Article

Separation of Nadolol Racemates by High pH Reversed-Phase Preparative Chromatography

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Abstract

The separation of nadolol racemates under high pH reversed-phase preparative chromatography is presented for the first time. Three Waters C18 adsorbents (XBridge, Shield and XSelect) are compared for the separation of nadolol racemates using ethanol:water:diethylamine solvent mixtures. Experimental and simulation results are presented to compare the separation performances at preparative scale using both the fixed-bed and the simulated moving bed operations. The Waters XBridge C18 adsorbent and an ethanol:water:diethylamine solvent mixture are selected as a good option for the separation of nadolol racemates. The validated methodology allows the separation of a multicomponent nadolol feed mixture composed by four stereoisomers into two pure racemates (two pairs of enantiomers). This work introduces the potential of using an initial achiral separation step in the global strategy for the complete multicomponent separation of the four nadolol stereoisomers.

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... Currently, nadolol is widely used to relieve cardiovascular disease (Arafah et al., 2020). It is a highly water-soluble substance that is not entirely metabolized by metabolic enzymes and is mainly excreted in the urine (Misaka et al., 2013). ...
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The separation of nadolol racemates under high pH reversed-phase using both the fixed-bed (FB) and the simulated moving bed (SMB) preparative chromatographic techniques is reported after the previous published work [1] where the Waters XBridge C18 adsorbent and an ethanol:water:diethylamine solvent mixture were validated to allow the separation of the multicomponent feed mixture composed by four nadolol stereoisomers into two pure racemates (two pairs of enantiomers). In this work, the experimental preparative separations using one commercial fixed-bed preparative HPLC Azura system equipped with one sole column of preparative dimensions (30 mm ID x 250 mm L) and one lab-scale SMB apparatus (the FlexSMB-LSRE pilot unit) equipped with six semi-preparative columns (19 mm ID x 100 mm L) are presented. Both systems use the Waters XBridge C18 adsorbent of 10 μm particle diameter. The screening of the mobile phase composition elected the 30:70:0.1 (v/v/v) ethanol:water:diethylamine solvent mixture to perform both FB and SMB preparative operations. A large set of experimental, modelling and simulation results are presented, including pulses, measurement and modelling of the adsorption equilibrium isotherms, and its validation through breakthroughs measurements. The modelling and simulation steps allowed the prediction and the optimization of both the FB and SMB operating conditions. For FB, using a feed concentration of 9 g/L of an equimolar mixture of the two nadolol racemates, both were recovered almost pure (at least 99.9%), with a global system productivity of 3.06 gfeed/(Lbed.hr) and a solvent consumption of 4.21 Lsolvent/gfeed. For SMB, the pilot unit’s pressure drops limits imposed a maximum internal flow-rate of only 5 mL/min and, for a nadolol feed concentration of 2 g/L, both racemates were recovered 100% pure, with a system productivity of 0.13 gfeed/(Lbed.hr) and a solvent consumption of 6.19 Lsolvent/gfeed. Additional simulation results showed that a SMB preparative unit can perform the 9 g/L nadolol racemate separation with a system productivity of 3.61 gfeed/(Lbed.hr) and a solvent consumption of only 1.95 Lsolvent/gfeed using the same average internal flow-rate as in FB operation. Even better SMB productivities can still be obtained using the same feed or solvent flow-rates as in FB operation if the internal SMB flow-rates are allowed and not limited by the system pressure drop. The experimental results presented in this work validate the strategy of separating a four nadolol stereoisomers mixture into two pure nadolol racemates, each one composed by a pair of nadolol enantiomers, using an achiral C18 adsorbent through FB and SMB chromatographic techniques. Each nadolol racemate can later be purified into pure nadolol stereoisomers using standard binary chiral FB and SMB chromatography. In this way, this works introduces a real and experimental solution for the complete multicomponent preparative separation of the four nadolol stereoisomers.
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The separation of high-purity compounds from traditional Tibetan medicines plays an important role in investigating their bioactivity. Nevertheless, it is often quite difficult to isolate compounds with high purity because of the complexity of traditional Tibetan medicines. In this work, an offline two-dimensional reversed-phase preparative method was successfully developed for the separation of high-purity compounds from Oxytropis falcata. Based on the analysis results, an ODS C18 prep column was used for first-dimensional preparation, and 14.8 g of the crude sample was separated into five fractions with a recovery of 74.6%. Then, an XAqua C18 prep column was used to isolate high-purity compounds in the second-dimensional preparation because its separation selectivity is different with the ODS C18 stationary phase. As a result, eight compounds in the crude sample were isolated in more than 98% purity. This is the first report of trans-cinnamic acid (1) and trifolirhizin (2) from Oxytropis falcata. This method has the potential to be an efficient separation method of high-purity compounds from Oxytropis falcata and it shows great promise for the separation of high-purity compounds from complex samples. This article is protected by copyright. All rights reserved.
