Influence of the particle porosity on chromatographic band profiles

The mass transfer kinetics of butyl benzoate, eluted on a monolithic RPLC column with methanol-water (65:35, v/v) as the mobile phase was investigated, using the perturbation method to acquire isotherm data and the mobile phase velocity dependence of the height equivalent to a theoretical plate of perturbation peaks to acquire kinetics data. The equilibrium isotherm of butyl benzoate is accounted for by the liquid-solid extended multilayer BET isotherm model. The total porosity of the column varies much with the butyl benzoate concentration, influencing strongly the parameters of its mass transfer kinetics and the profiles of the breakthrough curves. Using all these parameters, the general rate model of chromatography predicts band profiles and Van Deemter curves that are in excellent agreement with experimental results provided the influence of concentration on the porosity is properly taken into account. This agreement confirms the validity of the models selected for the isotherm and for the mass transfer kinetics.     

Optimal design of batch and simulated moving bed chromatographic separation processes

Preparative chromatography, especially simulated moving bed (SMB) chromatography, is a key technology for the separation of fine chemicals on a production scale. Most of the design methods for batch and SMB processes proposed in the open literature deal with the optimisation of the operating conditions for a given chromatographic unit only. Therefore, a comparison of the process economy may lead to incorrect results. In this contribution, an effective strategy for the optimal choice of all process parameters (operation and design parameters) is proposed. The main idea of this strategy is to apply a detailed and experimentally validated process model and to reduce the number of influencing parameters by introducing and optimising dimensionless process parameters. It is shown that there is an infinite choice of design and operating parameters to achieve maximum productivity or minimum separation costs and not at the maximum pressure drop only. The detailed design of the chromatographic unit and the selection of the operating conditions can be adjusted by considering the availability of columns and packing materials. As the model system, the separation of a racemic mixture (EMD53986) on Chiralpak AD was investigated. After complete optimisation of a batch and a SMB unit, a real comparison of the process economy can be achieved. Finally, the influences of two different objective functions, productivity and specific separation cost, are analysed.     

New hierarchically porous titania monoliths for chromatographic separation media

Separation media based on hierarchically porous titania (TiO₂) monoliths for high‐performance liquid chromatography (HPLC) have been successfully fabricated by the sol–gel process of titanium alkoxide in a mild condition utilizing a chelating agent and mineral salt. The as‐gelled TiO₂ monoliths were subjected to a simple solvent exchange process from ethanol (EtOH) to H₂O followed by drying and calcination. The resultant monolithic TiO₂ columns consist of anatase crystallites with the typical specific surface area of more than 200 m²/g. The resultant monolithic TiO₂ column calcined at 200 and 400°C exhibited a good separation performance for organophosphates as well as for polar benzene derivatives in the normal‐phase mode.     

Systematic optimisation of high-performance liquid chromatographic separation by varying the temperature, gradient, and stationary phase

Optimisation of the separation of a synthetic drug mixture by HPLC is performed by changing both continuous variables, i.e. mobile phase composition and temperature, and categorical variables, here the stationary phase. The retention of solutes is described on the basis of a general linear model in which the different columns are modelled by indicator variables. From the solute-specific retention models the global separation optimum is evaluated on the basis of multidimensional window diagrams using relative retentions of all peak pairs as the figure-of-merit.     

Experimental investigation of the effect of initial fuel particle shape, size and bed temperature on devolatilization of single wood particle in a hot fluidized bed

Considerable amount of investigation on the subject of devolatilization of wood is found in the open literature. However, a systematic study of the effect of initial particle size and shape, and bed temperature on devolatilization time and char yield of wood in a hot fluidized bed is still missing. This paper attempts to fill this gap through a systematic experimental investigation to determine the devolatilization time and char yield of a typical woody biomass, “Casuarina equisetifolia” particles of different initial sizes and shapes at various fluidized bed temperatures. Experiments are conducted using 10, 15, 20, and 25 mm Casuarina wood particles of three shapes, namely, cube, cylinder, and sphere at bed temperatures of 1023, 1123, and 1223 K.It is found that the initial wood particle size has the strongest influence on devolatilization time followed by the shape of initial wood particle and the bed temperature. Correlation for devolatilization time (τ_d) as a function of initial wood particle size (d_eq), sphericity (?), and bed temperature (T_b), is developed using 573 experimental data points exhibiting a correlation coefficient of 0.96 and predictions falling well within a deviation band of ±20%. The predictions of the present correlation are compared with the predictions of the existing correlations in literature for conditions also out of the present study and the deviation is found to be ±30%.Char yield, defined as the ratio of the residual mass at the end of devolatilization process to the initial mass of the wood particle is found to be in the range of 9-14% for all sizes, shapes, and bed temperatures. Char yield does not depict any definite trend with the variation of initial particle size, shape and bed temperature.     

