High-performance liquid chromatography of amino acids, peptides and proteins ☆LXXXVI. The influence of different displacer salts on the retention and bandwidth properties of proteins separated by isocratic anion-exchange chromatography (original) (raw)
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Journal of Chromatography A, 1988
This investigation deals with protein retention behaviour in high-performance anion-exchange chromatography in terms of the average distance of approach between the protein solute and the positively charged anion-exchange stationaryphase surface. The theoretical treatment is based on a modified Debye-Hiickel theory for spherical impenetrable ions, where the electrostatic potential energy has been related to the chromatographic capacity factor, k'. Results are presented for three globular proteins, eluted isocratically from a Mono-Q strong anion-exchange resin with sodium choride as the displacer salt by a mobile phase with pH in the range 5.5&9.60. Analysis of experimental retention data indicates that topographically predelined, charged regions on the protein surface, called ionotopes, control the orientation and approach distance of the protein solute.
Analytical Sciences, 1991
This study explores the physicochemical relationships which underlie the interactive processes between chaotropic or kosmotropic ions, proteins and coulombic chromatographic sorbents. Variations in the chaotropic and kosmotropic properties of the displacer ion and coion permit the protein-ligand electrostatic contact area, the protein conformation, the electrostatic topology and charge distribution of the protein to be systematically probed. Moreover, these data provide guidelines relevant not only to optimisation of a particular separation task but also pertinent to new sorbent design.
Peculiarities of gradient ion-exchange high-performance liquid chromatography of proteins
Journal of Chromatography A, 1996
Since the influence of column length on protein resolution in high-performance liquid chromatography (HPLC) is not clear, different viewpoints presented in the literature are analysed in detail. The influence of gradient steepness on the length of the working column part (Xo) or the part of a column in which the quasi-steady state is attained was studied. The equation for estimating the X o value was obtained for the general case of the retention model. It was shown that at steep gradients only a short part of the column is used as the working part on which all separation processes develop. The other part of a column is a ballast where the protein zone migrates in a regime of parallel transfer. These results form a theoretical basis for high-performance membrane chromatography. As was shown experimentally, this method makes it possible to perform protein separation at low gradient times with appropriate resolution, comparable with that of HPLC.
Journal of Chromatography A, 2008
The behavior of chromatographic columns packed with resins containing both weak and strong cation-exchange groups is investigated in order to obtain protein separations by means of internally generated pH gradients in response to step changes in buffer composition. A local equilibrium model is developed to predict pH transitions using non-adsorbed buffers, i.e. containing neutral and negatively charged buffering species, based exclusively on the resin titration curve. In agreement with experimental results, the model predicts practical, fairly linear gradients between pH 5 and 7, which are formed using suitable mixtures of acetate and phosphate buffers. The separation of mixtures of ovalbumin, albumin, and transferrin is used as a model system, but, unlike most previous work, we consider preparative conditions. Near baseline resolution is obtained with protein loads as high as 10 mg/mL and mobile phase velocities at high as 460 cm/h using porous, 70-m diameter particles. The peaks obtained with this approach are much sharper than could be obtained isocratically or using externally generated, unretained gradients as a result of the peak compression caused by the axial pH gradient formed along the column. Moreover, separation is obtained at very low ionic strengths (2-3 mS/cm). The effects of flow velocity, mobile phase composition, time of injection, and protein load on retention and elution pH are investigated systematically demonstrating a range of ways in which the separation can be controlled and optimized.
Separation of peptides by strong cation-exchange high-performance liquid chromatography
Journal of Chromatography A, 1985
The effects of pH and gradient conditions on the separation of a series of ten peptides (9-36 residues) and carboxamidomethylated troponin I (CM-TnI, 178 residues) on a new commercially available strong cation-exchange silica based 300-A column (Synchropak S300) were examined. The elution times of the peptides were linear with respect to their net charge at pH 3.0 and pH 6.5. The basic protein CM-TnI (pZ % 9.5) and peptides with net charges from + 2 to + 10 were separated with linear AB salt gradients varying from 5 mM to 10 mM B per min (A = 5 mM KH2P04 buffer, pH 6.5 or 3.0; B = 5 mM KH2P04 buffer, pH 6.5 or 3.0, containing 1 M KCl). All peptides and CM-TnI were eluted with KC1 concentrations below cu. 0.6 M. The advantage of performing cation-exchange chromatography over anionexchange chromatography was demonstrated for the separation of peptides which, while acidic or weakly basic at neutral pH, through protonation of the acidic functions results in positively charged peptides at pH 3.0. 0021-9673/85/%03.30 0 1985 Elsevier Science Publishers B.V.
