Mobile Phase Flow Velocity Research Articles

Development and Validation of Ultraviolet and Reverse Phase-high Performance Liquid Chromatographic Method for Estimation of Cilnidipine

Aim: The current experiment was to develop and validate a straight forward RP-HPLC methodology for the determination of Cilnidipine.
 Methodology: UV spectroscopy was used to estimate Cilnidipine. Action separation of Cilnidipine was achieved by employing a C18 column. Mobile phase combination of methanol: water (90:10 v/v) was tense at the flow of 1 ml/min. Detection was performed at 241 nm. Validation parameters were evaluated in line with the International conference on harmonization (ICH) Q2R1 guidelines.
 Results: The standardization curve was linear within the varying concentration of 2-10 mg/ml for Cilnidipine with parametric statistic (r2) equal to 0.999. The tactic was found to be accurate (101.66% recovery), precise (intraday, 1.65 and inter day, 1.38) and robust (% RSD was calculated to be 0.66, 0.58 and 0.81 for variation in mobile phase composition, wave length and flow velocity respectively) for the analysis of Cilnidipine.
 Conclusion: The developed method has passed all the validation tests and can be successfully applied to estimate the presence of Cilnidipine in bulk as well as in pharmaceutical formulations.

Numerical correction for asymmetrical peak profiles for moment analysis of chromatographic behavior

A numerical correction method is proposed for the moment analysis of some properties of chromatographic columns, even when the profiles of their elution peaks exhibit some asymmetry. Information on the retention equilibrium and the mass transfer kinetics of a C18-silica gel packing material is derived by the moment analysis from the experimental data obtained under three different sets of conditions: (1) the tailing peaks eluted from the column used; they are not corrected by the numerical method; (2) the same peaks are corrected by the numerical method, which provides the information on the column radial heterogeneity and on the efficiency at its center; and (3) symmetrical peaks having nearly Gaussian profiles eluted from another column packed with the same material; these peaks are not corrected by the numerical method. The analytical results obtained under the three different conditions are compared. The results demonstrate that the numerical correction method allows the determination of the information on the chromatographic behavior of columns from asymmetrical peak profiles, i.e., column efficiency at radial center, order of the polynomial functions for representing the radial distributions of mobile phase flow velocity and column efficiency, ratio of the flow velocity near the wall to that at the column center, and ratio of the column efficiency near the wall to that at the column center.

Robust naphthyl methacrylate monolithic column for high performance liquid chromatography of a wide range of solutes

An organic monolithic column based on the co-polymerization of 2-naphthyl methacrylate (NAPM) as the functional monomer and trimethylolpropane trimethacrylate (TRIM) as the crosslinker was introduced for high performance reversed-phase liquid chromatography (RPC). The co-polymerization was performed in situ in a stainless steel column of 4.6mm i.d. in the presence of a ternary porogen consisting of 1-dodecanol and cyclohexanol. This monolithic column (referred to as naphthyl methacrylate monolithic column or NMM column) showed high mechanical stability at relatively high mobile phase flow velocity indicating that the column has excellent hydrodynamic characteristics. To characterize the NMM column, different probe molecules including alkyl benzenes, and aniline, benzene, toluene and phenol derivatives were chromatographed on the column and the results in terms of k, selectivity and plate counts were compared to those obtained on an octadecyl silica (ODS) column in order to assess the presence of π–π and hydrophobic interactions on the NMM column under otherwise the same elution conditions. The NMM column offered additional π–π interactions with aromatic molecules in addition to hydrophobic interactions under RPC elution conditions. Run-to-run and column-to-column reproducibility of solute k values were evaluated, and percent relative standard deviation of <1% and ∼2–3.5%, respectively, were obtained. Six standard proteins were readily separated on the NMM column using shallow (30min at 1.0mL/min), steep (10min at 1.0mL/min) and ultra steep (1min at 3.0mL/min) linear gradient elution at increasing ACN concentration in the mobile phase using a 10cm×4.6mm i.d. column in case of shallow and steep linear gradients and a 3cm×4.6mm i.d. column for ultra steep linear gradient.

