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Lloyd R. Snyder - One of the best experts on this subject based on the ideXlab platform.
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Chapter 15 – Theory and practice of Gradient Elution liquid chromatography
Liquid Chromatography, 2017Co-Authors: John W. Dolan, Lloyd R. SnyderAbstract:Gradient Elution is the method of choice for the separation of samples with a wide retention range. This chapter reviews the basic theory and practical consideration for method development in Gradient Elution. Optimization requires selection of the range of solvent composition, solvent type, the Gradient time, and Gradient shape. The linear solvent strength model of Gradient Elution provides the tools for practical method development and computer-aided optimization of separations starting from a minimum number of trial experiments.
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Theory and Practice of Gradient Elution Liquid Chromatography
Liquid Chromatography, 2013Co-Authors: John W. Dolan, Lloyd R. SnyderAbstract:Abstract Gradient Elution is the method of choice for the separation of samples with a wide retention range. This chapter reviews the basic theory and practical consideration for method development in Gradient Elution. Optimization requires selection of the range of solvent composition, solvent type, the Gradient time, and Gradient shape. The linear solvent strength model of Gradient Elution provides the tools for practical method development and computer-aided optimization of separations starting from a minimum number of trial experiments.
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Encyclopedia of Analytical Chemistry - Gradient Elution Chromatography
Encyclopedia of Analytical Chemistry, 2012Co-Authors: John W. Dolan, Lloyd R. SnyderAbstract:Elution chromatography can be carried out in either isocratic or Gradient modes. In isocratic Elution, the mobile-phase composition is held constant during separation of the sample, e.g. 60% v acetonitrile–water. In Gradient Elution, the mobile-phase composition will be varied during sample separation, e.g. changing from 0 to 100% v acetonitrile–water. Gradient Elution requires special chromatographic equipment, as well as somewhat greater care on the part of the operator, but it has important advantages for many separations. Thus, in isocratic Elution (Figure 1a), sample peaks tend to “bunch up” at the beginning of the chromatogram (often with decreased resolution) and to broaden at the end of the chromatogram (with reduced detection sensitivity). Gradient Elution (Figure 1b), on the other hand, provides a more even spacing of peaks, similar widths throughout the chromatogram, and often a shorter run time. For these and other reasons, Gradient Elution is preferred for the separation of many samples.
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high performance Gradient Elution the practical application of the linear solvent strength model
2006Co-Authors: Lloyd R. Snyder, John W. DolanAbstract:High-performance Gradient Elution :the practical application of the linear-solvent-strength model , High-performance Gradient Elution :the practical application of the linear-solvent-strength model , مرکز فناوری اطلاعات و اطلاع رسانی کشاورزی
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peak compression in reversed phase Gradient Elution
Journal of Chromatography A, 2006Co-Authors: Uwe D Neue, D H Marchand, Lloyd R. SnyderAbstract:Previous reports suggest that peak widths in linear Gradient Elution are consistently larger than predicted by theory; however, if Gradient compression is ignored, experiment and theory are in reasonable agreement. This suggests that Gradient compression might represent an incorrect or poorly understood concept. In the present study, an experimental program was carried out to better understand the role of Gradient compression and the reason for past differences between experiment and theory. It is concluded that the concept of Gradient compression is correct.
M C García-alvarez-coque - One of the best experts on this subject based on the ideXlab platform.
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Study of the column efficiency using Gradient Elution based on Van Deemter plots.
