Torgny Fornstedt

A CLOSER STUDY OF CHIRAL RETENTION MECHANISMS

The retention mechanisms and the separation of enantiomers on the classes of chiral stationary phases which are made by bonding isolated groups on the surface of an adsorbent are discussed. It is s ...

Peak tailing and mass transfer kinetics in linear chromatography

The origin of peak tailing under linear conditions, at very low analyte concentrations, is investigated using the equations of the transport-dispersive model of chromatography. It is shown that a general explanation can be obtained by assuming the existence of two different types of adsorption sites having different equilibrium isotherms and different rates of mass transfer kinetics. Even if the experimental conditions are such that both mechanisms operate linearly, tailing can be observed if the mass transfer kinetics is much slower on one type of sites than on the other. The most pronounced and typical peak tailings occur when the slow type of adsorption sites give a smaller contribution to the retention than the fast type and if the rate constant of mass transfer for the slow sites is between 20 and 2000 times smaller than that of the fast sites. Axial dispersion (caused by molecular and eddy diffusion) dampens the effects. Therefore, peak tailing of kinetic origin will be more important on highly efficient columns than on mediocre ones. An important case in point is that of enantiomeric separations. Chiral phases are designed as two-types-of-sites phases since it is impossible to eliminate the general non-selective interactions between analytes and bonded groups. The chiral selective sites involve strong interactions between the stationary phase and at least one of the enantiomers, conditions which slow down mass transfers.

Apparent and True Enantioselectivity in Enantioseparations

The separation factor of two compounds in chromatography is the ratio of their equilibrium constants or retention factors. This parameter is universally employed to investigate their resolution and to optimize the experimental conditions of their analysis. In enantioseparations, the situation is more complex because there is a mixed retention mechanism. The retention factor is the sum of two contributions, one enantioselective, the other nonselective. Although both contribute to retention, the latter being identical for the two enantiomers and does not contribute to their separation. We show how these two contributions can be measured and how it becomes necessary to distinguish between the apparent, alpha(app), and the true, alpha(true), separation factors. The existence of nonselective sites is responsible for alpha(app) being less than alpha(true). Depending on the difference between these two factors, the more effective approach to improve a separation is either to increase the enantioselectivity or to reduce the nonselective interactions. Practical applications to separations of different beta-blockers on cellobiohydrolase are discussed. The apparent enantioselectivity of alprenolol is larger and increases faster with increasing pH than that of the more hydrophobic propranolol, in spite of the importance of hydrophobic interactions in the enantioselective mechanism. These two unexpected properties are discussed and explained.

Peak tailing and slow mass transfer kinetics in nonlinear chromatography

Peak tailing has two main origins in chromatography: (i) heterogenous mass transfer kinetics, (ii) heterogenous thermodynamics with overloading of a nonlinear isotherm. The individual effects of ea ...

An improved algorithm for solving inverse problems in liquid chromatography

Competitive adsorption isotherms must be measured in order to simulate and optimize modern continuous modes of liquid chromatography, such as simulated moving bed chromatography, in situations where experimental trial-and-error approaches are too complex and expensive. An attractive method for obtaining adsorption isotherms is to solve the inverse problem, i.e., to numerically estimate adsorption isotherm parameters so that the simulated batch separation coincides with actual experimental results. The chromatographic community refers to this as the inverse method and it has many advantages over conventional methods of adsorption isotherm determination. This work examines and proposes improvements to the four basic parts of the algorithm used in the inverse method, i.e., the partial differential equation solver routine, calculation of the Jacobian of the computer-simulated elution profiles with respect to the adsorption isotherm parameters, conversion of experimental detector response into individual concentration contributions and screening between different possible adsorption isotherm models.

Characterization of adsorption processes in analytical liquid-solid chromatography.

This review discusses nonlinear chromatographic methods of importance for proper characterization of the adsorption processes in analytical chromatographic systems, with focus on reversed-phase liquid chromatography. Linear methods such as the linear solvation energy relationship (LSER) method and the Snyder-Dolan hydrophobic-subtraction model will also be reviewed briefly. The nonlinear methods for adsorption isotherm determination and the tools for further treatment of the nonlinear adsorption data will be extensively treated in a way suitable for the general chromatographer. Applications of the various methods will be given and the outcome and conclusions will be discussed. Special emphasis will be placed on discussing the possibilities of combining linear and nonlinear methods in order to obtain a deeper and more complete investigation of the interactions in the actual phase system.

