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The total alkaloids from Nitraria sibirica leaves have been confirmed to exhibit significant protective effects against inflammatory renal injury, hypertension and albuminuria in angiotensin II-salt hypertension. In the present study, a separation method of pH-zone-refining counter-current chromatography was established for separation of the alkaloids from N. sibirica . The separation was performed with a solvent system of MtBE- n -BuOH-H 2 O (2:2:5, v / v ) at a flow rate of 2.0 mL/min. And 15 mM triethylamine (TEA) was added to the upper organic phase, while 10 mM hydrochloric acid was added to the lower aqueous phase. As a result, a new alkaloid, schobemine (5.6 mg), and a known alkaloid, nitraramine (5.0 mg), together with fractions A and B were obtained from the total alkaloids of N. sibirica . The fractions A and B were further purified by means of pH-zone-refining counter-current chromatography with solvent systems of n -hexane- n -BuOH-H 2 O (1.5:3.5:5, v / v ) and (2:3:5, v / v ), respectively. TEA (10 mM) was added to the upper phase, and 10 mM of HCl was added to the lower phase in above two solvent systems, respectively. As a result, a known alkaloid, schoberidine (5.0 mg), and a new alkaloid, schoberimine (3.0 mg) were obtained from fractions A and B, respectively. The purities of the compounds were measured by HPLC–ELSD, and their structures were identified by ESI–MS, 1D and 2D NMR. [ABSTRACT FROM AUTHOR]

Highlights: [•] Retention behavior of monoclonal IgG1 was investigated on IEC and HIC media. [•] Dynamics of both processes was dominated by kinetic effects. [•] A short-cut method was proposed for mathematical modeling. [•] A dynamic model was used for optimization of the integrated process IEC/HIC. [ABSTRACT FROM AUTHOR]

Abstract: In this study, resin adsorption as a means to separate and purify baicalin and wogonoside from extracts of Scutellaria baicalensis was investigated. Among the ten tested resins, the non-polar resin HPD-100 offered the best adsorption and desorption properties. Langmuir and Freundlich isotherms were used to describe the interactions between solutes and resin at different temperatures, and the equilibrium experimental data were well fitted to the two isotherms. Column packed with HPD-100 resin was used to perform dynamic adsorption and desorption tests to optimize the separation process. After one round treatment with HPD-100 resin, the contents of baicalin and wogonoside were 3.6-fold and 12.0-fold increased with recovery yields of 85.7% and 65.6%, respectively. In addition, a laboratory preparative-scale separation was carried out under the final conditions. The results showed that the preparative separation of baicalin and wogonoside can be easily and efficiently achieved via adsorption and desorption on HPD-100 resin. The developed method is a promising basis for large-scale preparation of baicalin and wogonoside from S. baicalensis extracts. [Copyright &y& Elsevier]

Abstract: Sample displacement chromatography (SDC) in reversed-phase and ion-exchange modes was introduced approximately twenty years ago. This method takes advantage of relative binding affinities of components in a sample mixture. During loading, there is a competition among different sample components for the sorption on the surface of the stationary phase. SDC was first used for the preparative purification of proteins. Later, it was demonstrated that this kind of chromatography can also be performed in ion-exchange, affinity and hydrophobic-interaction mode. It has also been shown that SDC can be performed on monoliths and membrane-based supports in both analytical and preparative scale. Recently, SDC in ion-exchange and hydrophobic interaction mode was also employed successfully for the removal of trace proteins from monoclonal antibody preparations and for the enrichment of low abundance proteins from human plasma. In this review, the principals of SDC are introduced, and the potential for separation of proteins and peptides in micro-analytical, analytical and preparative scale is discussed. [Copyright &y& Elsevier]

