Countercurrent Chromatography Using a Toroidal Coil Planet-Centrifuge: A Comparative Study of the Separation of Organelles Using Aqueous Two-Phase Partition

Abstract

A toroidal coil planet centrifuge is described and compared with other countercurrent chromatography (CCC) and countercurrent distribution (CCD) techniques. The basis of separation is partition in aqueous two-phase polymer systems, with each method assessed by fractionating rat liver organelles. The size and ease of operation of the toroidal coil planet centrifuge gave significant advantages over conventional CCD systems achieving equivalent resolution in a fraction of the time.     

Optimization of a high speed countercurrent chromatograph for analytical separations

The analytical capability of the high speed countercurrent chromatograph model 4000 (HSCCC-4000) has been improved by optimizing the dimensions of the multilayer coiled column. Using a two-phase solvent system of n-hexane – ethyl acetate – methanol – water (1:1:1:1) and a set of indole auxins as test samples, a series of studies was conducted to evaluate performance of coiled columns with i.d.s ranging from 0.1 to 0.55 mm. The studies on the stationary phase retention indicated that multilayer coils of 0.55 mm and 0.30 mm i.d. provide satisfactory retention of the stationary phase while the 0.10 mm i.d. column fails to yield reproducible retention. The best partition efficiencies were obtained from the 0.30 mm i.d. multilayer coil with a 6 ml capacity which produced theoretical plate numbers ranging from 5500 to 10500 with a resolution factor of 2.30. The feasibility of interfacing the HSCCC-4000 with a mass spectrometer is briefly discussed.     

一种用于大容量逆流色谱制备的立式多层螺旋管行星式离心机

Owing to no complications caused by solid supports, such as adsorptive sample loss and deactivation, tailing of solute peaks, and contamination, countercurrent chromatography (CCC) has been an area of intense research since the first introduction of CCC in 1970,[1] and various apparatus and broad applications have been advanced[2,3]. For these developments, the type-J synchronous planet centrifuge has received considerable attention, which relies not only on its relatively simple mechanic design, but also on its high partition efficiency and short elution time caused by mixing and settling for the efficient chromatographic separations. In the past, however, almost all of type-J centrifuges rotated slowly were disposed horizontally due to the original design and some experiments that gravis plays an important role at a low rotary speed as similar to type-V rotating multilayer helical tube in unit gravity[4-9]. In fact,we discovered that the upright apparatus holds more retention of stationary phase than the horiziontal aparatus when large standard tubings were used as mutilayer coil column and the aparatus was operated under same contions. We report here a new coil planet centrifuge with four upright cylindrical columns for large scale countercurrent chromatographic preparation. The design principle and apparatus of UCCC is as samilar to type-J multilayer coil planet centrifuge. Four uptight cylindrical column holders are symmetrically arranged around the centrifuge axis as similar to the type-J HSCCC with three horizontal multilayer coils connected in series[8] . A series of experiments indicat that upright CCC has many advantages over the horizontal CCC when using a large-bore tube as multilayer coil column for large scale countercurrent chromatographic separation.Upright CCC provide a versatile countercurrent chromatographic method for large-scale preparation from very crude sample. It has good preparative capacity and flexible suitability to various sample and two-phase system.The present apparatus not only can be operated at a high speed as similar as commonly used HSCCC for the system having short settling time and but also can be run at a low speed for the system having relative long settling time. Because of automatical control and seal-free flow through device, the uptight CCC apparatus may be readily scaled up to industrial preparation.     

Studies on the performance of different coiled column configurations for compact type-I countercurrent chromatography

Three types of novel coiled column configurations, i.e. a triangular coiled column and elliptical coiled columns I and II, were designed for type-I countercurrent chromatography and their performances were evaluated with two solvent systems each with suitable test samples. Three dinitrophenyl (DNP) amino acids (DNP-DL-glu, DNP-β-ala and DNP-L-ala) were separated with a moderately hydrophobic two-phase solvent system composed of hexane-ethyl acetate-metanol-0.1 M hydrochloric acid (1:1:1:1, v/v), while two dipeptides (tryptophyl-tyrosine and valyl-tyrosine) were separated with a polar solvent system composed of 1-butanol-acetic acid-water (4.75:0.25:5, v/v). The overall results indicated that the performance of compact type-I countercurrent chromatography was improved by elliptical coiled column I which was mounted with its maximum coil diameter perpendicular to the surface of the column holder. Hydrodynamic effects involved in these separations were discussed.     

