Cs. Horváth

Salt-mediated retention of proteins in hydrophobic-interaction chromatography : Application of solvophobic theory

Retention behavior in hydrophobic-interaction chromatography was examined within the framework of the solvophobic theory. The principal parameters which determine the effect of salt on the retention are salt molality and the molal surface tension increment of the salt. According to the theory, in the absence of special binding effects, increase in salt molality in the mobile phase or change of salt to one of greater molal surface tension increment will result in increased retention of proteins in hydrophobic chromatography. The theory is expanded to treat retention in gradient elution with linear decrease in salt concentration that is equivalent to linear increase in eluent strength. The results of the simple model lead to an expression with two parameters: the adjusted isocratic retention volume of the eluite with the gradient former and the slope of plot of logarithmic adjusted elution volume against salt molality, lambda. The latter parameter is linearly dependent on molal surface tension increment if no specific interactions between the eluite and the stationary phase and/or salt are present. In practice, deviations are to be expected from the predicted behavior due to such effects. The results of calculations are consistent with experimental results obtained with several proteins as the eluites and various salts in the eluent. Although unique values of the critical parameter lambda could not be obtained from the data, the trends showed that lambda is strongly correlated with the value of the molal surface tension increment. The prediction that increase in salt concentration in the initial eluent leads to increase in retention volume was found to be generally true, even when the isocratic retention volumes obtained with use of eluent having low salt concentration were small. Use of NaClO4 in the starting eluent led in some cases to decrease in retention volume with increase in the salt concentration at the beginning of the gradient elution. This effect may be due to specific binding effects.

High-speed high-performance liquid chromatography of peptides and proteins

Over the last thirty years the name HPLC has been synonymous with high-speed liquid chromatography and during the last ten years we have experienced a dramatic increase in the speed of analysis particularly as far as the separation of biological macromolecules, such as proteins, is concerned. With a solid grounding in the chromatographic theories, column technology has been mainly responsible for the advances in this field. Recent development shows that columns packed with micropellicular or gigaporous stationary phases of the bidisperse or the bimodal type facilitate rapid mass transfer between the mobile and stationary phases and thus can deliver high resolution separations in a very short time. This suggest that HPLC has the potential to be the prime analytical technique for on-line monitoring of biotechnological processes in real time. Further enhancement of the speed of separation comes from the use of elevated temperatures. The role of temperature in HPLC has largely been ignored and most commercial instruments are not equipped with appropriate temperature control. Results presented here strongly suggest, however, that elevated column temperature may find increasing use in the HPLC of large molecules. In such analytical applications temperature programming may also play a major role provided columns with low heat capacity, such as packed fused-silica capillaries, gain wider employment in HPLC.

Separation parameters via virtual migration distances in high-performance liquid chromatography, capillary zone electrophoresis and electrokinetic chromatography

Among the various differential migration processes of separation, high-performance liquid chromatography (HPLC) and capillary zone electrophoresis (CZE) have emerged as the two major high-performance analytical techniques for separation of complex biological substances. In both HPLC and CZE with electroendoosmotic flow, the differential migration process can be divided into a separative component, which involves selective interactions with the stationary phase or differences in the electrophoretic migration velocities, and a non-separative component representing migration by convection that does not contribute directly to separation. The introduction of virtual migration distances leads to an additivity relationship for the two components that is applicable to both of the above techniques and facilitates the recognition of the underlying similarities as well as the expression and comparison of the various separation parameters. Examination of the key migration parameters led to the characterization and the classification of the various modes of CZE with electroendoosmotic flow. The treatment was extended to the analysis of capillary electrochromatography and micellar electrokinetic chromatography; two hybrid processes which exhibit features borrowed from HPLC and CZE. The use of virtual migration distances also led to a consistent and unified description of the characteristic parameters of these separation systems.

High performance displacement chromatography of proteins: Separation of β-Lactoglobulins A and B

Summaryβ-Lactoglobulins A and B were separated by high performance displacement chromatography on an anion-exchanger column with chondroitin sulfate as the displacer. A sample of 100 mg containing a mixture of the two β-lactoglublins was separated on a column of 75×7.5 mm in a single chromatographic run. The separation process followed the rules of the classical displacement development: the two proteins formed contiguous rectangular bands and their concentrations were dependent on the displacer concentration. The results demonstrate that high performance displacement chromatography is a useful technique for the separation of proteins in preparative amounts with columns and instrumentation typically used in analytical HPLC. Furthermore, it has the potential to become the method of choice in large scale separation of proteins.

