Preparative Chromatographic Separation Research Articles

Influences of the constraints of a separation task on the optimal selection of a cation exchanger resin

One important step in designing preparative chromatographic separation units is the selection of a well-performing adsorbent. Such selection is often based on selectivity factors which are calculated based on experimentally determined adsorption isotherms. Influences of constraints of the separation tasks, like purity demand, on the optimal selection are usually not considered. In this contribution, influences of the constraints of a separation task on the optimal adsorbent were investigated. First, a previously published model-based optimisation of the adsorbent selection using correlations between structural adsorbent properties with model parameters were extended by influences of particle size and ionic form of resins (Leipnitz et al., 1610). Model parameters of glucose and xylose for cation exchanger resins in Na+- and Ca2+-form with varying degrees of cross-linking and particle sizes were experimentally determined in a sequential parameter determination approach (Altenhoner et al., 1997). Correlations between the structural properties of resins and the model parameters were derived and applied in optimisation case studies. Following this approach, optimal structural properties were identified, increasing the volume-specific productivity of a separation of glucose and xylose, significantly. Further, the purity demand and feed composition were varied in different case studies, revealing the influences of constraints of a separation task on structural properties of optimal resins.

Preparative chromatographic separation of neighboring lanthanides using amines as a pH adjusting additive for producing carrier-free 177Lu

Preparative chromatographic separation of neighboring lanthanides using amines as a pH adjusting additive for producing carrier-free 177Lu

Metal-Organic Frameworks for Practical Separation of Cyclic and Linear Polymers.

The purification step in the manufacturing of cyclic polymers is difficult as complete fractionation to eliminate linear impurities requires considerable effort. Here, we report a new polymer separation methodology that uses metal-organic frameworks (MOFs) to discriminate between linear and cyclic polyethylene glycols (PEGs) via selective polymer insertion into the MOF nanopores. Preparation of a MOF-packed column allowed analytical and preparative chromatographic separation of these topologically distinct pairs. In addition, gram-scale PEGs with only cyclic structures were successfully obtained from a crude reaction mixture by using MOF as an adsorbent.

Metal‐Organic Frameworks for Practical Separation of Cyclic and Linear Polymers

AbstractThe purification step in the manufacturing of cyclic polymers is difficult as complete fractionation to eliminate linear impurities requires considerable effort. Here, we report a new polymer separation methodology that uses metal‐organic frameworks (MOFs) to discriminate between linear and cyclic polyethylene glycols (PEGs) via selective polymer insertion into the MOF nanopores. Preparation of a MOF‐packed column allowed analytical and preparative chromatographic separation of these topologically distinct pairs. In addition, gram‐scale PEGs with only cyclic structures were successfully obtained from a crude reaction mixture by using MOF as an adsorbent.

Predicting intraparticle diffusivity as function of stationary phase characteristics in preparative chromatography

Diffusion inside pores is the rate limiting step in many preparative chromatographic separations and a key parameter for process design in weak interaction aqueous chromatographic separations employed in food and bio processing. This work aims at relating diffusion inside porous networks to properties of stationary phase and of diffusing molecules. Intraparticle diffusivities were determined for eight small molecules in nine different stationary phases made from three different backbone materials. Measured intraparticle diffusivities were compared to the predictive capability of the correlation by Mackie and Meares and the parallel pore model. All stationary phases were analyzed for their porosity, apparent pore size distribution and tortuosity, which are input parameters for the models. The parallel pore model provides understanding of the occurring phenomena, but the input parameters were difficult to determine experimentally. The model predictions of intraparticle diffusion were of limited accuracy. We show that prediction can be improved when combining the model of Mackie and Meares with the fraction of accessible pore volume. The accessible pore volume fraction can be determined from inverse size exclusion chromatographic measurements. Future work should further challenge the improved model, specifically widening the applicability to greater accessible pore fractions (> 0.7) with corresponding higher intraparticle diffusivities (Dp/Dm > 0.2). A database of intraparticle diffusion and stationary phase pore property measurements is supplied, to contribute to general understanding of the relationship between intraparticle diffusion and pore properties.

Model-based selection of the degree of cross-linking of cation exchanger resins for an optimised separation of monosaccharides

One of the main steps in designing preparative chromatographic separation units is the selection of a well-performing adsorbent. This is often based on expert knowledge or based on case studies of preselected adsorbents. Therefore, the selection is usually limited in terms of an optimised choice. In this contribution, a model-based optimisation of the selection of an adsorbent on the basis of correlations between structural adsorbent properties with model parameters of a transport dispersive model is proposed. Model parameters of glucose and xylose for five cation exchanger resins with varying degree of cross-linking are experimentally determined in a sequential approach. Void fractions and particle porosities were determined by pulse experiments with different tracers. Single-component isotherms were determined threefold via breakthrough curves with concentrations of up to 250 g l−1 at 60 °C. Mass transfer coefficients were determined by batch experiments. Correlations between the degree of cross-linking of the resins and the Henry coefficients as well as the mass transfer coefficients were derived and applied in an optimisation case study. Based on the derived mathematical formula, the process performance of experimentally not investigated resins were predicted. Further, the selection of a resin for a preparative monosaccharide separation was included into optimisation algorithms.