Computational investigation of longitudinal diffusion, eddy dispersion, and trans-particle mass transfer in bulk, random packings of core–shell particles with varied shell thickness and shell diffusion coefficient

In recent years, chromatographic columns packed with core–shell particles have been widely used for efficient and fast separations at comparatively low operating pressure. However, the influence of the porous shell properties on the mass transfer kinetics in core–shell packings is still not fully understood. We report on results obtained with a modeling approach to simulate three-dimensional advective–diffusive transport in bulk random packings of monosized core–shell particles, covering a range of reduced mobile phase flow velocities from 0.5 up to 1000. The impact of the effective diffusivity of analyte molecules in the porous shell and the shell thickness on the resulting plate height was investigated. An extension of Giddings’ theory of coupled eddy dispersion to account for retention of analyte molecules due to stagnant regions in porous shells with zero mobile phase flow velocity is presented. The plate height equation involving a modified eddy dispersion term excellently describes simulated data obtained for particle-packings with varied shell thickness and shell diffusion coefficient. It is confirmed that the model of trans-particle mass transfer resistance of core–shell particles by Kaczmarski and Guiochon [42] is applicable up to a constant factor. We analyze individual contributions to the plate height from different mass transfer mechanisms in dependence of the shell parameters. The simulations demonstrate that a reduction of plate height in packings of core–shell relative to fully porous particles arises mainly due to reduced trans-particle mass transfer resistance and transchannel eddy dispersion.

Influence of some operation variables on continuous separation process of orthogonal pressurized planar electrochromatography

Orthogonal pressurized planar electrochromatography (OPPEC) is the new separation technique, in which two simultaneous and orthogonal processes of electrochromatography/electrophoresis and chromatography proceed under external pressure. It means in the OPPEC separation system a mobile phase solution hydrodynamically flows against adsorbent layer of the chromatographic plate and electric field is orthogonally applied to it. This technique can be applied to analytical and preparative separations. In the paper we report influence of some operating variables on continuous preparative OPPEC separation of test dyes in system with HPTLC RP-18W plates from Merck and methanol–buffer mobile phase. The following operation variables are discussed: external pressure exerted on the adsorbent layer, mobile phase flow velocity, and the ratio of flow velocities of the mobile phase to the sample solution, voltage applied to electrodes, modifier concentration in the mobile phase, concentration and pH of buffer.

Influence of Radial Heterogeneity upon Columns of Different Lengths

A numerically calculated method was used to study the influence of radial heterogeneity upon columns of different lengths. In this study, we assumed the fourth-order polynomial distribution function of mobile phase flow velocity and the local efficiency. The results relating to column efficiencies and tailing factors show that long and high efficiency columns are influenced by radial heterogeneity more than the shorter and low efficiency columns are influenced. Radial heterogeneity has various effects on columns of different lengths. The results indicate that an extreme high efficiency column has a threshold of length. When the length exceeds the threshold, increasing length will not result in a significant increase in the column plate numbers. The influence of radial heterogeneity upon mobile phase flow velocity and local column efficiency also varies. The radial distribution of local efficiency can enhance the effects of the mobile phase flow velocity radial distribution.

Numerical Method for the Estimation of Column Radial Heterogeneity and of the Actual Column Efficiency from Tailing Peak Profiles

It is probably impossible to prepare high-performance liquid chromatography (HPLC) columns that have a completely homogeneous packing structure. Many reports in the literature show that the radial distributions of the mobile phase flow velocity and the local column efficiency are not flat, even in columns considered as good. A degree of radial heterogeneity seems to be a common property of all HPLC columns and an important source of peak tailing, which prevents the derivation of accurate information on chromatographic behavior from a straightforward analysis of elution peak profiles. This work reports on a numerical method developed to derive from recorded peak profiles the column efficiency at the column center, the degree of column radial heterogeneity, and the polynomial function that best represents the radial distributions of the flow velocity and the column efficiency. This numerical method was applied to two concrete examples of tailing peak profiles previously described. It was demonstrated that this numerical method is effective to estimate important parameters characterizing the radial heterogeneity of chromatographic columns.

Efficiency of the same neat silica column in hydrophilic interaction chromatography and per aqueous liquid chromatography

The dependencies on the mobile phase flow velocity of the efficiency of a column packed with shell particles of neat porous silica (Halo) was measured under two different sets of experimental conditions. These conditions corresponded to the retention mechanisms of per aqueous liquid chromatography (PALC) at low acetonitrile concentrations and of hydrophilic interaction chromatography (HILIC) at high acetonitrile concentrations. The results are compared. Small amounts of a diluted solution of caffeine were injected in order to record the chromatograms under strictly linear conditions. These efficiencies were measured in both water-rich (PALC retention mechanism) and acetonitrile-rich (HILIC mechanism) mobile phases for the same retention factors, between 0.25 and 2.5. The mobile phases were mixtures of acetonitrile and water containing neither supporting salt nor buffer component. At low retention factors, the efficiency of caffeine is better in the PALC than in the HILIC mode. For k ′ = 0.5 , the minimum reduced height equivalent to a theoretical plate (HETP) is close to 2.5 in PALC while it exceeds 5 in HILIC. The converse is true for high retention factors. For k ′ > 2.5 , the HETP is lower in HILIC than in PALC, because the major contribution to band broadening and peak tailing in this latter mode originates from the heterogeneous thermodynamics of retention and eventually restricts column performance in PALC. Most interestingly, the reduced HETP measured in HILIC for caffeine never falls below 4. This suggests that the mass transfer of caffeine between the multilayer adsorbed phase (due to the interactions of the strong solvent and the silanol groups) and the acetonitrile-rich bulk eluent is slow.