Stationary phase effects in reversed-phase chromatography II. Substituent selectivities for retention on various hydrocarbonaceous bonded phases

SummaryLinear free-energy relationships are manifest in the linear dependence of the logarithmic retention factor on the carbon number for homologous series in reversedphase chromatography and allow the comparison of the free-energy increments of substituents for retention of a given elute by different stationary phases. Three methods were used for such comparison. In the first two, methylene group selectivities obtained from retention data obtained with homologous series on different hydrocarbonanceous bonded phases were compared by using different approaches. In the third method the energetics of retention of the end groups such as phenyl-, bromo- and disulfide-moieties of homologous series were examined. The results show that at fixed mobile phase composition and temperature the free energy increments due to methylene groups upon retention by alkyl-silica stationary phases are identical. Only hydrocarbonaceous bonded phases with short (C1–C2) alkyl chains or functional groups of unusual shape show consistent differences from octyl- or octadecyl-silicas. The results broaden the theoretical basis for the development of quantitative structure-retention relationships to be used for the prediction or interpretation of retention data in reversed-phase chromatography.

Dynamics of capillary electrochromatography. II. Comparison of column efficiency parameters in microscale high-performance liquid chromatography and capillary electrochromatography.

In capillary electrochromatography (CEC) the flow of the mobile phase is generated by electrosmotic means in high electric field. This work compares band spreading measured experimentally in several packed capillaries with electrosmotic flow (EOF) and viscous flow under otherwise identical conditions. The data were fitted to the simplified van Deemter equation for the theoretical plate height, H = A + B/u + Cu, in order to evaluate parameters A and C in each mode of flow in the different columns. The ratio of these two parameters obtained with the same column in microscale HPLC (mu-HPLC) and CEC was used to quantify the attenuation of their contribution to band spreading upon changing from viscous flow (in mu-HPLC) to electrosmotic flow (in CEC). The capillary columns used in this study were packed with stationary phases of different pore sizes as well as retentive properties and measurements were carried out under different mobile phase conditions to examine the effects of the retention factor and buffer concentration. In the CEC mode, the value of both column parameters A and C was invariably by a factor of two to four lower than in the mu-HPLC mode. This effect may be attributed to the peculiarities of the EOF flow profile in the interstitial space and to the generation of intraparticle EOF inside the porous particles of the column packing. Thus, band spreading due to flow maldistribution and mass transfer resistances is significantly lower when the mobile phase flow is driven by voltage as in CEC, rather than by pressure as in mu-HPLC.

Investigation of operating parameters in high-performance displacement chromatography

The effect of operational parameters of displacement chromatography was examined in the separation of various mixtures such as that of the main hydrolysis products of methylfurylbutyrolactone, a potential anticancer drug, the diastereoisomers benzoyl-D- and benzoyl-L-phenylalanyl-L-alanyl-L-proline, as well as polyethylene glycol homologues containing 1-10 ethylene oxide units. The chromatograph was assembled from modules generally used in analytical high-performance liquid chromatography (HPLC) and the column effluent was analyzed by an on-line HPLC unit at 30-sec intervals. Octadecyl-silica was used throughout as the stationary phase. Derivatives of ethylene glycol and propylene glycol as well as tetrabutylammonium bromide and n-butanol were used as displacers. The throughput was used as the measure of efficiency. In the absence of axial dispersion, for a given separation various displacers are expected to yield the same efficiency if the slope of the operating line is kept the same by appropriate adjustment of displacer concentrations. In practice, however, the optimum slope of the operating line has to be determined experimentally as most available chromatographic systems depart from ideal behavior. The dependence of the throughput on the flow-rate and feed load also indicated the presence of non-equilibrium phenomena and the optimum value of these parameters was established experimentally. In most cases water was used as the carrier solvent but the separation of poorly soluble peptides required the use of hydro-organic carriers. Results obtained with octadecyl-silicas of different origin and a given displacer were found to vary significantly suggesting that even for stationary phases of the same type the selection of displacer requires special consideration. Most experiments were carried out with columns having dimensions customary in analytical HPLC. Increasing the inner diameter of the column did not result in the expected increase in throughout probably due to poor distribution of the sample at the column entrance. Therefore scaling-up the process requires careful engineering of inlet conditions. Throughput can be increased by connecting a small inner diameter column to the outlet of a large diameter preparative column. As theoretical predictions for ideal displacement chromatography do not hold in practice when axial dispersion is significant, optimization of the process requires experimental support. The results obtained in the separation of a variety of mixtures shed light on the most important operational aspects of displacement chromatography and suggest approaches to find optimum conditions.(ABSTRACT TRUNCATED AT 400 WORDS)

