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Georges Guiochon - One of the best experts on this subject based on the ideXlab platform.
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band broadening along gradient reversed phase columns a potential gain in resolution Factor
Journal of Chromatography A, 2014Co-Authors: Fabrice Gritti, Georges GuiochonAbstract:Abstract The advantages of using columns packed with stationary phases having a composition gradient so that Retention Factors increase toward the outlet and eluting them with an isocratic mobile phase may provide increased peak resolution in liquid chromatography. This approach is discussed from a theoretical viewpoint. The Retention Factor is assumed to increase linearly along the column. The peak width can be predicted under three different conditions: (1) the possible band compression is neglected (the Giddings model); (2) band compression is taken into account (the Poppe model); and (3) both band compression and extra-column effects are considered in the calculations. The impact of a stationary phase gradient on the resolution of a pair of compound difficult to separate (constant selectivity α = 1.05) is illustrated for a 3 mm × 100 mm silica monolithic column of the second generation (porosity ϵ t = 0.85, plate height H = 6 μm) operated with a new generation of liquid chromatograph (pre- and post-column volume variances of 10 and 5 μL 2 , respectively). The results show that the resolution Factor R S becomes optimum for a specific positive value of the Retention gradient along the column. This optimum depends on the Retention Factor at the column inlet.
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investigation of the axial heterogeneity of the Retention Factor of carbamazepine along an supercritical fluid chromatography column i linear conditions
Journal of Chromatography A, 2013Co-Authors: Fahimeh Kamarei, Fabrice Gritti, Georges GuiochonAbstract:Abstract The Retention behavior of carbamazepine eluted in SFC was studied by changing the back pressure between 100 and 200 bar and the volumetric flow rate between 0.5 and 5 mL/min. Carbamazepine was eluted under linear conditions from a silica column with 90:10% (V/V) mixtures of supercritical carbon dioxide and methanol at 310 K. The Retention Factor of carbamazepine decreases by 20% when the pressure drop along the column increases from 1 to 28 bar and the outlet pressure increases from 100 to 200 bar. The variation of the Retention Factor at low pressure drops (below 15 bar) is negligible (a few percent) but it is significant (exceeding 20%) for pressure drops larger than 15 bar. Under these latter conditions, the variation of the equilibrium constant along the column is significant at high pressure drops. Because the adsorption parameters depend on the local pressure, the extension of dynamic methods from LC to SFC should be made cautiously for the determination of adsorption isotherms. Our results suggest that the flow rate applied during the course of dynamic method measurements should be kept small. The determination of the adsorption parameters from measurements carried out at high flow rates might not be as accurate as it is when the column is axially uniform.
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peak compression Factor of proteins
Journal of Chromatography A, 2009Co-Authors: Georges GuiochonAbstract:An experimental protocol is proposed in order to measure with accuracy and precision the band compression Factor G(12)(2) of a protein in gradient RPLC. Extra-column contributions to bandwidth and the dependency of both the Retention Factor and the reduced height equivalent to a theoretical plate (HETP) on the mobile phase composition were taken into account. The band compression Factor of a small protein (insulin, MW kDa) was measured on a 2.1mm x 50mm column packed with 1.7 microm C(4)-bonded bridged ethylsiloxane BEH-silica particles, for 1 microL samples of dilute insulin solution (<0.05g/L). A linear gradient profile of acetonitrile (25-28% acetonitrile in water containing 0.1% trifluoroacetic acid) was applied during three different gradient times (5, 12.5, and 20 min). The mobile phase flow rate was set at 0.20 mL/min in order to avoid heat friction effects (maximum column inlet pressure 180 bar). The band compression Factor of insulin is defined as the ratio of the experimental space band variance measured under gradient conditions to the reference space band variance, which would be observed if no thermodynamic compression would take place during gradient elution. It was 0.56, 0.71, and 0.76 with gradient times of 5, 12.5, and 20 min, respectively. These Factors are 20-30% smaller than the theoretical band compression Factors (0.79, 0.89, and 0.93) calculated from an equation derived from the well-known Poppe equation, later extended to any Retention models and columns whose HETP depends on the mobile phase composition. This difference is explained in part by the omission in the model of the effect of the pressure gradient on the local Retention Factor of insulin during gradient elution. A much better agreement is obtained for insulin when this effect is taken into account. For lower molecular weight compounds, the pressure gradient has little effect but the finite Retention of acetonitrile causes a distortion of the gradient shape during the migration of its breakthrough front along the column. This phenomenon should be taken into account in the theoretical models.