Characterization of synthetic adsorbents with fine particle sizes for preparative-scale chromatographic separation

Synthetic adsorbents with fine particle sizes (15-30 microm) were manufactured. These adsorbents are made of poly(styrenedivinylbenzene) and polymethacrylate, and have the same chemical structure as analytical- (5-10 microm) or industrial- (200-600 microm) grade synthetic adsorbents. Both of them have very similar porous structure to those of analytical or industrial sizes, so that they can adsorb compounds of various molecular masses. Chromatographic separation characteristics of newly manufactured fine-particle grades of synthetic adsorbents were evaluated and compared to those of analytical or industrial adsorbents. Reasonable dependency of separation performance on particle size of synthetic adsorbents was obtained. Hydraulic properties of fine-grade adsorbents had also been measured in view of column operations. Furthermore, scalability and applicability of these adsorbents for preparative-scale chromatographic separation of bioactive compounds was evaluated. Separation of soybean isoflavones and tea catechin derivatives had revealed that fine-grade synthetic adsorbents could be well applied with scalability under the same elution conditions used for analytical use. Scalability up to a 22400-fold loading amount was achieved from a small column packed with analytical-grade adsorbent used for method development to a scale-up preparative column packed with fine-grade adsorbent used for preparative purification. These results showed the usefulness of the fine-grade synthetic adsorbents for more precise purification of bioactive compounds, including pharmaceuticals and functional food additives with higher recovery.     

An improved extraction chromatographic resin for the separation of uranium from acidic nitrate media

The preparation and characterization of a new extraction chromatographic resin exhibiting extraordinarily strong retention of hexavalent uranyl ion over a wide range of nitric acid concentrations and very high selectivity for U(VI) over Fe(III) and numerous other cations is described. This new material (designated U/TEVA-2) comprises a novel liquid stationary phase consisting of an equimolar mixture of diamyl amylphosphonate (DA[AP]) and Cyanex 923^® (a commercially available trialkyl-phosphine oxide, TRPO) sorbed on silanized silica or Amberchrom CG-71. Cyanex 923 is shown to be preferable to a related TRPO, Cyanex 925^®, due to its lower viscosity and higher selectivity for U(VI) over Fe(III). The retention of uranyl nitrate by the U/TEVA-2 resin, as measured by the k′ values (number of free column values to peak maximum) is >5000 from approximately 0.1 to 8 M HNO₃. The ability of the new resin to strongly and selectively retain U(VI) from such a wide range of acid concentrations, along with its favorable physical properties, make it a good candidate for application in the separation and preconcentration of U(VI) from complex environmental, biological, and nuclear waste samples for subsequent determination.     

Kinetic optimisation of the reversed phase liquid chromatographic separation of rooibos tea (Aspalathus linearis) phenolics on conventional high performance liquid chromatographic instrumentation

Rooibos tea, produced from the endemic South African shrub Aspalathus linearis, has various health-promoting benefits which are attributed to its phenolic composition. Generating reliable, quantitative data on these phenolic constituents is the first step towards documenting the protective effects associated with rooibos tea consumption. Reversed phase liquid chromatographic (RP-LC) methods currently employed in the quantitative analysis of rooibos are, however, hampered by limited resolution and/or excessive analysis times. In order to overcome these limitations, a systematic approach towards optimising the RP-LC separation of the 15 principal rooibos tea phenolics on a 1.8 μm phase using conventional HPLC instrumentation was adopted. Kinetic plots were used to obtain the optimal configuration for the separation of the target analytes within reasonable analysis times. Simultaneous optimisation of temperature and gradient conditions provided complete separation of these rooibos phenolics on a 1.8 μm C18 phase within 37 min. The optimised HPLC–DAD method was validated and successfully applied in the quantitative analysis of aqueous infusions of unfermented and fermented rooibos. Major phenolic constituents of fermented rooibos were found to be a phenylpropanoid phenylpyruvic acid glucoside (PPAG), the dihydrochalcone C-glycoside aspalathin, the flavones isoorientin and orientin, and a flavonol O-diglycoside tentatively identified as quercetin-3-O-robinobioside. Content values for PPAG, ferulic acid and quercetin-3-O-robinobioside in rooibos are reported here for the first time. Mass spectrometric (MS) and tandem MS detection were used to tentatively identify 13 additional phenolic compounds in rooibos infusions, including a new luteolin-6-C-pentoside-8-C-hexoside and a novel C-8-hexosyl derivative of aspalathin reported here for the first time.     

Continuous chromatographic separation process: simulated moving bed allowing simultaneous withdrawal of three fractions

Continuous counter-current chromatographic separation has been carried out in a simulated moving bed system (SMB). We have worked with a SMB pilot plant (8 columns, 4.4 litres of resin each) which allows the continuous withdrawal of two different fractions. A mixture of glucose-fructose has been separated. To calculate the concentration profile within the separator an axial dispersed plug flow model and an equilibrium stage model have been employed; software has been created to simulate the behaviour of the separator. The necessary parameters of the mode: the adsorption equilibrium constant, the height equivalent to a theoretical plate and the bed voidage, have been acquired experimentally from elution chromatography measurements. The results calculated by simulation give a good representation of the experimental concentration profiles; other separations like xylitol-arabitol have been simulated. The influence of some factors like desorbent flow-rate, feed flow-rate and the bed voidage have been studied using the software. Once the system has worked in a two withdrawal way, an extension of the pilot plant has been constructed so as to obtain a third one. The necessary parameters of the three withdrawal model will be studied.     

