Robert Arnell

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.

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.

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.

Development of the Tracer-Pulse Method for Adsorption Studies of Analyte Mixtures in Liquid Chromatography Utilizing Mass Spectrometric Detection

The tracer-pulse method provides the real adsorption data points directly from simple, straightforward calculations and is therefore a superior method for multicomponent adsorption isotherm determination in HPLC. Only one important problem has restricted its use so far: the tracer peaks are invisible using any conventional detection principle. We present a solution to this problem with an approach with a firm base in analytical chemistry, utilizing stable isotopes and mass spectrometric detection. The new approach was used for the determination of binary adsorption isotherms, and a systematic investigation was made of its main sources of error. With this modification, the tracer method can be a prime choice for future characterizations of multicomponent separation systems and of competitive drug binding studies.

Adsorption behaviour of a quinidine carbamate-based chiral stationary phase: Role of the additive

In this study, we incorporate the additive properties into the theoretical model of a general preparative chromatographic system; this is normally not done and this limits a proper process optimization. As a model phase system, we used the adsorption of 9H-fluoren-9-ylmethoxycarbonyl-allylglycine (Fmoc-allylglycine) enantiomers on a quinidine carbamate-based chiral stationary phase (anion exchanger) together with a methanol-glacial acetic acid-ammonium acetate eluent. The inverse method was used to measure the competitive adsorption isotherms of both the Fmoc-allylglycine enantiomers as well as the non-detectable additive acetic acid. It was concluded that this enantioselective preparative system is well described by a non-heterogeneous adsorption model and that the loading capacity is very high. The proposed model is valid over a wide range of additive concentrations, which is important for process optimization.

Effects of a strongly adsorbed additive on process performance in chiral preparative chromatography

The shapes of elution profiles are often significantly influenced by the presence of strongly adsorbed additives in the mobile phase. This aspect needs to be considered in quantitative optimization of preparative chromatography. The theoretical study carried out here is based on available thermodynamic information for the enantiomers of three beta-blockers, alprenolol, propranolol, and atenolol, on a teicoplanin chiral stationary phase (Chirobiotic T) using methanol/acetonitrile as the mobile phase and acetic acid/triethylamine as the additive. The properties of this strong additive made it possible to tune the binary elution profiles in any combination of the following apparent band shapes: anti-Langmuir/anti-Langmuir, anti-Langmuir/Langmuir and Langmuir/Langmuir. Optimization of the productivity and yield, when performing repetitive batch injections, was investigated using the equilibrium dispersive model. We show that it is important to consider the invisible additive perturbation peak when defining the cycle time and therefore a model-based optimization needs to take this into account. Furthermore, both productivity and yield could be improved for the two unusual shape combinations in comparison to the traditional Langmuir/Langmuir case.

A systematic investigation of algorithm impact in preparative chromatography with experimental verifications.

Computer-assisted optimization of chromatographic separations requires finding the numerical solution of the Equilibrium-Dispersive (ED) mass balance equation. Furthermore, the competitive adsorption isotherms needed for optimization are often estimated numerically using the inverse method that also solves the ED equations. This means that the accuracy of the estimated adsorption isotherm parameters explicitly depends on the numerical accuracy of the algorithm that is used to solve the ED equations. The fast and commonly used algorithm for this purpose, the Rouchon Finite Difference (RFD) algorithm, has often been reported not to be able to accurately solve the ED equations for all practical preparative experimental conditions, but its limitations has never been completely and systematically investigated. In this study, we thoroughly investigate three different algorithms used to solve the ED equations: the RFD algorithm, the Orthogonal Collocation on Finite Elements (OCFE) method and a Central Difference Method (CDM) algorithm, both for increased theoretical understanding and for real cases of industrial interest. We identified discrepancies between the conventional RFD algorithm and the more accurate OCFE and CDM algorithms for several conditions, such as low efficiency, increasing number of simulated components and components present at different concentrations. Given high enough efficiency, we experimentally demonstrate good prediction of experimental data of a quaternary separation problem using either algorithm, but better prediction using OCFE/CDM for a binary low efficiency separation problem or separations when the compounds have different efficiency. Our conclusion is to use the RFD algorithm with caution when such conditions are present and that the rule of thumb that the number of theoretical plates should be greater than 1000 for application of the RFD algorithm is underestimated in many cases.

A quest for the optimal additive in chiral preparative chromatography

Traditionally, the choice of acid/base additives used in chiral preparative chromatography has not been considered very important. However, it was recently demonstrated that strongly adsorbing additives can result in the most unexpected enantiomer band shapes in modern chiral preparative chromatographic systems. In the present study we demonstrate that, depending on the choice of additive, it is actually possible to obtain the following four binary band-shape compositions when a racemic mixture is injected: (i) anti-Langmuir/anti-Langmuir, (ii) anti-Langmuir/Langmuir, (iii) Langmuir/Langmuir and (iv) Langmuir/anti-Langmuir. Further, we made an advanced numerical investigation, in order to ascertain which one of the four band-shape compositions, is the most favourable one in preparative batch chromatography of a racemic mixture. We found that if the target for purification is either the first eluting enantiomer or both ones, the traditional Langmuir/Langmuir band-shape composition should be chosen. But, if only the second eluting enantiomer is to be purified the optimal situation is the anti-Langmuir/Langmuir band-shape composition. Thus, it was concluded that the best choice of additive depends on which enantiomer is of interest and it is useful to perform a thorough additive screening to find the optimal additive, giving the most advantageous peak shape composition and accordingly the best process performance for a particular separation problem.

Biotechnological Approach to the Synthesis of 9α-Hydroxylated Steroids

Abstract The steroid 9α‐hydroxylase gene has been cloned from Mycobacterium smegmatis into Escherichia coli BL21. Progesterone added to bioreactors was subjected to in vivo transformation into 9α‐hydroxyprogesterone. In 7 days, 43.6 mg 9α‐hydroxyprogesterone was formed from 53.8 mg/L progesterone. The enzyme also has shown evidence of processing 4‐androstene‐3,17‐dione in vivo. An extensive analytical method development, including LLE, HPLC‐DAD, MS, and NMR was performed to verify the product and to enable a quantitative analysis. Protocols for analytical and preparative separation have been developed, using binaphtol as internal standard. Both the growth pattern and the bioconversion rate were unaffected by the presence of binaphtol in the bioreactor. The enzyme was purified by immobilised metal affinity and ion exchange chromatography, resulting in low in vitro activity.