高速逆流色谱分离同分异构体

应用高速逆流色谱法对同分异构体的分离纯化进行研究。实验结果表明,以正己烷-乙酸乙酯-水(体积比为1∶10∶10)为两相溶剂系统,上相为固定相,下相为流动相,进行二次高速逆流色谱分离,可从茶多酚中分离出g级的儿茶素同分异构体——(-)-表没食子儿茶素没食子酸酯(EGCG)和(-)-没食子儿茶素没食子酸酯(GCG),其高效液相色谱纯度均在98%以上。选择四氯化碳-氯仿-甲醇-水(体积比为7∶3∶7∶3)为溶剂系统,下相为固定相,上相为流动相,经一次高速逆流色谱即可将药物中间体溴代苯胺同分异构体进行有效的分离。     

高速逆流色谱分离纯化EGCG3"Me的研究

首次采用高速逆流色谱法对经自制聚酰胺初步分离的表没食子儿茶素-3-(3″-O-甲基)没食子酸酯( EGCG3"Me)样品中的EGCG3"Me单体进行分离纯化.结果表明,选择水-甲醇-乙酸乙酯-正己烷(体积比5:2:9:1)为高速逆流色谱分离的两相溶剂系统,上相为固定相,下相为流动相,在主机转速700 r／min、流速2.0 mL/min、检测波长254 nm的条件下,可对EC CG3"Me实现良好分离.经高效液相色谱检测,分离纯化后的EC CG3"Me纯度可达90%以上,产物的收率为76.53%.     

半制备型液相色谱法分离纯化茶叶鲜叶中7种儿茶素类化合物

建立了从茶叶鲜叶中分离纯化7种儿茶素类化合物(没食子儿茶素(GC)、表没食子儿茶素(EGC)、儿茶素(C)、表没食子儿茶素没食子酸酯(EGCG)、表儿茶素(EC)、表没食子儿茶素3-O-(3-O-甲基)没食子酸酯(EGCG3"Me)和表儿茶素没食子酸酯(ECG))的半制备色谱法。铁观音鲜叶经甲醇超声浸提、浓缩、氯仿萃取后,向水相中加入碱式醋酸铅沉淀,得到茶多酚粗品。分别以甲醇-水、乙腈-水作为流动相,采用半制备色谱法纯化7种儿茶素类化合物,纯度均达到90%。此外,利用同样的方法分离纯化另外两种茶叶鲜叶中的7种儿茶素类化合物,得到相似的结果。该方法以溶剂提取、离子沉淀结合半制备色谱,适于简单、高效地同时分离制备多种儿茶素类化合物。     

高速逆流色谱法从蝙蝠蛾拟青霉中快速分离制备麦角甾醇纯品

建立了用高速逆流色谱从蝙蝠蛾拟青霉中高效、快速分离制备高纯度麦角甾醇的方法。将蝙蝠蛾拟青霉的乙酸乙酯提取物直接进行高速逆流色谱分离,考察了不同溶剂系统的分离效果。结果表明,最佳的溶剂系统为正己烷-乙酸乙酯-甲醇-水(体积比为6:1.7:6:0.3),以上相为固定相,下相为流动相,转速为850 r/min,流速为2 mL/min,检测波长为280 nm。制备所得的麦角甾醇经紫外光谱(UV)和高分辨质谱(HRMS)鉴定及与标准品对照定性;纯度经高效液相色谱(HPLC)分析为99.2%(峰面积归一化法)。该方法制备麦角甾醇简便、快速,所得产物的纯度高,适合于麦角甾醇对照品的制备。     

高速逆流色谱分离纯化EGCG3"Me的研究

首次采用高速逆流色谱法对经自制聚酰胺初步分离的表没食子儿茶素-3-(3″-O-甲基)没食子酸酯( EGCG3"Me)样品中的EGCG3"Me单体进行分离纯化.结果表明,选择水-甲醇-乙酸乙酯-正己烷(体积比5:2:9:1)为高速逆流色谱分离的两相溶剂系统,上相为固定相,下相为流动相,在主机转速700 r／min、流速2...     

