"Sweet space" strategy for solvent system selection in countercurrent chromatography: A case study on separation of polyunsaturated fatty acids from borage oil.

This study presents a comprehensive strategy for the selection and optimization of solvent systems in countercurrent chromatography (CCC) for the effective separation of compounds. With a focus on traditional organic solvent systems, the research introduces a "sweet space" strategy that merges intuitive understanding with mathematical accuracy, addressing the significant challenges in solvent system selection, a critical bottleneck in the widespread application of CCC. By employing a combination of volume ratios and graphical representations, including both regular and trirectangular tetrahedron models, the proposed approach facilitates a more inclusive and user-friendly strategy for solvent system selection. This study demonstrates the potential of the proposed strategy through the successful separation of gamma-linolenic acid, oleic acid, and linoleic acid from borage oil, highlighting the strategy's effectiveness and practical applicability in CCC separations.

Separation of bufadienolides from Helleborus thibetanus Franch. by a combination approach involving macroporous resin column chromatography and gradient countercurrent chromatography.

In this study, a combination approach involving macroporous resin (MR) column chromatography and gradient countercurrent chromatography (CCC) was employed to enrich and purify bufadienolides from the roots and rhizomes of Helleborus thibetanus Franch. Initially, a D101 MR-packed column chromatography was utilized for fractionation and enrichment of the bufadienolides, which were effectively eluted from the column using a 60% ethanol solution. CCC was subsequently introduced to separate the enriched product using the ethyl acetate/n-butanol/water (EBuWat, 4:1:5, v/v) and EBuWat (5:0:5, v/v) solvent systems in a gradient elution mode. As results, five bufadienolides, including 6.1 mg of hellebrigenin-3-O-ß-D-glucoside (1), 2.2 mg of tigencaoside A (2), 8.3 mg of deglucohellebrin (3), 3.5 mg of 14 ß-hydroxy-3ß-[ß-D-glucopyranosyl-(1→6)-(ß-D-glucopyranosyl)oxy]-5α-bufa-20,22-dienolide (4), and 3.0 mg of 14ß-hydroxy-3ß-[(ß-D-glucopyranosyl)oxy]-5α-bufa-20,22-dienolide (5), were effectively separated from 300 mg of the enriched product. The respective high-performance liquid chromatography purities were as follows: 95.2%, 75.8%, 85.7%, 82.3%, and 92.8%. This study provides valuable insights for the efficient enrichment and separation of bufadienolides from Helleborus thibetanus Franch.

Purification of triterpenoid saponins and 25R/25S-inokosterone from Achyranthes bidentata Bl. by high-speed countercurrent chromatography coupled silver nitrate coordination.

An effective method by high-speed countercurrent chromatography coordinated with silver nitrate for the preparative separation of sterones and triterpenoid saponins from Achyranthes bidentata Blume was developed. Methyl tert-butyl ether/n-butanol/acetonitrile/water (4:2:3:8, v/v/v/v) was selected for 20-hydroxyecdysone (compound 1), chikusetsusaponin IVa methyl ester (compound 4), 2'-glycan-11-keto-pigmented saponin V (compound 5), as well as a pair of isomers of 25S-inokosterone (compound 2) and 25R-inokosterone (compound 3), which were further purified by silver nitrate coordinated high-speed countercurrent chromatography. What is more, dichloromethane/methanol/isopropanol/water (6:6:1:4, v/v/v/v) was applied for calenduloside E (compound 6), 3ß-[(O-ß-d-glucuronopyranosyl)-oxy]-oleana-11,13-dien-28-oic acid (compound 7), zingibroside R1 (compound 8) and chikusetsusaponin IVa (compound 9). Adding Ag+ to the solvent system resulted in unique selectivity for 25R/25S isomers of inokosterone, which increased the complexing capability and stability of Ag+ coordinated 25S-inokosterone, as well as the α value between them. These results were further confirmed by the computational calculation of geometry optimization and frontier molecular orbitals assay. Comprehensive mass spectrometry and nuclear magnetic resonance analysis demonstrated the structures of the obtained compounds.

