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Herein, ultrasound-assisted mixture extraction (UAME) and online extraction solution concentration (OESC) were conducted to extract products from crops and plants. These techniques were coupled with parallel countercurrent chromatography (PCCC) and applied for continuous extraction and online isolation of chemical constituents from Phellinus vaninii. The UAME instrument comprises extraction and solution separation chambers. It provides higher extraction efficiency and fewer impurities and is suitable for processing various sample matrices. The OESC device comprises a spray nozzle, concentrating cylinder, and hot-blast air nozzle. The mechanical parameters for UAME and OESC were optimized, and the operation of online UAME and OESC coupled with PCCC was described. Raw plant materials were extracted using a two-phase extractant comprising petroleum-ethyl acetate-ethanol-water (0.5:2.0:0.5:2.0, v/v/v/v). The aqueous and organic phases were then concentrated using the OESC technique. Two CCC runs were conducted for preparatory work. After extraction and online concentration, the concentrate was pumped into the CCC for separation. During PCCC separation, continuous automated extraction and concentration were still conducted. When the first cycle of the UAME/OESC/PCCC was completed, followed by the initiation of the second cycle, and the process was continued. Six target compounds with purities exceeding 97.22% were successfully separated using the CCC solvent systems comprising n-hexane-ethyl acetate-acetonitrile-water (5.5:2.5:5.0:0.4, v/v/v/v) and n-butanol-ethanol-water (4.5:1.3:6.5, v/v/v). Compared with conventional extraction methods, the proposed UAME/OESC/PCCC method has higher efficiency, facilitates high-purity separation of analytes, and offers opportunity for automation and systematic preparation of natural products., (Copyright © 2021 Elsevier B.V. All rights reserved.)

In our recent study, the design and performance of type-I counter-current chromatography (CCC) were changed and improved by multilayer coil. In the present study, the performance of protein separation using the multilayer coil configuration in type-I CCC was investigated under various flow rates. In order to overcome the noise signal interrupting the target peaks, a hollow fiber membrane dialyzer was inserted between CCC column outlet and inlet of the monitor. The separation was performed with a two-phase solvent system composed of polyethylene glycol 1000/dibasic potassium phosphate each at 12.5% (w/w) in deionized water and lysozyme and myoglobin were used as the test samples. The retention of stationary phase in the head to tail elution mode was lower than that in the tail to head elution mode, but Rs (peak resolution) was opposite. The intermittently pressed tubing can efficiently improve the performance of protein separation by type-I CCC with the multilayer coil. The best Rs was 1.54 and obtained at the flow rate of 0.10 ml/min under a revolution speed of 200 rpm. The present result indicated that type-I CCC can be as a potential powerful tool of protein separation for the first time., (Copyright © 2018 Elsevier B.V. All rights reserved.)

A simple and efficient method based on ultrafiltration liquid chromatography-mass spectrometry (UFLC-MS) was applied to rapidly screen and identify ligands for lactate dehydrogenase (LDH) from the flowers of Pueraria lobata, and the compounds were assessed for anti-stroke activity using a PC12 cell model. Seven major isoflavones, kakkalide, 3'-hydroxy puerarin, puerarin, puerarin xyloside, tectoridin, tectorigenin, and ononin, were identified as potent LDH inhibitors. A continuous online method, which consisted of microwave-assisted extraction and countercurrent chromatography (MAE-CCC), was newly developed for scaled-up production of these compounds with high purity and efficiency. This novel approach, using UFLC-MS coupled with MAE-CCC and a PC12 cell model, provided a powerful tool for screening, extraction, and separation of LDH inhibitors from complex samples, and a useful platform for the large-scale production of functional food and nutraceutical ingredients., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Vine tea (Ampelopsis grossedentata), a widely used healthy tea, beverage and herbal medicine, exhibited strong antioxidant activity. However, systematic purification of antioxidants, especially for those with similar structures or polarities, is a challenging work. Here, we present a novel at-line hyphenation of high-speed countercurrent chromatography with Sephadex LH-20 column chromatography (HSCCC-Sephadex LH-20 CC) for rapid and efficient separation of antioxidants from vine tea target-guided by 1,1-diphenyl-2-picryl-hydrazyl radical-high performance liquid chromatography (DPPH-HPLC) experiment. A makeup pump, a six-port switching valve and a trapping column were served as interface. The configuration had no operational time and mobile phase limitations between two dimensional chromatography and showed great flexibility without tedious sample-handling procedure. Seven targeted antioxidants were firstly separated by stepwise HSCCC using petroleum ether-ethyl acetate-methanol-water (4:9:4:9, v/v/v/v) and (4:9:5:8, v/v/v/v) as solvent systems, and then co-eluted antioxidants were on-line trapped, concentrated and desorbed to Sephadex LH-20 column for further off-line purification by methanol. It is noted that six elucidated antioxidants with purity over 95% exhibited stronger activity than ascorbic acid (VC). More importantly, this at-line hyphenated strategy could sever as a rapid and efficient pathway for systematic purification of bioactive components from complex matrix., (Copyright © 2016 Elsevier B.V. All rights reserved.)

