Antibiotic purification from fermentation broths by counter-current chromatography: analysis of product purity and yield trade-offs

Counter-current chromatography (CCC) is a low pressure, liquid–liquid chromatographic technique which has proven to be a powerful purification tool for the high-resolution fractionation of a variety of active pharmaceutical compounds. The successful integration of CCC into either existing or new manufacturing processes requires the predictable purification of target compounds from crude, fermentation-derived, feed streams. This work examines the feasibility of CCC for the purification of fermentation-derived erythromycin A (EA) from its structurally and chemically similar analogues. At the laboratory scale, the effect of feed pre-treatment using either clarified, forward extracted (butyl acetate) or back extracted broth on EA separation was investigated. This defined the degree of impurity removal required, i.e. back extracted broth, to ensure a reproducible elution profile of EA during CCC. Optimisation and scale-up of the separation studied the effects of mobile phase flow (2–40 mlmin^–1) and solute loading (0.1–10 g) on the attainable EA purity and yield. The results in all cases demonstrated a high attainable EA purity (>97% w/w) with throughputs up to 0.33 kgday^–1. Secondly, a predictive scale-up model was applied demonstrating, that from knowledge of the solute distribution ratio of EA (K_EA) at the laboratory scale, the EA elution time at the pilot scale could be predicted to within 3–10%, depending upon the solute injection volume. In addition, this study has evaluated a fractionation diagram approach to visually determine the effects of key operational variables on separation performance. This resulted in accurate fraction cut-point determination for a required degree of product purity and yield. Overall, the results show CCC to be a predictable and scaleable separation technique capable of handling real feed streams.     

高速逆流色谱法从白芍中分离纯化五没食子酰基葡萄糖

本文建立高速逆流色谱(HSCCC)方法,从白芍粗提物中分离纯化五没食子酰基葡萄糖.分别采用正己烷-乙酸乙酯-甲醇-水体积比0.5∶5∶1∶5及0.5∶5∶0.5∶5混合溶剂作为两相溶剂体系,上相为固定相,下相为流动相,转速为800 rpm,流速为2.0 mL/min,用HPLC检测及ESI-MS进行验证.经过两次HSCCC分离纯化,得到五没食子酰基葡萄糖纯度为95.7％.     

高速逆流色谱法分离制备丹酚酸B

采用高速逆流色谱法分离纯化丹参水溶性成分丹酚酸类物质,制备丹酚酸B化学对照品。分离采用的溶剂系统为正己烷-乙酸乙酯-水-甲醇(1.5:5:5:1.5),上相做固定相,下相做流动相,流速为1.7 mL/min,仪器转速850 rpm,进样量80 mg,纯度用HPLC方法测定。结果表明:一次分离可制备63.4 mg丹酚酸B,其纯度为98.6%。该方法操作简单,可作为高纯度丹酚酸B化学对照品的制备分离方法。     

A novel design of simulated moving bed (SMB) chromatography for separation of ketoprofen enantiomer

A simulated moving bed (SMB) chromatography system is a powerful tool for preparative scale separation, which can be applied to the separation of chiral compound. We have designed our own lab-scale SMB chromatography using 5 HPLC pumps, 6 stainless steel columns and 4 multi-position valves, to separate a racemic mixture of ketoprofen in to its enantiomers. Our design has the characteristics of the low cost for assembly for the SMB chromatography and easy repair of the unit, which differs from the designs suggested by other investigators. It is possible for the flow path through each column to be independently changed by computer control, using 4 multi-position rotary valves and 5 HPLC solvent delivery pumps. In order to prove the operability of our SMB system, attempts were made to separate the (S)-ketoprofen enantiomer from a ketoprofen racemic mixture. The operating parameters of the SMB chromatography were calculated for ketoprofen separation from a batch chromatography experiment as well as by the triangle theory. With a feed concentration of 1 mg/mL, (S)-ketoprofen was obtained with a purity of 96% under the calculated operating conditions.     