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Exact peak compression Factor in linear gradient elution. I. Theory.
Journal of chromatography. A, 2008Co-Authors: Fabrice Gritti, Georges GuiochonAbstract:The only existing expression for the peak compression Factor in linear gradient elution chromatography assumes that the linear-solvent-strength model (LSSM) applies to the Retention of the compound studied, that the column efficiency is independent of the mobile phase composition, and that, during gradient elution, the relative Retention Factor of a compound inside its band varies linearly with the distance from the band center. Because the Retention Factors of many analytes in reversed-phase liquid chromatography do not rigorously follow the LSSM, we extend the theoretical approach of Poppe et al. to the prediction of peak compression Factors in linear gradient elution chromatography for any Retention model, when column efficiency varies with the mobile phase composition. Only the contribution of the chromatographic column to the peak compression was taken into account, the contribution of the dwell volume being neglected. A second restriction is the linearity of the relative Retention Factor as a function of the position along the band width inside the column. These constraints could be the sources for the difference observed between experimental and theoretical values of peak compression Factors. When the Retention Factor varies steeply with the mobile phase composition, such as with proteins or large peptides in RP-HPLC, it is found that the thermodynamic compression term, which tends to sharpen the peak, is coupled with the column dispersion term, which tends to broaden the peak. This coupling term acts as an apparent dispersion term, contributing to broaden the peak. This result is consistent with the measurements of peak compression Factors found in the literature.
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influence of the errors made in the measurement of the extra column volume on the accuracies of estimates of the column efficiency and the mass transfer kinetics parameters
Journal of Chromatography A, 2006Co-Authors: Fabrice Gritti, Georges Guiochon, Attila FelingerAbstract:Abstract The influences of the errors made in the measurement of the extra-column volume of an instrument on the accuracies of the estimates made of the column efficiency and of the parameters of the mass transfer kinetics were investigated from an experimental point of view. A standard HP1090 apparatus (extra-column volume, ≈ 50 μ L) was used to measure the efficiency of a Sunfire-C18 RPLC column (column hold-up volume, ≈ 1.50 mL). The first and second moments of the peaks of phenol (a retained compound) and of thiourea (a practically non-retained compound) were measured at six different temperatures between 22 and 78 °C, for flow rates between 0.10 and 4.70 mL/min (i.e., for linear velocities between 0.025 and 1.179 cm/s). Each series of measurements was successively made with the instrument being fitted with and without the column. The experimental HETP data must be corrected for the solute dispersion in the connected tubes in order properly to assess the true column efficiency. Even with a modern, high performance instrument, the dispersion of a non-retained compound is essentially due to the band broadening phenomena that take place in the extra-column volumes, the sum of all these extra-column band broadening contributions accounting for more than 80% of the total band broadening measured. The contribution of the sampling device is particularly deleterious since, for a 2 μ L injection, the maximum solute concentration in the peak that enters into the column is nearly ten-fold lower than that of the sample. Nevertheless, the impact of the extra-column volumes on the estimates of the kinetic parameters (e.g., molecular diffusion coefficient D m and effective particle diffusivity D e ) remains negligible. Obviously, the relative error made on the column efficiency of a retained compound depends much on its Retention Factor. It decreases from 8 to 1% when the Retention Factor increases from 5 to 17.
Fabrice Gritti - One of the best experts on this subject based on the ideXlab platform.