Elution behavior of drugs in high-speed counter-current chromatography using on-column complexation with metal ions.

In this study, determination of (nitrogen containing) drugs by on-column complexation with metal ions in high-speed counter-current chromatography (HSCCC) was investigated. Bromazepam (BMP) was strongly retained in the organic upper stationary phase (UP) of the two-phase solvent system composed of tert-butyl methyl ether-acetonitrile-water (2:2:3, v/v/v) by eluting the aqueous lower mobile phase (LP) at a flow rate of 2 mL min-1. On the other hand, BMP (200 µg mL-1) was eluted faster without retention to the organic UP with the two-phase system containing 100 µg mL-1 of copper ions (CuCl2) because a very polar BMP-Cu2+ complex was immediately formed in the aqueous LP. The dramatic change in the retention behavior of BMP resulted from on-column complexation. The on-column complexation in HSCCC was further investigated for five (nitrogen containing) drugs and seven metal ions. In the result, tizanidine and phentolamine formed complexes with Al3+, Fe2+, Co2+, Ni2+, Cu2+, and Zn2+, ambroxol formed complexes with Al3+, Fe2+, and Cu2+, but voriconazole formed no complexes with all metal ions tested.

Enhancing the purification of crocin-I from saffron through the combination of multicolumn countercurrent chromatography and green solvents.

Crocin-I, a valuable natural compound found in saffron (Crocus sativus L.), is the most abundant among the various crocin structures. Developing a cost-effective and scalable purification process to produce high-purity crocin-I is of great interest for future investigations into its biological properties and its potential applications in the treatment of neurological disorders. However purifying crocin-I through single-column preparative chromatography (batch) poses a yield-purity trade-off due to structural similarities among crocins, meaning that the choice of the collection window sacrifices either yield in benefit of higher purity or vice versa. This study demonstrates how the continuous countercurrent operating mode resolves this dilemma. Herein, a twin-column MCSGP (multicolumn countercurrent solvent gradient purification) process was employed to purify crocin-I. This study involved an environmentally friendly ethanolic extraction of saffron stigma, followed by an investigation into the stability of the crocin-I within the feed under varying storage conditions to ensure a stable feed composition during the purification. Then, the batch purification process was initially designed, optimized, and subsequently followed by the scale-up to the MCSGP process. To ensure a fair comparison, both processes were evaluated under similar conditions (e.g., similar total column volume). The results showed that, at a purity grade of 99.7%, the MCSGP technique demonstrated significant results, namely + 334% increase in recovery + 307% increase in productivity, and - 92% reduction in solvent consumption. To make the purification process even greener, the only organic solvent employed was ethanol, without the addition of any additive. In conclusion, this study presents the MCSGP as a reliable, simple, and economical technique for purifying crocin-I from saffron extract, demonstrating for the first time that it can be effectively applied as a powerful approach for process intensification in the purification of natural products from complex matrices.

Effective separation of maslinic acid and oleanolic acid from olive pomace using high-speed shear off-line coupled with high-speed countercurrent chromatography and their antibacterial activity test.

In this work, a high-speed shear extraction off-line coupling high-speed countercurrent chromatography method was developed to separate maslinic acid and oleanolic acid from olive pomace. To improve extraction efficiency, the polar disparity between maslinic acid and oleanolic acid necessitated the concurrent utilization of both polar and non-polar solvents during high-speed shear extraction. Then, the high-speed shear extraction was directly feed to high-speed countercurrent chromatography for subsequently separation. A total of 250 min were needed to complete the extraction and separation process. This yielded two molecules from 3.3 g of defatted olive pomace: 7.2 mg of 93.8 % pure maslinic acid and 2.3 mg of 90.1 % pure oleanolic acid, both determined by HPLC at 210 nm. Furthermore, the compounds exhibited inhibitory activity against Escherichia coli and Staphylococcus aureus. At a concentration of 100 µg/mL, its efficacy in inhibiting hyaluronidase was comparable to that of the standard drug indomethacin. Compared with the conventional separation method, this coupled technique reduced the whole time due to the direct injection of sample extraction solution. This technique provides a useful approach for the separation of natural products with significant polarity differences.