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An analytical reversed-phase high-performance liquid chromatography (HPLC) method for the detection and quantitative determination of two genotoxic impurities at ppm level present in the vortioxetine manufacturing process is described. Applying the concept of threshold of toxicological concern, a limit of 75 ppm each for both genotoxic impurities was calculated based on the maximum daily dose of active pharmaceutical ingredients. The novel reversed-phase HPLC method with photochemically induced fluorescence detection was developed on XSELECT Charged Surface Hybrid Phenyl-Hexyl column using the mobile phase consisted a mixture of 10 mM ammonium formate pH 3.0 and acetonitrile. The elution was performed using an isocratic composition of 48:52 (v/v) at a flow rate of 1.0 mL/min. The photochemically induced fluorescence detection is based on the use of UV irradiation at 254 nm through measuring the fluorescence intensity at 300 nm and an excitation wavelength of 272 nm to produce fluorescent derivatives of both genotoxic impurities. The online photochemical conversion and detection is easily accomplished for two expected genotoxic impurities and provides a sufficiently low limit detection and quantification for the target analysis.
Article
Chiralpak IA adsorbent is used for both analytical and preparative chromatographic separation of nadolol stereoisomers. The results include a complete screening of the mobile phase composition for both the baseline resolution of all four nadolol stereoisomers (analytical separation) and the simulated moving bed (SMB) pseudo-binary separation of the most retained stereoisomer. The experimental results show that analytical baseline resolution of nadolol stereoisomers can be achieved using alcohol/hydrocarbon and alcohol/acetonitrile solvent mixtures. The 10%ethanol/90%acetonitrile mixture is presented as the one that presents baseline resolution with lower retention. For the preparative pseudo-binary separation, pure ethanol, pure methanol, alcohol/acetonitrile, and alcohol/tetrahydrofuran mixtures proved to allow good separation results. The 100%methanol/0.1%diethylamine solvent composition was selected to perform the experimental SMB separation. Using a 10 g/L total feed concentration, the more retained stereoisomer was recovered at the extract outlet stream with 99.5% purity, obtaining a system productivity of 1.98 gL−1 h−1 and requiring a solvent consumption of 3.13 L/g of product. Comparing these results with the ones recently presented by Ribeiro et al. (2013), this work shows that the Chiralpak IA chiral adsorbent is an interesting alternative to Chiralpak AD for the separation of nadolol stereoisomers at both analytical and preparative scales. Chirality 00:000–000, 2016.
Article
This month's "Column Watch" examines the topic of phase collapse in reversed-phase liquid chromatography. Phase collapse is best described as a dewetting phenomenon. It can cause a total loss of retention and chromatographic problems such as peak tailing, non-reproducible retention times and gradient regeneration delays. The authors offer suggestions for avoiding phase collapses and regenerating collapsed phases.
Article
Last month's "Column Watch" discussed the phenomenon of phase collapse and ways to solve the problem and successfully separate polar analytes using mobile-phase systems with a very high percentage of water. In this month's column, Majors and Przybyciel describe columns that can deal with this difficult situation and suggest some commercial columns for separating polar compounds in highly aqueous environments.
Article
Triapine is an inhibitor of ribonucleotide reductase (RNR). Studies have shown that triapine significantly decreases the activity of RNR and enhanced the radiation-mediated cytotoxicity in cervical and colon cancer. In this work, we have developed and validated a selective and sensitive LC-MS/MS method for the determination of triapine in human plasma. In this method, 2-[(3-fluoro-2-pyridinyl)methylene] hydrazinecarbothioamide (NSC 266749) was used as the internal standard (IS); plasma samples were prepared by deproteinization with acetonitrile; tripaine and the IS were separated on a Waters Xbridge Shield RP18 column (3.5 µm; 2.1 × 50 mm) using a mobile phase containing 25.0% methanol and 75.0% ammonium bicarbonate buffer (10.0 mm, pH 8.50; v/v); column eluate was monitored by positive turbo-ionspray tandem mass spectrometry; and quantitation of triapine was carried out in multiple-reaction-monitoring mode. The method developed had a linear calibration range of 0.250-50.0 ng/mL with correlation coefficient of 0.999 for triapine in human plasma. The IS-normalized recovery and the IS-normalized matrix factor of triapine were 101-104% and 0.89-1.05, respectively. The accuracy expressed as percentage error and precision expressed as coefficient of variation were ≤±6 and ≤8%, respectively. The validated LC-MS/MS method was applied to the measurement of triapine in patient samples from a phase I clinical trial. Copyright © 2015 John Wiley & Sons, Ltd. Copyright © 2015 John Wiley & Sons, Ltd.
