Recent advances in counter-current chromatography

During the past several years, counter-current chromatography (CCC) technology has been advanced to cover a broad spectrum of applications, from large-scale preparative to analytical-scale separations. These advances include liquid-liquid dual CCC, foam CCC and partition of macromolecules with aqueous-aqueous polymer phase systems. For these developments the synchronous coil planet centrifuge scheme has been used, which relies on a relatively simple mechanical design. Future developments in CCC may be focused on the improvement of the more intricate non-synchronous coil planet centrifuge scheme which has a greater potential for the separation of biopolymers and cell particles.     

Continuous counter-current extraction on an industrial sample using dual-flow counter-current chromatography

Both batch and continuous separations were performed on an industrial liquor using a specially built continuous counter-current extraction centrifuge. Changing the flow regime for different batch separations showed that the elution of components from the respective ends of the coil depends on the flow rates of both upper and lower phases. It was shown that, within the scope of the study, the elution of the components was not affected by the concentration of the injected reaction liquor and more importantly that continuous processing with a counter-current chromatography centrifuge was feasible. This research represents an important step forward in making continuous counter-current chromatography (or true moving bed chromatography) accessible for the pharmaceutical industry.     

How to realize the linear scale-up process for rapid purification using high-performance counter-current chromatography

This study used the isolation of six constituents from Selaginella tamariscina as an example to demonstrate how to achieve rapid and predictable linear scale-up processes in both normal- and reversed-phase high-performance counter-current chromatography. After systematic optimization of solvent systems, sample concentration, sample loading volume, rotation speed and flow rate on the analytical Mini-DE centrifuge, the optimized parameters obtained were directly transferred to the preparative Midi-DE centrifuge, with nearly the same purities, resolutions and elution times but with 50 times the throughput. Amentoflavone (446.7 mg, 97.8%), robustaflavone (21.6 mg, 89.4%), bilobetin (80.7 mg, 92.7%), hinokiflavone (15.1 mg, 85.5%), isocryptomerin (34.8 mg, 89.6%) and an apigenin-diglucoside (46.3mg, 96.4%) were obtained with amounts and purities shown in parentheses as analysed by HPLC. The process, therefore, offers an efficient and rapid method of obtaining sufficient quantities of target compounds with significantly increased throughput after a linear scale-up.     

Rapid purification and scale-up of honokiol and magnolol using high-capacity high-speed counter-current chromatography

In this paper, a rapid separation approach has been developed using high-capacity high-speed counter-current chromatography (high-capacity HSCCC) to isolate and purify honokiol and magnolol, which are the main bioactive constituents from Houpu. The optimization of the solvent selection process, sample loading volume and flow rate is systematically studied using analytical high-capacity HSCCC. The optimized parameters obtained rapidly at analytical scale were used for a 1000 x scale-up preparative run using pilot scale high-capacity HSCCC in a MAXI-DE centrifuge. A crude sample of 43 g was successfully separated and the fractions were analysed by high-performance liquid chromatography (HPLC). This large scale preparative single step run yielded 16.9 and 19.4 g of honokiol and magnolol with purities of 98.6 and 99.9%, in only 20 min. This is the first time that high-performance counter-current chromatography has been used to purify multiple gram grade bioactive compounds in less than 1h and at such high concentrations of final products (10.8 g/l for magnolol and 7.0 g/l for honokiol).     

高速逆流色谱在分离纯化木蝴蝶活性成分中的线性放大

利用高速逆流色谱分离纯化中草药木蝴蝶乙酸乙酯粗提物中的黄酮类活性成分,并将分离规模从分析型线性放大到制备型,以获得大量的活性成分,为进一步的药物筛选提供物质基础。实验在分析型高速逆流色谱上对分离参数进行了系统优化,并将优化条件放大到制备型高速逆流色谱上对911.6 mg木蝴蝶乙酸乙酯粗提物进行分离,得到5种化合物,经高效液相色谱、电喷雾电离质谱和核磁共振氢谱、碳谱分析鉴定,分别为白杨素(160.9 mg,纯度为97.3%)、黄芩素(130.4 mg,纯度为97.6%)、黄芩素-7-O-葡萄糖苷(314.0 mg,纯度为98.3%)、黄芩素-7-O-双葡萄糖苷(179.1 mg,纯度为99.2%)和一种新的白杨素双葡萄糖苷(21.7 mg,纯度为98.8%)。该放大过程不仅将处理量提高了53倍,还保持了在分析型设备上的分离度和分离时间。该工作为天然产物的研究提供了一个高效的分离纯化方法。     