An effective high-speed counter-current chromatography (HSCCC) method was established for the preparative isolation and purification of astaxanthin from Phaffia rhodozyma. With a two-phase solvent system composed of n-hexane-acetone-ethanol-water (1:1:1:1, v/v/v/v), 100mg crude extract of P. rhodozyma was separated to yield 20.6mg of astaxanthin at 92.0% purity. By further one step silica gel column chromatography, the purity reached 99.0%. The chemical structure of astaxanthin was confirmed by thin layer chromatography (TLC), UV spectroscopy scanning, high performance liquid chromatography with a ZORBAX SB-C18 column and a Waters Nova-pak C18 column, and ESI/MS/MS., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Two new prenylated flavonoids, sinoflavonoids A-B, were isolated from the dried fruits of Sinopodophyllum emodi by silica gel column chromatography (SGCC) and high-speed counter-current chromatography (HSCCC). The 95% ethanol extract was partitioned with petroleum ether, dichloromethane, ethyl acetate, and n-butanol in water, respectively. The ethyl acetate fraction was pre-separated by SGCC with a petroleum ether-acetone gradient. The eluates containing target compounds were further separated by HSCCC with n-hexane-ethyl acetate-methanol-water (4:6:4:4, v/v). Finally, 17.3mg of sinoflavonoid A and 25.9mg of sinoflavonoid B were obtained from 100mg of the pretreated concentrate. The purities of sinoflavonoid A and sinoflavonoid B were 98.47% and 99.38%, respectively, as determined by HPLC. Their structures were elucidated on the basis of spectroscopic evidences (HR-ESI-MS, (1)H-NMR, (13)C-NMR, HSQC, HMBC). The separation procedures proved to be efficient, especially for trace prenylated flavonoids., (Copyright © 2014 Elsevier B.V. All rights reserved.)

So far, research workers have built several math models of counter-current chromatography (CCC), such as cell model, eluting counter-current distribution model, continuous-stirred tank reactors model, and probability model. Based on plate theory and Van't Hoff equation, a new temperature dependence plate model has been established in this paper. When temperature was taken into consideration, the error of retention time was significantly reduced. The relationship between temperature and partition coefficient K can be summarized into linear equations (logK=A-B/T), which can be introduced to the plate model where A and B are constant and T is the temperature in Kelvin. K values decreased with the increasing temperature. Since resolution values (R) are related to K values, the change of temperature finally leads to the change of resolutions between different compounds. K values at different temperatures of a certain compound in a given system were calculated according to the equation above. New program with a temperature parameter was applied in prediction of experimental results, which diminished the error in modelling prediction. Besides, it is difficult for the CCC work when two compounds have similar K values. However, separation can be achieved by changing temperature, which can be directed by this new model., (Copyright © 2013 Elsevier B.V. All rights reserved.)

The performance of protein separation using the figure-8 column configuration in centrifugal counter-current chromatography was investigated under various flow rates and revolution speeds. The separation was performed with a two-phase solvent system composed of polyethylene glycol 1000/potassium phosphate each at 12.5% (w/w) in water and with lysozyme and myoglobin as test samples. In order to improve tracing of the elution curve, a hollow fiber membrane dialyzer was inserted at the inlet of the UV detector. The results showed that the retention of stationary phase (Sf) and resolution (Rs) increased with decreased flow rate and increased revolution speed. The highest Rs of approximately 1 was obtained at a flow rate of 0.01 mL/min under a revolution speed of 1200 rpm with a 3.4 mL capacity column., (Copyright © 2011 Elsevier B.V. All rights reserved.)

