Rapid purification and scale-up of tilianin using counter-current chromatography with rectangular horizontal tubing.

In counter-current chromatography (CCC), linear scale-up is an ideal amplification strategy. However, when transferring from analytical to predictable preparative processes with high throughput, linear scale-up would be challenging due to limitations imposed by differences in instrument parameters, such as gravitational forces, tubing cross-section area, tubing length, column volume and flow rate. Some effective scale-up strategies have been studied for different instrument parameters, but so far, these scale-up works have only been tested on standard circular (SC) tubing. The previous research of our group found that rectangular horizontal (RH) tubing can double the separation efficiency compared with conventional SC tubing, and has industrial production potential. This paper used the separation of tilianin from Dracocephalum moldavica L. as an example to demonstrate how to scale up the optimized process from analytical SC tubing to preparative RH tubing. After systematic optimization of solvent systems, sample concentration and flow rate on the analytical CCC, the optimized parameters obtained were successfully transferred to the preparative CCC. The results showed that a crude sample of 2.07 g was successfully separated using a solvent system of n-hexane - ethyl acetate - ethanol - water (1:4:1:5, v/v/v/v) in reversed phase mode, and the three consecutive separations produced a total of 380 mg tilianin in 75 min with high purities of 98.3%, as analyzed by HPLC. The total throughput achieved from the analytical to semi-preparative scale was improved by 138 times (from 12 mg/h to 1.66 g/h), while the column volume was increased by only 46.5 times (from 15.5 mL to 720 mL). This is the successful application of CCC for the separation and purification of tilianin. Given that SC tubing is the traditional configuration for CCC columns, this study is a necessary step to prove the applicability of RH tubing columns for routine use and potential large-scale industrial applications.

Development of ultrasound-assisted mixture extraction and online extraction solution concentration coupled with countercurrent chromatography for the preparation of pure phytochemicals from Phellinus vaninii.

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.

Bioassay-Guided Separation of Centipeda minima Using Comprehensive Linear Gradient Centrifugal Partition Chromatography.

A comprehensive linear gradient solvent system for centrifugal partition chromatography (CPC) was developed for the bioassay-guided isolation of natural compounds. The gradient solvent system consisted of three different ternary biphasic solvents types: n-hexane-acetonitrile-water (10:2:8, v/v), ethyl acetate-acetonitrile-water (10:2:8, v/v), and water-saturated n-butanol-acetonitrile-water (10:2:8, v/v). The lower phase of the n-hexane-acetonitrile-water (10:2:8, v/v) was used as the stationary phase, while its upper phase, as well as ethyl acetate-acetonitrile-water (10:2:8), and water-saturated n-butanol-acetonitrile-water (10:2:8, v/v) were pumped to generate a linear gradient elution, increasing the mobile phase polarity. We used the gradient CPC to identify antioxidant response elements (AREs), inducing compounds from Centipeda minima, using an ARE-luciferase assay in HepG2 cells, which led to the purification of the active molecules 3-methoxyquercetin and brevilin A. The developed CPC solvent systems allow the separation and isolation of compounds with a wide polarity range, allowing active molecule identification in the complex crude extract of natural products.

Batch chromatography with recycle lag. I-Concept and design.

This is the first of a two-part study in which we explore the concept of batch chromatography with recycle lag, concluding with the design, construction, and experimental validation of a prototype that embodies the physical realization of this concept. Moreover, the apparatus is simple to set up in particular in view of large-scale applications. Here the theory behind batch chromatography with recycle lag is revisited and extended, with emphasis on the mathematical formulation and procedure for deriving the single-column batch analogue of any variant of multicolumn simulated countercurrent chromatography. By resorting to selected examples, namely GE Healthcare Bio-science's three-column periodic countercurrent chromatography, Novasep's sequential multicolumn chromatography, and a few hypothetical multicolumn processes, we discuss how the theory can be operationalized. Finally, we conclude by describing the design of a device or apparatus-an eluate recycling device (ERD)-to physically realize the proposed concept. The ERD implements an approximate "first in, first out" method for organizing and manipulating the to-be-recycled fractions of eluate collected from the chromatography column, where the oldest (first) amount fluid, or 'head' of the fraction, is the first to exit and be recycled to the column.

Ionic liquid-modified countercurrent chromatographic isolation of high-purity delphinidin-3-rutinoside from eggplant peel.