An estimation of the column efficiency made by analyzing tailing peak profiles

It has been shown previously that most columns are not radially homogeneous but exhibit radial distributions of the mobile phase flow velocity and the local efficiency. Both distributions are best approximated by fourth-order polynomial, with the velocity in the column center being maximum for most packed columns and minimum for monolithic columns. These distributions may be an important source of tailing of elution peaks. The numerical calculation of elution peaks shows how peak tailing is related to the characteristics of these two distributions. An approach is proposed that permits estimations of the true efficiency and of the degree of column radial heterogeneity by inversing this calculation and using the tailing profiles of the elution peaks that are experimentally measured. This method was applied in two concrete cases of tailing peak profiles that had been previously reported and were analyzed by applying this new inverse approach. The results obtained prove its validity and demonstrate that this numerical method is effective for deriving the true column efficiency from experimental tailing profiles.

Optimization of the thermal environment of columns packed with very fine particles

The apparent reduced HETP (height equivalent to a theoretical plate) of columns packed with very fine 1.7μm BEH-C18particles were measured under isocratic conditions, for different thermal conditions applied to the surface wall (column left in still-air at laboratory temperature, column placed in the oven of the Acquity UPLC chromatograph heated at different temperatures, or column immersed in a water stream at different controlled temperatures). The sample was the low-molecular weight, highly diffusive compound naphtho[2,3-a]pyrene. The mobile phase was either pure acetonitrile or a mixture of acetonitrile and water (85/15, v/v). The plot of the measured HETPs versus the mobile phase flow velocity begins to deviate from the classical isothermal van Deemter curve or the advantages of using very fine particles begins to disappear when the heat power released by the friction of the stream percolating through the packed bed exceeds 4W per unit length of column (m). These effects are minimum when (1) the temperature of the column wall is not controlled and the wall remains in contact with air; or (2) a suitable difference between the temperature of the column wall (hot) and the temperature of the entering liquid (cooler) is set by the analyst.

Capillary electrochromatography with monolithic silica column: I. Preparation of silica monoliths having surface-bound octadecyl moieties and their chromatographic characterization and applications to the separation of neutral and charged species.

Monolithic silica columns with surface-bound octadecyl (C18) moieties have been prepared by a sol-gel process in 100 microm ID fused-silica capillaries for reversed-phase capillary electrochromatography of neutral and charged species. The reaction conditions for the preparation of the C18-silica monoliths were optimized for maximum surface coverage with octadecyl moieties in order to maximize retention and selectivity toward neutral and charged solutes with a sufficiently strong electroosmotic flow (> 2 mm/s) to yield rapid analysis time. Furthermore, the effect of the pore-tailoring process on the silica monoliths was performed over a wide range of treatment time with 0.010 M ammonium hydroxide solution in order to determine the optimum time and conditions that yield mesopores of narrow pore size distribution that result in high separation efficiency. Under optimum column fabrication conditions and optimum mobile phase composition and flow velocity, the average separation efficiency reached 160 000 plates/m, a value comparable to that obtained on columns packed with 3 microm C18-silica particles with the advantages of high permeability and virtually no bubble formation. The optimized monolithic C18-silica columns were evaluated for their retention properties toward neutral and charged analytes over a wide range of mobile phase compositions. A series of dimensionless retention parameters were evaluated and correlated to solute polarity and electromigration property. A dimensionless mobility modulus was introduced to describe charged solute migration and interaction behavior with the monolithic C18-silica in a counterflow regime during capillary electrochromatography (CEC )separations. The mobility moduli correlated well with the solute hydrophobic character and its charge-to-mass ratio.