Abstract: The growing demand on plasmid DNA (pDNA) manufacture for therapeutic applications requires a final product with higher quality and quantity, spending the least time. Most of the current processes for pDNA production use at least one chromatographic step, which often constitutes a key-step in the purification sequence. Monolithic stationary phases are new alternatives to the conventional matrices, which offer fast separation of pDNA due to their excellent mass transfer properties and their high binding capacity for large molecules, as pDNA. However, the efficient recovery of pure pDNA focuses on a suitable balance of the feedstock, adsorbent and mobile phase properties. To satisfy the increasing demand for pharmaceutical grade plasmids, we developed a novel downstream process which overcomes the bottlenecks of common lab-scale techniques while complying with all regulatory requirements. This work reports an integrative approach using the carbonyldiimidazole monolith to efficiently purify the supercoiled (sc) pDNA active conformation from other plasmid topologies and Escherichia coli impurities present in a clarified lysate. The monolith specificity and selectivity was also assessed by performing experiments with plasmids of several sizes of 2.7, 6.05 and 7.4 kilo base pairs (kbp), verifying the applicability to purify different plasmids. Hence, the process yield of the pDNA purification step using the CDI monolith was 89%, with an extremely reduced level of impurities (endotoxins and gDNA), which was reflected in good transfection experiments of the sc plasmid DNA sample. Overall, the analytical results and transfection studies performed with the pDNA sample purified with this monolithic enabling technology, confirmed the suitability of this pDNA to be used in pharmaceutical applications. [Copyright &y& Elsevier]

Abstract: A microwave-assisted extraction (MAE) method is presented for the extraction of xanthones, α-mangostin and γ-mangostin from Garcinia mangostana. The MAE conditions including extraction temperature, liquid/solid ratio, extraction time and concentration of ethanol were optimized with an orthogonal test, and 5g sample was extracted with the optimized conditions. The crude extraction of MAE was successfully isolated and purified by high-speed counter-current chromatography (HSCCC) with a two-phase solvent system composed of petroleum ether–ethyl acetate–methanol–water (0.8:0.8:1:0.6, v/v) in one-step separation. The separation yielded 75mg of α-mangostin at 98.5% purity, and 16mg of γ-mangostin at 98.1% purity from 360mg crude extract of G. mangostana in less than 7h. The purity of the two xanthones was determined by HPLC. Their structures were further identified by ESI-MS, 1H NMR and 13C NMR. [Copyright &y& Elsevier]

Abstract: Acid/base mobile phase modifiers affect enantioseparations in ways that are not fully understood yet, for the lack of systematic studies. This makes chiral analysis of some pharmaceuticals difficult to reproduce. Once a column has been exposed to a modifier, the selectivity of certain pairs of enantiomers may change, for the better or the worse. We study the behavior of five enantiomeric pairs, three which are highly sensitive to the addition of certain modifiers and two that have little sensitivity to these modifiers. Their use permits the determination of the extent of the memory effect response on individual columns. The selectivity of 4-chlorophenylalanine methyl and ethyl ester, and of ketoprofen improve as a solution of ethanesulfonic acid is percolated through the column. As a result, these pairs are most useful for the determination of the extent of acid memory effect on a column. The selectivity of propranolol HCl and, to a lesser degree, Tröger''s base increases as a solution of diisopropylethylamine is percolated through the column. The separation of each one of these five pairs is inversely affected by the percolation of the opposite acid/base solution. We used trans-stilbene oxide (TSO) as a ‘standard’ to determine the column stability because no memory effect is observed for it (its retention, enantioselectivity, and resolution remain constant). Understanding whether a column is under the influence of the memory effect is critical to both the analysis of pharmaceutical ingredients and to the development of preparative purification techniques for racemic mixtures. Thus, columns that were unreliable for method development and method transfer, due to the memory effect and a lack of proper solvent exposure records, can now be used. [Copyright &y& Elsevier]

Abstract: In the first part of this work adsorption isotherm parameters were acquired to describe the migration of recombinant streptokinase in Butyl Sepharose columns at different salt concentrations. Based on these results, a simulated moving bed (SMB) chromatographic process was designed and realised, which exploits a two-step salt gradient and allows the continuous separation of streptokinase from contaminants present in a clarified Escherichia coli cell lysate solution. This second part describes the design of the three-zone open-loop gradient SMB process applying both equilibrium theory and an equilibrium stage model and presents results of a series of experiments aiming to obtain pure streptokinase. Moreover, the potential of the SMB process and the design approach are evaluated. [Copyright &y& Elsevier]

