One-step purification of histone deacetylase from Escherichia coli cell-lysate by counter-current chromatography using aqueous two-phase system

Aqueous-aqueous two-phase (AATP) systems composed of polyethylene glycol (PEG) (molecular mass, M(r):1000-8000) and dextran (M(r):40,000) were evaluated for purification of maltose binding protein tagged-histone deacetylase (MBP-HDAC) by counter-current chromatography (CCC). CCC purification of an MBP-HDAC from Escherichia coli cell-lysate was successfully demonstrated with a 7.0% PEG 3350-10% dextran T40 system containing 10 mM potassium phosphate buffer at pH 9.0. After CCC purification, both polymers in the CCC fractions were easily removed by ultrafiltration in a short period of time. The collected fractions containing target protein were analyzed by an HPLC-based in vitro assay as well as sodium dodecyl sulfate polyacrylamide gel electrophoresis. MBP tag was digested from fusion HDAC during the CCC separation and native HDAC was purified by one-step operation with well preserved deacetyl enzyme activity.     

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.     

Screening of antioxidant phenolic compounds in Chinese Rhubarb combining fast counter‐current chromatography fractionation and liquid chromatography/mass spectrometry analysis

In this paper, an effective method combing fast elution‐extrusion counter‐current chromatography (CCC) and LC/MS for rapid screening of antioxidative phenolic compounds in Chinese Rhubarb is presented. An integrated three‐coil CCC column (40 mL each coil) was used to accomplish the optimization of biphasic liquid system. In a single run (approximately 40 min), the solvent system composed of n‐hexane/ethyl acetate/methanol/water (1:1:1:1, v/v) was selected as optimum CCC liquid system for fast fractionation of the crude ethanol extract. With a 140 mL‐capacity CCC instrument, 100 mg Chinese Rhubarb extract was separated under the optimized conditions, producing six fractions in only 100 min. The quantities of each fraction were ～15 mg. In addition, each fraction was subjected to antioxidant activity assay and characterized by LC/MS analysis. Fifty compounds, including phenolic acids, phenolic glucosides and hydroxyanthraquinones, were detected by LC/MS/MS analysis. As a result, gallic acid together with Fr I showed excellent antioxidant activity, which was well consistent with previous studies and exhibited great potential for natural drug discovery program of the present method.     

pH-zone-refining counter-current chromatography: Origin,mechanism, procedure and applications

Since 1980, high-speed counter-current chromatography (HSCCC) has been used for separation and purification of natural and synthetic products in a standard elution mode. In 1991, a novel elution mode called pH-zone refining CCC was introduced from an incidental discovery that an organic acid in the sample solution formed the sharp peak of an acid analyte. The cause of this sharp peak formation was found to be bromoacetic acid present in the sample solution which formed a sharp trailing border to trap the acidic analyte. Further studies on the separation of DNP-amino acids with three spacer acids in the stationary phase revealed that increased sample size resulted in the formation of fused rectangular peaks, each preserving high purity and zone pH with sharp boundaries. The mechanism of this phenomenon was found to be the formation of a sharp trailing border of an acid (retainer) in the column which moves at a lower rate than that of the mobile phase. In order to facilitate the application of the method, a new method was devised using a set of retainer and eluter to form a sharp retainer rear border which moves through the column at a desired rate regardless of the composition of the two-phase solvent system. This was achieved by adding the retainer in the stationary phase and the eluter in the mobile phase at a given molar ratio. Using this new method the hydrodynamics of pH-zone-refining CCC was diagrammatically illustrated by three acidic samples. In this review paper, typical pH-zone-refining CCC separations were presented, including affinity separations with a ligand and a separation of a racemic mixture using a chiral selector in the stationary phase. Major characteristics of pH-zone-refining CCC over conventional HSCCC are as follows: the sample loading capacity is increased over 10 times; fractions are highly concentrated near saturation level; yield is improved by increasing the sample size; minute charged compounds are concentrated and detected at the peak boundaries; and elution peaks are monitored with a pH flow meter for compounds with no chromophore. Since 1994, over 70 research papers on pH-zone-refining CCC have been published with the trends increasing in the recent years.     

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.     

Preparative enantioseparation of propafenone by counter‐current chromatography using di‐n‐butyl l‐tartrate combined with boric acid as the chiral selector

This paper extends the research of the utilization of borate coordination complexes in chiral separation by counter‐current chromatography (CCC). Racemic propafenone was successfully enantioseparated by CCC with di‐n‐butyl l ‐tartrate combined with boric acid as the chiral selector. The two‐phase solvent system was composed of chloroform/ 0.05 mol/L acetate buffer pH 3.4 containing 0.10 mol/L boric acid (1:1, v/v), in which 0.10 mol/L di‐n‐butyl l ‐tartrate was added in the organic phase. The influence of factors in the enantioseparation of propafenone were investigated and optimized. A total of 92 mg of racemic propafenone was completely enantioseparated using high‐speed CCC in a single run, yielding 40–42 mg of (R)‐ and (S)‐propafenone enantiomers with an HPLC purity over 90–95%. The recovery for propafenone enantiomers from fractions of CCC was in the range of 85–90%.     