Centrifugal partition chromatography – A review of recent applications and some classic references

Countercurrent chromatography, based on liquid–liquid partitioning, has many technological variants. One of them is centrifugal partition chromatography, introduced by Wataru Murayama and Kanichi Nunogaki in 1982. This technique, like other countercurrent chromatography techniques, is based on the phenomenon of liquid–liquid partitioning between two immiscible liquid phases that stay at equilibrium. But the significant difference between this technique and others is the retention mechanism of stationary phase. In the case of centrifugal partition chromatography, this mechanism is based on hydrostatic force, formed by the centrifugal field in the rotor in one‐axis centrifuge. Sometimes that allows more control of stationary phase, for example, when aqueous two‐phase and other difficult solvent systems are used. However, the efficiency of the separation in centrifugal partition chromatography is also affected by a variety of parameters dependent on the sample properties in the solvent system, physical properties of the solvent system, parameters of the instrument, and the method. This article includes also recent ideas for improvements to the technique and broadening its application (e.g., (multiple) dual‐mode or elution–extrusion procedure, pH‐zone‐refining centrifugal partition chromatography, ion‐exchange centrifugal partition chromatography, online and offline coupling of centrifugal partition chromatography).     

Evaluation of Analytical Countercurrent Chromatographs: High-Speed Countercurrent Chromatograph-4000 Vs. Analytical Toroidal Coil Centrifuge

Abstract

Performance of two countercurrent chromatographic models, high speed countercurrent chromatograph (HSCCC-4000) and analytical toroidal coil centrifuge (TCC), is evaluated in terms of theoretical plate number, resolution factor and separation time to assess their analytical capability. A series of experiments was conducted to investigate the effects of internal diameter and length of the coiled column, and flow rate of the mobile phase on the separation of indole auxins in two-phase solvent systems composed of n-hexane-ethyl acetate-methanol-water at different volume ratios. The three components of indole auxins were completely resolved in 16 min with a HSCCC system equipped with a multilayer coil of a 0.55 mm I.D. PTFE tube with theoretical plates ranging from 1290 to 829. Similar separation was achieved in 24 min with a TCC systems equipped with a 0.3 mm I.D. PTFE tube with theoretical plates ranging from 1811 to 969. It is concluded that both systems have comparable analytical capability at the present stage of development.     

Efficient separation of tocopherol homologues in vegetable oil by ionic‐liquid‐based countercurrent chromatography using a non‐aqueous biphasic system

Tocopherol homologues are important fat‐soluble bioactive compounds with high nutritional value. However, it is of great challenge to separate these homologues because of their high structural similarities. In this work, ionic‐liquid‐based countercurrent chromatography was used for the separation and purification of tocopherol homologues. Conventional countercurrent chromatography and ionic‐liquid‐based countercurrent chromatography solvent systems were evaluated in respect of partition coefficient, separation factor, and stationary phase retention factor to separate these targets. Kind of ionic liquids, amount of ionic liquid, and sample amount were systematically optimized. A novel countercurrent chromatography non‐aqueous biphasic system composed of n‐hexane‐methanol‐1‐butyl‐3‐methylimidazolium chloride was established. The baseline separation of tocopherol mixtures was obtained in one cycle process. The ionic liquid played a key role in the countercurrent chromatography separation, which resulted in difference of partition behavior of individual tocopherol in the whole system through different hydrogen‐bonding affinity. Finally, n‐hexane‐methanol‐1‐butyl‐3‐methylimidazolium chloride (5:5:3, v/v) water‐free biphasic system was successfully applied to separate tocopherol homologues from vegetable oil that was not achieved beforehand. This method can be widely employed to separate many similar molecules such as tocotrienols, tocomonoenols, and marine‐derived tocopherol in food samples.     

