Improved High-Speed Countercurrent Chromatograph with Three Multilayer Coils Connected in Series. III. Evaluation of Semianalytical Column with Various Biological Samples and Inorganic Elements

Abstract

The partition efficiency of the counter-current chromatographic centrifuge was improved by mounting a set of three multilayer coils prepared from a 1.07 mm I.D. and ca 300 m length of PTFE (polytetrafluoroethylene) tube with a total capacity of 270 ml. The high performance of the present apparatus has been successfully demonstrated in separations of various testing samples which include DNP (dinitrophenyl) amino acids, indole auxins, tetracycline derivatives, flavonoids, bacitracin, triterpenoic acids, and lanthanoid chlorides.     

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.     

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.     

Gram Quantity Separation of DNP (Dinitrophenyl) Amino Acids with Multi-Layer Coil Countercurrent Chromatography (CCC)

Abstract

The efforts have been successfully made to extend the preparative capability of the high-speed CCC scheme with a multi-layer large capacity coiled column. The apparatus is a table top model of a horizontal flow-through coil planet centrifuge which produces a synchronous planetary motion of the column holder. The separation column was prepared from a single piece of 70 m long, 2.6 mm i.d., PTFE tubing coiled around the spool-shaped holder to form multiple layers of the coil with a total capacity of about 400 ml. The performance of the apparatus was assessed with a standard set of DNP amino acid samples and a two-phase solvent system composed of chloroform, acetic acid and 0.1N HCl (2:2:1). Preparative capability of the method was evaluated in terms of the retention level of the stationary phase and peak resolution for various sample size ranging from 0.05g to 2g. The effects of sample volume, sample concentration and the choice of the sample diluent on the separation were studied. The results indicated that both the retention level and the peak resolution tend to decrease with the increase of the sample volume applied at a given concentration. For separation of 1 gram quantity, best results were obtained by applying the sample dissolved in a small volume (10 ml) consisting of equal amounts of the two phases. Overall results indicate that the present scheme is capable of efficient separation for gram quantity of samples in a short period of time. The preparative capability may be further increased by the use of a larger-diameter and/or longer coil.     

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.     

Separation of Alkaloids Extracted from Stephania Tetranda S. Moore by Analytical High-Speed Countercurrent Chromatography

Abstract

High-Speed countercurrent chromatography is a recently developed separation method which has been remarkably improved in both partition efficiency and separation time. In the present study, this advanced countercurrent chromatographic method was applied to separation of sample mixture containing tetrandrine, fangchinoline, and cyclanoline originally extracted from

Stephania tetrandra S. Moore. Separations were performed with a two-phase solvent system composed of n-hexane/ethyl acetate/methanol/water in two different elution modes. Sample mixture containing 3 mg of alkaloids was efficiently separated in 100 min. The peak fraction of each component was analyzed with a mass spectrometer for structure identification.     

Improved high-speed counter-current chromatograph with three multilayer coils connected in series. II. Separation of various biological samples with a semi-preparative column

The semipreparative capability of the newly developed high-speed counter-current chromatograph equipped with a set of three multilayer coils has been demonstrated in separations of a variety of biological samples including triterpenoic acids, indole auxins, bacitracin, flavonoids and tetracycline derivatives, each with a suitable two-phase solvent system. The sample quantities ranging from 50 to 500 mg were efficiently separated within a few hours. The separation of tetracycline derivatives was remarkably improved by adding ammonium acetate to the solvent system.     

Optimization of Solvent Mixture Compositions for High Speed Countercurrent Distribution

Abstract

Seven solvents considered appropriate for use with relatively sensitive biosynthetic products undergoing separation by multilayer coil planet centrifuge high speed countercurrent distribution (HSCCD) have been evaluated in a new and systematic procedure for optimal solvent selection. the new method was illustrated by a complete HSCCD separation of five closely related dipeptides.     

Partition Efficiency of High-Pitch Locular Multilayer Coil for Countercurrent Chromatographic Separation of Proteins Using Small-Scale Cross-Axis Coil Planet Centrifuge and Application to Purification of Various Collagenases with Aqueous-Aqueous Polymer Phase Systems