Uncovering the Power of HSCCC: Separation of Diastereomeric and Regioisomeric Styrylpyrones from the Stem Bark of Goniothalamus leiocarpus.

The plant Goniothalamus leiocarpus of the Annonaceae family is used as an alternative medicine in tropical regions. Applying high-speed counter current chromatography (HSCCC), eight new bioactive styrylpyrone isomers, including 6R,7S,8R,2'S-goniolactone B (1), 6S,7S,8S,2'S-goniolactone B (2), 6R,7R,8R,2'S-goniolactone B (3), 6R,7S,8S,2'S-goniolactone C (4), 6R,7S,8R,2'S-goniolactone C (5), 6S,7R,8S,2'S-goniolactone C (6), and two positional isomers, 6R,7R,8R,2'S-goniolactone G (7) and 6S,7R,8R,2'S-goniolactone G (8), were isolated from a chloroform fraction (2.1 g) of G. leiocarpus, which had a prominent spot by TLC analysis. The structures of the new compounds were elucidated by MS, NMR, IR, and UV spectra, and their absolute configurations were determined by Mosher's method, ECD, and X-ray diffraction analysis. The isolates are characteristic components found in plants of the genus Goniothalamus and consist of two structural moieties: a styrylpyrone and a dihydroflavone unit. The isolation of the eight new compounds demonstrates the effectiveness of HSCCC in separating the isomers of natural styrylpyrone. In a bioactivity assessment, compounds 1 and 6 exhibited cytotoxic effects against the human colon carcinoma cell lines LS513 and SW620 with IC50 values ranging from 1.6 to 3.9 µM. Compounds 1, 2, 7, and 8 showed significant synergistic activity against antibiotic-resistant Staphylococcus aureus strains.

Application of two-dimensional reversed phase countercurrent chromatography × high-performance liquid chromatography to bioactivity-guided screening and isolation of α-glucosidase inhibitors from Rheum palmatum L.

In the present work, comprehensive two-dimensional reversed-phase countercurrent chromatography × reversed-phase liquid chromatography combined (2D RPCCC × RPLC) with 2D microfraction bioactive evaluation was employed to screen and isolate α-glucosidase inhibitors from Rheum palmatum L. Countercurrent chromatography was employed to improve 2D analysis and preparative separation. A selected biphasic solvent system composed of petroleum ether/ethyl acetate/methanol/water with gradient elution mode was used for the first dimension RPCCC separation (1D RPCCC). Solid-phase extraction was applied to eliminate interfering polar compounds before the second dimension analysis (2D RPLC). 76 components were shown in 2D contour plot in UV 280 nm. 11 Candidates were separated by a scaled-up CCC and identified by 1H NMR and 13C NMR, including anthraquinones, flavonoids, stilbenes, phenols, and glucoside derivatives. In addition, it was found that two components, resveratrol-4'-O-(6″-galloyl)glucoside (36) and lyciumaside (43) were identified as natural α-glucosidase inhibitors in Rheum palmatum L. for the first time.

Efficient discovery of active isolates from Dioscorea spongiosa by the combination of bioassay-guided macroporous resin column chromatography and high-speed counter-current chromatography.

In the present study, twelve compounds from Dioscorea spongiosa were successfully purified by an efficient technique combined bioassay-guided fractionation macroporous resin column chromatography (MRCC) pretreatment and high-speed counter-current chromatography (HSCCC) separation for the first time. Then, D101 MRCC was used to fractionate the crude extract into five parts, which further applied the bioassay-guided fractionation strategy to screen the active fractions of 2 and 4. As for the separation, 200 mg Fr.2 was purified by HSCCC using EtOAc/n-BuOH/H2 O (2:2:3, v/v), leading to annulatomarin (1), dioscoresides C (2), diosniponol C (3), methyl protodioscin (4), pseudoprotodioscin (5), protogracillin (6), as well as 200 mg Fr.4 yielding montroumarin (7), dioscorone A (8), diosniponol D (9), protodioscin (10), gracillin (11), and dioscin (12) using CH2 Cl2 /MeOH/H2 O (3:3:2, v/v) with the purities over 95.0%. Finally, the isolates were assayed for their anti-inflammatory, urico-lowering, and anti-diabetic activities in vitro, which indicated that the steroidal saponins of 5, 6, and 11 showed all these three activities.