Delphinidin-3-rutinoside, a high-value of anthocyanin, was isolated and purified by ionic liquid (IL)-modified countercurrent chromatography (CCC) from waste peel of eggplant (Solanum melongena), one of the most common vegetables consumed all around the world. Different conventional CCC and IL-CCC solvent systems were evaluated in respect of partition coefficient (K), separation factor (α), and stationary phase retention factor (Sf ) to separate polar target and other components. Basic solvent system, kind of ILs, and amount of ILs were systematically optimized by totally K-targeted strategy, which drastically reduced the experimental effort. Finally, a novel CCC two-phase solvent system (methyl tert-butyl ether-butanol-acetonitrile-1% trifluoroacetic acid water-1-butyl-3-methylimidazolium hexafluorophosphate ([C4 MIM][PF6 ]) [2:4:1:5:0.2; v/v/v/v/v]) was successfully established and applied. The baseline separation of target fraction was obtained in one cycle process. The purity of delphinidin-3-rutinoside was over 99%. Moreover, the distribution behavior of different kinds of ILs in biphasic solvent system and the removal method of ILs were explored. The results showed that hydrophobic IL significantly improved the partition of polar anthocyanin in organic solvent system, thereby the separation resolution and stationary phase retention through introducing intermolecular forces. This IL-modified CCC strategy may be applied for the separation of other anthocyanins from variety of natural food resources and waste.

Hydrophobic and hydrophilic interactions in countercurrent chromatography.

Countercurrent chromatography (or counter-current chromatography, CCC) is a unique support-free liquid-liquid partition chromatography. Since it was invented by Y. Ito in 1960s, CCC has been widely accepted and applied as popular separation and purification technique for natural and synthetic complex. However, up to date there is little study to address on hydrophobic and hydrophilic interactions in CCC process, although hydrophobic interaction chromatography (HIC) and hydrophilic interaction chromatography (or hydrophilic interaction liquid chromatography, HILIC) as solid-support chromatographic techniques are widely applied at different stages of downstream processing. In fact, hydrophobic and hydrophilic interactions might be more popular in CCC separation than that in the liquid chromatography. For example, adding small solvents or additives in two-phase solvent systems may change significantly hydrophobic or hydrophilic interactions between solvents and solutes. Normally, CCC separation employs a light and hydrophobic organic phase as the stationary phase, and a heavy and hydrophilic aqueous phase as the mobile phase. Hydrophobic interactions between the solvent system and solutes (targets) will increase the partition coefficients (K values) of solutes and lengthen retention time, while hydrophilic interactions will reduce the K values and separation time. In this work, we aim to provide a general insight on the hydrophobic and hydrophilic interactions in CCC separation. We also highlight the current advances in utilizing the hydrophobic and hydrophilic interactions for K-targeted CCC separation and purification.

Continuous separation of maslinic and oleanolic acids from olive pulp by high-speed countercurrent chromatography with elution-extrusion mode.

In this work, a continuous high-speed countercurrent chromatography method has been developed on the basis of elution-extrusion mode and this method was successfully applied to the separation of maslinic and oleanolic acid from the extract of olive pulp. In the process of 'elution', the sample solution was continuously loaded into the column and the maslinic acid was steadily eluted out in this step while highly retained oleanlic acid always stayed in the column. In the process of 'extrusion', the oleanlic acid was pushed out of the column with the stationary phase. In this way, we achieved a large sample loading. A total of 120 mL sample solution (about 89.55% of the column volume) which contains 600 mg olive pulp extract was pumped in the apparatus by a constant-flow pump and the maslinic and oleanolic acids were largely separated within 120 min. Both of these two compounds presented high yields and high purities (271.6 mg for maslinic acid with 86.7% and 83.9 mg oleanolic acids with 83.4%).

Isolation and Purification of a Neuroprotective Phlorotannin from the Marine Algae Ecklonia maxima by Size Exclusion and High-Speed Counter-Current Chromatography.

Phlorotannins are polyphenolic metabolites of marine brown algae that have been shown to possess health-beneficial biological activities. An efficient approach using a combination of high-speed counter-current chromatography (HSCCC) and size exclusion chromatography with a Sephadex LH-20 has been successfully developed for the isolation and purification of a neuroprotective phlorotannin, eckmaxol, from leaves of the marine brown algae, Ecklonia maxima. The phlorotannin of interest, eckmaxol, was isolated with purity >95% by HSCCC using an optimized solvent system composed of n-hexane-ethyl acetate-methanol-water (2:8:3:7, v/v/v/v) after Sephadex LH-20 size exclusion chromatography. This compound was successfully purified in the quantity of 5.2 mg from 0.3 kg of the E. maxima crude organic extract. The structure of eckmaxol was identified and assigned by NMR spectroscopic and mass spectrometric analyses. The purification method developed for eckmaxol will facilitate the further investigation and development of this neuroprotective agent as a drug lead or pharmacological probe. Furthermore, it is suggested that the combination of HSCCC and size exclusion chromatography could be more widely applied for the isolation and purification of phlorotannins from marine algae.