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band broadening along gradient reversed phase columns a potential gain in resolution Factor
Journal of Chromatography A, 2014Co-Authors: Fabrice Gritti, Georges GuiochonAbstract:Abstract The advantages of using columns packed with stationary phases having a composition gradient so that Retention Factors increase toward the outlet and eluting them with an isocratic mobile phase may provide increased peak resolution in liquid chromatography. This approach is discussed from a theoretical viewpoint. The Retention Factor is assumed to increase linearly along the column. The peak width can be predicted under three different conditions: (1) the possible band compression is neglected (the Giddings model); (2) band compression is taken into account (the Poppe model); and (3) both band compression and extra-column effects are considered in the calculations. The impact of a stationary phase gradient on the resolution of a pair of compound difficult to separate (constant selectivity α = 1.05) is illustrated for a 3 mm × 100 mm silica monolithic column of the second generation (porosity ϵ t = 0.85, plate height H = 6 μm) operated with a new generation of liquid chromatograph (pre- and post-column volume variances of 10 and 5 μL 2 , respectively). The results show that the resolution Factor R S becomes optimum for a specific positive value of the Retention gradient along the column. This optimum depends on the Retention Factor at the column inlet.
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investigation of the axial heterogeneity of the Retention Factor of carbamazepine along an supercritical fluid chromatography column i linear conditions
Journal of Chromatography A, 2013Co-Authors: Fahimeh Kamarei, Fabrice Gritti, Georges GuiochonAbstract:Abstract The Retention behavior of carbamazepine eluted in SFC was studied by changing the back pressure between 100 and 200 bar and the volumetric flow rate between 0.5 and 5 mL/min. Carbamazepine was eluted under linear conditions from a silica column with 90:10% (V/V) mixtures of supercritical carbon dioxide and methanol at 310 K. The Retention Factor of carbamazepine decreases by 20% when the pressure drop along the column increases from 1 to 28 bar and the outlet pressure increases from 100 to 200 bar. The variation of the Retention Factor at low pressure drops (below 15 bar) is negligible (a few percent) but it is significant (exceeding 20%) for pressure drops larger than 15 bar. Under these latter conditions, the variation of the equilibrium constant along the column is significant at high pressure drops. Because the adsorption parameters depend on the local pressure, the extension of dynamic methods from LC to SFC should be made cautiously for the determination of adsorption isotherms. Our results suggest that the flow rate applied during the course of dynamic method measurements should be kept small. The determination of the adsorption parameters from measurements carried out at high flow rates might not be as accurate as it is when the column is axially uniform.
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relationship between trans column eddy diffusion and Retention in liquid chromatography theory and experimental evidence
Journal of Chromatography A, 2010Co-Authors: Fabrice GrittiAbstract:Abstract Experimental results demonstrate that trans-column eddy diffusion depends on the Retention of compounds. The combination of elution profiles recorded in different points of the exit column cross-section and of the height equivalent to a theoretical plate (HETP) of small molecules clearly show a strong link between Retention and column performance in liquid chromatography. These results validate a new model of trans-column eddy diffusion in packed columns. The contribution to the column HETP of the trans-column eddy diffusion term decreases with increasing Retention Factor from k′ = 0 to k′ = 3 above which it becomes negligible. The best column performance in RPLC is observed for the most retained compounds. This is due to the combination of the lack of a residual trans-column eddy diffusion contribution and the vanishing contribution of the instrument to band broadening.
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Exact peak compression Factor in linear gradient elution. I. Theory.
Journal of chromatography. A, 2008Co-Authors: Fabrice Gritti, Georges GuiochonAbstract:The only existing expression for the peak compression Factor in linear gradient elution chromatography assumes that the linear-solvent-strength model (LSSM) applies to the Retention of the compound studied, that the column efficiency is independent of the mobile phase composition, and that, during gradient elution, the relative Retention Factor of a compound inside its band varies linearly with the distance from the band center. Because the Retention Factors of many analytes in reversed-phase liquid chromatography do not rigorously follow the LSSM, we extend the theoretical approach of Poppe et al. to the prediction of peak compression Factors in linear gradient elution chromatography for any Retention model, when column efficiency varies with the mobile phase composition. Only the contribution of the chromatographic column to the peak compression was taken into account, the contribution of the dwell volume being neglected. A second restriction is the linearity of the relative Retention Factor as a function of the position along the band width inside the column. These constraints could be the sources for the difference observed between experimental and theoretical values of peak compression Factors. When the Retention Factor varies steeply with the mobile phase composition, such as with proteins or large peptides in RP-HPLC, it is found that the thermodynamic compression term, which tends to sharpen the peak, is coupled with the column dispersion term, which tends to broaden the peak. This coupling term acts as an apparent dispersion term, contributing to broaden the peak. This result is consistent with the measurements of peak compression Factors found in the literature.