Rapid screening and target-guided isolation of antioxidants from German chamomile by 2,2-diphenyl-1-picrylhydrazyl-ultra-high-performance liquid chromatography-mass spectrometry coupled with off-line two-dimensional high-speed countercurrent chromatography.

German chamomile is one of the most effective herbal elements used in anti-allergic products and as an antioxidant. Herein, the antioxidant activity of different extract fractions of German chamomile was initially evaluated using an off-line 2,2-diphenyl-1-picrylhydrazyl spectrophotometric assay. The ethyl acetate extract demonstrated the highest efficacy in scavenging free radicals. Based on this, a rapid screening and separation method using ultra-high-performance liquid chromatography combined with the 2,2-diphenyl-1-picrylhydrazyl assay was implemented to identify antioxidants in the ethyl acetate fraction of German chamomile flowers. Ten potential radical scavengers were tentatively screened from German chamomile using a target-guided isolating approach with off-line two-dimensional high-speed countercurrent chromatography and the structures of the compounds were analyzed and identified. Ultimately, 10 radical scavengers were obtained from the ethyl acetate extract with a purity quotient exceeding 90%. The results demonstrated the effectiveness and reproducibility of this method for isolating potential antioxidants from complex mixtures in a targeted manner. This strategy can be applied to the target-guided isolation of complex mixtures of natural products with broad K-values and similar structures.

Comparison of two different multiple dual-mode counter-current chromatograph for separation of ketoconazole enantiomers.

In this work, two different multiple dual-mode (MDM) counter-current chromatography methods, conventional MDM and modified MDM elution modes, were compared for the chiral separation of the ketoconazole enantiomers. The biphasic solvent system which consisted of n-hexane: isobutyl acetate: 0.1 mol/L phosphate buffer (2:4:6, v/v) (pH = 8.5) was employed as stationary phase and mobile phase. And the hydroxypropyl-ß-cyclodextrin (HP-ß-CD) with a concentration of 100 mmol/L was dissolved in the phosphate buffer, as the chiral selector. Under two different methods, dual-mode (DM) elution was performed to determine the time of the transformed phase roles and multiple cycles were performed to isolate ketoconazole, respectively. The result indicated that the modified MDM elution had a significant improvement on the separation, increasing the resolution from 0.51 to 1.19, while the resolution was increased from 0.40 to 0.79 by the conventional MDM elution. Ultimately, baseline separation of ketoconazole enantiomers was essentially achieved by high-speed counter-current chromatography under optimized modified MDM separation conditions. The final recoveries of the two enantiomers, R-(K) and S-(K), were 92.5 % and 83.3 %, respectively, corresponding to enantiomeric excess values of 99.0 % and 97.0 %, as determined by HPLC.

A new combined technology: Macroporous adsorption resin and high speed counter current chromatography for hydroxytyrosol separation from olive leaf enzymatic hydrolysate.

Due to three free hydroxyl groups, hydroxytyrosol (HT) presents strong bioactivity and has broad food and drug application prospects. However, there is no good separation and purification technology. In this study, separation and purification technology of HT from the ethyl acetate extraction of enzymatic hydrolysate from olive leaf (EEEH) was investigated with macroporous adsorption resin (MAR) and high-speed counter-current chromatography (HSCCC) and the separation factors were optimized. First, the adsorption properties of eight MARs (AB-8, S-8, D-101, X-5, XAD-1, XAD-5, NKA-Ⅱ, H-103) for HT enrichment were studied. The results showed that H-103 macroporous resin was adsorbent, sample concentration was 1.5 mg/mL, eluent was 30 % ethanol solution, sample loading rate was 3.0 BV/h, elution velocity was 2.0 BV/h, and HT purity of EEEH was increased from 10.23 % to 40.78 %. Then, solvent systems were examined according to partition coefficients of target component and petroleum ether: ethyl acetate: methanol: water (4:6:4:6, v/v) system was chosen. The critical experimental parameters of HSCCC were optimized as following: revolution speed was 1200 rpm and flow rate was 3 mL/min. The HT purity of macroporous resin purified EEEH was increased from 40.78 % to 85.7 %. Therefore, MAR-HSCCC combined technology could be a very effective approach to separate and purify HT from EEEH.