Abstract: A preparative high-speed counter-current chromatography (HSCCC) method for isolation and purification of four prenylflavanones from the crud extract after microbial biotransformation of kurarinone was developed successfully using a stepwise elution with two-phase solvent system composed n-hexane–ethyl acetate–methanol–water at the volume ratios of 1:1:0.7:1 (v/v) and 1:1:1.2:1 (v/v). A total of 7mg of 4a,5a-dihydroxy norkurarinone (1), 11mg of 7-methoxyl-4a,5a-dihydroxy norkurarinone (2), 14mg of 6a-hydroxykurarinone (3) and 10mg of norkurarinone (4) were obtained in one-step separation from 520mg of the crude biotransformation sample with purities of 94.0%, 97.8%, 99.5% and 98.7%, respectively. Among them, compounds 1–3 are novel compounds, and their chemical structures were identified on the basis of the extensive spectral methods such as UV, IR, HR-MS and NMR. [ABSTRACT FROM AUTHOR]

Abstract: In this paper, a simple method for the rapid extraction, separation and purification of bergenin from Ardisia crenata sims and Rodgersia sambucifolia hemsl by microwave-assisted extraction (MAE) coupled with high-speed counter-current chromatography (HSCCC) was developed. The MAE conditions were optimized and 2.0g sample was extracted using 60% (v/v) aqueous methanol as extraction solvent with liquid/solid ratio of 10/1(mL/g) at 60°C for 15min. The crude extract of MAE was separated and purified directly by HSCCC using ethyl acetate–n-butanol–water (3:2:5, v/v/v) solvent system. In less than 3.5h, 18.6 or 25.0mg of bergenin was obtained from 160mg crude extract of A. creanta or R. sambucifolia in one-step separation, respectively. The purity of bergenin was over 99% determined by HPLC and its chemical structure was further identified by ESI-MS, 1H NMR and UV. The results indicate that microwave-assisted extraction coupled with high-speed counter-current chromatography is very suitable for the extraction, separation and purification of bergenin from A. creanta and R. sambucifolia. [ABSTRACT FROM AUTHOR]

Abstract: High-speed countercurrent chromatography (HSCCC) was applied to preparative separation and purification of punicalin from pomegranate husk by one-step separation. The two-phase system consisted of n-butyl alcohol–ethyl acetate–water (4:1:5, v/v/v) was employed. The revolution speed of the separation column, flow rate of the mobile phase and separation temperature were 800rpm, 2ml/min and 25°C respectively. About 100mg amount of crude extract was separated, yielding 13.1mg of punicalin at a high purity of over 96%. The mass recovery was more than 90%. The product was assessed by analytical HPLC and characterized by MS, 1H NMR and 13C NMR. [Copyright &y& Elsevier]

Abstract: High-speed counter-current chromatography (HSCCC) and preparative high-performance liquid chromatography (prep-HPLC) were successively used for the separation of epigallocatechin and flavonoids from Hypericum perforatum L. The two-phase solvent system composed of ethyl acetate–methanol–water (10:1:10, v/v) was used for HSCCC. About 900mg of the crude extract was separated by HSCCC, yielding 7.8mg of quercitrin at a purity of over 97%, 12.6mg of quercetin at a purity of over 93%, and 38.9mg of a mixture of hyperoside, isoquercitrin and miquelianin constituting over 97% of the fraction. A mixture of epigallocatechin and avicularin pooled from three HSCCC runs, a total amount of 54.3mg, was further separated by prep-HPLC yielding 23.4mg of epigallocatechin and 15.3mg of avicularin each at a purity of over 97%. [Copyright &y& Elsevier]