Article
A general design methodology for chromatographic three fraction separation by application of the three column intermittent simulated moving bed (3C-ISMB) cascade is proposed and experimentally validated by studying the purification of an intermediately retained stereoisomer of nadolol, from an equimolar mixture of its four stereoisomers. The theoretical part shows that the 3C-ISMB cascade can be easily designed by applying Triangle Theory. Moreover, a re-scaling approach for the second stage is proposed so as to account for the fact that the feed flow rates to stage 2 are generally higher as compared to stage 1 due to dilution in the latter. Scaling the columns of the second stage accordingly enables to run both stages under optimal conditions with respect to switching time and step ratio, which is an important advantage as compared to integrated ternary processes. The experimental part starts with studying the linear adsorption behavior of nadolol in heptane/ethanol/DEA on Chiralpak AD for varying ratios of heptane and ethanol. Based on that, a solvent composition of Hept/EtOH/DEA 30/70/0.3 (v/v/v) is selected and the competitive multi-component Langmuir isotherm of the quaternary mixture is determined by frontal analysis. The resulting isotherm parameters are used to design several first stage experiments aiming at removal of the most retained component. The resulting ternary intermediate product is reprocessed in several second stage experiments studying various configurations. Finally, the dilution of the intermediate product with Hept/DEA yielding a solvent composition of Hept/EtOH/DEA 60/40/0.3 (v/v/v) is examined showing that the resulting increase in retention is beneficial for final product purities. Moreover, the reduction in viscosity compensates for the dilution as it enables higher flow rates. Dilution of the intermediate product is hence the best option, yielding highest overall cascade productivity (2.10gl(-1)h(-1)) and highest product purity (97.8%) requiring a specific solvent consumption of 12l/g of product. Copyright © 2014 Elsevier B.V. All rights reserved.
Article
Differences in the system constants of the solvation parameter model, discontinuities in retention factor plots (log k against volume fraction of organic solvent) and retention factor correlation plots are used to study the retention mechanism on XTerra MS C18, XBridge C18 and XBridge Shield RP18 stationary phases with acetonitrile–water and methanol–water mobile phases containing from 10 to 70% (v/v) organic solvent. Wetting of XBridge C18 at 10 and 20% (v/v) acetonitrile is incomplete and is responsible for small changes in the retention mechanism. The intermolecular interactions responsible for retention on XTerra MS C18 and XBridge C18 are similar with minor differences in cavity formation and hydrogen-bonding interactions responsible for small selectivity differences. On the other hand, for bulky solutes there are large changes in retention at low volume fractions of organic solvent (<40% v/v) associated with steric repulsion on the XTerra MS C18 stationary phases that is absent for XBridge C18. Selectivity differences are more apparent for XBridge C18 and XBridge Shield RP18. For acetonitrile-water mobile phases cavity formation in the solvated XBridge Shield RP18 is slightly more difficult and hydrogen-bond acid and base interactions are more important than for XBridge C18. With methanol–water mobile phases it becomes slightly easier to form a cavity in the solvated XBridge RP18 compared with XBridge C18. In addition, the methanol-water solvated XBridge RP18 is a stronger hydrogen-bond base and more dipolar/polarizable than XBridge C18. These variations in selectivity justify the use of XBridge C18 and XBridge Shield RP18 as complementary stationary phases for method development.
Article
The pharmaceutical industry is now directed to the market of more safety and efficient drugs, based on single enantiomers. Ketoprofen, still used as a racemic pharmaceutical drug, belongs to the profens class, one of the most representatives of the non-steroidal anti-inflammatory drugs. This work presents the chiral separation of ketoprofen enantiomers by simulated moving bed technology, using a laboratory scale unit (the FlexSMB-LSRE®) with six columns, packed with the Chiralpak AD® stationary phase (20 µm). A comparative study between a mobile phase composed by a traditional high hydrocarbon content (10%ethanol/90%n-hexane/0.01%TFA) and a strong polar organic composition (100%ethanol/0.01%TFA) is presented. The study includes the measurement of the adsorption isotherms, elution and frontal chromatography experiments, carried out on a SMB column for both compositions. The results obtained allowed the prediction and optimization of the SMB operation. Using pure ethanol as solvent and a racemic feed concentration of 40 g/L, purities above 98.6% on both outlet streams were obtained, with a productivity of 3.84 gfeed/(Lbed.hr) and a solvent consumption of 0.78 Lsolvent/gfeed. The results obtained in the experimental separation of ketoprofen enantiomers by SMB chromatography indicates that pure ethanol presents better performances than the classic high hydrocarbon content composition.