One-step isolation of γ-oryzanol from rice bran oil by non-aqueous hydrostatic countercurrent chromatography

The value-added γ-oryzanol was purified in one step from crude rice bran oil (RBO) using a preparative hydrostatic countercurrent chromatography (hydrostatic CCC) method, operating in the dual mode. The fractionation was performed using a non-aqueous biphasic solvent system consisting of heptane-acetonitrile-butanol (1.8:1.4:0.7, v/v/v), leading rapidly to the target compounds. Transfer of the analytical CCC method to large-scale isolation was also carried out yielding a high quantity-high purity fraction of γ-oryzanol. In addition, a fraction of hydroxylated triterpene alcohol ferulates (polar γ-oryzanol) was clearly separated and obtained. Furthermore, a fast HPLC-APCI(±)-HRMS method was developed and applied for the identification of γ-oryzanol as well as the polar γ-oryzanol in RBO and the resulting fractions. The purity of γ-oryzanol fraction was estimated as 97% based on HPLC-APCI-HRMS analysis.     

Preparative isolation and purification of senkyunolide-I, senkyunolide-H and ferulic acid from Rhizoma Chuanxiong using counter-current chromatography

Three active compounds, senkyunolide-I, senkyunolide-H and ferulic acid (FA), were successfully isolated and purified from the extracts of Rhizoma Chuanxiong by counter-current chromatography (CCC). Based on the principle of the partition coefficient values (k) for target compounds and the separation factor (α) between target compounds, the two-phase solvent system that contains n-hexane-ethyl acetate-methanol-water at an optimized volume ratio of 3:7:4:6 v/v was selected for the CCC separation, and the lower phase was employed as the mobile phase in the head-to-tail elution mode. In a single run, 400 mg of the crude extract yielded pure senkyunolide-I (6.4 mg), senkyunolide-H (1.7 mg) and FA (4.4 mg) with the purities of 98, 93 and 99%, respectively. The CCC fractions were analyzed by high-performance liquid chromatography, and the structures of the three active compounds were identified by MS and (1)H NMR.     

Preparative isolation and purification of amides from the fruits of Piper longum L. by upright counter-current chromatography and reversed-phase liquid chromatography

A versatile counter-current chromatography (CCC) with upright type-J multilayer coil planet centrifuge, named upright CCC, was applied to the isolation and purification of amides from Piper longum L., which is widely used as an anodyne and a treatment for stomach disease in China. After the saponification by KOH of the ethanol extracts solution of 15 kg of crude drug "Piper Longi Fructus", the fruits of P. longum L., the solution was extracted with light petroleum and 500 g of red crude oil was obtained. Using 2.5 g of red crude oil as sample, the preparative upright CCC with a two-phase system composed of light petroleum (bp 60-90 degrees C)-ethyl acetate-tetrachloromethane-methanol-water (1:1:8:6:1, v/v) was successfully performed, which yielded nine fractions. Then these fractions were further purified by use of reversed-phase liquid chromatography (RPLC) with a glass column of 500 x 10 mm i.d. packed with reversed-phase silica gel. As a result, nine target amides with over 95% purity, i.e., 50 mg of (2E,4E)-N-isobutyl-eicosa-2,4-dienamide, 150 mg of (2E,4E,14Z)-N-isobutyl-eicosa-2,4,14-trienamide, 110 mg of (2E,4E,12Z)-N-isobutyl-ocatadeca-2,4,12-trienamide, 50 mg of guineensine, 60 mg of pipernonaline, 75 mg of pellitorine, 63 mg of piperine, 45 mg of piperanine, and 40 mg of piperlonguminine were isolated, respectively. Structures of all compounds were identified by electrospray ionization MS, electron impact ionization MS, one- and two-dimensional NMR spectra.     

Purification of the seven tetranortriterpenoids in neem (Azadirachta indica) seed by counter-current chromatography sequentially followed by isocratic preparative reversed-phase high-performance liquid chromatography

Counter-current chromatography (CCC) sequentially followed by isocratic preparative reversed-phase high-performance liquid chromatography was used to isolate the seven bio-actives (azadirachtin A, azadirachtin B, azadirachtin H, desacetylnimbin, desacetylsalannin, nimbin and salannin) from the seed concentrate (NSC) of the neem tree (Azadirachta indica A. Juss). Reproducible, narrow polarity range, high purity fractions were obtained from repeated injections of the NSC (700 mg loadings/injection), on to a relatively small volume CCC coil (116 mL). The CCC biphasic solvent system chosen was hexane:butanol:methanol:water (1:0.9:1:0.9, v/v). A mass balance of injected material showed that 95+% were recovered.     