Detailed Phytochemical Characterization of Bergamot Polyphenolic Fraction (BPF) by UPLC-DAD-MS and LC-NMR.

Bergamot ( Citrus bergamia) is cultivated in Southern Italy almost exclusively to produce the prized essential oil, a top note in several perfumes. The juice of bergamot, until recently poorly studied, is the object of a growing scientific interest due to its claimed activity to treat metabolic syndrome. The aim of this investigation was a detailed characterization of bergamot juice polyphenolic fraction (BPF) based on a UPLC-DAD-MS analysis complemented by preparative chromatographic separations, followed by NMR characterization of the isolated compounds. The combination of these techniques efficiently covered different classes of secondary metabolites, leading to the identification of 39 components, several of which had never been reported from bergamot. One of them, bergamjuicin (35), is a new flavanone glycoside, whose structure has been determined by MS and NMR techniques. The reported results could provide a guide for future routine analyses of BPF, a material of great nutraceutical and industrial interest.

Combination of Enantioselective Preparative Chromatography and Racemization: Experimental Demonstration and Model-Based Process Optimization

Conventional enantioselective preparative chromatographic separation using columns packed with chiral stationary phase is characterized by a 50% yield constraint. Racemization of the undesired enan...

Enzymatic Kinetic Resolution of Secondary Alcohols Using an Ionic Anhydride Generated In Situ.

We developed a method for the resolution of secondary alcohols using an ionic anhydride acylating agent prepared directly in the reaction medium containing the biocatalyst Candida antarctica lipase B (CALB). NMR studies showed that mixing all components at the same time does not interfere with the coupling reaction or the enzymatic activity. After optimization of the reaction conditions, the method allowed the resolution of a number of substrates in very high conversions (46-48 %) and enantiomeric ratios (E>170) along with an easy recovery of both enantiomers without the need for preparative chromatographic separation. Additionally, both the starting ionic acid and the biocatalyst could be recovered and reused up to nine cycles without significant loss of enantioselectivity.

Separation of Nadolol Stereoisomers Using Chiralpak IA Chiral Stationary Phase.

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 10g/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 3.13L/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 28:399-408, 2016. © 2016 Wiley Periodicals, Inc.

Sialic acid-specific affinity chromatography for the separation of erythropoietin glycoforms using serotonin as a ligand.

Recombinant human erythropoietin (rhEPO) is an important CHO cell-derived glycoprotein and the degree of sialylation of this hormone is crucial for its in vivo bioactivity. In order to improve the purification process serotonin as a potential affinity ligand was tested for preparative chromatographic separation of rhEPO glycoforms into fractions of different degrees of sialylation. Therefore, two chromatographic matrices were prepared by immobilizing serotonin on CNBr- and NHS-Sepharose™. First it was shown both matrices bind rhEPO only in its sialylated form. Results indicate that binding is pH independent between pH 3.5 to 8 suggesting it is not only based on electrostatic interactions. Second, after optimal binding conditions were identified, semi-purified rhEPO was loaded onto both matrices and eluted using a stepwise elution gradient of sodium chloride. For comparison same affinity purification experiments were performed using wheat germ agglutinin-coupled agarose, a lectin known for its affinity towards sialylated glycoproteins. To monitor changes in N-glycan fingerprint, eluate fractions were analyzed by multiplexed capillary gel electrophoresis coupled to laser-induced fluorescence (xCGE-LIF). For the serotonin matrices an increasing degree of sialylation was observed from the first to the third elution fraction while purity of rhEPO could be increased at the same time. The late elution fractions of serotonin-coupled CNBr- and NHS-Sepharose™ also showed an overall sialylation degree exceeding that of the starting material. In contrast, for rhEPO bound to wheat germ agglutinin-coupled agarose, no distinct change in the degree of sialylation could be observed after elution. Overall, these encouraging results highlight the potential of serotonin as a chromatographic ligand for the improvement of pharmaceutical purification processes of rhEPO.