Efficient and fast screening and separation based on computer-aided screening and complex chromatography methods for lipoxygenase inhibitors from Ganoderma lucidum.

INTRODUCTION: Accurate screening and targeted preparative isolation of active substances from natural medicines have long been technical challenges in natural medicine research. OBJECTIVES: This study outlines a new approach for improving the efficiency of natural product preparation, focusing on the rapid and accurate screening of potential active ingredients in Ganoderma lucidum and efficient preparation of lipoxidase inhibitors, with the aim of providing new ideas for the treatment of Alzheimer's disease with G. lucidum. METHODS: The medicinal plant G. lucidum was selected through ultrafiltration coupled with liquid chromatography and mass spectrometry (UF-LC-MS) and computer-assisted screening for lipoxygenase (LOX) inhibitors. In addition, the inhibitory effect of the active compounds on LOX was studied using enzymatic reaction kinetics, and the underlying mechanism is discussed. Finally, based on the earlier activity screening guidelines, the identified ligands were isolated and purified through complex chromatography (high-speed countercurrent chromatography and semi-preparative high-performance liquid chromatography). RESULTS: Five active ingredients, ganoderic acids A, B, C2, D2, and F, were identified and isolated from G. lucidum. We improved the efficiency and purity of active compound preparation using virtual computer screening and enzyme inhibition assays combined with complex chromatography. CONCLUSION: The innovative methods of UF-LC-MS, computer-aided screening, and complex chromatography provide powerful tools for screening and separating LOX inhibitors from complex matrices and provide a favourable platform for the large-scale production of bioactive substances and nutrients.

Target-guided isolation and purification of cyclooxygenase-2 inhibitors from Meconopsis integrifolia (Maxim.) Franch. by high-speed counter-current chromatography combined with ultrafiltration liquid chromatography.

Meconopsis integrifolia (Maxim.) Franch. is used extensively in traditional Tibetan medicine for its potent anti-inflammatory properties. In this study, six cyclooxygenase-2 (COX-2) inhibitors were purified from M. integrifolia using high-speed counter-current chromatography guided by ultrafiltration liquid chromatography (ultrafiltration-LC). First, ultrafiltration-LC was performed to profile the COX-2 inhibitors in M. integrifolia. The reflux extraction conditions were further optimized using response surface methodology, and the results showed that the targeted COX-2 inhibitors could be well enriched under the optimized extraction conditions. Then the six target COX-2 inhibitors were separated by high-speed countercurrent chromatography with a solvent system composed of ethyl acetate/n-butanol/water (4:1:4, v/v/v. Finally, the six COX-2 inhibitors, including 21.2 mg of 8-hydroxyluteolin 7-sophoroside, 29.6 mg of 8-hydroxyluteolin 7-[6'''-acetylallosyl-(1→2)-glucoside], 42.5 mg of Sinocrassoside D3, 54.1 mg of Hypolaetin 7-[6'''-acetylallosyll-(l→2)-3''-acetylglucoside, 30.6 mg of Hypolaetin 7-[6'''-acetylallosyll-(l→2)-6''-acetylglucoside and 17.8 mg of Hypolaetin were obtained from 500 mg of sample. Their structures were elucidated by 1 H-NMR spectroscopy. This study reveals that ultrafiltration-LC combined with high-speed counter-current chromatography is a robust and efficient strategy for target-guided isolation and purification of bioactive molecules. It also enhances the scientific understanding of the anti-inflammatory properties of M. integrifolia but also paves the way for its further medicinal applications.

A novel strategy by combining foam fractionation with high-speed countercurrent chromatography for the rapid and efficient isolation of antioxidants and cytostatics from Camellia oleifera cake.