Cooperation of solid granule and solvent as porogenic agents Novel porogenic mode of biporous media for protein chromatography

A novel porogenic mode, cooperation of solid granule and solvent, has been introduced to prepare a biporous medium for protein chromatography. The matrix, a ternary copolymer of glycidyl methacrylate, triallylisocyanurate and divinylbenzene, was produced by a simple in situ polymerization with granules of sodium sulfate and cyclohexanol and dodecanol as porogenic agents. Functionalized with diethylamine, the resin (denoted as Resin C) was used as an anion exchanger. The pore structure, specific surface area and chromatographic properties of Resin C were determined and compared with those of the resin with only the solvents as porogen (Resin A) and the resin with only the salt granules as porogen (Resin B). The results indicated that Resin C contained regions of micropores with a maximum at approximately 55 nm and regions of macropores with a distinct maximum near 340 nm, which swelled to about 1 μm in aqueous solution. Compared with Resins A and B, the biporous medium Resin C simultaneously possessed a high specific surface area of 37.2 m 2/g and a low back-pressure at mobile phase flow velocity up to 720 cm/h. The result of dynamic porosity showed that mobile phase was able to convectively flow through the macropores in Resin C. The dynamic adsorption capacity of Resin C for bovine serum albumin was as high as 57.0 mg/ml column volume (95.0 mg/g wet resin), basically identical to its static capacity, while that of Resin A was only 1.95 mg/ml column volume (3.12 mg/g wet resin), about 3% that of its static capacity. In addition, the column efficiency of Resin C was comparable to that of Resin B, but much higher than that of Resin A, indicating that the mass transfer behavior of proteins in the column was greatly improved by convective flow through the macropores.

Influence of the column radial heterogeneity on the determination of single-component isotherms by the elution by characteristic point method

The influence of the column radial heterogeneity on the determination of equilibrium isotherm data by the elution by characteristic point (ECP) method is studied using nondimensional numerical calculations and taking into account typical radial distributions of the mobile phase flow velocity and the column efficiency across a column. Overloaded elution peaks were calculated with the equilibrium-dispersive model as a function of four dimensionless parameters, the number of theoretical plates at the center of the column, the Langmuir equilibrium constant, the retention and the loading factors. The influence of the mass transfer resistances and the radial heterogeneity of the column on the ECP data was analyzed by comparing the true isotherm and the one estimated from the diffuse profile of overloaded peaks. The results provide information on the accuracy of the ECP method. The error made increases with increasing degree of radial heterogeneity. This error can be corrected by using the results of the nondimensional numerical calculations, allowing a further extension of the applicability of the ECP method.

Determination of the lumped mass transfer rate coefficient by frontal analysis

The validity of measurements of the lumped mass transfer rate coefficient ( k m,L) is studied on the basis of experimental data acquired under Langmuir isotherm conditions, in reversed-phase liquid chromatography. Two different methods were used, the perturbation method and frontal analysis. Accurate values of k m,L can be properly obtained by the perturbation method because, with this method, the chromatographic processes take place under locally linear isotherm conditions. Values of k m,L can also be derived from the breakthrough curves obtained in frontal analysis. Because the contribution of axial dispersion to band broadening was larger than that of the mass transfer resistances, the apparent axial dispersion coefficient ( D a) was first derived from the breakthrough curve by applying the equilibrium–dispersive model. Then, the value of k m,L was calculated from D a. The values of k m,L determined by the two methods were in close agreement in the range of nondimensional Langmuir equilibrium constants ( r=1/[1+ K L C 0]) between 0.32 and 0.85, irrespective of the mobile phase flow velocity. Thus, frontal analysis can be used for kinetic studies of the mass transfer in chromatographic columns.

DISPLACEMENT THIN-LAYER CHROMATOGRAPHY

Thin-layer chromatography has several advantages in comparison to column chromatography, e.g., simultaneous separation of several samples, easy realization of two-dimensional separation, direct observation of both the chromatographic development and the separation using colored compounds, sensitive and easy detection of the spots with specific spray reagents. The entire procedure, i.e., loading, development, and evaluation are relatively simple. An additional feature is the optimization possibilities of mobile phase flow velocity, based on the complexity of driving force where capillarity results in a concave flow profile that may be counterbalanced by convex profile of laminar flow. Displacement thin-layer chromatography has the advantages of planar arrangement of the stationary phase, and opens new possibilities for analytical separations.This paper outlines basic features of planar displacement chromatography, including differences from elution chro-matography, and that of column displacement chromatography, the possibility of using spacer displacement chromatography. Parameters limiting displacement chromatography, and their optimization to achieve better separation are discussed.