Abstract: The proanthocyanidin extract from tea (Camellia sinensis) leaves was purified for the further study of the biological role of proanthocyanidins in blister blight leaf disease of tea, which is caused by the fungus Exobasidium vexans. An aqueous acetone extract of proanthocyanidins prepared from healthy tea leaves was partially purified using Sephadex LH-20 chromatography. The crude proanthocyanidin extract obtained was fractionated with high-speed counter-current chromatography (HSCCC) using the solvent system n-hexane–EtOAc–MeOH–water (1:5:1:5). The purity of the each isolated fraction after a single HSCCC run was evaluated by high-performance liquid chromatography (HPLC). Seven fractions of high purity were isolated. The identity of the compound present in each fraction isolated was established using electrospray ionization mass spectrometry (ESI-MS) and nuclear magnetic resonance (NMR) spectroscopy. Five proanthocyanidins and two flavanol digallates, (−)-epigallocatechin digallate (EGCDG) and (−)-epicatechin digallate (ECDG) were isolated. Comparison of spectral data of the proanthocyanidins isolated with those previously reported indicated that all five were known B-type proanthocyanidins with 2,3-cis stereochemistry in both the upper (u-unit) and the terminal (t-unit) units, and 4R configuration of the C-ring in the u-unit. The proanthocyanidins were established to be dimers composed of (−)-epigallocatechin gallate (EGCG), (−)-epicatechin gallate (ECG) and (−)-epiafzelechin gallate (EAG) units with the following structures: EGCG-(4β→6)-EGCG, ECG-(4β→6)-EGCG, EGCG-(4β→6)-ECG, EAG-(4β→6)-EGCG, ECG-(4β→6)-ECG by analysis of spectral data. Therefore HSCCC offers a powerful method for the separation of a group of closely related naturally occurring compounds. [Copyright &y& Elsevier]

Abstract: Apple pomace, a by-product in the processing of apple juice, was investigated as a potential source of polyphenols. Two methods of separation and purification of polyphenols from apple pomace extract were established by combination of gel chromatography with high-speed counter-current chromatography (HSCCC) and solvent extraction with HSCCC, respectively. The optimal separation was performed on a Sephadex LH-20 column using gradient aqueous ethanol as eluting solvent from 0% to 100% in increments of 10%. HPLC analysis indicated that main polyphenols existed in fractions eluted between 40% and 50% aqueous ethanol. The fractions of interest from column were separated by HSCCC with the solvent system hexane–ethyl acetate–1% aqueous acetic acid (0.5:9.5:10, v/v/v). Ethyl acetate fractionation of the apple pomace extract followed by direct HSCCC separation by the same solvent system in the volume ratio of 1:9:10 also produced a good separation of the main polyphenols of interest. Six high-purity polyphenols were achieved tentatively and identified by HPLC/MS: chlorogenic acid (1, m/z 354), quercetin-3-glucoside/quercetin-3-glacaside (2, m/z 464), quercetin-3-xyloside (3, m/z 434), phloridzin (4, m/z 436), quercetin-3-arabinoside (5, m/z 434), and quercetin-3-rhamnoside (6, m/z 448). These results provided a preliminary foundation for further development and exploration of apple pomace. [Copyright &y& Elsevier]

Abstract: Although the medicinal plant and food Nigella glandulifera Freyn has been researched for decades, isobenzofuranones have never been isolated before. Two isobenzofuranone derivatives and two saponins were successfully separated and purified from seeds of N. glandulifera Freyn by high-speed counter-current chromatography (HSCCC) with the optimized two-phase solvent system, n-hexane-ethyl acetate–methanol–water (7:3:5:5, v/v). Salfredin B11 (22.1mg, HPLC purity 95.3%), 5, 7-dihydroxy-6-(3-methybut-2-enyl) isobenzofuran-1(3H)-one (18.9mg, HPLC purity 97.3%) and crude sample 2 (555mg) were separated from 600mg of ethyl acetate extract of N. glandulifera Freyn. Following a cleaning-up step by chromatography on Sephadex LH-20, hederagenin (12mg) and 3-O-[β-d-xylopyranosyl-(1→3)-α-l-rhamnopyranosyl-(1→2)-α-l-arabinopyranosyl]-hederagenin (45mg) were separated from sample 2. All of the fractions before peak II were collected and subjected to a Sephadex LH-20 column and eluted by methanol, two of triterpene saponins (12mg of hederagenin and 45mg of 3-O-[β-d-xylopyranosyl-(1→3)-α-l-rhamnopyranosyl-(1→2)-α-l-arabinopyranosyl]-hederagenin) were isolated. The structures of peak fractions were identified by IR, electron ionization MS, 1H NMR and 13C NMR. 5, 7-Dihydroxy-6-(3-methybut-2-enyl) isobenzofuran-1(3H)-one was isolated for the first time from higher plant and salfredin B11 was isolated for the first time in this plant. [Copyright &y& Elsevier]