Characterization of lysozyme adsorption in cellulosic chromatographic materials using small-angle neutron scattering

Measurements of the nanoscale structure of chromatographic adsorbents and the associated distribution of sorbed protein within the media can facilitate improvements in such media. We demonstrate a new technique for this purpose using small-angle neutron scattering (SANS) to characterize the nano- to microscale structure of the chromatographic media and sorbed protein under conditions relevant for preparative chromatographic separations. The adsorption of lysozyme on cellulosic S HyperCel™ (Pall Corporation), a strong cation exchanger, was investigated by SANS. The scattering spectrum is reduced to three contributions arising from (1) the chromatographic medium, (2) discrete protein molecules, and (3) the distribution of sorbed protein within the medium. These contributions are quantified for a range of protein loadings. The total concentration of protein in the chromatographic media can be quantified from the SANS spectrum and the protein is observed to retain its tertiary structure upon adsorption, within the resolution of the method. Further analysis of the SANS spectra shows that protein adsorption is uniform in the media. These measurement techniques provide new and valuable nanoscale information about protein sorption in chromatographic media.

Modeling Preparative Chromatographic Separation of Heavy Rare Earth Elements and Optimization of Thulium Purification

Rare Earth Elements are in growing demand globally. This paper presents a case study of applied mathematical modeling and multi objective optimization to optimize the separation of heavy Rare Earth Elements, Terbium-Lutetium, by means of preparative solid phase extraction chromatography, which means that an extraction ligand, HDEHP, is immobilized on a C18 silica phase, and nitric acid is used as an eluent. An ICP-MS was used for online detection of the Rare Earths. A methodology for calibration and optimization is presented, and applied to an industrially relevant mixture. Results show that Thulium is produced at 99% purity, with a productivity of 0.2 - 0.5 kg Tm per m3 stationary phase and second, with Yields from 74% to 99%.

Drug synthesis methods and manufacturing technology preparative chromatographic separation of ibuprofen enantiomers on Whelk-O1 chiral stationary phase

The influence of experimental conditions (temperature, sample volume, mobile-phase composition) on the preparative separation of ibuprofen enantiomers on chiral stationary phase (S,S)-Whelk-O1 with hexane:EtOH eluents modified with acetic acid was studied. It was established that the effects of these factors on the elution profiles were complex with the patterns disturbed by overlap with the systemic peak. The importance of the last factor decreased with increasing alcohol concentration in the mobile phase. The possibility of optimizing the preparative separation with respect to both purity and yield of the target enantiomers was discussed.

Active flow management in preparative chromatographic separations: A preliminary investigation into enhanced separation using a curtain flow inlet fitting and segmented flow outlet fitting

Active flow management in the form of curtain flow sample introduction and segmented outlet flow control has been shown to enable sample to elute through a chromatography column under the principles of the "infinite diameter column". Such an elution process avoids the detrimental effects of the heterogeneity of particle-packed chromatographic columns by injecting the sample directly into the radial core region of the column, thus avoiding wall effects. The process described herein illustrates how the principles of the infinite diameter column can be applied using conventional injection devices suitable for long-term analysis that requires robust protocols. Using this approach, sensitivity in separation was 2.5 times greater than conventional chromatography, yielding a product at twice the concentration. Benefits of curtain flow injection are thus relevant to both preparative-scale and analytical-scale separations.

Preparative Chromatographic Purification and Separation of Individual Jujubasaponins from Zizyphi Spinosi Semen

Zizyphi Spinosi Semen is well known as a kind of herb for the treatment of insomnia and anxiety. However, due to the lack of purified compounds for functional tests, limited data proves pharmacological activities of each jujubasaponin monomer. In this study, we developed a novel method for the preparative separation and purification of jujuboside A, A1, B, and B1, simultaneously, from Zizyphi Spinosi Semen. Macroporous resin and reversed-phase flash chromatography was successfully used and gave a good resolution of each jujubasaponins monomer. Jujuboside A, A1, B, and B1 with a purity of over 98% were yielded.

Optimization of preparative chromatographic separation of multiple rare earth elements

This work presents a method to optimize multi-product chromatographic systems with multiple objective functions. The system studied is a neodymium, samarium, europium, gadolinium mixture separated in an ion exchange chromatography step. A homogeneous Langmuir Mobile Phase Modified model is calibrated to fit the experiments, and then used to perform the optimization task. For the optimization a multi-objective Differential Evolution algorithm was used, with weighting based on relative value of the components to find optimal operation points along the Pareto front. The objectives of the Pareto front are weighted productivity and weighted yield with purity as an equality constraint. A prioritizing scheme based on relative values is applied for determining the pooling order. A simple rule of thumb for pooling strategy selection is presented. The multi-objective optimization gives a Pareto front which shows the rule of thumb, as a gap in one of the objective functions.