Movement of components in reversed-phase chromatography : I. Mobile phase space with multi-component eluents

Abstract The retention volume of a peak that is neither retained nor excluded from the mobile phase space in the column is required for determination of retention (capacity) factor and related thermodynamic quantities. In liquid chromatography with multi-component eluents, however, the mobile phase space is theoretically indeterminate owing to interaction between the individual components and the stationary phase and therefore definition and measurement of the mobile phase necessitates that an appropriate convention be found. Here the following four conventions are examined: (i) “all mobile phase components are present in the solvation layer”; (ii) “no solvation layer exists”; (iii) “one given eluent component is not present in the solvation layer”; and (iv) “the most weakly bound solvent component is not present in the solvation layer”. Whereas all conventions appear to be acceptable for a thermodynamic description of the interaction between mobile phase components and the stationary phase in most cases, they yield different values of retention factors and associated thermodynamic properties for eluites. Furthermore, the use of some conventions can give rise to practical problems, e.g. , experimental determination of void volume by use of the first convention is ill-defined in general and the second and third conventions can lead to negative values for the mobile phase space. The present work suggests that the fourth convention is free of these problems and provides retention factor values which are more appropriate for use in liquid chromatography than those obtained by the other conventions.

Movement of components in reversed-phase chromatography : II. Eigenpeaks in reversed-phase chromatography with silica-bound hydrocarbonaceous stationary phases: effect of the eluite structure

Abstract Eigenpeaks of the eluent appear in the column effluent upon perturbing the equilibrium composition of the eluent at the column inlet, e.g., by sample introduction. Eigenpeaks move down the column with concentration velocities which are the eigenvectors given by the characteristic solutions of the mass balance equations for the system. The eigenpeak height, as obtained by monitoring the refractive index of the effluent, is related to both the mobile phase composition and chemical structure of the eluite. Eigenpeaks obtained experimentally with eluites of different chemical structure and eluents commonly used in reversed-phase chromatography were examined in order to relate their height to salvation phenomena which occur in the column: (i) solvation of eluite by mobile phase components and (ii) change in solvation of stationary phase and eluite upon eluite binding. The eluite was dissolved in the eluent proper, and the eigenpeak height was found to be proportional to the mass of eluite over the range of sample loads investigated. The relationship observed between the molecular structure of eluite and the height of the attendant eigenpeaks is explained on the basis of the solvation model that takes into account the predominant different solvation effects by using slightly or strongly retained eluites. The results obtained with both binary and ternary eluents could be accounted for by the solvation model. Isomeric eluites which were eluted together in the chromatographic system under study showed significantly different eigenpeak patterns that could be used for identification. The results of this study show that solvation phenomena involving the eluite manifest themselves by the magnitude of the eigenpeak signals that convey a novel type of chromatographic information for possible use in the evaluation of solvation processes as well as for identification of components of elute pairs that are refractory to separation.

Protein surface area and retention in hydrophobic interaction chromatography

SummaryMolecular surface areas accessible to a 4 Å diameter spherical probe were calculated from crystallographic data for five proteins: α-chymotrypsinogen A, lysozyme, trypsinogen, ribonuclease A and ribonuclease S. The retention factors of various proteins were measured on stationary phases having polyether- and phenylligates and with aqueous eluents containing (NH4)2SO4, Na2SO4 or NaCl at pH 7.0. The logarithmic retention factors were plotted against the salt molality and the hydrophobic interaction parameters evaluated from the limiting slopes of the plots at high salt concentrations for the proteins in the chromatographic systems investigated. The hydrophobic interaction parameters thus obtained were linear in both the molecular surface areas of the proteins and the molal surface tension increments of the salts. The experimental results obtained with these relatively simple proteins of known molecular structure, which were available in high purity, support earlier theoretical predictions for the dependence of the hydrophobic interaction parameter on the surface area of the protein and the surface tension raising effect of the salt.