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influence of the errors made in the measurement of the extra column volume on the accuracies of estimates of the column efficiency and the mass transfer kinetics parameters
Journal of Chromatography A, 2006Co-Authors: Fabrice Gritti, Georges Guiochon, Attila FelingerAbstract:Abstract The influences of the errors made in the measurement of the extra-column volume of an instrument on the accuracies of the estimates made of the column efficiency and of the parameters of the mass transfer kinetics were investigated from an experimental point of view. A standard HP1090 apparatus (extra-column volume, ≈ 50 μ L) was used to measure the efficiency of a Sunfire-C18 RPLC column (column hold-up volume, ≈ 1.50 mL). The first and second moments of the peaks of phenol (a retained compound) and of thiourea (a practically non-retained compound) were measured at six different temperatures between 22 and 78 °C, for flow rates between 0.10 and 4.70 mL/min (i.e., for linear velocities between 0.025 and 1.179 cm/s). Each series of measurements was successively made with the instrument being fitted with and without the column. The experimental HETP data must be corrected for the solute dispersion in the connected tubes in order properly to assess the true column efficiency. Even with a modern, high performance instrument, the dispersion of a non-retained compound is essentially due to the band broadening phenomena that take place in the extra-column volumes, the sum of all these extra-column band broadening contributions accounting for more than 80% of the total band broadening measured. The contribution of the sampling device is particularly deleterious since, for a 2 μ L injection, the maximum solute concentration in the peak that enters into the column is nearly ten-fold lower than that of the sample. Nevertheless, the impact of the extra-column volumes on the estimates of the kinetic parameters (e.g., molecular diffusion coefficient D m and effective particle diffusivity D e ) remains negligible. Obviously, the relative error made on the column efficiency of a retained compound depends much on its Retention Factor. It decreases from 8 to 1% when the Retention Factor increases from 5 to 17.
Heba Elmansi - One of the best experts on this subject based on the ideXlab platform.
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analysis of four antimigraine drugs in two ternary mixtures by sweeping micellar electrokinetic chromatography with Retention Factor gradient effect and dynamic ph junction
Microchemical Journal, 2016Co-Authors: F Belal, Mohie Sharaf K Eldin, M M Tolba, Mohamed I Elawady, Heba ElmansiAbstract:Abstract A new, robust, and reliable sweeping-micellar electrokinetic chromatography (sweeping-MEKC) method with dynamic pH junction and Retention Factor gradient effect (RFGE) is developed for the simultaneous determination of four different analytes in two ternary mixtures of pharmaceutical importance. The studied analytes are drotaverine hydrochloride and caffeine combined with paracetamol (Mixture-I) or dipyrone (Mixture-II). Different experimental parameters are carefully investigated in order to achieve the highest possible resolution and sensitivity in a short analysis time. A special interest is given to the effect of sample matrix composition on the attainable enrichment efficiency with a focus on dynamic pH junction and RFGE. Up to 8 times increase in sensitivity is achieved by the modification of the sample matrix. The theoretical considerations of the underlying effects regarding the separation and the enrichment processes are thoroughly discussed. A full validation study of the developed method based on the pharmacopeial guidelines is performed. The method is successfully applied to the analysis of the studied drugs in their co-formulated or single-ingredient tablet preparations with a total run time of less than 11 min.