The isolation of 1,2,3,4,6-penta-O-galloyl-beta-D-glucose from Acer truncatum Bunge by high-speed counter-current chromatography

High-speed counter-current chromatography (HSCCC) was successfully used for the isolation and purification of 1,2,3,4,6-penta-O-galloyl-beta-D-glucose from the ethyl acetate extract of the leaves of Acer truncatum Bunge using a two-phase system composed of n-hexane-ethyl acetate-methanol-water at a volume ratio of (0.25:5:1:5, v/v/v/v) for the first time. Each injection of 80 mg crude extract yielded 7.25 mg of pure 1,2,3,4,6-penta-O-galloyl-beta-D-glucose. High-performance liquid chromatography (HPLC) analyses of the CCC fraction revealed that the purity of 1,2,3,4,6-penta-O-galloyl-beta-D- glucose was over 95%.     

Isolation and purification of series bioactive components from Hypericum perforatum L. by counter-current chromatography

Counter-current chromatography (CCC) combined with pre-separation by ultrasonic solvent extraction was successively used for the separation of series bioactive compounds from the crude extract of Hypericum perforatum L. The petroleum ether extract was separated by the solvent system of n-heptane-methanol-acetonitrile (1.5:0.5:0.5, v/v) and n-heptane-methanol (1.5:1, v/v) in gradient elution, yielding a phloroglucinol compound, hyperforin with HPLC purity over 98%. The ethyl acetate extract was separated by using the solvent system composed of hexane-ethyl acetate-methanol-water (1:1:1:1 and 1:3:1:3, v/v) in gradient through both reverse phase and normal phase elution mode, yielding a naphthodianthrone compound, hypericin with HPLC purity about 95%. The n-butanol extract was separated with the solvent system composed of n-butanol-ethyl acetate-water (1:4:5 and 1.5:3.5:5, v/v) in elution and back-extrusion mode, yielding two of flavones, rutin and hyperoside, with HPLC purity over 95%. HPLC-MS, reference sample and UV spectrum were selectively used in separation to search for target compounds from HPLC-DAD profiles of different sub-extracts. The structures of isolated compounds were further identified by ESI-MS, 1HNMR and 13CNMR.     

Chiral HPLC Separation and Modeling of Four Stereomers of DL‐Leucine‐DL‐Tryptophan Dipeptide on Amylose Chiral Column

Chiral high‐performance liquid chromatography (HPLC) separation and modeling of four stereomers of DL‐leucine‐tryptophan DL‐dipeptide on AmyCoat‐RP column are described. The mobile phase applied was ammonium acetate (10 mM)‐methanol‐acetonitrile (50:5:45, v/v). The flow rate of the mobile phases was 0.8 mL/min with UV detection at 230 nm. The values of retention factors for LL‐, DD‐, DL‐, and LD‐ stereomers were 2.25, 3.60, 5.00, and 6.50, respectively. The values of separation and resolution factors were 1.60, 1.39, and 1.30 and 7.76, 8.05, and 7.19. The limits of detection and quantitation were ranging from 1.0–2.3 and 5.6–14.0 μg/mL. The simulation studies established the elution orders and the mechanism of chiral recognition. It was seen that π–π connections and hydrogen bondings were the main forces for enantiomeric resolution. The reported chiral HPLC method may be applied for the enantiomeric separation of DL‐leucine‐DL‐tryptophan in unknown matrices. Chirality 28:642–648, 2016. © 2016 Wiley Periodicals, Inc.     

Stereoselective HPLC separation of alvimopan on cellulose‐based immobilized polysaccharide as a chiral stationary phase

Chiral separation by normal phase high performance liquid chromatography is one of the most powerful technique to quantify the chiral purity of the compounds. In this study, a novel, simple, and specific analytical method was proposed to ascertain the chiral purity of alvimopan (ALV). The normal phase HPLC method was developed based on cellulose tris (3,5‐dichlorophenylcarbamate) stationary phase. The separation of ALV isomers achieved by using column CHIRALPAK IC (250 × 4.6 mm, 5 μm), mobile phase n‐hexane: isopropyl alcohol: ethanol: diethylamine (650:200:150:5 v/v), column oven temperature 30°C, flow rate 1.0 mL min^?1, injection volume was 10 μL, chromatographic response monitored at 273 nm. The developed method was validated as per the ICH guidelines and found precise, accurate, and linear. The advantage of the method is a good separation of ALV isomers within 35 minutes of the analysis time. Therefore, this method is suitable for routine determination of chiral purity of ALV active pharmaceutical ingredient.     