Journal of Chromatography A, 2018Co-Authors: T. Alvarez-segura, E. Cabo-calvet, Juan José Baeza-baeza, M C García-alvarez-coqueAbstract:Performance of chromatographic columns is of major importance in the development of more efficient separation methods. So far, a common practice is to study the column behavior in isocratic Elution by modifying the flow rate and fitting the theoretical plate height values versus the mobile phase linear velocity, according to the Van Deemter equation. In this work, an approach is presented to extend the measurement of efficiency to linear Gradient Elution, where the mean retention factor is kept constant at each assayed flow. This avoids a possible source of uncertainty due to the change in the distribution equilibria profile, and makes the mean interactions with the stationary phase in Gradient Elution similar to those in isocratic Elution. The approach was applied to Zorbax Eclipse XDB C18 and Chromolith SpeedROD C18 columns, using four sulphonamides as probe compounds. The results are compared with those obtained in isocratic Elution, and Gradient Elution where the Gradient program is kept constant while the flow is varied, giving rise to changes in the mean retention factor at each flow rate. The mean compression factor obtained experimentally was higher than expected, with mean values of 0.98 and 1.02 for the Zorbax and Chromolith columns, whereas the predicted values were 0.87 and 0.92, respectively. Better efficiencies were obtained at lower flow and higher Gradient slope. The resolution shows similar values in isocratic and Gradient Elution when the mean retention factor is kept constant. When the Gradient slope is constant, the resolution is usually smaller, although it improves at higher flows.
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Optimisation of Gradient Elution with serially-coupled columns Part II: Multi-linear Gradients.
Journal of chromatography. A, 2014Co-Authors: C Ortiz-bolsico, J R Torres-lapasió, M C García-alvarez-coqueAbstract:An interpretive approach is reported for the simultaneous optimisation of resolution and analysis time in the separation of complex samples by reversed-phase liquid chromatography, using serially-coupled columns of different lengths and nature, and multi-linear Gradient Elution. The results complement those reported in JCA 1350 (2014) 51 for single linear Gradients, using combinations of five columns: three C18 columns of different characteristics, a cyano column and a phenyl column. The relationships between the logarithm of the retention factor (lnk) and the organic solvent content (φ), and between the peak half-widths and the retention times, for each analyte, were determined using isocratic retention times. From the established correlations, the Gradient retention time was predicted using the fundamental equation for Gradient Elution (modified to be used for serially-coupled columns), and with this information, the peak profile. Two chromatographic goals (maximal peak purity and minimal analysis time) were simultaneously evaluated using the Pareto optimality concept. The rigorous predictive system can be easily applied to columns from different manufacturers and succeeds at different levels: single and combined columns, using isocratic and Gradient Elution. The computation time for Gradient Elution optimisation was significantly reduced to practical values with the help of genetic algorithms (GAs).
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Optimisation of Gradient Elution with serially-coupled columns. Part I: single linear Gradients.
Journal of Chromatography A, 2014Co-Authors: C Ortiz-bolsico, J R Torres-lapasió, M C García-alvarez-coqueAbstract:An interpretive approach is reported for the simultaneous optimisation of resolution and analysis time in the separation of complex samples by reversed-phase liquid chromatography, using serially-coupled columns of different lengths and nature, and multi-linear Gradient Elution. The results complement those reported in JCA 1350 (2014) 51 for single linear Gradients, using combinations of five columns: three C18 columns of different characteristics, a cyano column and a phenyl column. The relationships between the logarithm of the retention factor (lnk) and the organic solvent content (φ), and between the peak half-widths and the retention times, for each analyte, were determined using isocratic retention times. From the established correlations, the Gradient retention time was predicted using the fundamental equation for Gradient Elution (modified to be used for serially-coupled columns), and with this information, the peak profile. Two chromatographic goals (maximal peak purity and minimal analysis time) were simultaneously evaluated using the Pareto optimality concept. The rigorous predictive system can be easily applied to columns from different manufacturers and succeeds at different levels: single and combined columns, using isocratic and Gradient Elution. The computation time for Gradient Elution optimisation was significantly reduced to practical values with the help of genetic algorithms (GAs).
A Pappalouisi - One of the best experts on this subject based on the ideXlab platform.
