bearing one hydrophobic naphthyl end group (Nap-MPEG) on β-cyclodextrin polymer (poly-β-CD) immobilized on a silica support, under isocratic elution conditions and using water as mobile phase. Studies of retentions and theoretical plate heights H were conducted at infinite dilution by comparing the behavior of Nap-MPEGs having different molecular weight (750, 1000 and 5000 g/mol). The larger is its molecular size, the lower is the retention of the polymer. The linear increase of H with mobile phase velocity reveals slow mass-transfer kinetics arising from the restricted diffusion into the pores of the support. The complexation constants between the Nap-MPEGs and β-CD in solution (around 500 M −1) were determined from the decrease of retention observed by adding increasing concentrations of hydroxypropyl β-CD into the eluent. The peak profiles in mass-overload conditions were studied by fitting a model based upon bi-Langmuir kinetics which assumes a non-uniform support having two types of binding sites and apparent adsorption rate constants are used to describe mass-transfer kinetics. A three-parameter adsorption equilibrium isotherm was sufficient to account for the modifications of peak shapes observed when increasing amounts of polymer were injected. This result indicates an interaction with a heterogeneous poly-β-CD support mainly composed of low affinity groups, non-saturable in the range of polymer concentration studied. An upper limit was estimated for the equilibrium constant (<1000 M −1) characterizing the affinity of Nap-MPEG for the non-saturable sites of the poly-β-CD support. Large affinity constants (8–9 × 10 4 M −1) were found for the interaction of Nap-MPEGs with a small percentage of active sites.

Practical Constraints in the Kinetic Plot Representation of Chromatographic Performance Data: Theory and Application to Experimental Data

It is demonstrated 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 microm 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 Lmax = 30 cm. In the plate number range that remains accessible, the investigated sub-2 mum particle columns in any case perform (at least for the presently considered parabene separation) better than the 3.5 mum 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.

Hydrodynamic flow and electroosmotic flow in zirconia‐packed capillaries

Fused-silica capillaries were packed with Zirchrom-PBD stationary phase for application in CEC, nanoLC and pseudoelectrochromatography (PEC). Acido-basic properties of zirconia can be used to control the EOF even if the zirconia particles were coated by polybutadiene. As for native zirconia, the EOF is pH-dependent and the pI is close to pH 5. The mixed-mode pressure-voltage technique induced a modulation of the mobile-phase velocity as well as an electrophoretic migration of the solutes in order to improve the resolution of the separation. A significant increase of the flow appeared when both hydrodynamic and EOFs were in the same direction. But an important reduction of the electroosmotic velocity was observed when the hydrodynamic flow and EOF were opposed in Zirchrom-PBD columns. This behaviour has been observed at high or low pH on several columns. Separations of neutral and charged compounds have been performed with these columns in PEC mode.

Faster axial band dispersion in a monolithic silica column than in a particle-packed column

The contribution of molecular diffusion to peak broadening was studied in a reversed-phase HPLC system, consisting of a monolithic silica C 18 column and methanol–water mobile phase. Study on the band broadening effect of holding a solute in a column or elution at very low linear velocity of mobile phase allowed facile determination of the contribution of the molecular diffusion term. Less obstruction against molecular diffusion, or the faster axial band dispersion in a monolithic silica column than in a particle-packed column, was found both in mobile phase and in stationary phase.

Capillary Electrochromatography on Methacrylate Based Monolithic Columns: Evaluation of Column Performance and Separation of Polyphenols

Fused-silica capillary columns (100 µm I.D.) englobing a porous monolithic stationary phase were prepared by in situ copolymerization of 2-ethylhexyl methacrylate, ethylene glycol dimethacrylate and 2-acrylamido-2-methylpropanesulfonic acid (AMPS) in the presence of a porogenic mixture containing 1-propanol, 1,4 butanediol and water. The influence of the monomers ratio and the porogen solvent composition as well as the content of AMPS in the polymerization mixture on column total porosity and efficiency was investigated to attain minimum HETP values for the reversed-phase capillary electrochromatography separation of bioflavonoids. For the most promising column, the van Deemter plots, in both μ-HPLC and CEC, were also evaluated. In CEC the reduced plate height was found almost constant (1.6–2.0) within the range of linear mobile phase velocity between 0.2–2.0 mm s−1. The chemical and mechanical stabilities of the monolithic column over a wide range of buffer pH (2-10) and time were satisfactory. Furthermore, the effects of mobile phase parameters, such as buffer concentration and organic modifier content, on the electroosmotic flow were studied systematically. CEC separations of standard mixtures of polyphenols, including flavonols, flavanones and flavanones-7-O-glycosides, were accomplished in less than 8 min. The CEC separation of the major flavanone glycoside constituents in the extract from a freshly squeezed grapefruit juice was also reported.