pH-Zone-refining counter-current chromatography was successfully applied for the preparative separation of alkaloids from Dactylicapnos scandens. The two-phase solvent system was composed of petroleum ether-ethyl acetate-methanol-water (3:7:1:9, v/v), where 20 mM of triethylamine (TEA) was added to the upper phase as a retainer and 5 mM of hydrochloric acid (HCl) to the aqueous phase as an eluter. In this experiment, the apparatus with an adjustable length of the separation column was used for the separation of alkaloids from D. scandens and the resolution of the compounds can be remarkably improved by increasing the length of the separation column. As a result, 70 mg protopin, 30 mg (+) corydine, 120 mg (+) isocorydine and 40 mg (+) glaucine were obtained from 1.0 g of the crude extracts and each with 99.2%, 96.5%, 99.3%, 99.5% purity as determined by HPLC. The chemical structures of these compounds were confirmed by positive ESI-MS and (1)H NMR., (Copyright © 2011 Elsevier B.V. All rights reserved.)

An on-line method based upon dynamic microwave-assisted extraction (DMAE) coupled with high-speed counter-current chromatography (HSCCC) was developed for continuous isolation of nevadensin from Lyeicnotus pauciflorus Maxim. The DMAE parameters were optimized by means of the Box-Behnken design. The maximum extraction yield was achieved using 30:1 ml/g of liquid-solid ratio, 10 ml/min of solvent flow rate and 200 W of microwave power. The crude extracts were then separated by HSCCC with a two-phase solvent system composed of n-hexane-ethyl acetate-methanol-water (7:3:5:5, v/v/v/v). 13.0mg of nevadensin was isolated from 15.0 g original sample by HSCCC with five times sample injection in 12h, and the isolation yield of nevadensin was 0.87 mg/g. The average purity of nevadensin was higher than 98.0%. The chemical structure of collected fraction was identified by HPLC, ESI-MS and (1)H NMR. The results indicated that this on-line method was effective and fast for high-throughput isolation of nevadensin from L. pauciflorus Maxim., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Punicalagin, the main ingredient of pomegranate (Punica granatum L.) husk, is a high molecular weight polyphenolic compound. It has shown remarkable pharmacological activities attributed in the presence of dissociable OH groups. To isolate punicalagin, previous methods included labor intensive and expensive solid phase extractions by column chromatography (C-18, polyamides, dellulose, Sephadex Lipophilic LH-20, Diaion HP20). High-speed countercurrent chromatography (HSCCC) was used for isolation and purification of punicalagin from pomegranate husk. Using preparative HSCCC about a 350 mg amount of the crude extract was separated, yielding 105 mg of punicalagin at a high-purity of over 92%. Eighty milligrams of gallic acid was simultaneously separated as another product, at a purity of 75%.

New aqueous-aqueous two-phase systems composed of relatively low molecular weight polymers such as polyethylene glycol (PEG) (Mr: 1000-4000) and dextran (Mr: 10,000 and 40,000) were evaluated for purification of proteins by counter-current chromatography (CCC). The compositions of aqueous two-phase systems were optimized by measuring parameters such as viscosity and volume ratio between the two phases. CCC purification of a glucosyltransferase (GTF) from Streptococcus mutans (SM) cell-lysate was successfully demonstrated with a 7.5% PEG 3350-10% dextran T40 system containing 10mM potassium phosphate buffer at pH 9.0. After CCC purification, both PEG and dextran contained in the CCC fractions were easily removed by ultrafiltration in a short period of time. The fractionated column contents containing GTF were analyzed by enzymatic activity as well as sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. The recovery of the enzyme from CCC fraction was over 95% as estimated by enzymatic activities.

Scutellarin, a flavone glycoside, popularly applied for the treatment of cardiopathy, has been purified in two-step purification by high-speed counter-current chromatography (HSCCC) from Erigeron breviscapus (vant.) Hand. Mazz. (Deng-zhan-hua in Chinese), a well-known traditional Chinese medicinal plant for heart disease. Two solvent systems, n-hexane-ethyl acetate-methanol-acetic acid-water (1:6:1.5:1:4, v/v/v/v/v) and ethyl acetate-n-butanol-acetonitrile-0.1% HCl (5:2:5:10, v/v/v/v) were used for the two-step purification. The purity of the collected fraction of scutellarin was 95.6%. This study supplies a new alternative method for purification of scutellarin.