Partition efficiency of the high-pitch locular multilayer coil was evaluated in countercurrent chromatographic (CCC) separation of proteins with an aqueous-aqueous polymer phase system using the small-scale cross-axis coil planet centrifuge (X-axis CPC) fabricated in our laboratory. The separation column was specially made by high-pitch (ca 5 cm) winding of 1.0 mm I.D., 2.0 mm O.D. locular tubing compressed at 2 cm intervals with a total capacity of 29.5 mL. The protein separation was performed using a set of stable proteins including cytochrome C, myoglobin, and lysozyme with the 12.5% (w/w) polyethylene glycol (PEG) 1000 and 12.5% (w/w) dibasic potassium phosphate system (pH 9.2) under 1000 rpm of column revolution. This high-pitch locular tubing yielded substantially increased stationary phase retention than the normal locular tubing for both lower and upper mobile phases. In order to demonstrate the capability of the high-pitch locular tubing, the purification of collagenase from the crude commercial sample was carried out using an aqueous-aqueous polymer phase system. Using the 16.0% (w/w) PEG 1000 - 6.3% (w/w) dibasic potassium phosphate - 6.3% (w/w) monobasic potassium phosphate system (pH 6.6), collagenase I, II, V and X derived from Clostridium hystolyticum were separated from other proteins and colored small molecular weight compounds present in the crude commercial sample, while collagenase N-2 and S-1 from Streptomyces parvulus subsp. citrinus were eluted with impurities at the solvent front with the upper phase. The collagenase from C. hystolyticum retained its enzymatic activity in the purified fractions. The overall results demonstrated that the high-pitch locular multilayer coil is effectively used for the CCC purification of bioactive compounds without loss of their enzymatic activities.     

Protein separation by nonsynchronous coil planet centrifuge with aqueous-aqueous polymer phase systems

Counter-current chromatographic separation of proteins was performed using a rotary-seal-free nonsynchronous coil planet centrifuge (CPC) fabricated in our laboratory. This apparatus has a unique feature that allows a freely adjustable rotational rate of the coiled separation column at a given revolution speed. The separation was performed using a set of stable proteins including cytochrome c, myoglobin and lysozyme with two different types of aqueous-aqueous polymer phase systems, i.e., PEG (polyethylene glycol) 1000-dibasic potassium phosphate, and PEG 8000-dextran T500 in 5 mM potassium phosphate buffer. Using a set of multilayer coiled columns prepared from 0.8 mm I.D. PTFE tubing with different volumes (11, 24, 39 ml), the effect of the column capacity on the partition efficiency was investigated under a given set of experimental conditions. Among these experiments, the best separation of proteins was attained using the 39 ml capacity column with a 12.5% (w/w) PEG 1000-12.5% (w/w) dibasic potassium phosphate system at 10 rpm of coil rotation under 800 rpm. With lower phase mobile at 0.2 ml/min in the head-to-tail elution, the resolution between cytochrome c and myoglobin was 1.6 and that between myoglobin and lysozyme, 1.9. With upper phase mobile in the head-to-tail elution, the resolution between lysozyme and myoglobin peaks was 1.5. In these two separations, the stationary phase retention was 35.0 and 33.3%, respectively. Further studies were carried out using a pair of eccentric coil assemblies with 0.8 mm I.D. PTFE tubing at a total capacity of 20 ml. A comparable resolution was obtained using both lower and upper phases as a mobile phase in a head-to-tail elution. The results of our studies demonstrate that the nonsynchronous CPC is useful for protein separation with aqueous-aqueous polymer phase systems.     

Separation of S-Triazine Herbicides by Countercurrent Chromatography

Abstract

Countercurrent chromatography (CCC) has been successfully applied for the separation of four chlorinated s-triazine derivatives, namely, simazine, atrazine, propazine, and trietazine, which are widely used as herbicides. Of several solvent systems investigated, n-hexane-ethyl acetate-methanol-water (8:2:5:5) gave the best range of the partition coefficient values because of the satisfactory solubility for all samples and therefore was used for the separation of s-triazines. Two types of countercurrent chromatographs, high speed CCC, and horizontal flow-through coil planet centrifuge, were used for s-triazines separation, and the identity of the separated fractions was unequivocally established by mass spectrometry. The potential practical application of CCC to herbicide analysis is discussed.     

Application of pH-Zone-Refining Countercurrent Chromatography in the Preparative Separation of Alkaloids from Traditional Chinese Medicine

pH-Zone-refining countercurrent chromatography (CCC) is a recently developed new preparative separation method based on conventional CCC. The method uses a retainer base (or acid) in the stationary phase to retain the analyses in the column and an eluent acid (or base) to elute the analyses according to their pKa values and hydrophobicities. It produces a succession of highly concentrated rectangular peaks with minimum overlap similar to those observed in displacement chromatography. pH-zone-refining CCC has important advantages over the conventional CCC including an over 10-fold increase (up to 10 gram or more) in sample loading capacity, high concentration of fractions, and concentration of minor impurities. pH-zone-refining CCC has been successfully applied to the preparative separation of a variety of compounds including both acidic and basic derivatives of amino acid, hydroxyxanthene dyes, peptides, alkaloids, indole auxins, structural, geometrial and optical isomers.     

High-Speed Countercurrent Chromatography of Dipeptides on a Polar Two-Phase Solvent System

Abstract

The performance of the high-speed countercurrent chomatograph was evaluated by separating dipeptide samples on a polar biphasic solvent composed of n-butanol-acetic acid-water (4:1:5, v/v/v). Best results were obtained with a set of multilayer coils of small helical diameter by eluting with the upper nonaqueous phase in a head to tail direction at a flow rate of 1 or 2 ml/min. Four components were completely resolved in 5 to 11 hours. Other types of coiled columns such as a multilayer coil with large helical diameter (in a commercial model) and eccentric dual-layer coil assemblies mounted on a horizontal coil planet centrifuge also yielded satisfactory separations.     