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analysis of four antimigraine drugs in two ternary mixtures by sweeping micellar electrokinetic chromatography with Retention Factor gradient effect and dynamic ph junction
Microchemical Journal, 2016Co-Authors: F Belal, Mohie Sharaf K Eldin, M M Tolba, Mohamed I Elawady, Heba ElmansiAbstract:Abstract A new, robust, and reliable sweeping-micellar electrokinetic chromatography (sweeping-MEKC) method with dynamic pH junction and Retention Factor gradient effect (RFGE) is developed for the simultaneous determination of four different analytes in two ternary mixtures of pharmaceutical importance. The studied analytes are drotaverine hydrochloride and caffeine combined with paracetamol (Mixture-I) or dipyrone (Mixture-II). Different experimental parameters are carefully investigated in order to achieve the highest possible resolution and sensitivity in a short analysis time. A special interest is given to the effect of sample matrix composition on the attainable enrichment efficiency with a focus on dynamic pH junction and RFGE. Up to 8 times increase in sensitivity is achieved by the modification of the sample matrix. The theoretical considerations of the underlying effects regarding the separation and the enrichment processes are thoroughly discussed. A full validation study of the developed method based on the pharmacopeial guidelines is performed. The method is successfully applied to the analysis of the studied drugs in their co-formulated or single-ingredient tablet preparations with a total run time of less than 11 min.
Giorgio Carta - One of the best experts on this subject based on the ideXlab platform.
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gradient elution behavior of proteins in hydrophobic interaction chromatography with a u shaped Retention Factor curve under overloaded conditions
Journal of Chromatography A, 2018Co-Authors: Arch Creasy, Joseph V Lomino, Giorgio CartaAbstract:Abstract The gradient elution hydrophobic interaction chromatography of a monoclonal antibody that exhibits U-shaped Retention as a function of the ammonium sulfate concentration is investigated for overloaded conditions at protein loads up to 30% of the column equilibrium binding capacity. Protein load and gradient slope affect both elution peak shape and protein recovery during the gradient. Higher protein loads result in tailing peaks with near 100% recovery that transition to fronting peaks and incomplete recovery as the protein load is reduced. The gradient slope also affects peak shape and recovery. Tailing peaks with lower recovery are obtained with sharper gradients and the most tailing peak and lowest recovery are obtained when step elution rather than gradient is implemented. Modeling the chromatographic elution based on independently determined adsorption isotherms as a function of protein and ammonium sulfate concentration predicts results in agreement with the experimental trends confirming that the unusual chromatographic behavior observed is due to the U-shaped protein binding as a function of the ammonium sulfate concentration. Although less pronounced than in the dilute limit, the U-shaped binding still produces peak shapes and recovery losses as a function of gradient slope that differ from those seen for systems where Retention is a monotonic function of salt concentration.
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gradient elution behavior of proteins in hydrophobic interaction chromatography with u shaped Retention Factor curves
Journal of Chromatography A, 2018Co-Authors: Arch Creasy, Joseph V Lomino, Gregory Barker, Anurag Khetan, Giorgio CartaAbstract:Abstract Protein Retention in hydrophobic interaction chromatography is described by the solvophobic theory as a function of the kosmostropic salt concentration. In general, an increase in salt concentration drives protein partitioning to the hydrophobic surface while a decrease reduces it. In some cases, however, protein Retention also increases at low salt concentrations resulting in a U-shaped Retention Factor curve. During gradient elution the salt concentration is gradually decreased from a high value thereby reducing the Retention Factor and increasing the protein chromatographic velocity. For these conditions, a steep gradient can overtake the protein in the column, causing it to rebind. Two dynamic models, one based on the local equilibrium theory and the other based on the linear driving force approximation, are presented. We show that the normalized gradient slope determines whether the protein elutes in the gradient, partially elutes, or is trapped in the column. Experimental results are presented for two different monoclonal antibodies and for lysozyme on Capto Phenyl (High Sub) resin. One of the mAbs and lysozyme exhibit U-shaped Retention Factor curves and for each, we determine the critical gradient slope beyond which 100% recovery is no longer possible. Elution with a reverse gradient is also demonstrated at low salt concentrations for these proteins. Understanding this behavior has implications in the design of gradient elution since the gradient slope impacts protein recovery.
Mohamed I Elawady - One of the best experts on this subject based on the ideXlab platform.