Influence of column radial heterogeneity on peak fronting in linear chromatography

Using numerical calculations of elution peak profiles, an explanation of the fronting behavior of elution peaks in linear chromatography was found in certain radial distributions of the mobile phase flow velocity and local bed efficiency. Fronting peaks are observed only if the flow velocity is higher in the wall region than in the center part of the column and the local efficiency is lower near the wall than in the center. By contrast, tailing or symmetrical peaks are observed if only the flow velocity or the local efficiency are radially heterogeneous. The degree of peak fronting increases with increasing amplitude of the radial distributions. The influence of the radial heterogeneity of the flow velocity on the degree of peak fronting is more severe for high than for low efficiency columns. An equation is suggested to correlate peak fronting behavior for columns of different efficiencies and a procedure proposed for the estimation of the radial distributions of the flow velocity and the local efficiency by analyzing some characteristics of asymmetric peaks.

Estimation of the column radial heterogeneity from an analysis of the characteristics of tailing peaks in linear chromatography

An estimation procedure of the radial heterogeneity of the distributions of the mobile phase flow velocity and of the local column efficiency was derived from a comparison of experimental data and of the characteristics of these tailing peaks calculated numerically. The analysis of these characteristics indicated that the radial heterogeneity of the packing density of a column is a cause of peak tailing. This analysis showed also that it is possible to correlate the radial fluctuations of the flow velocity and the apparent efficiency of different columns. The comparison of experimental data and calculated results suggested also a correlation between the radial distributions of the flow velocity and of the column efficiency. Previous experimental data concerning the radial heterogeneity of column packings validated the correlation. Information on the degree of heterogeneity of the radial distributions of the flow velocity and the column efficiency were obtained by analyzing the peak width and the asymmetry factor of elution peaks. It was also found that the exponentially modified Gaussian distribution cannot provide a comprehensive representation of all types of tailing profiles in chromatography.

Shock layer thickness and optimum linear velocity in displacement chromatography

The width of the mixed zones between two successive bands in the isotachic train represents the loss in recovery yield achieved in displacement chromatography. Intuitively, this width depends on the mobile phase flow velocity, but no systematic study of this effect has yet been performed. On the other hand, constant pattern behavior and the theory of shock layer are well known in chemical engineering. Using this approach, and assuming competitive Langmuir isotherm behavior, an analytical equation is derived which relates the shock layer thickness (SLT) in displacement chromatography and the column design and operating parameters. Using this equation, it is possible to investigate the dependence of the SLT between two consecutive bands in the isotachic train on the mobile phase velocity, the concentration and the retention factor of the displacer and the separation factor of the two components. In displacement chromatography, the optimum mobile phase linear velocity ( u opt s) for minimum shock layer thickness, or maximum recovery yield depends not only on the coefficients of axial dispersion and mass transfer resistance of the two components, as does the optimum mobile phase velocity ( u opt L) in linear chromatography, but also on the retention factor and the concentration of the displacer. The results of the study of this analytical equation are in excellent agreement with those of numerical calculations.

The shock layer thickness, a new approach to the study of column performance in non-linear chromatography: I. Optimum linear velocity in frontal analysis

In non-linear chromatography, it is common to observe very steep profiles. This happens for overloaded elution bands, for frontal analysis breakthrough curves and for the band profiles of the isotachic train in displacement chromatography. These regions where the concentration vary very rapidly are called shock layers. The relationship between the thickness of the shock layer in frontal analysis and the coefficients of the conventional terms of the plate height equation were studied experimentally. The shock layer theory of Rhee and co-workers permits the simple determination of the optimum linear velocity for minimum shock layer thickness in the case when the adsorption behavior of the feed components is described by the Langmuir model. The optimum linear velocity in frontal analysis is not only a function of the coefficients of the axial dispersion and the mass transfer resistance terms, and the retention factor ( k 0 ) ́ , as in linear chromatography, but also a function of the plateau concentrations and the second Langmuir parameter of the isotherm, b. Depending on the retention factor, the optimum velocity in frontal analysis may be larger, but is most often much smaller than in linear chromatography. Experimental results are in excellent agreement with the prediction of the theory. If they could be extended to displacement chromatography, these findings would explain some apparent contradictions found in the literature regarding the influence of the mobile phase flow velocity on the degree of separation between bands achieved in displacement chromatography, and clarify certain controversies.

A simplex optimization of the experimental parameters in preparative liquid chromatography

A study of the optimization of the experimental conditions for the purification or extraction of pure compounds by liquid chromatography is presented. Optimum values of the parameters of overloaded elution are derived for maximum production rate, using a Simplex algorithm and the procedure previously described for the simulation of the elution profiles of binary mixtures. The mobile phase flow velocity and the sample size have been optimized together in a first step, simulating the procedure followed in practice, when a column is available. In a second part, the influence of the column length and the average particle size of the packing material on the column performance as well as the trade-offs between the production rate and the yield are discussed.