Application of High Speed Countercurrent Chromatography/Thermospray Mass Spectrometry for the Analysis of Bio-Active Triterpenoic Acids from Boswellia Carterh

Abstract

The versatility and high resolving power of countercurrent chromatography has been demonstrated with a newly developed analytical high speed planet centrifuge system (HSCCC). Interfacing countercurrent chromatography with mass spectrometry (MS) provides a new analytical methodology which Integrates the advantages of countercurrent chromatography with the low detection limit and identification capability of mass spectrometry. The capability of thermo-spray HSCCC/MS has been successfully demonstrated in the identification of plant alkaloids and lignans. In this paper, the technigue proved useful in identifying and validating the bio-active and structural closely related triterpenoid carboxylic acids from a crude extract of Boswellia carterii (Burseraceae). Thermospray HSCCC/MS can become a useful and complementary method to thermospray HPLC/MS for the analysis of nonvolatile or thermally unstable molecules.     

Separation of solanesol in tobacco leaves extract by slow rotary counter-current chromatography using a novel non-aqueous two-phase solvent system

Non-aqueous solvent system composed of sunflower oil-ethanol was found to be suitable for the separation of lower polarity components. Employing this solvent system, 3 g of tobacco leaves extract containing 15% solanesol was separated using a slow rotary counter-current chromatograph equipped with an 1100 mL column made of 5.7 mm I.D. convoluted PTFE tube. The separation yielded 1.5 g of yellow oily product in which solanesol occupied 26.8% determined by HPLC. The tobacco tar and other main impurities in the tobacco leaves extract were removed after slow rotary counter-current chromatographic separation. The separation processing may be developed as a technique for producing a solanesol product with food grade.     

Toroidal Coil Countercurrent Chromatography: A Fast Simple Alternative to Countercurrent Distribution Using Aqueous Two Phase Partition: Principles,Theory, and Apparatus

Abstract

The principles, theoretical basis and equipment for continuous two phase toroidal coil chromatography are described. Rat liver homogenates were subjected to analytical subcellular fractionation by toroidal coil chromatography in a phase mixture of 3.3% (w/w) dextran T500, 5.4% (w/w) poly(ethylene glycol) 6000, 10 mM sodium phosphate-phosphoric acid buffer, pH 7.4, in 0.26 M sucrose containing 0.05 mM Na₂EDTA and 1 mM ethanol. The distribution of organelles, as reflected by their marker enzymes, was compared to that obtained by discrete counter-current partition in a 17 transfer apparatus. Toroidal coil chromatography showed enhanced resolution of certain organelles. In particular, almost complete separation of plasma membrane from endoplasmic reticulum was achieved and some resolution of plasma membrane from lysosomes was obtained. It is concluded that toroidal coil chromatography offers a potentially useful alternative approach to organelle separation techniques.     

Preparative Purification of Peptides by Countercurrent Chromatography on the Ito Coil Planet Centrifuges

Abstract

For the purification of up to 1000 mg of synthetic peptides by countercurrent chromatography, the coil planet centrifuges have proven useful in the research laboratory. Besides the earlier described horizontal flow-through coil planet centrifuge which chromatographs substances in all solvent systems at room temperature, the multi-layer coil planet centrifuge affords more rapid chromatography. However, separations using n-butanol, especially suited for peptides, have to be conducted at elevated temperatures. A new machine that has characteristics of both of the foregoing instruments is the compact horizontal flow-through coil planet centrifuge which promises rapid chromatography with full retention of the stationary phase.     

Toroidal Coil Countercurrent Chromatography in the Affinity Partitioning of Nicotinic Cholinergic Receptor Enriched Membranes

Abstract

A variation on the aqueous polymer phase partition method, affinity partitioning, has proved suitable for the preparative scale purification of binding site enriched membrane fragments. The full resolving potential of the affinity partitioning technique often requires the utilization of multiple extraction procedures such as countercurrent distribution. In this report, we evaluate the combination of a newly developed countercurrent purification technique, toroidal coil chromatography, with affinity partitioning. This approach provides an efficient method for purification and characterization of membrane bound nicotinic cholinergic receptors. The relative merits of the toroidal coil chromatography technology and the more conventional thin-layer countercurrent distribution techniques are compared.     

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.     

Cell Separations on the Countercurrent Chromatograph

Abstract

Separation of cells differing only subtly has been achieved by partitioning between the two phases formed by solution of dextran and polyethylene glycol in water. Cell populations which have related, but not identical, surface properties seldom exhibit sufficiently different partition behavior to be separated in a single extraction. In such cases, repeated partitions are carried out via countercurrent distribution or countercurrent chromatography to effect the separation. Potential advantages of countercurrent chromatography are its ease and rapidity of operation. In the present work we describe our approach to determining and possibly improving the efficiency of polymer phase partitioning by countercurrent chromatography.