Development of an high-performance liquid chromatographic simulated moving bed separation from an industrial perspective

A binary test mixture consisting of cyclopentanone and cycloheptanone is used for the performance evaluation of a pilot-scale simulated moving bed unit. The involved adsorption equilibria and the kinetic behavior are discussed in detail. The results of the test runs are evaluated using the recently introduced "triangle theory" which allows to account for the overload conditions prevailing under preparative chromatographic conditions and to select optimal operating conditions. Under optimized conditions the separation of 735 g test mixture/kg stationary phase per day with purities >99.9% for extract and raffinate stream has been achieved.     

Double-emulsion templated macroporous cellulose microspheres as a high-performance chromatographic media for protein separation

Cellulose - Cellulose microspheres are commonly chromatographic media yet seriously limited in biomacromolecules separation and purification due to the slow mass transfer kinetics resulting from...     

Convective interaction media short monolithic columns: enabling chromatographic supports for the separation and purification of large biomolecules

New therapeutics that are being developed rely more and more on large and complex biomacromolecules like proteins, DNA, and viral particles. Manufacturing processes are being redesigned and optimized both upstream and downstream to cope with the ever-increasing demand for the above target molecules. In downstream processing, LC still represents the most powerful technique for achieving high yield and high purities of these molecules. In most cases, however, the separation technology relies on conventional particle-based technology, which has been optimized for the purification of smaller molecules. New technologies are, therefore, needed in order to push the downstream processing ahead and into the direction that will provide robust, productive, and easy to implement methods for the production of novel therapeutics. New technologies include the renaissance of membranes, various improvements of existing technologies, but also the introduction of a novel concept--the continuous bed or monolithic stationary phases. Among different introduced products, Convective Interaction Media short monolithic columns (SMC) that are based on methacrylate monoliths exhibit some interesting features that make them attractive for these tasks. SMC can be initially used for fast method development on the laboratory scale and subsequently efficiently transferred to preparative and even more importantly to industrial scale. A brief historical overview of methacrylate monoliths is presented, followed by a short presentation of theoretical considerations that had led to the development of SMC. The design of these columns, as well as their scale-up to large units, together with the methods for transferring gradient separations from one scale to another are addressed. Noninvasive methods that have been developed for the physical characterization of various batches of SMC, which fulfill the regulatory requirements for cGMP production, are discussed. The applications of SMC for the separation and purification of large biomolecules, which demonstrate the full potential of this novel technology for an efficient downstream processing of biomolecules, are also presented.     

Liquid chromatographic separation of the thyroid hormones

The liquid Chromatographic separation of 3,3',5-triiodothyronine, 3,3',5'-triiodothyronine, and thyroxine with a nonpolar stationary phase was studied as a function of pH, temperature, organic content of the mobile phase, and ionic strength using aqueous phosphate—acetonitrile, aqueous phosphate—methanol, and aqueous phosphate— n-propanol mobile phase systems. It was demonstrated that the quality of the thyroid hormone separations, as determined by normalized peak capacity values, was unchanged with temperature, remained relatively constant with increasing ionic strength, and was affected to the greatest extent by changes in pH and organic modifier content of the mobile phase. Chromatographic behavior of the compounds studied as a function of these variables was found to be consistent with existing Chromatographic theory and/or empirical observations. Recommended conditions are aqueous phosphate—methanol mobile phase, pH 2–5 (aqueous portion), and high temperature (60–70°C).     

Three novel high performance affinity chromatographic media for the separation of antithrombin III from human plasma.

Novel monodisperse, non-porous, cross-linked poly (glycidyl methacrylate) beads (PGMA) were employed as the support for high performance affinity chromatography. Heparin was covalently attached to PGMA beads by three different coupling methods. Heparin-PGMA-I was prepared by directly coupling amino-groups of heparin with PGMA. Heparin-PGMA-II and III were prepared by the coupling of heparin to amino-PGMA, which was obtained by amination of PGMA. Heparin-PGMA-II was prepared by coupling the carboxyl groups of heparin to amino-PGMA by using water-soluble carbodiimide as coupling reagent, and heparin-PGMA-III was prepared by the reductive amination of heparin and amino-PGMA with sodium cyanoborohydride. The heparin contents of heparin-PGMA-I, II and III were 1.6, 10.2 and 1.0 mg/g beads, respectively. Their affinity capacities for antithrombin III were investigated. Their binding activity to antithrombin III was not proportional to the content of heparin immobilized, and heparin-PGMA-I was the most efficient affinity medium for antithrombin III. The resultant affinity media presented minimal non-specific interaction with other proteins and can be used in a wide pH range. All the three heparin-PGMA beads were exploited for the separation of antithrombin III from human plasma. The purity of antithrombin III obtained was higher than 90%, which was confirmed by high performance size exclusion chromatography.