逆流溶质出峰顺序的预测

 根据逆流保留方程可知,溶质的出峰顺序主要取决于其在互不混溶的两液相间分配系数的大小顺序。以饱和及不饱和脂肪酸乙酯、2,4 二硝基苯胺基脂肪醇和对硝基苯基葡萄糖甙等结构较为简单、极性差别较大的溶质系列为研究对象,应用修正的通用基团活度系数(UNIFAC)(Dortmund)模型,通过相平衡计算,预测了它们在含水或不含水的溶剂体系中分配系数的变化趋势,并与前人的工作进行了对比。结果表明,由该方法预测出的相似结构溶质分配系数的顺序基本上与实验值相同,其中对饱和脂肪酸乙酯在己烷 乙腈(体积比为1∶1)体系中分配系数的预测最好。     

Model for spiral columns and stationary phase retention in synchronous coil planet centrifuges

Stationary phase retention in a synchronous coil planet centrifuge or high-speed counter-current chromatography (CCC) relies on the interplay of hydrostatic (tangential and normal centrifugal) and hydrodynamic (Archimedean screw and mobile phase drag) forces. By offering a set of quantitative or semi-quantitative theoretical frameworks, this work has resolved fundamental questions such as "in the absence of mobile phase flow, how is the distribution of the two phases in a CCC column determined?" and "for Type-J CCC, do the helical and the spiral columns lead to similar performance?"     

Scaling up of high-speed countercurrent chromatographic apparatus with three columns connected in series for rapid preparation of (−)-epicatechin

In the present study, compact high-speed countercurrent chromatographic apparatus was constructed with three columns connected in series. Two sets of columns were prepared from 10 mm and 12 mm I.D. tubing to form 12 L and 15 L capacities, respectively. Performance of these columns was compared for the separation of (−)-epicatechin gallate (ECG) from a tea extract by flash countercurrent chromatography (FCCC). In each separation, 200 g of the tea extract in 1600 mL of mobile phase was loaded onto the column. The 12 L column gave 7.5 L (35 g of ECG) and the 15 L column gave 9 L (40 g of ECG) of ECG solution without impurities. The ECG solution was directly hydrolyzed by tannase into (−)-epicatechin. The hydrolysate was purified by flash chromatography on AB-8 macroporous resin to give 52 g of EC (purity 99.1%). This scaled up apparatus could be used for the industrial separation of natural products.     

The generally useful estimate of solvent systems method facilitates off-line two-dimensional countercurrent chromatography for isolating compositions from Siraitia grosvenorii roots

Solvent system selection is a crucial and the most time-consuming step for successful countercurrent chromatography separation. A thin-layer chromatography-based generally useful estimate of solvent systems method has been developed to simplify the solvent system selection. We herein utilized the method to select a solvent system for off-line two-dimensional countercurrent chromatography to separate chemical compositions from a complex fraction of the Siraitia grosvenorii root extract. The first-dimensional countercurrent separation using chloroform/methanol/water (10:5.5:4.5, v/v/v) yielded four compounds with high purity and three mixture fractions (Fr I, III, and VII). The second-dimensional countercurrent separation conducted on Fr I, III, and VII using the hexane/ethyl acetate/methanol/water (4:6:6:4, 3:7:3:7, v/v/v) and chloroform/methanol/water (10:9:6, v/v/v) solvent systems, respectively, produced another four compounds. Four triterpenoids and four lignans were finally isolated, including two novel compounds. Hence, the generally useful estimate of solvent systems method is a feasible and efficient approach for selecting an applicable solvent system for separating complex samples. In addition, the off-line two-dimensional countercurrent chromatography method can improve both the peak resolution and the capacity of countercurrent chromatography.     

Separation and purification of intermediates for the preparation of naproxen from synthetic mixtures by countercurrent chromatography

Three key intermediates in the preparation of the nonsteroidal anti‐inflammatory drug naproxen were successfully separated and purified with high purity from synthetic mixtures by countercurrent chromatography with a selected biphasic solvent system. The biphasic solvent system composed of n‐hexane/ethyl acetate/methanol/water (9:1:9:1, v/v/v/v) was selected according to partition performance of the three components using thin‐layer chromatography. Fifty milligrams of the synthetic mixture after the three‐step reaction was injected into a preparative countercurrent chromatography separation column and yielded 3.5, 14.0, and 8.0 mg of three key intermediates with 95.0, 99.0, and 98.0% purity, and the recovery of each component was 65.2, 71.2, and 69.6%, respectively. The results indicated that countercurrent chromatography is an efficient alternative and economical method for the separation and purification of intermediate components from synthetic mixtures.     