The Impact of Mobile Phase Pressure and Velocity on the Development of Chromatograms in TLC and OPLC—a Comparison

Overpressured‐layer chromatography (OPLC) has been widely recommended in many scientific publications as a special thin‐layer chromatographic technique with forced flow of mobile phase that largely out‐performs classical thin‐layer chromatography (TLC) with its spontaneous low‐pressure capillary flow. Some authors even claim that OPLC can be regarded as a successful bridge between TLC and high‐performance liquid chromatography (HPLC). The essential difference between OPLC and TLC is the nature of the force that, in each of these two modes of planar chromatography, pushes the mobile phase through the pores of the stationary phase. It was the objective of this study to compare the impact of capillary and forced flow on analyte retention and separation quality. As test analytes, we selected three different hydrocarbons, tetralin, phenanthrene, and anthracene; these are, respectively, mono‐, bi‐, and tricyclic aromatic compounds with no functionality and, hence, are unable to participate in lateral interactions that might eclipse the basic effects of retention. In the most advanced OPLC systems, development of the thin‐layer chromatograms resembles that in HPLC as closely as possible (e.g., the stationary phase bed is preconditioned with the mobile phase and the samples are applied on‐line to the sorbent layer, without interrupting the flow of mobile phase and without drying the initial spots). With the Cobrabid OPLC apparatus used in this study, however, the only possibility was to develop dry layers with the dried spots of samples applied off‐line. Therefore, the only difference between development of TLC and OPLC chromatograms was, in fact, the pressure (and consequently the flow rate) of the mobile phase. Surprisingly enough, values of the retardation factor (R F) obtained for our test analytes by OPLC were always substantially lower than those obtained by TLC, which, under the conditions of our experiment, was proof of the poorer selectivity of OPLC compared with TLC. Two physical explanations (either alternative, or complementary) are offered to explain how the elevated pressure of the mobile phase in OPLC results in much lower numerical values of R F than in TLC.

Linear Velocity Surge Caused by Mobile-Phase Compression as a Source of Band Broadening in Isocratic Ultrahigh-Pressure Liquid Chromatography

A linear velocity surge caused by mobile-phase compression was investigated as a source of band broadening for ultrahigh-pressure liquid chromatography (UHPLC). To measure the effect of compression on mobile-phase velocity, ionic sample fronts were monitored using a contactless conductivity detector, as they migrated through long packed capillaries. Mobile-phase compression was found to cause an abnormally high linear velocity surge as the pressure was applied to the inlet of the column. An empirical equation was developed to describe the mobile-phase flow velocity during and after compression. Data fits to this equation were then used to determine the amount of additional variance caused by mobile-phase compression. For a 10/90 v/v acetonitrile/water mobile phase, the velocity surge occurred over roughly 10-15% of the column length. In addition, the velocity surge caused by mobile-phase compression was found to be capable of causing a 50% increase in the measured van Deemter C-terms for reversed-phase UHPLC.

Mobile-phase velocity — a tool for separation of alkaloids by OPLC

Extracts of tertiary and quaternary alkaloids from Chelidonium majus L. have been separated by OPLC. Samples of different volumes were developed on SiO 2 layers in a BS-50 Personal OPLC chamber using different mobile-phase velocities from 100 to 400 µL min −1 . The effect of mobile-phase velocity in OPLC on properties such as retardation factors, reproducibility, efficiency, number of theoretical plate, HETP , and resolution, was investigated.

Comparison of the efficiency of microparticulate and monolithic capillary columns

A comparison is made between the efficiency of microparticulate capillary columns and silica and polymer-based monolithic capillary columns in the pressure-driven (high-performance liquid chromatography) and electro-driven (capillary electrochromatography) modes. With packed capillary columns similar plate heights are possible as with conventional packed columns. However, a large variation is observed in the plate heights for individual columns. This can only be explained by differences in the quality of the packed bed. The minimum plate height obtained with silica monolithic capillary columns in the HPLC mode is approximately 10 microm, which is comparable to that of columns packed with 5-microm particles. The permeability of wide-pore silica monoliths was found to be much higher than that of comparable microparticulate columns, which leads to much lower pressure drops for the same eluent at the same linear mobile phase velocity. For polymer-based monolithic columns (acrylamide, styrene/divinyl benzene, methacrylate, acrylate) high efficiencies have been found in the CEC mode with minimum plate heights between 2 and 10 microm. However, in the HPLC mode minimum plate heights in the range of 10 to 25 microm have been reported.

Novel biporous polymeric stationary phase for high-speed protein chromatography

A novel rigid biporous bead (BiPB) had been fabricated by double emulsification to prepare a (w/o)/w emulsion and a subsequent polymerization. The polymerization of monomers, glycidyl methacrylate and ethylene glycol dimethacrylate, was initiated with benzoin ethyl ether by ultraviolet irradiation. The BiPB with an average diameter of 42.8 microm was characterized to possess two types of pores, i.e., micropores (20-100nm) and superpores (300-4000nm). Its specific surface area was determined to be 41.9m2/g, about 20% smaller than that of a microporous bead (MiPB) (52.1 m2/g). Flow hydrodynamic experiments showed that the BiPB column had smaller backpressure and plate height than those of the MiPB column at a given flow rate. Derivatized with diethylamine (DEA), the static adsorption capacity of the DEA-BiPB was about 7% smaller than that of the DEA-MiPB for BSA (bovine serum albumin). However, frontal analysis demonstrated that the dynamic binding capacity of the DEA-BiPB column was 1.6-2.4 times higher than that of the DEA-MiPB at high flow rate range of 1200-2400cm/h. Moreover, separation of a model protein mixture (myoglobin and BSA) was conducted at mobile phase velocities up to 3000cm/h to compare the performance of the two stationary phases. All the results indicate that the BiPB contains interconnected flowthrough pores and the BiPB column is promising for high-speed protein chromatography.