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retention models for isocratic and Gradient Elution in reversed phase liquid chromatography
Journal of Chromatography A, 2009Co-Authors: P Nikitas, A PappalouisiAbstract:One- and multi-variable retention models proposed for isocratic and/or Gradient Elution in reversed-phase liquid chromatography are critically reviewed. The thermodynamic, exo-thermodynamic or empirical arguments adopted for their derivation are presented and discussed. Their connection to the retention mechanism is also indicated and the assumptions and approximations involved in their derivation are stressed. Special attention is devoted to the fitting performance of the various models and its impact on the final predicted error between experimental and calculated retention times. The possibility of using exo-thermodynamic retention models for prediction under Gradient Elution is considered from a practical point of view. Finally, the use of statistical weights in the fitting procedure of a retention model and its effect on the calculated Elution times as well as the transferability of retention data among isocratic and Gradient Elution modes are also examined and discussed.
Csaba Horváth - One of the best experts on this subject based on the ideXlab platform.
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Temperature programming and Gradient Elution in reversed-phase chromatography with packed capillary columns.
Journal of Chromatography A, 1997Co-Authors: Michelle Hong Chen, Csaba HorváthAbstract:Abstract The two major anisocratic Elution modes were compared in reversed-phase chromagraphy with 180 μm I.D. fused-silica capillary columns acked with 6 μm Zorbax SB ODS-silica. By evaluating the retention factors of alkylbenzenes at acetonitrile concentrations varying from 60 to 80% (v/v) in the aqueous eluent and in the temperature range of 30–80°C, it was found that a 5°C change in column temperature and a 1% change in acetonitrile concentration have almost the same effect on retention. This is illustrated by the almost identical chromatograms of an alkylbenzene sample obtained by temperature programming and by Gradient Elution under the same conditions otherwise and by simulation of the trajectories of the eluent peaks moving down the column. The results suggest that in reversed-phase HPLC with packed capillary columns temperature programming offers an alternative to Gradient Elution in a relatively narrow range of the required Elution strength. Thermodynamic data from isocratic chromatographic measurements were used to predict the retention times of alkylbenzenes in reversed-phase chromatography with temperature programming at different heating rates and column inlet pressures. Temperature programming was used to separate β-lactoglobulins A and B by reversed-phase chromatography. It was also employed concomitantly with Gradient Elution to enhance the separation of a mixture of four standard proteins. The results indicate that temperature programming could serve as an adjunct to Gradient Elution by means of fine retention tuning to bring about or increase the resolution of closely related macromolecules.
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Capillary Electrochromatography with Gradient Elution
Analytical Chemistry, 1997Co-Authors: Christian G. Huber, Gargi Choudhary, Csaba HorváthAbstract:A capillary electrochromatograph incorporating a Gradient-forming system generally employed in HPLC is described, and the use of Gradient Elution in reversed phase electrochromatography is demonstrated by the separation of PTH-amino acids and steroid hormones. The Gradient former employs two reciprocating displacement pumps to control the composition of the eluent in the reservoir at the column inlet with time in a controlled manner. Thus, the composition of the mobile phase flowing through the column and driven by electrosmotic forces can be changed with time in a controlled fashion as customary in HPLC with Gradient Elution. The design of the system allows also for isocratic Elution by pumping the eluent of constant composition through the cavity at the column inlet and thus continuously supplying fresh buffer. The eluent Gradient is generated by the two pumps and a 10 μL mixer. From there the liquid passes at a flow rate of 0.1−0.2 mL/min through the 17 μL cavity housing the column inlet and an electro...
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Gradient Elution Chromatography
Chromatographic and Membrane Processes in Biotechnology, 1991Co-Authors: Firoz D. Antia, Csaba HorváthAbstract:Theoretical principles of Gradient Elution in both linear and nonlinear chromatography are discussed. In analytical applications, where linear chromatography prevails, the theory of Gradient linear Elution is well developed. On the other hand, in preparative applications, where the high concentrations encountered force the adsorption isotherms into the nonlinear range, Gradient Elution theory is as yet in its infancy. In this work, the governing principles of the theory, its limitations, and its application in some special cases are outlined.
Christian Frech - One of the best experts on this subject based on the ideXlab platform.