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analysis of four antimigraine drugs in two ternary mixtures by sweeping micellar electrokinetic chromatography with Retention Factor gradient effect and dynamic ph junction
Microchemical Journal, 2016Co-Authors: F Belal, Mohie Sharaf K Eldin, M M Tolba, Mohamed I Elawady, Heba ElmansiAbstract:Abstract A new, robust, and reliable sweeping-micellar electrokinetic chromatography (sweeping-MEKC) method with dynamic pH junction and Retention Factor gradient effect (RFGE) is developed for the simultaneous determination of four different analytes in two ternary mixtures of pharmaceutical importance. The studied analytes are drotaverine hydrochloride and caffeine combined with paracetamol (Mixture-I) or dipyrone (Mixture-II). Different experimental parameters are carefully investigated in order to achieve the highest possible resolution and sensitivity in a short analysis time. A special interest is given to the effect of sample matrix composition on the attainable enrichment efficiency with a focus on dynamic pH junction and RFGE. Up to 8 times increase in sensitivity is achieved by the modification of the sample matrix. The theoretical considerations of the underlying effects regarding the separation and the enrichment processes are thoroughly discussed. A full validation study of the developed method based on the pharmacopeial guidelines is performed. The method is successfully applied to the analysis of the studied drugs in their co-formulated or single-ingredient tablet preparations with a total run time of less than 11 min.
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analysis of four antimigraine drugs in two ternary mixtures by sweeping micellar electrokinetic chromatography with Retention Factor gradient effect and dynamic ph junction
Microchemical Journal, 2016Co-Authors: F Belal, Mohie Sharaf K Eldin, M M Tolba, Mohamed I Elawady, Heba ElmansiAbstract:Abstract A new, robust, and reliable sweeping-micellar electrokinetic chromatography (sweeping-MEKC) method with dynamic pH junction and Retention Factor gradient effect (RFGE) is developed for the simultaneous determination of four different analytes in two ternary mixtures of pharmaceutical importance. The studied analytes are drotaverine hydrochloride and caffeine combined with paracetamol (Mixture-I) or dipyrone (Mixture-II). Different experimental parameters are carefully investigated in order to achieve the highest possible resolution and sensitivity in a short analysis time. A special interest is given to the effect of sample matrix composition on the attainable enrichment efficiency with a focus on dynamic pH junction and RFGE. Up to 8 times increase in sensitivity is achieved by the modification of the sample matrix. The theoretical considerations of the underlying effects regarding the separation and the enrichment processes are thoroughly discussed. A full validation study of the developed method based on the pharmacopeial guidelines is performed. The method is successfully applied to the analysis of the studied drugs in their co-formulated or single-ingredient tablet preparations with a total run time of less than 11 min.
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processes involved in sweeping under inhomogeneous electric field conditions as sample enrichment procedure in micellar electrokinetic chromatography
Journal of Chromatography A, 2012Co-Authors: Mohamed I Elawady, Carolin Huhn, Ute PyellAbstract:Abstract Sweeping under inhomogeneous electric field conditions has been described as a process that includes stacking or destacking of the micelles when entering the sample zone, sweeping of analytes by the stacked or destacked micelles, and destacking or stacking of the swept analyte zone. However, there is ongoing debate that not only the Retention Factor of the analyte but also the electric conductivity of the sample solution or the concentration of an added salt can have an impact on the enrichment efficiency. Revisiting the equations describing sweeping, a Factor θ (phase ratio shift Factor) is defined to quantitatively describe the change of the Retention Factor between the sample and separation zones. The influence of the sample matrix composition on the experimentally obtained sweeping efficiency is studied with SDS as pseudostationary phase taking parabens, benzamide and anilines as model analytes. To this end, a robust and reliable method for the assessment of the sweeping efficiency is developed. The values obtained via this method are very precise and agree well with theoretically predicted ones. The results obtained for varied buffer concentration and varied concentration of NaCl in the sample solution show that under the conditions of our experimental study, the approximation of assuming θ to be equal to the reciprocal value of the field strength enhancement Factor γ is valid. Accordingly, the sweeping efficiency for neutral analytes is independent of the electric conductivity of the sample matrix. It is also shown that under specific conditions unexpectedly high enrichment Factors are obtained which are ascribed to the focusing of neutral analytes by micellar transient isotachophoresis (mtITP). The results obtained in this study can be used as a guide for better understanding of the sweeping process and the Factors affecting the sweeping efficiency in micellar electrokinetic chromatography (MEKC).