固相萃取/超高压液相色谱-质谱/质谱联用技术测定土壤与灌溉水中的矮壮素及缩节胺

采用固相萃取技术对土壤和灌溉用水中的矮壮素及缩节胺进行富集处理,并对固相萃取柱的类型和淋洗条件进行优化,采用超高压液相色谱-质谱/质谱联用技术对富集样品进行分离检测.结果表明,灌溉水中矮壮素和缩节胺的加标水平为0.05、0.10、0.20μg/L时,回收率分别为73%～83%和76%～81%,相对标准偏差分别为9.2%...     

Separation of anthraquinone compounds from the seed of Cassia obtusifolia L. using recycling counter‐current chromatography

Recycling counter‐current chromatography (CCC) together with step‐gradient CCC and medium‐pressure liquid chromatography (MPLC) was employed to separate nine anthraquinone compounds from Cassia obtusifolia L. in this study. The results showed that recycling CCC is a powerful tool for compounds that are difficult to separate with common elution mode. CCC was the better option for crude material while MPLC had advantage for the final tuning. The combination of recycling CCC and MPLC could simplify the method exploring process in the separation process. The structures of these compounds were identified according to their mass spectra, by ¹H‐NMR and compared with standard compounds.     

Efficient Separation of Phytochemicals from Muehlenbeckia volcanica (Benth.) Endl. by Polarity-Stepwise Elution Counter-Current Chromatography and Their Antioxidant,Antiglycation, and Aldose Reductase Inhibition Potentials

Muehlenbeckia volcanica (Benth.) Endl. (M. volcanica), native to South America, is a traditional Peruvian medicinal plant that has multi-therapeutic properties; however, no phytochemicals have been identified from it yet. In this study, a five-step polarity-stepwise elution counter-current chromatography (CCC) was developed using methanol/water (1:5, v/v) as the stationary phase and different ratios of n-hexane, ethyl acetate, and n-butanol as mobile phases to separate the compounds from the 70% methanol extract of M. volcanica, by which six compounds with a wide range of polarities were separated in a single run of CCC and were identified as gallic acid, protocatechuic acid, 4,4′-dihydroxy-3,3′-imino-di-benzoic acid, rutin, quercitrin, and quercetin. Then, two compounds from the fractions of stepwise elution CCC were separated using conventional high-speed CCC, pH-zone-refining CCC, and preparative high-performance liquid chromatography, and identified as shikimic acid and miquelianin. These compounds are reported from M. volcanica for the first time. Notably, except for shikimic acid, all other compounds showed anti-diabetic potentials via antioxidant, antiglycation, and aldose reductase inhibition. The results suggest that the polarity-stepwise elution CCC can be used to efficiently separate or fractionate compounds with a wide range of polarities from natural products. Moreover, M. volcanica and its bioactive compounds are potent anti-diabetic agents.     

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.     

Mathematical model of computer-programmed intermittent dual countercurrent chromatography applied to hydrostatic and hydrodynamic equilibrium systems

Dual high-speed countercurrent chromatography (dual CCC) literally permits countercurrent flow of two immiscible solvent phases continuously through the coiled column for separation of solutes according to their partition coefficients. Application of this technique has been successfully demonstrated by separation of analytes by gas–liquid and liquid–liquid two-phase systems. However, the method cannot be directly applied to the system with a set of coiled columns connected in series, since the countercurrent process is interrupted at the junction between the columns. However, this problem can be solved by intermittent dual CCC by eluting each phase alternately through the opposite ends of the separation column. This mode of application has an advantage over the conventional dual CCC in that the method can be applied to all types of CCC systems including hydrostatic equilibrium systems such as toroidal coil CCC and centrifugal partition chromatography. Recently, the application of this method to high-speed CCC (hydrodynamic system) has been demonstrated for separation of natural products by Hewitson et al. using a set of conventional multilayer coil separation columns connected in series. Here, we have developed a mathematical model for this intermittent dual CCC system to predict retention time of the analytes, and using a simplified model system the validity of the model is justified by a series of basic studies on both hydrodynamic and hydrostatic CCC systems with a computer-programmed single sliding valve. The present method has been successfully applied to spiral tube assembly high-speed CCC (hydrodynamic system) and toroidal coil CCC (hydrostatic system) for separation of DNP-amino acid samples with two biphasic solvent systems composed of hexane–ethyl acetate–methanol–0.1 M hydrochloric acid (1:1:1:1 and 4:5:4:5, v/v).     