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Evaluation of differences between dual salt‐pH Gradient Elution and mono Gradient Elution using a thermodynamic model: Simultaneous separation of six monoclonal antibody charge and size variants on preparative‐scale ion exchange chromatographic resin
Biotechnology Progress, 2018Co-Authors: Yi Feng Lee, Matthias Jöhnck, Christian FrechAbstract:The efficiencies of mono Gradient Elution and dual salt-pH Gradient Elution for separation of six mAb charge and size variants on a preparative-scale ion exchange chromatographic resin are compared in this study. Results showed that opposite dual salt-pH Gradient Elution with increasing pH Gradient and simultaneously decreasing salt Gradient is best suited for the separation of these mAb charge and size variants on Eshmuno® CPX. Besides giving high binding capacity, this type of opposite dual salt-pH Gradient also provides better resolved mAb variant peaks and lower conductivity in the Elution pools compared to single pH or salt Gradients. To have a mechanistic understanding of the differences in mAb variants retention behaviors of mono pH Gradient, parallel dual salt-pH Gradient, and opposite dual salt-pH Gradient, a linear Gradient Elution model was used. After determining the model parameters using the linear Gradient Elution model, 2D plots were used to show the pH and salt dependencies of the reciprocals of distribution coefficient, equilibrium constant, and effective ionic capacity of the mAb variants in these Gradient Elution systems. Comparison of the 2D plots indicated that the advantage of opposite dual salt-pH Gradient system with increasing pH Gradient and simultaneously decreasing salt Gradient is the noncontinuous increased acceleration of protein migration. Furthermore, the fitted model parameters can be used for the prediction and optimization of mAb variants separation in dual salt-pH Gradient and step Elution. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:973-986, 2018.
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Solvent modulated linear pH Gradient Elution for the purification of conventional and bispecific antibodies: Modeling and application.
Journal of Chromatography A, 2015Co-Authors: Simon Kluters, Mathias Hafner, Thomas Von Hirschheydt, Christian FrechAbstract:Classical ion-exchange chromatography using a linear salt Gradient to elute the adsorbed protein at fixed pH is the most common method to separate product-related impurities during downstream processing of biopharmaceuticals. Linear pH Gradient Elution provides a useful alternative by separating proteins in a linear pH Gradient at fixed salt concentration. Although linear pH Gradient Elution provides excellent selectivity, it is rarely encountered in industrial purification processes. Here, a stoichiometric displacement model is used to characterize pH Gradient Elution based on simple linear Gradient Elution experiments. Protein retention behavior is described with respect to the pH dependencies of the characteristic binding charge and the equilibrium constant of the ion exchange reaction. Furthermore, the influence of solvent composition using PEG as a mobile phase modifier is investigated. Validity and applicability of the model are demonstrated for the purification of a conventional monoclonal antibody from soluble aggregates and for a novel bispecific antibody format containing a unique product-related impurity profile. pH step Elution protocols are derived from model calculations without further optimization experiments necessary.
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Modeling of salt and pH Gradient Elution in ion-exchange chromatography
IEEE Journal of Solid-state Circuits, 2014Co-Authors: Michael Schmidt, Mathias Hafner, Christian FrechAbstract:The separation of proteins by internally and externally generated pH Gradients in chromatofocusing on ion-exchange columns is a well-established analytical method with a large number of applications. In this work, a stoichiometric displacement model was used to describe the retention behavior of lysozyme on SP Sepharose FF and a monoclonal antibody on Fractogel SO3 (S) in linear salt and pH Gradient Elution. The pH dependence of the binding charge B in the linear Gradient Elution model is introduced using a protein net charge model, while the pH dependence of the equilibrium constant is based on a thermodynamic approach. The model parameter and pH dependences are calculated from linear salt Gradient Elutions at different pH values as well as from linear pH Gradient Elutions at different fixed salt concentrations. The application of the model for the well-characterized protein lysozyme resulted in almost identical model parameters based on either linear salt or pH Gradient Elution data. For the antibody, only the approach based on linear pH Gradients is feasible because of the limited pH range useful for salt Gradient Elution. The application of the model for the separation of an acid variant of the antibody from the major monomeric form is discussed.