Separation of Substances in Rotating Coiled Columns: From Trace Elements to Microparticles

The potentialities of rotating coiled columns in countercurrent chromatography (CCC) and centrifugal field-flow fractionation (CFFF) are demonstrated. A rotating coiled column is a fluoroplastic or steel coil wound around a rigid cylindrical drum, which revolves about its axis and, at the same time, revolves around the central axis of the device called planet centrifuge. The stationary (liquid, solid, or heterogeneous) phase is retained in the column because of the centrifugal force field, and the mobile liquid phase is continuously pumped through the column. The methods for recovery, separation, and preconcentration of various trace elements in geological samples and high-purity substances with the use of two-phase liquid systems (CCC) are developed. Procedures are proposed for the continuous sequential extraction of various element species from soil and for the recovery of polycyclic aromatic hydrocarbons from sewage sludge with the use of natural suspensions or solid particulates as stationary phases. It is also shown that rotating coiled columns can be used in a new field, microparticle fractionation by CFFF.     

Plant Hormone Analysis by Countercurrent Chromatography

Abstract

Countercurrent chromatography (CCC) has been successfully applied for the separation of plant hormones; namely, indole auxins, gibberellins, cytokinins and abscisic acid. In our present study three different types of CCC devices were evaluated for their performance in separation of plant hormones with a special emphasis on analysis and purification of abscisic acid (ABA). A large-scale preparative CCC apparatus consisting of a slowly rotating coil assembly was used for preliminary separations of ABA from a large volume of crude plant extracts. The toroidal coil planet centrifuge (CPC) for analytical-scale separations was subsequently applied for purification of ABA, the final confirmation being obtained by HPLC and combined gas chromatographic-mass spectrometric method. This two-step procedure utilizing preparative CCC and toroidal CPC was successfully applied for determination of ABA content in several plant tissues. A recently introduced high-speed CCC apparatus was tested for semipreparative separation of ABA and indole-3-acetic acid. The method yielded high peak resolution within 2 hours.     

高速逆流色谱研究进展

综述了近年来高速逆流色谱（HSCCC）在分析、半制备和制备分离天然产物、蛋白质、抗生素、无机物等领域的研究和应用进展,总结了适用于HSCCC的溶剂体系,并展望了HSCCC与质谱联用、pH区带逆流色谱和离子对逆流色谱新技术的应用前景。     

The Efficiency of Substance Separation in Countercurrent Liquid Chromatography

The effect of hydrodynamic parameters and the specific features of instrument design on the efficiency of substance separation in countercurrent liquid chromatography (CCC) was studied using a constant retention factor of the stationary phase in the column. The study was conducted with the separation of benzyl alcohol and p-cresol in a two-phase liquid system heptane–ethyl acetate–methanol–water (1.4 : 0.6 : 1 : 1) in as an example. It was shown that the peak resolution is improved with an increase in the rotational speed of the column and a decrease in the flow rate of the mobile phase. The best peak separation was attained using columns for which the ratio of the column rotation radius to the radius of column revolution was 0.615. It was shown that countercurrent chromatography allows the separation of substances with low partition constants (K < 1) in dilute solutions. The volume of the test sample may be up to 15% of the total volume of the chromatography column.     

Separation of polar betalain pigments from cacti fruits of Hylocereus polyrhizus by ion-pair high-speed countercurrent chromatography