Multistage continuous countercurrent diafiltration for formulation of monoclonal antibodies.

There is growing interest in the development of fully integrated and continuous biomanufacturing processes for the production of monoclonal antibody products. A recent study has demonstrated the feasibility of using a two-stage countercurrent diafiltration (DF) process for continuous product formulation, but this system did not provide sufficient levels of buffer exchange for most applications. The objective of this study was to design and test a three-stage countercurrent DF system that could achieve at least 99.9% buffer exchange over 24 hr of continuous operation. Experimental data were obtained using concentrated solutions of human immunoglobulin G as a model protein, with the extent of vitamin B12 removal used to track the extent of DF. Pall Cadence™ inline concentrators with Delta 30 kD regenerated cellulose membranes were used in the three stages to achieve high conversion in a single pass. The three-stage system showed stable operation with >99.9% vitamin B12 removal and a minimal increase in pressure over the full 24 hr. Modules were effectively cleaned using sodium hydroxide, with nearly complete recovery of water permeability. A simple economic analysis was presented that accounts for the trade-offs between quantity of buffer used and membrane costs for this type of countercurrent staged DF process. The results provide important insights to the design and operation of a continuous process for antibody formulation.

Closed-loop recycling dual-mode counter-current chromatography. A theoretical study.

Closed-loop recycling dual-mode counter-current chromatography (CLR DM CCC) processes consist of two successive separation stages: separation of solutes in the recycling closed-loop with mobile x-phase and separation of solutes in the counter-current mode with mobile y-phase. Several variants of the implementation of this separation method can be developed: the closed-loop recycling stage may consist of one or several successive separation steps; all components of a mixture can pass through both stages of separation or individual components may be withdrawn from the system at different stages. In this study, such separation processes are theoretically investigated, and simple equations for simulation presented. These equations can help to simulate and select a suitable separation scheme for a given mixture of components. Several examples of separation by CLR DM CCC method are discussed. Examples of simulation are presented in "Mathcad" program. It is shown that CLR DM CCC allows separation of solutes with similar partition coefficients and separation of complex mixtures containing solutes with widely different partition coefficients, providing concentrated fractions of the separated solutes.

Hollow fiber countercurrent dialysis for continuous buffer exchange of high-value biotherapeutics.

Buffer exchange, desalting, and formulation of high-value biotherapeutics are currently performed using batch diafiltration (DF); however, this type of tangential flow filtration process may be difficult to implement as part of a fully continuous biomanufacturing process. The objective of this study was to explore the potential of using countercurrent dialysis for continuous protein formulation and buffer exchange. Experiments were performed using concentrated solutions of immunoglobuin G (IgG) with commercially available hollow fiber dialyzers having 1.5 and 1.8 m2 membrane surface area. More than 99.9% buffer exchange was obtained over a range of conditions, as determined from the removal of a model impurity (vitamin B12 ). The dialyzers were able to process more than 0.5 kg of IgG per day in an easily scalable low-cost process. In addition, buffer requirements were less than 0.02 L of buffer per gram IgG, which is several times less than that used in current batch DF processes. These results clearly demonstrate the potential of using low-cost hollow fiber dialyzers for buffer exchange and product formulation in continuous bioprocessing. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2763, 2019.

The effect of increasing centrifugal acceleration/force and flow rate for varying column aspect ratios on separation efficiency in Counter-Current Chromatography.

Increasing column/tubing aspect ratio has been shown in a feasibility study to improve column efficiency when operating in reversed phase mode. This paper contains a thorough investigation on how increases in mobile phase flow and centrifugal force field affect stationary phase retention and column efficiency (as measured by the resolution between adjacent peaks) for columns wound with rectilinear tubing of different aspect ratio. The study uses a Mini CCC instrument operating from 1500 to 2100 rpm (126-246 g) to compare three columns with the same cross-sectional area but different aspect ratio - rectangular horizontal (force field perpendicular to the flat side - aspect ratio 3.125); square (aspect ratio 1.0) and rectangular vertical (flat side parallel with force field - aspect ratio 0.32). Columns are compared by measuring stationary phase retention, resolution and normalized resolution for 3 different mobile phase flow rates 2, 4 and 8 ml/min in both normal phase and reversed phase modes. The results with rectilinear tubing are compared to conventional circular tubing with the same cross-sectional area. The results show that resolution increases with aspect ratio and that at the highest aspect ratio the highest flow rate can maintain a high efficiency only if the highest g-field of 246 g is used. When comparing the rectangular horizontal tubing which gave the best results with conventional circular tubing with the same cross-sectional area a 45% improvement was found in reversed phase mode and a 51% improvement in normal phase mode over the conventional circular cross-section tubing. In other words, a rectangular horizontally wound bobbin with half the length of tubing can achieve the same result as a circular one. These are very significant results for halving separation times analytically or enabling designers to produce new instruments of the same capacity with a much-reduced size.