Potential of adsorption isotherm measurements for closer elucidating of binding in chiral liquid chromatographic phase systems

The human body is a chiral environment and many drugs are chiral and interact differently depending on the type of enantiomer. Therefore, the interest in analytical and preparative separations of enantiomers has steadily increased over the years. LC is today the most important technique in analytical laboratories worldwide. The key to understand the separation system lies in the adsorption isotherm, which describes the equilibrium distribution of solutes between the mobile and stationary phases. By measuring adsorption isotherms in chiral phase systems, a deeper interpenetration concerning enantioselective and non-selective binding energies and adsorption processes is possible. Furthermore, this data provides the core information needed to optimize preparative chromatographic processes for purification of single enantiomers. However, the measurement of adsorption isotherms is a delicate matter and there are many dangerous pitfalls that may produce erroneous results and even wrong mechanistic conclusions. This review summarizes the most relevant methods and a workflow will be given for avoiding the common pitfalls and obtaining reliable data. Several applications from the literature are also treated to give insight in what information can potentially be obtained from using this methodology.

Peak tailing and mass transfer kinetics in linear chromatography - Dependence on the column length and the linear velocity of the mobile phase

Although peak tailing in analytical (i.e., linear) chromatography is often considered to be associated merely with slow mass transfer kinetics, we show that it results rather from the use of too sh ...

Theoretical study of the accuracy of the elution by characteristic points method for bi-Langmuir isotherms

The bi-Langmuir equation has recently been proven essential to describe chiral chromatographic surfaces and we therefore investigated the accuracy of the elution by characteristic points method (ECP) for estimation of bi-Langmuir isotherm parameters. The ECP calculations was done on elution profiles generated by the equilibrium-dispersive model of chromatography for five different sets of bi-Langmuir parameters. The ECP method generates two different errors; (i) the error of the ECP calculated isotherm and (ii) the model error of the fitting to the ECP isotherm. Both errors decreased with increasing column efficiency. Moreover, the model error was strongly affected by the weight of the bi-Langmuir function fitted. For some bi-Langmuir compositions the error of the ECP calculated isotherm is too large even at high column efficiencies. Guidelines will be given on surface types to be avoided and on column efficiencies and loading factors required for adequate parameter estimations with ECP.

Approach for Reliable Evaluation of Drug Proteins Interactions Using Surface Plasmon Resonance Technology

The surface plasmon resonance (SPR) biosensor was recently introduced to the analytical biochemical society for measuring small drug-protein interactions. However, the technique has many times been used without specifying the type of enantiomeric form of the chiral drug measured and/or with using a too narrow drug concentration range resulting in biased values of binding coefficients and sometimes even assumptions about single-site bindings although the binding in reality comprises a multisite interaction. In this study we will give guidelines for reliable experimental and methodological approaches to avoid these pitfalls. For this purpose, we also introduce a new tool, based on physical chemistry, to the sensor community; the calculation of the adsorption energy distribution (AED). The AED-calculations reveal the degree of heterogeneity directly from the SPR raw data and thus guide us into a narrower selection of probable models before the rival model fitting procedure. We demonstrate how to measure reliable equilibrium data for the two typically different cases: drug binding to (i) transport (plasma) proteins and to (ii) a target protein. Both the binding of the chiral beta-blocker propranolol to alpha(1)-acid glycoprotein (AGP) and that of the anticoagulant warfarin to human serum albumin were heterogeneous, with a few strong enantioselective sites and many weak nonselective sites. We also demonstrate how the multisite binding rapidly falsely turns to single-site as the concentration range is narrowed and how adding dimethyl sulfoxide to the buffer affects multisite drug-protein data. The binding of the enantiomers of the thrombin inhibitor melagatran was investigated on both thrombin and the transport proteins, revealing clear enantioselectivity for thrombin in favor of the active enantiomer, but almost similar binding properties for both enantiomers to the transport protein AGP. The AED-calculations verified that both these system has a unimodal energy distribution and are best described with a homogeneous adsorption model.