Abstract: This paper describes intermittent counter-current extraction, a novel method of using a conventional twin column counter-current chromatograph to either split a sample into two groups of compounds or extract and enrich a target compound from a crude extract. The first method is demonstrated by splitting a model mixture of four compounds into two groups. The second method is demonstrated by the extraction and enrichment of a high value target compound, triptolide, from a Chinese herbal medicine crude extract of Tripterygium wilfordii Hook. f., where it is found at low concentration (2%). This was achieved by retaining and enriching the target compound within the column while washing away all other components of the crude material. The success of the first method allowed the second method to be carried out without the need for costly preliminary experiments with the high value sample. 188mg of triptolide at greater than 98% purity was separated from 9.2g of crude extract, using 10l of solvent in a 3-h separation. [Copyright &y& Elsevier]

Abstract: A large-scale purification of salvianolic acid B from Salvia miltiorrhiza Bunge is presented. The method development began with selection of the solvent system, then optimization of the operating parameters and ended up with linear scale-up from an analytical to a preparative instrument. Three factors were used for method optimization and scale-up estimation: purity, process throughput and process efficiency. Preparation was achieved using a two-phase solvent system comprising hexane–ethyl acetate–methanol–acetic acid–water (1:5:1.5:0.00596:5, v/v). This preparation yielded 475mg of salvianolic acid B with a purity of 96.1% from 1.5g of crude extract. The process throughput of crude was 2.23g/h while process efficiency per gram of target compound was 0.769g/h. Two factors—process environmental risk factor and process evaluation factor were used for evaluation of the separation process. [Copyright &y& Elsevier]

Abstract: High-speed counter-current chromatography (HSCCC) was applied to preparative separation and purification of dl-tetrahydropalmatine from Corydalis yanhusuo by a one-step separation. The two-phase system consisted of petroleum ether–ethyl acetate–methanol–water (15:30:21:20, v/v) was employed. An orthogonal design was used for optimizing the revolution speed of the separation column, flow rate of the mobile phase and separation temperature, which was 850rpm, 1.2ml/min and 20°C, respectively. HPLC analysis of the fractions collected by preparative HSCCC of 200mg of crude extracts showed that the purity of dl-tetrahydropalmatine (8.6mg) was 96.4%. The chemical identity of the component was confirmed by 1H NMR and EI-MS. [Copyright &y& Elsevier]

By selecting the different combination schemes, a simple, fast and highly efficient method for separation and purification of nerve growth factor (NGF) from venom of Chinese cobra is reported in this paper. This purification process consists of a two-step chromatographic separation on DEAE-Sepharose F.F. anion-exchange medium followed by a Sephadex G-50 gel filtration. On reducing and non-reducing sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), the nerve growth factor obtained with this process proved to be homogeneous and its molecular weight was separately estimated to be approximately 14.5 and 29.0 kD, which was consistent with that reported in literature; and on high performance size-exclusion chromatography and reversed-phase chromatography, its purity was about 99%. The yield of this purification method was 0.51% and the nerve growth factor obtained had the activity of eliciting neurite outgrowth from chick embryonic dorsal root ganglia. The optimum concentration of nerve growth factor was 5–100 ng/ml and the minimal concentration eliciting neurite outgrowth from chick embryonic dorsal root ganglia was 5.0 ng/ml. [Copyright &y& Elsevier]