Camellia oleifera cake is a by-product, which is rich in functional chemical components. However, it is typically used as animal feed with no commercial value. The purpose of this study was to isolate and identify compounds from Camellia oleifera cake using a combination of foam fractionation and high-speed countercurrent chromatography (HSCCC) and to investigate their biological activities. Foam fractionation with enhanced drainage through a hollow regular decahedron (HRD) was first established for simultaneously enriching flavonoid glycosides and saponins for further separation of target compounds. Under suitable operating conditions, the introduction of HRD resulted in a threefold increase in enrichment ratio with no negative effect on recovery. A novel elution-extrusion countercurrent chromatography (EECCC) coupled with the consecutive injection mode was established for the successful simultaneous isolation of flavonoid glycosides and saponins. As a result, 38.7 mg of kaemferol-3-O-[2-O-D-glucopyranosyl-6-O-α-L-rhamnopyranosyl]-ß-D-glucopyranoside (purity of 98.17%, FI), 70.8 mg of kaemferol-3-O-[2-O-ß-D-xylopyranosyl-6-O-α-L-rhamnopyranosyl]-ß-D-glucopyranoside (purity of 97.52%, FII), and 560 mg of an oleanane-type saponin (purity of 92.32%, FIII) were separated from the sample (900 mg). The present study clearly showed that FI and II were natural antioxidants (IC50 < 35 µg/mL) without hemolytic effect. FIII displayed the effect of inhibiting Hela cell proliferation (IC50 < 30 µg/mL). Further erythrocyte experiments showed that this correlated with the extremely strong hemolytic effect of FIII. Overall, this study offers a potential strategy for efficient and green isolation of natural products, and is beneficial to further expanding the application of by-products (Camellia oleifera cake) in food, cosmetics, and pharmacy.

High-speed countercurrent chromatography with offline detection by electrospray mass spectrometry and nuclear magnetic resonance detection as a tool to resolve complex mixtures: A practical approach using Coffea arabica leaf extract.

INTRODUCTION: Many secondary metabolites isolated from plants have been described in the literature owing to their important biological properties and possible pharmacological applications. However, the identification of compounds present in complex plant extracts has remained a great scientific challenge, is often laborious, and requires a long research time with high financial cost. OBJECTIVES: The aim of this study was to develop a method that allows the identification of secondary metabolites in plant extracts with a high degree of confidence in a short period of time. MATERIAL AND METHODS: In this study, an ethanolic extract of Coffea arabica leaves was used to validate the proposed method. Countercurrent chromatography was chosen as the initial step for extraction fractionation using gradient elution. Resulting fractions presented a variation of compounds concentrations, allowing for statistical total correlation spectroscopy (STOCSY) calculations between liquid chromatography coupled with high-resolution tandem mass spectrometry (LC-HRMS/MS) and NMR across fractions. RESULTS: The proposed method allowed the identification of 57 compounds. Of the annotated compounds, 20 were previously described in the literature for the species and 37 were reported for the first time. Among the inedited compounds, we identified flavonoids, alkaloids, phenolic acids, coumarins, and terpenes. CONCLUSION: The proposed method presents itself as a valid alternative for the study of complex extracts in an effective, fast, and reliable way that can be reproduced in the study of other extracts.

Separation of polysaccharides from Lycium barbarum L. by high-speed countercurrent chromatography with aqueous two-phase system.