Application of analytical and preparative high-speed counter-current chromatography for the separation of Z-ligustilide from a crude extract of Angelica sinensis

Z-Ligustilide was separated and purified from the traditional Chinese medicinal plant Angelica sinensis by high-speed counter-current chromatography (HSCCC). Analytical HSCCC was first used for the systematic selection of the two-phase solvent system. Preparative HSCCC separation was performed with a two-phase solvent system composed of petroleum ether (60-90 degrees C)-ethanol-water at an optimum volume ratio of 10:17:10 (v/v). A total of 38 mg Z-ligustilide at 98.8% purity was obtained in one step from 200 mg crude extract as determined by HPLC analysis. The structure of the target compound was identified by electron impact ionisation mass spectrometry.     

应用高速逆流色谱分离桑枝酚类成分

建立了高速逆流色谱(HsCCC)分离制备高纯度的桑枝酚类成分的新方法.分离条件如下:溶剂系统为正己烷-乙酸乙酯-甲醇冰(1∶1∶1∶2,v/v),上相为固定相,下相为流动相;流速2.0 mL/min;转速900rpm;进样量75 mg.收集得到三个高纯度化合物,经HPLC、MS、1H和13C NMR等分别鉴定为反式氧化白藜芦醇(25.2mg),反式白藜芦醇(7.4 mg)和桑辛素M(29.1 mg).高速逆流色谱可以高效分离桑枝成分,方法简便,技术可行,优于传统的柱色谱法.     

Separation of supercoiled and open-circular plasmid DNA by liquid-liquid counter-current chromatography

We report for the first time the use of liquid-liquid counter-current chromatography (CCC) for the preparative scale fractionation of plasmid DNA. Almost complete fractionation of supercoiled and open circular plasmid DNA (6.9 kb) could be achieved using a phase system comprising 12.5% (w/w) PEG 600 and 18% (w/w) K₂HPO₄. Experiments were carried out on a Brunel J-type CCC machine (100 ml PTFE coil) at a mobile phase flow rate of 0.5 ml min^{– 1} and a rotational speed of 600 rpm. Compared to conventional HPLC techniques the capacity of CCC is not limited by the surface area of resin available for adsorption. Symbols: C _b, Concentration of plasmid in lower phase (g ml^–1); C _t, Concentration of plasmid in upper phase (g ml^–1); CV, Total volume of mobile phase present in the coil and connecting leads (ml); K, Equilibrium solute partition coefficient (K=C _t/C _b); OC, Open circular plasmid; SC, Supercoiled plasmid; S _f, Percentage stationary phase retention (S _f=V _s/V _c); t _s, Time for phase separation (s); V _b, Volume of bottom phase (ml); V _c, Coil volume (ml); V _m, Volume of mobile phase present in coil at equilibrium (ml); V _r, Volume ratio of two phases (V _r=V _t/V _b); V _s, Volume stationary phase present in coil at equilibrium (ml); V _t, Volume of top phase (ml); V _tot, Total volume of phase system (ml).     

发酵法生产紫杉醇的检测分析

目的:采用高效液相色谱法对发酵液中的紫杉醇进行测定。方法:将紫杉醇产生菌发酵产物经乙酸乙酯萃取得测定样品,高效液相色谱分析方法为苯基柱(250mm×4.6mm,5μm),流动相乙腈-甲醇-水(36∶4∶60),流速1mL/min,检测波长227nm,进样体积20μL,柱温室温。结果:紫杉醇与干扰物可达到基线分离,在2.2～110μg/mL范围内,紫杉醇的峰面积与浓度线性关系良好,相关系数0.9996,平均回收率为99.55%,RSD为0.60%。结论:与使用C18柱色谱条件相比,该分析方法灵敏度高,不需要复杂的样品前处理过程,仪器配置不复杂、操作方便、准确性高,可有效地检测发酵液中紫杉醇的含量。     