Flow rate gradient high-speed counter-current chromatography separation of five diterpenoids from Triperygium wilfordii and scale-up

In this paper, high-speed counter-current chromatography (HSCCC) instruments with different gravitational forces were applied for the separation of bioactive compounds from Triperygium wilfordii Hook.f. The critical parameters including sample concentration, sample volume and flow rate were first optimized on an analytical Mini-DE HSCCC system, and then scaled up to a preparative TBE 300A HSCCC system. Although this scale-up process was performed using different CCC instruments with different centrifuges and gravitational forces, the same resolutions were obtained and the elution time could be predictable. Five diterpenoid compounds and one unknown compound were separated from Triperygium wilfordii Hook.f. by HSCCC with a two-phase solvent system composed of n-hexane-ethyl acetate-methanol-water (HEMW) (3:2:3:2, v/v/v/v). This one-step flow gradient separation produced triptonide (25 mg), isoneotriptophenolide (77 mg), hypolide (83 mg), unknown compound (1 mg), triptophenolide (42 mg), triptonoterpene methyl ether VI (37 mg) from 320 mg crude extract with purities of 98.2%, 96.6%, 98.1%, 95.3%, 95.1%, and 96.5%, respectively. Their purities and structures were identified by high-performance liquid chromatography, mass spectrometry and NMR. This paper demonstrates that analytical CCC plays an important role in optimizing parameters and scale-up process when analytical CCC and preparative CCC are supplied by different manufacturers with different gravitational forces, and the scale-up process from analytical CCC to preparative CCC is still predictable.     

Universal counter-current chromatography modelling based on counter-current distribution

There is clearly a need for a model which is versatile enough to take into account the numerous operating modes and pump out procedures that can be used with counter-current chromatography (CCC). This paper will describe a universal model for counter-current chromatography based on counter-current distribution. The model is validated with real separations from the literature and against established CCC partition theory. This universal model is proven to give good results for isocratic flow modes, as well as for co-current CCC and dual flow CCC, and will likely also give good results for other modes such as intermittent CCC.     

An overview of recent progress in multiple dual-mode counter-current chromatography

Counter-current chromatography is a chromatographic separation and purification technique being developed. The development of different elution modes has significantly contributed to this field. Multiple dual-mode elution is a method developed based on dual-mode elution, which consists of a series of changing cycles of the phase role and the direction by switching between normal and reverse elution modes of counter-current chromatography. This dual-mode elution method takes full advantage of the liquid nature of stationary and mobile phases of counter-current chromatography and effectively improves the separation efficiency. So, this unique elution mode has gained extensive attention for separating complex samples. This review mainly describes and summarizes in detail its development, applications, and characteristics in recent years. Meanwhile, its advantages, limitations, and future outlook also have been discussed in this paper.     

Two-dimensional counter-current chromatography: 1st traditional counter-current chromatography, 2nd acid-base elution counter-current chromatography

An on-line column-switching counter-current chromatography (CCC) with solid-phase trapping interphase is presented in this paper. The large volume injection is avoided using solid-phase trapping interphase. Thereby, totally different chemical composition biphasic solvent system can be utilized to enhance system orthogonality. In the present work, a 140 mL-capacity CCC instrument was used in the first dimension, and a parallel three-coil CCC centrifuge (40 mL each coil) was used in the second dimensional separation allowing three injections at the same time. With biphasic solvent system composed of n-hexane: ethyl acetate: methanol: water (1:1:1:1, v/v), five well-separated fractions were obtained in the first dimension. Two fractions were online concentrated and further separated in the second dimension with solvent system composed of methyl tert-butyl ether: acetonitrile: water (2:2:3, v/v), where trifluoroacetic acid (10 mM) was added to the upper organic phase as a retainer and triethylamine (10mM) to the aqueous mobile phase as an eluter. Four hydroxyanthraquinones were successfully separated and purified from Chinese medicinal plant Rheum officinale in only one step.     