Article
Aqueous mobile phases above pH 8 often cause premature column failure, limiting the utility of silica-based columns for applications requiring high pH. Previous studies suggest that covalently bound silane ligands are hydrolyzed and removed by high-pH mobile phases. However, we found that the siloxane bonds for certain monomeric silanes are hydrolyzed very slowly from silica supports at pH 9–10. Therefore, bonded-phase packing degradation at high pH is a result mainly of silica support dissolution. The rate of column degradation for C18 columns is influenced not only by the type and purity of silica support, but also by the nature of the silane stationary phase. We found different rates of degradation for several commercial C18 columns. The relative rates of silica dissolution for these packings were determined by chemically measuring the silicate formed during column purging at high pH. The type and concentration of mobile phase organic modifier also significantly influences column degradation at high pH. Certain silica-based C18 packings can be used for long periods at pH 9 without significant changes in chromatographic properties. Results of this study better define the practical utility and limitations of silica-based columns in high pH environments.
Article
The separation of the four nadolol stereoisomers on Chiralpak® AD by chiral liquid chromatography was carried out at both analytical and preparative scales. A screening of possible mobile-phase compositions was performed using different alcohol-hydrocarbon mixtures. The results obtained confirm the use of 20:80:0.3 ethanol-hexane-diethylamine reported by McCarthy (1994) but introduce other possibilities for the complete resolution of the four nadolol stereoisomers at analytical scale, namely, the mixtures 30-40:70-60:0.3 ethanol-heptane-diethylamine. Additionally, this work describes how retention and resolution depend on the ethanol content in hexane and heptane mixtures. The separation of nadolol stereoisomers is also carried out at preparative scale and different alcohol-hydrocarbon compositions are proposed, depending on the target component to be obtained. Particularly, this work presents the experimental separation of the more retained nadolol stereoisomer (RSR-nadolol) by simulated moving bed (SMB) chromatography using an 80:20:0.3 ethanol-heptane-diethylamine mobile phase. For a 2 g/l feed concentration, RSR-nadolol is 100% recovered at the extract outlet stream, 100% pure, and with a system productivity of 0.65 g(RSR-nadolol) /(l(bed) (.) h) and a solvent consumption of 9.6 l(solvent) /g(RSR-nadolol) . Chirality, 2013. © 2013 Wiley Periodicals, Inc.
Article
(-)-Epigallocatechin gallate (EGCG) was purified from tea polyphenol by a two-step simulated moving bed (SMB) chromatographic process with C18-bonded silica gel as stationary phase and a mixture of methanol and water as mobile phase, removing the more retained components firstly and the less retained components secondly. An open-loop 3-zone SMB approach was used, a gradient of solvent strength in zones I and II was formed by different methanol dosage in purge and desorbent solutions and pre-equilibration was reached by the elution at the end of zone III. Under the approximation including linear adsorption and ideal chromatographic model, the operating parameters of the simulated moving bed were selected according to the "triangle theory". In the first SMB separation, a solution of tea polyphenol with 55.5% purity of EGCG was used for feeding and a raffinate solution with 92.2% purity and 99.7% recovery of EGCG was obtained. The raffinate solution was concentrated for feeding in the second SMB separation and an extract solution with 97.8% purity and 99.8% recovery of EGCG was obtained.
Article
The influence of dispersive effects (axial mixing and mass-transport resistance) on the performance of simulated moving bed (SMB) units is studied through detailed modeling. The results are represented in the space of the dimensionless operating parameters defined as the ratios between the fluid and the simulated stationary phase flow rate in the different sections of the SMB unit. The complete separation region calculated by numerical simulations is compared to the one obtained through equilibrium theory (i.e., by assuming ideal behavior and neglecting dispersive effects). The differences are shown to be relatively small when realistic values of the process parameters are used; however, they must be taken into account for fine-tuned process optimization. This proves the usefulness of the equilibrium theory approach to provide a first solution that can then be refined through detailed model simulations. A thorough analysis of the optimal design of the operating conditions when only one compound is required pure is carried out and a rather interesting asymmetric behavior is evidenced and explained. Finally, the usefulness of the theoretical findings is assessed by using them to discuss and explain a set of experimental data reported in the literature.
Article
Nadolol, a β-blocker used in the management of hypertension and angina pectoris, has three chiral centers and is currently marketed as an equal mixture of its four stereoisomers. Resolution of three of the four stereoisomers of nadolol was obtained by HPLC, with a complete separation of the most active enantiomer, (RSR)-nadolol, on a column packed with perphenyl carbamoylated β-cyclodextrin (β-CD) immobilized onto silica gel. The h-root method without the introduction of dummy species was presented and applied to determine the nonlinear competitive Langmuir isotherms of the three components of nadolol. In this method, h-root transformation was applied directly to the n-component nonstoichiometric system, without introduction of dummy species. The experiments consist of linear elution and nonlinear frontal chromatography. The solid film linear driving force model was used to simulate the response of a column to a pulse injection in the nonlinear region. The experimental and simulated profiles matched well, which confirmed the validity of the obtained Langmuir isotherm coefficients. On the basis of the isotherm obtained, complete (1, 2) and (2, 3) simulated moving bed (SMB) separation regions were determined to separate the ternary mixture of nadolol into different fractions.