A comprehensive study to protein retention in hydrophobic interaction chromatography
Journal of chromatography. B, Analytical technologies in the biomedical and life sciences, 2016
The effect of different kosmotropic/chaotropic salt systems on retention characteristics of intact proteins has been examined in hydrophobic interaction chromatography (HIC). The performance was assessed using different column chemistries, i.e., polyalkylamide, alkylamine incorporating hydrophobic moieties, and a butyl chemistry. Selectivity in HIC is mainly governed by the salt concentration and by the molal surface tension increment of the salt. Typically, a linear relationship between the natural logarithm of the retention factor and the salt concentration is obtained. Using a 250mm long column packed with 5μm polyalkylamide functionalized silica particles and applying a 30min linear salt gradient, a peak capacity of 78 was achieved, allowing the baseline separation of seven intact proteins. The hydrophobicity index appeared to be a good indicator to predict the elution order of intact proteins in HIC mode. Furthermore, the effect of adding additives in the mobile phase, such as ...
Journal of Chromatography A, 2010
Typical mobile phase employed in hydrophobic interaction chromatography contains cosmotropic salts, which promote retention and simultaneously reduce the protein solubility in the mobile phase. To increase mass overloading in the separation process the protein can be dissolved in a sample-solvent with concentration of salt lower than that in the mobile phase or in salt free solutions. However, this methodology may cause band splitting and band deformation, which results in yield losses. In this study, these phenomena were analyzed based on the retention behavior of two model proteins, i.e., lysozyme and bovine serum albumin. Retention of these proteins was accompanied by strong band broadening originated from slow rates of mass transfer and/or of adsorption-desorption process involving the protein conformational changes. The mass transport resistances and unfolding kinetics were found to contribute to the sample-solvent effects. To avoid band deformations the process variables such as the salt concentration and temperature were adjusted in such a way that complete resolution between band profile of the sample-solvent and the protein was achieved. For the process simulation a dynamic model, which accounted for underlying kinetics was used. General guidelines of the process design were developed.
Binding and elution behavior of proteins on strong cation exchangers
Journal of Chromatography A, 2009
This work provides a broad survey of binding and elution behavior of proteins on strong cation exchangers. Four proteins comprising two monoclonal antibodies, lysozyme, and cytochrome c were used as models in the investigation. Seven chromatography resins with different base matrices were compared. Dynamic binding capacity as a function of salt concentration was examined for a monoclonal antibody and lysozyme. Elution behavior as a function of gradient slope was modeled to determine the characteristic charge, essentially a measure of the number of sites involved in binding, for each protein on each resin. Trends with respect to dynamic binding capacity and elution behavior are analyzed and discussed.
Journal of Chromatography A, 1993
In large-scale chromatography, process optimisation is one of the key elements for success. This paper presents a method for determining the optimum operating parameters for affinity and ion-exchange chromatographic columns when used for protein purification. Based on a mathematical mode1 developed as part of our association investigations, computer programs have been developed to describe the dynamic relationships acting within the chromatographic system. Two basic operating parameters, the flow-rate and the effluent concentration at which the adsorption stage is terminated, can be optimised to give a maximum production rate. The sample loading volume and the processing time then can be determined. The effect of washing conditions on the production rate and the yield is also discussed. Examples are given for a specific system where the optimisation is based on the yield and the percentage utilisation of the column capacity. Contour plots are generated to aid the determination of the range of controlling parameters, and to guide further system design.
Journal of Chromatography A, 2009
We investigated the mechanisms involved in the retention of various peptides on a stationary phase embedded with a quaternary ammonium group (BS C23), by high-performance liquid chromatography. This was compared with peptide retention on a conventional reversed-phase C18 (RP C18) column under isocratic conditions, to understand better the various mechanisms involved. Chromatographic characterization of the two stationary phases with "model" compounds showed that BS C23 is less hydrophobic than RP C18 and induces electrostatic interaction (attraction or repulsion) with ionized compounds. If reversed-phase partitioning was the predominant retention phenomenon, for both stationary phases, the retention mechanisms in BS C23 provided different selectivity to that of RP C18. Electrostatic attraction or repulsion was clearly observed between peptides and the permanent positively charged group embedded in BS C23 depending on the pH. For most of the peptides, a weak anion-exchange mechanism was observed on the quaternary ammonium-embedded stationary phase if mobile phases at neutral pH and low ionic strengths were employed.