Size-fractionation of silver nanoparticles using ion-pair extraction in a counter-current chromatograph

Size separation of silver nanoparticles was investigated in counter-current chromatography (CCC) based on a unique step-gradient extraction process. Carboxylate anions were modified on silver nanoparticles to produce water-dispersible nanoparticles. The aqueous nanoparticles were readily transferred to the organic phase (toluene/hexane = 1:1, v/v) together with the phase transfer catalyst, tetraoctylammonium bromide (TOAB), owing to the ion-pair adduct formation between silver nanoparticle anions and tetraoctylammonium cations. Smaller nanoparticles were found to be more readily transferred to the organic phase compared to larger nanoparticles. Various concentrations of TOAB in the organic elution phase were used in the CCC extraction experiments. It appeared that a concentration of 0.02 mM of TOAB was adequate to achieve optimum separation and recovery for the aqueous Ag nanoparticle sample (1.5 mg) in the CCC extraction experiments. Samples of 15.8 ± 5.3 nm were separated; the distributions of four fractions collected were 13.7 ± 1.9, 14.1 ± 3.5, 19.2 ± 4.3, and 22.2 ± 4.9 nm. Compared with the stepwise extraction performed in this study, the step-gradient extractions using CCC provided much better size discrimination.     

Using the liquid nature of the stationary phase in countercurrent chromatography. IV. The cocurrent CCC method

The retention volumes of solutes in countercurrent chromatography (CCC) are directly proportional to their distribution coefficients, K(D) in the biphasic liquid system used as mobile and stationary phase in the CCC column. The cocurrent CCC method consists in putting the liquid "stationary" phase in slow motion in the same direction as the mobile phase. A mixture of five steroid compounds of widely differing polarities was used as a test mixture to evaluate the capabilities of the method with the biphasic liquid system made of water/methanol/ethyl acetate/heptane 6/5/6/5 (v/v) and a 53 mL CCC column of the coil planet centrifuge type. It is shown that the chromatographic resolution obtained in cocurrent CCC is very good because the solute band broadening is minimized as long as the solute is located inside the "stationary" phase. Pushing the method at its limits, it is demonstrated that the five steroids can still be (partly) separated when the flow rate of the two liquid phases is the same (2 mL/min). This is due to the higher volume of upper phase (72% of the column volume) contained inside the CCC column producing a lower linear speed compared to the aqueous lower phase linear speed. The capabilities of the cocurrent CCC method compare well with those of the gradient elution method in HPLC. Continuous detection is a problem due to the fact that two immiscible liquid phases elute from the column. It was partly solved using an evaporative light scattering detector.     

高效液相色谱-串联质谱法测定动物源性食品中矮壮素残留

建立了高效液相色谱-串联质谱测定动物源性食品中矮壮素残留的分析方法。样品经含1%（v/v）乙酸的乙腈溶液提取、正己烷脱脂、阳离子固相萃取柱（PCX）净化,采用Venusil MP C18（2）色谱柱（150 mm×2.1 mm,3 μm）分离,以乙腈和0.1%（v/v）甲酸水溶液为流动相进行梯度洗脱,采用电喷雾电离、正离子模式扫描,多反应监测模式（MRM）检测,基质匹配标准曲线内标法定量。结果表明：矮壮素在0.200~500 μg/L范围内呈良好线性,相关系数（r²）均不低于0.9993,方法的定量限为0.500 μg/kg;以猪肉、牛肉、羊肉、鸡肉、鸡蛋、猪肾、牛肝、羊肾、鸡肝、牛奶为基质,矮壮素的平均加标回收率为93.4%~101%,相对标准偏差为2.3%~8.0%。该方法基质干扰小,灵敏度高,准确可靠,适用于动物源性食品中矮壮素残留的定量检测。     

Application of counter-current chromatography as a new pretreatment method for the determination of polycyclic aromatic hydrocarbons in environmental water

Counter‐current chromatography (CCC) was investigated as a new sample pretreatment method for the determination of trace polycyclic aromatic hydrocarbons (PAHs) in water environmental samples. The experiment was performed with a non‐aqueous binary two‐phase solvent system composed of n‐heptane and acetonitrile. The CCC column was first filled with the upper stationary phase, and then a large volume of water sample was pumped into the column while the CCC column was rotated at 1600 rpm. Finally, the trace amounts of PAHs extracted and enriched in the stationary phase were eluted out by the lower mobile phase and determined by gas chromatography–flame ionization detector (GC‐FID) or gas chromatography–mass spectrometry (GC‐MS). The enrichment and cleanup of PAHs can be fulfilled online by this method with high recoveries (84.1–103.2%) and good reproducibility (RSDs: 4.9–12.2%) for 16 EPA PAHs under the optimized CCC pretreatment conditions. This method has been successfully applied to determine PAHs in lake water where 8 PAHs were detected in the concentration of 40.9–89.9 ng/L. The present method is extremely suitable for the preparation of large volume of environmental water sample for the determination of trace amounts of organic pollutants including PAHs as studied in this paper.     

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.