Vortex counter-current chromatography

A novel counter-current chromatographic system is developed by mounting a vortex column on a type-I coil planet centrifuge. The column is fabricated from a high-density polyethylene disk (16 cm diameter and 5 cm thick) by making multiple holes of various diameters (3-12.5 mm) each arranged in a circle and connected with narrow transfer ducts. The performance of this vortex column is tested with three different two-phase solvent systems with a broad range in hydrophobicity. The results indicated that the smallest diameter column (3mm diameter, 120 units with 42.8 ml capacity) yielded the best separation with the height equivalent to a theoretical plate of 2 cm compared with 20 cm required by the conventional multilayer coil column of high-speed CCC. By avoiding the use of an Archimedean Screw Force, the system shows a low column pressure which would permit safe operation of a large preparative column without a risk of leakage of solvent and column damage.     

Industrial scale-up of countercurrent chromatography: predictive scale-up

This study describes how scale-up in countercurrent chromatography (CCC) can be simply predicted on a process scale CCC device by running a preliminary analytical-sized sample and having knowledge of the stationary-phase retention at scale-up conditions. Results have shown that simple experimentation can lead within a day to a process with the capability of several kilograms per day (tons per year) compound yield, and that this is feasible with benchtop CCC units.     

Evaluation of dual flow counter-current chromatography and intermittent counter-current extraction

The aim of this research is to compare two continuous extraction technologies, intermittent counter-current extraction (ICcE) and dual flow counter-current chromatography (DFCCC), in terms of loading and throughput using the GUESSmix, and show the advantages and disadvantages of the two methods. A model sample containing caffeine, vanillin, naringenin and carvone, with a total load of 11.2 g, was employed with a hexane-ethyl acetate-methanol-water (2:3:2:3) phase system to evaluate an ICcE method on a preparative (912 ml coil volume) DE-Midi instrument. While DFCCC was carried out on a specially designed preparative (561 ml coil volume) bobbin installed in a similar Midi instrument case. While similar throughputs of 7.8 g/h and 6.9 g/h were achieved for the ICcE and DFCCC methods respectively, ICcE was demonstrated to have a number of advantages over DFCCC.     

High-temperature continuous counter-current gas-liquid chromatography

In continuous counter-current gas-liquid chromatography, which has a high resolving power and is suitable for the large-scale purification of organic solvents, it is very important that the samples that can be applied extend from easily separable to more difficult to separate, such as azeotropes, low-volatility compounds and stereoisomers. A system was designed and constructed for high-temperature operation up to 200°C, and was applied to the separation of dimethyl and diethyl phthalate, trans- and cis-decahydronaphthalene and cis-decahydronaphthalene and tetrahydronaphthalene. It was confirmed that over 99% of purity could be achieved for dimethyl and diethyl phthalate, trans-decahydronaphthalene, and tetrahydronaphthalene.     

Optimisation & scale-up of affinity chromatography

When affinity chromatography is used on the large scale, economics dictate that the process must be optimised to ensure high throughputs and high adsorbent utilisation. The optimisation of an affinity chromatography process starts with the selection of both the ligand and matrix components of the affinity adsorbent. The relative merits of using highly selective ligands and small particle matrices in the development of high performance chromatography systems is discussed. Once the adsorbent has been chosen the parameters that characterise its interaction with the adsorbate must be characterised from small scale experiments. The method is illustrated in a series of experiments that show the influence of a number of factors involved in the preparation of immobilised monoclonal antibodies on the subsequent performance of the immunosorbents. These parameters together with computer simulations of the chromatography process can be used to design and optimise the operation of large scale systems. Attention has to be given to the optimisation of the adsorption, washing and elution phases of the process. Apart from initial process design, it is also necessary to optimise the chromatographic separation during the run. This involves the need to be able to measure the chromatographic performance in an on-line manner and a number of approaches to achieve this are described.     