Article
This article describes a systematic miniplant-based approach to rapid development of simulated moving bed (SMB) chromatography applications. The methodology involves analysis of single-column pulse tests to screen adsorbents and operating conditions and to determine initial values of profile advancement factors used to specify flow rates for an initial SMB miniplant experiment. A lumped-parameter linear driving force rate-based model is developed by fitting process data from a single miniplant run. The data are fit in a two-step procedure involving initial determination of effective adsorption isotherm constants as best-fit parameters with subsequent adjustment of calculated mass transfer coefficients to refine the data fit. The resulting simulation is used to guide further miniplant work and minimize experimental effort. The methodology is illustrated with miniplant data for a binary protein separation showing excellent agreement between model results and process data generated over a wide range of operating conditions. © 2009 American Institute of Chemical Engineers AIChE J, 2009
Article
Modeling strategies for simulated moving bed adsorbers were studied to compare the simulated moving bed model (SMB) that considers the real shift of the injection and collection points to the true moving bed approach (TMB) that considers liquid and solid flow in opposite directions. The prediction of these two models is compared in terms of steady-state performance, steady-state internal concentration profiles, and transient behavior of the extract and raffinate purities. The influence of the degree of subdivision of the bed in the SMB model predictions is also analyzed and compared with the TMB performance. Model results are compared with experimental results obtained for the chromatographic separation of binaphthol enantiomers.
Article
The Simulated Moving Bed (SMB) concept has been applied to the separation of different mixtures as a continuous counter current separation process, avoiding several problems related with solid motion. The aim of this work is to present some relevant examples of SMB separations corresponding to the two major ages in the use of the SMB concept, here named “old” and “new” applications. The “old” applications of SMB technology in the petrochemical industry are still important, with large and highly productive units; and the “new” applications of the second “age” of SMB concept are from the fine chemical, pharmaceutical and biochemistry areas, associated with the demand of high purity products during the last 10 years. Different examples are presented for different ages: a UOP Parex ® process for the “old”, modelled with the equivalent True Moving Bed (TMB) approach; and a chiral resolution for the “new”, modelled by the real SMB model. Some of the latest developments are also mentioned: the non conventional techniques as the Varicol ® process, PowerFeed, Modicon, M3C or Enriched Extract-SMB (EE-SMB), MultiFeed (MF), Outlet Streams Swing (OSS) or Pseudo-SMB, involving considerable changes in the SMB concept itself. The use of the last optimization/modelling packages for the development of design techniques, either at the conception stage as well as for performance improvements of existing units is emphasized.
Article
Nadolol, a β-blocker drug used in the management of hypertension and angina pectoris, has three chiral centers and is currently marketed as an equal mixture of four stereoisomers. Resolution of three of the four stereoisomers of nadolol was obtained previously by HPLC, with a complete separation of the most active enantiomer (RSR)-nadolol, on a column packed with perphenyl carbamoylated β-cyclodextrin (β-CD) immobilized onto silica gel.Continuous separation of enantiomer (RSR)-nadolol from its racemate (which is a ternary mixture in the chromatographic system of this study) in both 2-raffinate and 2-extract configuration of five-zone SMB was studied. Same experimental setup was applied to both configurations by modifying SMB controlling program accordingly. Separation performances of the five-zone SMB were investigated for both 2-raffinate and 2-extract configurations and same safety factors were applied to investigate the effect of m3–m2 (or m4–m3) on the separation performance systematically. The desired enantiomer of nadolol can be produced with a high purity and yield in 2-raffinate configuration compared with that in 2-extract configuration.
Article
The present study investigated a robust method for the preparation of four flavone C-glycosides, i.e. orientin, homoorientin, vitexin and isovitexin, which were prepared from an ethanol aqueous extract, i.e. antioxidant of bamboo leaves (AOB), by AB-8 resin-based column chromatography and preparative high-performance liquid chromatography (HPLC) using a mobile phase consisting of 10% and 15% (v/v) of acetonitrile and 1% acetic acid. These flavone C-glycosides were further purified by the drowning-out crystallization method using methanol and water as drowning-out anti-solvents and salting-out agents, respectively. The purity was assessed by analytical HPLC and the confirmation of chemical structures was performed by IR, MS, NMR and UV spectroscopy. Orientin (49 mg), homoorientin (142 mg), vitexin (15 mg) and isovitexin (62 mg) were prepared from 6.5 g of crude column chromatography fraction obtained from 5 L of AOB concentrated solution. The present method is robust and suitable for preparing available quantities of pure flavone C-glycosides and the quantification of orientin, homoorientin, vitexin and isovitexin in bamboo leaves.