Polar betacyanin pigments together with betaxanthins from ripe cactus fruits of Hylocereus polyrhizus (Cactaceae) were fractionated by means of preparative ion-pair high-speed countercurrent chromatography (IP-HSCCC) also using the elution–extrusion (EE) approach for a complete pigment recovery. HSCCC separations were operated in the classical ‘head-to-tail’ mode with an aqueous mobile phase. Different CCC solvent systems were evaluated in respect of influence and effectiveness of fractionation capabilities to separate the occurring pigment profile of H. polyrhizus. For that reason, the additions of two different volatile ion-pair forming perfluorinated carboxylic acids (PFCA) were investigated. For a direct comparison, five samples of Hylocereus pigment extract were run on preparative scale (900 mg) in 1-butanol–acetonitrile–aqueous TFA 0.7% (5:1:6, v/v/v) and the modified systems tert.-butyl methyl ether–1-butanol–acetonitrile–aqueous PFCA (2:2:1:5, v/v/v/v) using 0.7% and 1.0% trifluoroacetic acid (TFA) or heptafluorobutyric acid (HFBA) in the aqueous phase, respectively. The chemical affinity to the organic stationary CCC solvent phases and in consequence the retention of these highly polar betalain pigments was significantly increased by the use of the more lipophilic fluorinated ion-pair reagent HFBA instead of TFA. The HFBA additions separated more effectively the typical cacti pigments phyllocactin and hylocerenin from betanin as well as their iso-forms. Unfortunately, similar K_D ratios and selectivity factors α around 1.0–1.1 in all tested solvent systems proved that the corresponding diastereomers, 15S-type pigments cannot be resolved from the 15R-epimers (iso-forms). Surprisingly, additions of the stronger ion-pair reagent (HFBA) resulted in a partial separation of hylocerenin from phyllocactin which were not resolved in the other solvent systems. The pigments were detected by means of HPLC-DAD and HPLC-electrospray ionization–MS using also authentic reference materials.     

Preparative isolation of diterpenoids from Salvia bowleyana Dunn roots by high-speed countercurrent chromatography combined with high-performance liquid chromatography

The root of Salvia bowleyana Dunn (Lamiaceae) is used as a traditional Chinese medicine that has multiple therapeutic effects. In this study, an efficient strategy was developed to separate diterpenoid compounds, which are the main active ingredients in Salvia bowleyana Dunn roots, from complex crude extracts by high-speed countercurrent chromatography combined with preparative high-performance liquid chromatography. A two-phase solvent system comprising n-hexane–ethyl acetate–methanol–water (7:3:7:3, v/v/v/v) was selected for high-speed countercurrent chromatographic separation. Three major diterpenoids, 6α-hydroxysugiol ( 7 ), sugiol ( 8 ), and 6, 12-dihydroxyabieta-5,8,11,13-tetraen-7-one ( 9 ) were obtained at purities of 98.9, 95.4, and 96.2%, respectively, and minor diterpenoids were enriched via one-step separation. The enriched minor diterpenoids were further purified by continuous preparative high-performance liquid chromatography to yield two new norabietanoids ( 1 , 6 ) and four known compounds ( 2 – 5 ). The structures of these new compounds were determined using NMR spectroscopy, high-resolution electrospray ionization mass spectrometry, and electronic circular dichroism spectroscopy. The results suggest that high-speed countercurrent chromatography combined with preparative high-performance liquid chromatography efficiently isolates diterpenoids, including minor components, from complex natural products.     

高效逆流色谱法制备苏木中的氧化巴西木素

氧化巴西木素是苏木的主要化学成分之一,具有广泛的药理活性且常作为染色剂应用于各行各业。采用传统柱色谱法进行分离,不仅会造成色谱柱材料的污染,也会造成活性成分的损失。故采用高效逆流色谱(HPCCC)对苏木中的活性化合物氧化巴西木素进行分离纯化。苏木乙醇提取物经乙酸乙酯萃取后直接进行高效逆流色谱分离。首先利用基于薄层色谱的常用溶剂体系估算法和摇瓶法结合高效逆流色谱分析模式进行溶剂体系筛选。结果表明,最佳溶剂体系为氯仿-甲醇-水(4∶3∶2, v/v/v)。高效逆流色谱以反相模式为洗脱模式,主机转速为1600 r/min,流速为10 mL/min,分离温度为25 ℃,检测波长为285 nm,在氯仿-甲醇-水(4∶3∶2, v/v/v)溶剂体系下,从500 mg苏木乙酸乙酯萃取物中一次性分离制备得到15.2 mg纯度为95.6%的氧化巴西木素及一微量成分caesappanin C。采用高效逆流色谱分离制备氧化巴西木素,可避免苏木中的活性成分氧化巴西木素对色谱柱中的固体填充材料染色和难以洗脱等问题,并缩短分离纯化工作时间,提高工作效率。故可将高效逆流色谱应用到苏木中其他色素化合物或其他染料植物的分离制备工艺研究中。