高速逆流色谱分离制备蛹虫草中虫草素和N6-(2-羟乙基)-腺苷

采用高速逆流色谱（HSCCC）技术从蛹虫草子实体粗提物中分离制备高纯度虫草素和N⁶-(2-羟乙基)-腺苷。利用高效液相色谱（HPLC）测定目标产物在溶剂体系中的分配系数,优化HSCCC分离虫草素和N⁶-(2-羟乙基)-腺苷的溶剂体系,确定了以乙酸乙酯-正丁醇-1.5%氨水（1:4:5,V/V/V）为HSCCC的两相溶剂体系,并运用此溶剂体系,上相为固定相,下相为流动相,主机转速850r/min,流动相流速为1.5mL/min,检测波长为254nm条件下进行分离制备,在250min内从200mg蛹虫草子实体粗提物中一步分离得到10.8mg纯度99%的虫草素和6.1mg 纯度98%的N⁶-(2-羟乙基)-腺苷。该方法简便、快速,为虫草素和N⁶-(2-羟乙基)-腺苷的大量制备建立了基础。     

An HPLC method for the pharmacokinetic study of daidzein-loaded nanoparticle formulations after injection to rats

This study was aimed at developing a simple HPLC method for the detection of daidzein in rat plasma. Daidzein was extracted from rat plasma with ethylparaben as internal standards (IS). Chromatographic separation of daidzein and IS was achieved by a Dikma Dimonsil C18 column (200 mm × 4.6mm) with the mobile phase consisting of methanol-water (55:45, v/v) at a flow rate of 1.0 mL/min. The injection volume was 20 μL and the detecting wavelength was 249 nm. The calibration curve was linear over a concentration range from 0.05 to 5 μg/mL, and the accuracy was within a range of 93.4-126.2%. This HPLC method was applied successfully to the pharmacokinetic study of two kinds of daidzein-loaded poly(lactide-co-glycolide) (PLGA) nanoparticles (D-NPs) and daidzein suspension after intravenous injection in rats. Significant differences in main pharmacokinetic parameters of daidzein suspension and D-NPs were observed.     

Purification of optical imaging ligand-Cybesin by high-speed counter-current chromatography

Fluorescent Cybesin (Cypate-Bombesin Peptide Analogue Conjugate) was synthesized from Indocyanine Green (ICG) and the bombesin receptor ligand as a contrast agent for detecting pancreas tumors. However, the LC-MS analysis indicated that the target compound was only a minor component in the reaction mixture. Since preparative HPLC can hardly separate such a small amount of the target compound directly from the original crude reaction mixture without a considerable adsorptive loss onto the solid support, high-speed counter-current chromatography (HSCCC) was used for purification since the method uses no solid support and promises high sample recovery. A suitable two-phase solvent system composed of hexane/ethyl acetate/methanol/methyl t-butyl ether/acetonitrile/water) at a volume ratio of 1:1:1:4:4:7 was selected based on the partition coefficient of Cybesin (K≈0.9) determined by LC-MS. The separation was performed in two steps using the same solvent system with lower aqueous mobile phase. From 400 mg of the crude reaction mixture the first separation yielded 7.7 mg of fractions containing the target compound at 12.8% purity, and in the second run 1 mg of Cybesin was obtained at purity of 94.0% with a sample recovery rate of over 95% based on the LC-MS analysis.     

紫茎泽兰9-羰基-10,11-去氢泽兰酮分布积累动态

9-羰基-10,11-去氢泽兰酮为紫茎泽兰（Eupatorium adenophorum）的主要致肝脏毒性成分及杀虫的生物活性成分。从紫茎泽兰叶片中分离提纯得到9-羰基-10,11-去氢泽兰酮（Euptox A）标准品,建立了高效液相色谱法测定紫茎泽兰中Euptox A含量的分析方法。采用C18反相色谱柱,柱温30°C,以甲醇-水（60：40,v/v）为流动相、流速为0.8 mL.min–1、检测波长为255 nm进行测定。Euptox A在紫茎泽兰中的添加回收率为97.3%–103.7%,检测限为0.4μg.g–1。利用建立的方法测定Euptox A在紫茎泽兰体内分布与积累的动态变化规律。结果表明,Euptox A主要分布在紫茎泽兰的叶片中,且在营养生长期积累量高,生殖生长期积累量低。该方法快速、简捷,可用于紫茎泽兰原料及其产品中Euptox A成分的测定。     