Article
This work presents the experimental and simulation results obtained for the optimization of the mobile phase composition for the preparative separation of flurbiprofen enantiomers by liquid chromatography using an amylose-based chiral stationary phase (Chiralpak AD). The experimental work carried out includes solubility and adsorption isotherm measurements and pulse and breakthrough experiments under preparative conditions. The simulation work predicts the operation of a simulated moving bed (SMB) system for the separation of flurbiprofen enantiomers to compare the productivity and solvent consumption performances, for the different mobile phase compositions and using the experimental data obtained. This paper presents a new and different case study (flurbiprofen) of the one recently reported by the authors (ketoprofen enantiomers [A. Ribeiro, N. Graça, L. Pais, A. Rodrigues, Preparative separation of ketoprofen enantiomers: choice of mobile phase composition and measurement of competitive adsorption isotherms, Sep. Purif. Technol. 61 (2008) 375–383]), to clearly show that the optimization of the mobile phase composition for preparative chiral separation requires an individualized study, since different results are obtained even for enantiomers systems of the same family.
Article
The objective of this paper is to study the separation of enantiomers of 1,1′-bi-2-naphthol in 3,5-dinitrobenzoyl phenylglycine bonded to silica gel, using heptane–isopropanol (72:28) as eluent by simulated moving bed chromatography (SMB). A model for the prediction of the cyclic steady state performance of the SMB, based on the analogy with the true moving bed, is developed assuming axial dispersion flow, linear driving force approximation for intraparticle mass transfer and multicomponent adsorption equilibria. The SMB package allows the simulation of the pilot unit. The effect of several operating parameters on the SMB performance is analyzed. The performance is characterized by purity, recovery, solvent consumption and adsorbent productivity. The package is an important tool for learning and training operators, allowing the choice of best operating conditions. The operation of the SMB pilot unit was carried out for the separation of racemic mixtures using a 8-column configuration. Purities and recoveries higher than 95% in the extract and raffinate were obtained. Model and experimental results are compared and the package is also used to predict the steady state internal profiles for the SMB operation in good agreement with experimental results.
Article
The simulated moving bed (SMB) technology is receiving more and more attention as a convenient technique for the production scale continuous chromatographic separation of fine chemicals. Characteristic features of SMBs are improved performances with respect to preparative chromatography and nonlinear competitive adsorption behavior. The selection of the operating conditions to achieve high separation performances under nonlinear conditions is acknowledged to be the major problem in running a SMB unit for a new application. This problem is solved in this paper, where a general theory is developed which provides explicit criteria for the choice of the operating conditions of SMB units to achieve the prescribed separation of a mixture characterized by both constant selectivity Langmuir isotherms and variable selectivity modified Langmuir isotherms. The space of the operating parameters, i.e. the fluid to solid flow-rate ratios, is divided in regions with different separation regimes. The effect of increasing nonlinearity of the system on the operating conditions and the separation performances, namely desorbent requirement, enrichment, productivity and robustness of the separation, is thoroughly analyzed. The obtained results are shown to provide a very convenient tool to find both optimal and robust operating conditions of SMB units. Finally, a comparison between model predictions and experimental data dealing with the resolution of different racemic mixtures assesses the reliability and accuracy of the obtained theoretical findings.
Article
The present work intends to investigate how mobile phase composition influences the adsorption behavior of ketoprofen enantiomers (a nonsteroidal anti-inflammatory drug) on an amylose-based chiral stationary phase (Chiralpak AD). Three mobile phase compositions were studied: the usual 20% ethanol/80% n-hexane mixture and two pure mobile phases; methanol and ethanol. Pulse and breakthrough experiments under preparative conditions were carried out in order to measure and test adsorption isotherms. The results obtained show that, for preparative separations, pure ethanol is a better mobile phase than the usual 20% ethanol/80% n-hexane mixture: it allows higher solubility of the racemate, lower retention times, and also a higher selectivity at high enantiomer concentrations. These are aspects of crucial importance when the final goal is to achieve high productivity preparative separations, as it is shown for the simulated moving bed (SMB) operation.
Article
The application of counter-current and simulated counter-current process schemes to adsorption separations is reviewed with emphasis on industrial-scale operations and mathematical modelling.