Phytochemical and antimicrobial studies on Detarium microcarpum Guill and Sperr (Caesalpinioceae) seeds coat

Ninety percent aqueous methanol extract of the seeds coat (32.324%, w/w) ofDetarium microcarpum was screened against clinical isolates of Staphylococcus aureus, Bacillus subtilis, Escherichia coli, Pseudomona aeruginosa, Klebsiellapneumonia, Salmonella paratyphi and Candida albicans. A broad spectrum antimicrobial activity at the test concentration of 10 mg/ml comparable with the control drugs chloramphenicol and penicillin G for bacteria and nystatin for the mould C. albicans was observed. On bio-guided fractionation, the ethyl acetate soluble fraction (ES) retains the observed spectrum of antimicrobial activity. Preparative thin layer chromatographic separation of the active ESF on silica gel 60G using butan-2-one : chloroform : acetic acid : water (40:40:2:1 v/v/v/v) gave eight constituent bands: B1 (Rf = 0.000), B2 (Rf = 0.079), B3 (Rf = 0.250), B4 (Rf = 0.286), B5 (Rf = 0.500), B6 (Rf = 0.686), B7 (Rf = 0.814), and B8 (Rf = 1.000). B1 retained the observed antimicrobial activity spectrum, B2 and B3 showed residual inhibition against B. subtilis and B4 - B8 were inactive. Phytochemical screening indicated the presence steroidal saponins and flavonoids in B1, and flavonoids in B2 and B3. The UV-visible spectrum of B2 and B3 revealed the characteristic benzopyrone maxima (236 – 300 nm). Against the selected bacteria, MIC range of B1 of 1.0488 -2.8887 mg/ml was observed comparable to those of standard control (1.2357 - 3.3420 mg/ml) and chloramphenicol (2.0840 -3.9630 mg/ml) which is suggestive of resistant strains. This study revealed the antimicrobial principles in the seed coat of D. microcarpum to be steroidal saponins and flavonoids with the possibility of synergistic action. Key words: Detarium microcarpum, seed coat, antimicrobial activity, benzopyrone, flavonoids, steroidal saponins, phytoalexins.

Hydrophilic Interaction Chromatography Reduces the Complexity of the Phosphoproteome and Improves Global Phosphopeptide Isolation and Detection

The diversity and complexity of proteins and peptides in biological systems requires powerful liquid chromatography-based separations to optimize resolution and detection of components. Proteomics strategies often combine two orthogonal separation modes to meet this challenge. In nearly all cases, the second dimension is a reverse phase separation interfaced directly to a mass spectrometer. Here we report on the use of hydrophilic interaction chromatography (HILIC) as part of a multidimensional chromatography strategy for proteomics. Tryptic peptides are separated on TSKgel Amide-80 columns using a shallow inverse organic gradient. Under these conditions, peptide retention is based on overall hydrophilicity, and a separation truly orthogonal to reverse phase is produced. Analysis of tryptic digests from HeLa cells yielded numbers of protein identifications comparable to that obtained using strong cation exchange. We also demonstrate that HILIC represents a significant advance in phosphoproteomics analysis. We exploited the strong hydrophilicity of the phosphate group to selectively enrich and fractionate phosphopeptides based on their increased retention under HILIC conditions. Subsequent IMAC enrichment of phosphopeptides from HILIC fractions showed better than 99% selectivity. This was achieved without the use of derivatization or chemical modifiers. In a 300-microg equivalent of HeLa cell lysate we identified over 1000 unique phosphorylation sites. More than 700 novel sites were added to the HeLa phosphoproteome.

Studies of the chemical composition of birch bark extracts (Cortex betula) from the Betulaceae family

This report presents the results obtained from chemical studies of the composition of dried birch bark extracts (DBBE) from Betula pubescens Ehrh. analyzed by GLC, HPLC, and IR and PMR spectroscopy. The extract contained the following groups of natural compounds: terpenoids (75.2%) and their esters (fatty acid esters of betulinol and lupeol, 4.4%), ether oils (0.08%), hydrocarbons (6.3%) and their epoxides (1.0%), steroids (β-sitosterol, 2.7%), tannins (2.1%), flavonoids (1.56%-mainly kaempferol, its 7-methyl ester, quercetin, the 4-methyl ester of naringenin, etc.), hydroxycoumarins (0.85%-umbelliferone, esculetin, etc.), and a number of unidentified compounds (about 4.0%). The major components of DBBE were triterpenoids and hydrocarbons. Preparative chromatographic separation of fractions containing triterpenoids and hydrocarbons yielded isolated samples which allowed the following compounds to be identified: the terpenoids betulinol, isobetulinol, lupeol, lupenone, betulonic aldehyde, betulonic acid, betulinic acid, and platanic acid, and the hydrocarbons α-santalene, β-trans-bergamotene, and α-trans-bergamotene, and their epoxy derivatives. The chemical composition of DBBE varied depending on the preparation method used, this particularly affecting the content of the major component betulinol, which varied from 54 to 82%. These studies led to the development of a contemporary preparative technology for betulinol with a yield of 95%, along with production of many of its acyl derivatives (for example, the diacetate, succinate, benzoate, etc.) with quite high yields (95–98%); betulinic acid was synthesized in mild conditions with a yield of at least 99.0%. These compounds are currently subject to pharmacological studies.