The traditional method for isolation and purification of polysaccharides is time-consuming. It often involves toxic solvents that destroy the function and structure of the polysaccharides, thus limiting in-depth research on the essential active ingredient of Lycium barbarum L. Therefore, in this study, high-speed countercurrent chromatography (HSCCC) and aqueous two-phase system (ATPS) were combined for the separation of crude polysaccharides of Lycium barbarum L. (LBPs). Under the optimized HSCCC conditions of PEG1000-K2HPO4-KH2PO4-H2O (12:10:10:68, w/w), 1.0 g of LBPs-ILs was successfully divided into three fractions (126.0 mg of LBPs-ILs-1, 109.9 mg of LBPs-ILs-2, and 65.4 mg of LBPs-ILs-3). Moreover, ATPS was confirmed as an efficient alternative method of pigment removal for LBPs purification, with significantly better decolorization (97.1 %) than the traditional H2O2 method (88.5 %). Then, the different partitioning behavior of LBPs-ILs in the two-phase system of HSCCC was preliminarily explored, which may be related to the difference in monosaccharide composition of polysaccharides. LBPs-ILs-1 exhibited better hypoglycemic activities than LBPs-ILs-2 and LBPs-ILs-3 in vitro. Therefore, HSCCC, combined with aqueous two-phase system, was an efficient separation and purification method with great potential for separating and purifying active polysaccharides in biological samples.

Modeling of analytical, preparative and industrial scale counter-current chromatography separations.

Analytical, preparative and industrial scale counter-current chromatography (CCC) processes differ in the volumes of the loaded solution of components to be separated and in the design of the equipment. Preliminary mathematical modeling is necessary for selection of the optimal design and operation mode of these CCC separations. This study aims to compare simulations of CCC separations at different scales, using an exact description based on the model of equilibrium cells and a much simpler approximate solution based on the Gaussian distribution. Equations for modeling CCC separations of different scales and examples of simulation these separations are presented. It is shown that the discrepancy between the two simulations increases with an increase in the volume of the loaded solution of the components and a decrease in the number of equilibrium cells of a CCC device. In analytical and preparative separations, which are based on complex centrifugal devices, and relatively small sample volumes are injected, approximate equations can be used to simulate various options of CCC separation. In industrial-scale CCC separations, large volumes of the solution of components may be loaded, and as we have proposed previously, these separations can be based on a cascade of mixer-settler extractors. In this case, a more accurate mathematical description based on the cell model equations should be used for modeling.

Evaluation of dynamic control of continuous capture with periodic counter-current chromatography under feedstock variations.

Multi-column periodic counter-current chromatography is a promising technology for continuous antibody capture. However, dynamic changes due to disturbances and drifts pose some potential risks for continuous processes during long-term operation. In this study, a model-based approach was used to describe the changes in breakthrough curves with feedstock variations in target proteins and impurities. The performances of continuous capture of three-column periodic counter-current chromatography under ΔUV dynamic control were systematically evaluated with modeling to assess the risks under different feedstock variations. As the concentration of target protein decreased rapidly, the protein might not breakthrough from the first column, resulting in the failure of ΔUV control. Small reductions in the concentrations of target proteins or impurities would cause protein losses, which could be predicted by the modeling. The combination of target protein and impurity variations showed complicated effects on the process performance of continuous capture. A contour map was proposed to describe the comprehensive impacts under different situations, and nonoperation areas could be identified due to control failure or protein loss. With the model-based approach, after the model parameters are estimated from the breakthrough curves, it can rapidly predict the process stability under dynamic control and assess the risks under feedstock variations or UV signal drifts. In conclusion, the model-based approach is a powerful tool for continuous process evaluation under dynamic changes and would be useful for establishing a new real-time dynamic control strategy.

Large-scale separation of alkaloids from Corydalis decumbens by pH-zone-refining centrifugal partition chromatography and their anticomplement activity.

Centrifugal partition chromatography in the pH-zone-refining mode was successfully applied to the separation of alkaloids from the crude extract of Corydalis decumbens. The experiment was performed with a two-phase solvent system composed of petroleum ether-ethyl acetate-ethanol-water (5:5:3:7, v/v/v/v) where triethylamine (10 mM) was added to the stationary phase and hydrochloric acid (10 mM) to the mobile phase. From 1.6 g of the crude extract, 43 mg protopine, 189 mg (+)-egenine, and 158 mg tetrahydropalmatine were obtained with a purity of 98.2%, 94.6%, and 96.7%, respectively. Tetrahydropalmatine showed an interesting anticomplement effect with CH50 0.11 and AP50 0.25 mg/mL, respectively. In a mechanistic study, tetrahydropalmatine interacted with C1, C3, C4, and C5 components in the complement activation cascade.