Functionalities tuned enantioselectivity of phenylcarbamate cyclodextrin clicked chiral stationary phases in HPLC

The mixed chloro‐ and methyl‐ functionalities can greatly modulate the enantioselectivities of phenylcarbamate cyclodextrin (CD) clicked chiral stationary phases (CSPs). A comparison study is herein reported for per(4‐chloro‐3‐methyl)phenylcarbamate and per(2‐chloro‐5‐methyl)phenylcarbamate β‐CD clicked CSPs (i.e., CCC4M3‐CSP and CCC2M5‐CSP). The enantioselectivity dependence on column temperature was studied in both normal‐phase and reversed‐phase mode high performance liquid chromatography (HPLC). The thermodynamic study revealed that the stronger intermolecular interactions can be formed between CCC4M3‐CSP and chiral solutes to drive the chiral separation. The higher enantioselectivities of CCC4M3‐CSP were further demonstrated with the enantioseparation of 17 model racemates in HPLC.     

Preparative separation of helvolic acid from the endophytic fungus <Emphasis Type="Italic">Pichia guilliermondii</Emphasis> Ppf9 by high-speed counter-current chromatography

High-speed counter-current chromatography (HSCCC) was applied for preparative separation of helvolic acid from the crude extract of the endophytic fungus Pichia guilliermondii Ppf9, associated with the medicinal plant Paris polyphylla var. yunnanensis for the first time. The two-phase solvent system consisted of n-hexane-ethyl acetate-methanol-water (4.5:4.5:5.0:5.0, v/v) appending with phosphoric acid (0.2%, v/v) was employed. The revolution speed of the separation column, flow rate of the mobile phase and separation temperature of the apparatus were 800 rpm, 3 ml min(-1) and 25°C, respectively. About 6.8 mg of helvolic acid was successfully obtained from 450 mg of the crude extract by HSCCC within 4 h separation procedure, and its purity reached to 93.2% according to the HPLC analysis. The product was further characterized by MS, (1)H-NMR and (13)C-NMR spectra.     

Evaluation of the performance of protein separation in figure-8 centrifugal counter-current chromatography

The performance of protein separation using the figure-8 column configuration in centrifugal counter-current chromatography was investigated under various flow rates and revolution speeds. The separation was performed with a two-phase solvent system composed of polyethylene glycol 1000/potassium phosphate each at 12.5% (w/w) in water and with lysozyme and myoglobin as test samples. In order to improve tracing of the elution curve, a hollow fiber membrane dialyzer was inserted at the inlet of the UV detector. The results showed that the retention of stationary phase (Sf) and resolution (Rs) increased with decreased flow rate and increased revolution speed. The highest Rs of approximately 1 was obtained at a flow rate of 0.01 mL/min under a revolution speed of 1200 rpm with a 3.4 mL capacity column.     

Separation of cephalosporin C and desacetyl cephalosporin C by high speed counter-current chromatography in aqueous two-phase systems

Summary In the recovery of cephalosporin C (CPC) from fermentation broth, the separation of desacetyl cephalosporin C (DAC) is a major concern. Multistage extraction in aqueous two-phase systems, mainly PEG/ammonium sulfate systems, proved to be promising. In preparative scale operation, high speed counter-current chromatography (HSCCC) with eccentric columns was used with aqueous two-phase systems to obtain baseline resolution of CPC and DAC. Solvents (e.g. 5% acetone) or neutral salts (e.g. 1.45% KSCN) added into aqueous two-phase systems enhanced the separation efficiency. Operation parameters of HSCCC such as rotational speed and mobile phase flow rate can affect the retention of the stationary phase and HETP.