Type-I counter-current chromatography with the multilayer coil for protein separation.

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.

Direct from solid natural products to pure compounds in a single step: Coupling online extraction with high-speed counter-current chromatography.

Extraction is the most important step in the purification of bioactive compounds from natural products. This study introduces a simple online extraction strategy coupled with high-speed counter-current chromatography for efficient extraction and purification of bioactive components from solid natural products. For online extraction strategy, 1.0 g of ground Mangnolia officinalis or Piper nigrum was loaded into a guard column, which was then positioned on the manual injection valve instead of the sample loop. Bioactive components were directly extracted by the mobile phase of high-speed counter-current chromatography, and then transferred into high-speed counter-current chromatography for purification. In addition, the compatibility of the developed methodology for direct purification of bioactive components from fresh M. officinalis was successfully demonstrated. Obviously, in comparison with traditional offline heat-reflux extraction, online extraction avoided the instrument, time, solvent, and energy consumption, and purified two phenolic compounds (honokiol and magnolol) from M. officinalis and three alkaloids (piperyline, piperine, and piperanine) from P. nigrum with high extraction efficiency. The superiority of the developed methodology is to establish an easy, rapid, and efficient technique for the purification of a wide variety of bioactive components from solid natural products.

Industrial countercurrent chromatography separations based on a cascade of centrifugal mixer-settler extractors.

In hydrometallurgy, traditional extraction technologies, in particular, for isolation and purification of rare-earth metals include a number of processing steps using up to hundreds of mixer-settler extractors. These technologies could be greatly simplified by using the methods of countercurrent chromatography (CCC) separation. However, the current CCC equipment cannot process large volumes of feed material formed during the industrial production of these metals. In this paper, the cascade of centrifugal mixer-settler extractors assembled as a multi-stage unit is suggested for industrial application of CCC and discussed.

Simultaneous concentration and separation of target compounds from multicomponent mixtures by closed-loop recycling countercurrent chromatography.

Closed-loop recycling countercurrent chromatography (CLR CCC) with multiple sample injection has been shown to provide simultaneous concentration and separation of target compounds from multicomponent mixtures. Previous analysis of CLR CCC with multiple sample injections has been limited to the ideal recycling model, which neglects the effects caused by the pump and connecting lines. In this study, an analysis of the process is carried out based on the non-ideal recycling model: recycling chromatograms at two points of the closed-loop - the inlet of the column (A) and the outlet of the column (B) - are considered. The sample is repeatedly introduced at the inlet of the column when the circulating peak of target compound passes point A. Analytical expressions are developed, allowing the design and simulation of different variants of simultaneous separation and concentration of target compounds from multicomponent mixtures. Examples of separation of target compounds from three and five-component mixtures are discussed. Experimental results are presented demonstrating a reasonable agreement between the theory and the experiment. Due to its ability to concentrate individual solutes, CRL CCC with multiple sample injections can become an efficient analytical method to determine minor components in complex mixtures.

Preparative enantioseparation of loxoprofen precursor by recycling countercurrent chromatography with hydroxypropyl-ß-cyclodextrin as a chiral selector.