High-speed counter-current chromatography of apple procyanidins

Apple procyanidins were separated by high-speed counter-current chromatography using a type-J multilayer coil planet centrifuge. Several two-phase solvent systems with a wide range of hydrophobicities from a non-polar hexane system to polar n-butanol systems were evaluated their performance in terms of the partition coefficient and the retention of the phase. The best separation of procyanidins B and C was achieved with a two-phase solvent system composed of n-butanol-methyl tert.-butyl ether-acetonitrile-0.1% trifluoroacetic acid (2:4:3:8) using the lower phase as a mobile at a flow-rate of 1.0 ml/min.     

Enantioseparations in counter-current chromatography and centrifugal partition chromatography

Examples of chiral separations in counter-current chromatography (CCC) and centrifugal partition chromatography (CPC) are not numerous, due to the difficulty of finding chiral selectors highly selective in the liquid phase as well as a combination of solvents that does not destroy the selectivity and retains the capacity to elute chiral isomers of interest. New ideas and new chiral selectors generally come from other separation techniques, as will be highlighted in this review.     

Service life of counter-current chromatography coils

A multilayer coil of PTFE tubing, which failed after being used each workday for about 3 years in a type J centrifuge, was examined. Two types of defects were found. One, called crazes, occurs throughout the coil and does not leak initially, but may eventually lead to a short, axially oriented slit. Another, called indentations, is seen primarily in the innermost and other nearby layers. They are elongated, about 5 mm, indentations, usually on the central side of the tubing. These eventually crack and leak. PTFE tubing is permeable to air and hexane and expands by more than 1% when immersed in hexane, heptane or chloroform for a few days. It is suggested that the crazes result from exposure of the somewhat flexible tubing to the undulating centripetal force field in the coil-planet centrifuge, especially when further softened by solvent absorption. The indentations may result from carriage of the excess tubing length, created by solvent absorption, from the coil periphery to the coil center by the centripetal force field, which continuously travels from the peripheral tail to the central head of the coil. A 1% increase in coil length creates 74 cm of excess tubing in the 160-ml coils examined in this study. It is suggested that fluorinated ethylene propylene (FEP) tubing, especially when etched on the outside, may provide more stable CCC coils, since its expansion when exposed to organic solvents is 0.1 or less than that of PTFE.     

Separation of acetins by counter-current chromatography

In this work we report the purification of a crude acetin mixture into mono‐, di‐ and triacetin by countercurrent chromatography. The process was initially tested on a small, semi‐preparative scale (0.5 g) to determine its efficiency. The process was then scaled up to accommodate 2.5 g of crude reaction products containing a mixture of the acetins. The solvent system ethyl acetate/n‐butanol/water 1:0.2:1 was used in all separation procedures. Mono‐, di‐ and triacetins were separated similarly in the semi‐preparative and preparative runs.     

Modelling counter-current and dual counter-current chromatography using longitudinal mixing cell and eluting counter-current distribution models

Two well known approaches are considered to analyze the processes of counter-current and dual counter-current chromatography: the longitudinal mixing cell model and the Craig's counter-current distribution model. The cell model represents perfectly mixed, equally sized cells in series. The number of cells characterizes the rates of longitudinal mixing in the stationary and mobile phases. In the eluting counter-current distribution (CCD) model, the CCC process is considered as a continuous form of Craig's counter-current distribution. For a cascade of equilibrium stages theoretical elution profiles of the CCC process by using the CCD and cell model approaches have been compared. It is shown that in general, distribution functions of the CCD and cell models differ. It is established that the distribution of a solute between two solvent phases in the dual CCC process is determined by the extraction factor c, the total number of equilibrium stages n and the position of the sample inlet m by the equation Q(x)=(1-c(m))/(1-c(n+1)) with c=F(2)K(D)/F(1) (K(D), F(1), F(2) and Q(x) are the distribution constant, the phase flow-rates and the portion of solute eluted by the first phase, respectively).