Article
This work revisits the exceptionally rapid evolution of the technology of chromatographic columns and the important progress in speed of analysis and resolution power that was achieved over the last ten years. Whereas columns packed with 10 and 5 μm fully porous particles dominated the field for nearly thirty years (1975-2000), it took barely six years to see the commercialization of monolithic silica rods (2000), their raise to fame and decay to oblivion, the development of finer fully porous particles with size down to 1.7 μm (2006), and of sub-3 μm superficially porous particles (2006). Analysis times and plate heights delivered by columns packed with these recent packing materials have then been improved by more than one order of magnitude in this short period of time. This progress has rendered practically obsolete the age-old design of LC instruments. For low molecular weight compounds, analysts can now achieve peak capacities of 40 peaks in about 15s with a hold-up time of the order of 1.5s , in gradient elution, by operating columns packed with sub-3 μm shell particles at elevated temperatures, provided that they use optimized high pressure liquid chromatographs. This is the ultimate limit allowed by modern instruments, which have an extra-column band broadening contribution of 7 μL² at 4.0 mL/min and data acquisition rate of 160 Hz. The best 2.1 mm × 50 mm narrow-bore columns packed with 1.7 μm silica core-shell particles provide peaks that have a variance of 2.1 μL² for k=1. Finally, this work discusses possible ways to accelerate separations and, in the same time perform these separations at the same level of efficiency as they have today. It seems possible to pack columns with smaller particles, probably down to 1 μm and operate them with current vHPLC equipments for separations of biochemicals. Analyses of low molecular weight compounds will require new micro-HPLC systems able to operate 1mm I.D. columns at pressures up to 5 kbar, which would eliminate the heat friction problems, and providing extra-column band broadening contributions smaller than 0.1 μL². Alternatively, a new generation of vHPLC systems with minimal extra-column contributions of less than 0.5 μL² could run 2.1mm I.D. columns if these latter were to be packed with high heat conductivity materials such as core-shell particles made with an alumina or gold core.
Article
The aim of this study was to determine the influence of nonionic surfactants on drug permeability using the phospholipid vesicle-based permeation assay (PVPA), which excludes other than trans-membrane diffusion pathways. Barrier integrity was monitored both by electrical resistance and permeability measurement of the hydrophilic marker calcein. Permeability of the model drugs ketoprofen and nadolol across the PVPA-barrier was measured by HPLC-UV. Micelle association of the model drugs was determined using ultrafiltration, whereby micelle-bound drug and molecular drug were separated. The nonionic surfactant poloxamer 188 was demonstrated not to affect barrier integrity. Drug permeability was found depressed in the presence of poloxamer 188 in a concentration-dependent manner. Both drugs were found to associate with poloxamer 188 micelles. The extent of the decrease in permeability correlated mostly, but not in all cases, with the fraction of micelle-bound drug. Micelle association was one important but not the only factor affecting drug permeability across the PVPA-barrier.
Article
A stepwise method development strategy has been employed to develop a robust HPLC method to resolve several closely eluting structurally related impurities in an active pharmaceutical ingredient (API). This strategy consisted of automated column screening, optimization of the most critical chromatographic parameters, DryLab(®) modeling, and experimental verification of optimized separation conditions. DryLab(®) was used to predict an optimized gradient profile and separation temperature and these predictions were verified experimentally. A discussion of the accuracy of these predictions is presented. The robustness of the method was verified and the ability of DryLab(®) to predict, with reasonable accuracy, the outcome of such robustness studies was also examined. Once the robustness was established by the DryLab(®) predictions the remainder of the subsequent verification by experiment becomes a simple reiterative exercise. This study also demonstrates that factors such as column chemistry and critical chromatographic parameters can have a profound and oftentimes interrelated effect on the chromatographic separation of isomers, bromo analogs and other structurally very similar impurities. Therefore, it is critical to adopt a rational strategy, as demonstrated here, to evaluate the interplay of these factors, thereby greatly enhancing method development efficiency.
Article
This paper reports unusual on-column degradations of aniline compounds on Waters XBridge Shield RP18 column when ammonium hydroxide in water and acetonitrile were used as mobile phases in liquid chromatography. The change of the level of on-column degradation of a model compound (Compound 1) with time was observed in the first fifteen injections when started at 60 °C. During a subsequent cooling program from 60 °C to 10 °C with a 10 °C interval, the levels of the degradation products of Compound 1 changed with the change of temperature and reached a maximum at 40 °C. The on-column degradation of Compound 1 was observed when started at 10 °C in the first injection, however, the magnitude of the change of the level of on-column degradation of Compound 1 with time in the first fifteen injections was much smaller than that at 60 °C. During a subsequent heating program from 10 to 60 °C with a 10 °C interval, the levels of the degradation products of Compound 1 increased with the increase in temperature but without a maximum. The change of the degradation product levels of this model compound in the heating process is not super-imposable with that in the cooling process, which demonstrates the degree of the degradation also depends on the heating or cooling process. Column history studies demonstrated that the on-column degradation of Compound 1 changed dramatically on the used columns at both starting temperatures while the dependency of heating and cooling processes on on-column degradation still existed. The unusual on-column degradation of Compound 1 on the used columns can be regenerated in a very similar fashion with an acetic acid column-wash procedure, but is not identical to that on the new column. Similar degradations of other commercially available aniline compounds were also observed with this high pH aqueous mobile phase system.