Recycling countercurrent chromatography was successfully applied to the resolution of 2-(4-bromomethylphenyl)propionic acid, a key synthetic intermediate for synthesis of nonsteroidal anti-inflammatory drug loxoprofen, using hydroxypropyl-ß-cyclodextrin as chiral selector. The two-phase solvent system composed of n-hexane/n-butyl acetate/0.1 mol/L citrate buffer solution with pH 2.4 (8:2:10, v/v/v) was selected. Influence factors for the enantioseparation were optimized, including type of substituted ß-cyclodextrin, concentration of hydroxypropyl-ß-cyclodextrin, separation temperature, and pH of aqueous phase. Under optimized separation conditions, 50 mg of 2-(4-bromomethylphenyl)propionic acid was enantioseparated using preparative recycling countercurrent chromatography. Technical details for recycling elution mode were discussed. The purities of both the S and R enantiomers were over 99.0% as determined by high-performance liquid chromatography. The enantiomeric excess of the S and R enantiomers reached 98.0%. The recovery of the enantiomers from eluted fractions was 40.8-65.6%, yielding 16.4 mg of the S enantiomer and 10.2 mg of the R enantiomer. At the same time, we attempted to enantioseparate the anti-inflammatory drug loxoprofen by countercurrent chromatography and high-performance liquid chromatography using a chiral mobile phase additive. However, no successful enantioseparation was achieved so far.

A multilayer coil in type-I counter-current chromatography.

In the present study, a novel model of type-I counter-current chromatography was developed to simplify the separation column manufacture and extend its application ranges. In this system, a multilayer coil was used as the separation column of type-I counter-current chromatography for the first time. The chromatographic performance of this apparatus was evaluated in terms of retention of the stationary phase (Sf), theoretical plate (N) and peak resolution (Rs) using dipeptides and DNP-amino acids as test samples with two classic solvent systems composed of 1-butanol-acetic acid-water (4:1:5, v/v) (BAW) and hexane-ethyl acetate-methanol-0.1 M HCl (1:1:1:1, v/v) (HEMW), respectively. The results indicated that the performance was optimal at the revolution speed of 200 rpm. The lower flow rate was more beneficial to retention of stationary phase and peak resolution. Over all results of our study revealed that the new type-I counter-current chromatography with a multilayer coil has a high application potential.

Magnetic nanoparticles and high-speed countercurrent chromatography coupled in-line and using the same solvent system for separation of quercetin-3-O-rutinoside, luteoloside and astragalin from a Mikania micrantha extract.

A new in-line method of magnetic nanoparticles (MNPs) coupled with high-speed countercurrent chromatography (HSCCC) using a same solvent system during the whole separation process was established to achieve the rapid separation of flavonoids from Mikania micrantha. The adsorption and desorption capacities of five different MNPs for flavonoid standards and Mikania micrantha crude extract were compared and the most suitable magnetic nanoparticle Fe3O4@SiO2@DIH@EMIMLpro was selected as the in-line MNP column. An in-line separation system was established by combining this MNP column with HSCCC through a six-way valve. The comparison between two solvent systems n-hexane-ethyl acetate-methanol-water (3:5:3:5, v/v) and ethyl acetate-methanol-water (25:1:25, v/v) showed that the latter solvent system was more suitable for simultaneously in-line separating three flavonoids quercetin-3-O-rutinoside, luteoloside and astragalin from Mikania micrantha. The purities of these three compounds with the ethyl acetate-methanol-water solvent system were 95.13%, 98.54% and 98.19% respectively. Results showed the established in-line separation system of MNP-HSCCC was efficient, recyclable and served to isolate potential flavonoids with similar polarities from natural complex mixtures. The in-line combination of magnetic nanoparticles with high-speed countercurrent chromatography eluting with the same solvent system during the whole separation process was established for the first time.

Isolation of sutherlandins A, B, C and D from Sutherlandia frutescens (L.) R. Br. by counter-current chromatography using spiral tubing support rotors.

Spiral countercurrent-chromatography has great potential for improving the capacity and efficiency of purification of secondary metabolites, and here we describe applications useful for the isolation of flavonoids from the widely used South African medicinal plant, Sutherlandia frutescens (L.) R. Br. In the spiral tubing support rotor, STS-4 for high-speed counter-current chromatography, several polar butanol aqueous solvent systems were selected using a logK plot, and the novel flavonol glycosides (sutherlandins A-D) were well separated by the optimized solvent system (ethyl acetate:n-butanol:acetic acid:water; 5:1:0.3:6 by vol.). The yield of purified flavonoids from 0.9g extract varied from 8.6mg to 54mg of the sutherlandins for a total of 85.3mg. The same extract was fractionated in the new STS-12 rotor of the same outside dimensions but with more radial channels forming 12 loops of the tubing instead of 4. The rotor holds more layers and increased length of tubing. From 0.9g extract the STS-12 rotor yielded more recovery of 110.4mg total with amounts varying from 11.2mg to 64mg of the sutherlandins and apparent increased separation efficiency as noted by less volume of each fraction peak. Thus from 1-g amounts of extract, good recovery of the flavonoids was achieved in the butanol aqueous solvent system.