Article
It has recently been demonstrated, using mathematical models, how peculiar overloaded band profiles of basic compounds are due to the local pH in the column when using low capacity buffers. In this study, overloaded peak shapes resulting after injection of carefully pH matched samples close to the pK(a) of the chosen solute are investigated primarily on two columns; one hybrid silica C18 column (Kromasil Eternity) and one purely polymeric column (PLRP-S), the latter lacking C18 ligands. It was found that distorted peaks of the basic test compound appear even though there is no difference in pH between the injected sample solution and the eluent; the previous explanation to why these effects occur is based on a pH mismatch. Thus, the unusual band shapes are not due to an initial pH difference. Furthermore, it was observed that the effect does not appear on polymeric columns without C18 ligands, but only on columns with C18 ligands, independently of the base matrix (silica, hybrid silica, polymeric).
Article
This review considers some of the difficulties encountered with the analysis of ionised bases using reversed-phase chromatography, such as detrimental interaction with column silanol groups, and overloading which both lead to poor peak shapes. Methods of overcoming these problems in reversed-phase (RP) separations, by judicious selection of the column and mobile phase conditions, are discussed. Hydrophilic interaction chromatography is considered as an alternative method for the separation of some basic compounds.
Article
A method for the quantitative determination of seven fluoroquinolone antibacterial agents (FQs) used in beekeeping, viz. ciprofloxacin, norfloxacin, ofloxacin, pefloxacin, danofloxacin, enrofloxacin, and difloxacin, in royal jelly samples was developed on the basis of high performance liquid chromatography with fluorescence detection. Sample preparation included deproteination, ultrasonic-assisted extraction with a mixed inorganic solution of monopotassium phosphate (KH(2)PO(4)) and ethylenediaminetetraacetic acid disodium salt (Na(2)EDTA), and clean-up on a solid-phase extraction cartridge. The extraction procedure was optimized with regard to the amount of inorganic solvent and the duration of sonication for royal jelly as a complicated matrix. Overall recoveries for FQs ranged from 85.9 to 99.1% for royal jelly with standard deviations between 2.79 and 6.27%. Limits of quantification were 2-40 ng/g for seven FQs in royal jelly. A total of 57 real royal jelly samples collected from beekeepers and supermarkets were analyzed. The three most abundant honeybee-use FQs, i. e. ofloxacin, ciprofloxacin, and norfloxacin, were determined in some royal jelly samples in concentrations ranging from 11.9 to 55.6 ng/g. Unexpectedly, however, difloxacin was found at concentrations of about 46.8 ng/g in one sample although it is rarely used in beekeeping. The presented method was successfully applied to quantify FQs in real royal jelly samples.
Article
The design of a simulated moving bed involves thermodynamic, kinetic and hydrodynamic aspects and requires the optimisation of several variables: plant design variables, such as the column length and diameter, and operating variables, among them four independent flow-rates, the feed concentration and the switch time. In this work we develop an algorithm to design both the unit and its operating conditions, with an overall view on equilibrium properties, efficiency and hydrodynamics, using a simple equilibrium stage model. In this way we determine the parameters leading to the highest possible productivity for a given separation, only requiring the knowledge of the equilibrium isotherms, the Van Deemter equation and a correlation for pressure drop. The algorithm has been used to investigate the effect on the separation performance of some parameters, such as particle size and required product purity, which are not considered by equilibrium theory. The results have been compared with the predictions of equilibrium theory and the observed deviations have been put in evidence and discussed.
Article
Nadolol, a beta-blocker used in the management of hypertension and angina pectoris, has three chiral centers and is currently marketed as an equal mixture of its four stereoisomers. Enantiomeric separation of nadolol by high-performance liquid chromatography was studied on a column packed with novel heptakis (6-azido-6-deoxy-2, 3-di-O-phenylcarbamolyted) beta-cyclodextrin bonded chiral stationary phase. The retention behavior and resolution of nadolol enantiomers were investigated and discussed with respect to the mobile phase composition and flow rate, pH, ionic strength, and temperature. The optimal separation condition was found; the mobile phase contained 80% buffer solution (1% triethylamine acetate, pH 5.5) and 20% methanol with 0.3 ml/min mobile phase flow rate at a temperature of 20 degrees C. At the optimal conditions, resolution of three stereoisomers of nadolol was obtained with a complete separation of the most active enantiomer, (RSR)-nadolol. Thermodynamic properties including enthalpy and entropy change of binding to the CSP for the enantiomeric separation were also determined.