高速逆流色谱法分离制备中药诃子中的没食子酸

利用高速逆流色谱法从100 mg诃子醇提物中一次性分离制备得到8.6 mg没食子酸。通过分析型高速逆流色谱对5种溶剂系统进行筛选,确定以正己烷-乙酸乙酯-甲醇-水(体积比为1:5:1:5)为两相溶剂体系并放大到制备型上,以上相为固定相,下相为流动相,在主机转速850 r/min、流动相流速2 mL/min、检测波长254 nm的条件下进行分离制备,获得4个分离峰(组分Ⅰ、Ⅱ、Ⅲ、Ⅳ)。经高效液相色谱检测,按照面积归一法计算,其中组分Ⅲ的纯度达96.40%。经电喷雾电离质谱分析,并结合与没食子酸标准品的高效液相色谱测定结果的对比,确定组分Ⅲ为没食子酸。该方法简便、快速、重复性好,适合于诃子中没食子酸的分离制备。     

大孔树脂-高速逆流色谱分离纯化薇甘菊中的黄酮类化合物（英文）

该文建立了大孔树脂-高速逆流色谱分离薇甘菊中黄酮类物质的方法。分离条件为:采用大孔树脂AB-8,洗脱液为50%(v/v)乙醇水溶液,高速逆流色谱溶剂体系为正丁醇-乙酸-水(4∶1∶5,v/v)。从薇甘菊中分离到4种黄酮类物质:槲皮素-3-O-芸香糖苷(纯度90.2%)、山奈酚-3-O-芸香糖苷(纯度98.55%)、木犀草苷(纯度98.33%)和紫云英苷(纯度99.23%)。建立的大孔树脂-高速逆流色谱方法简单、高效,可扩展应用于从其他植物中分离黄酮类物质。     

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。     

高速逆流色谱法从秦艽地上部分制备分离龙胆苦苷

采用高速逆流色谱法分离纯化秦艽地上部分中的龙胆苦苷。溶剂系统为V(氯仿)∶V(甲醇)∶V(水)=4∶4∶2,上相为固定相,下相为流动相,转速为800r/min,流速为2mL/min。所得产物经LC-MS分析为龙胆苦苷,纯度经高效液相色谱分析为94.0%(峰面积归一化法)。在保证分离效果的前提下,增大进样量,摸索制备分离龙胆苦苷的高速逆流色谱条件,为用高速逆流色谱法大量制备龙胆苦苷提供了一个可行方法。     

夏天无生物碱的高速逆流色谱分离纯化

采用pH-区带精制逆流色谱与常规高速逆流色谱相结合的方法快速分离纯化夏天无总生物碱.利用pH-区带精制逆流色谱对夏天无总生物碱进行分离,以正己烷-乙酸乙酯-甲醇-水(5∶ 5∶ 2∶ 8, V/V, 上相加5 mmol/L三乙胺,下相加5 mmol/L HCl)为溶剂系统,上样量3.0 g,分离得到1个混合物和3个高纯度的生物碱单体:原阿片碱(375 mg)、苏元胡碱甲(362 mg)和比枯枯灵(246 mg), 其纯度分别为97.5%,95.6%和97.1%.所得混合物850 mg经常规高速逆流色谱二次分离,以正己烷-乙酸乙酯-甲醇-水(1∶ 0.8∶1.1∶ 1, V/V)为溶剂系统,得到比枯枯灵(105 mg)和四氢巴马亭(470 mg)单体,纯度分别为99.1%和99.7%.从该药材分得苏元胡碱甲,两种制备分离模式相结合,提高了夏天无生物碱的分离效率.     

Large-scale isolation of flavan-3-ol phloroglucinol adducts by high-speed counter-current chromatography

Flavan-3-ol phloroglucinol adducts were synthesised through acid catalysed degradation of a procyanidins-rich grape seed extract in the presence of phloroglucinol. The reaction mixture (3.3 g) was fractionated without further sample preparation using the all-liquid chromatographic technique of high-speed counter-current chromatography (HSCCC). Selected solvent systems were hexane-ethyl acetate-methanol-water (0.1:5:0.1:5, v/v/v/v) and (1.5:10:1.5:10, v/v/v/v). The fractions obtained were found to contain almost pure compounds, in some cases final purification was achieved by preparative HPLC. The so-obtained pure standards of (+)catechin-(4alpha-->2)-phloroglucinol, (-)epicatechin-(4beta-->2)-phloroglucinol, (+)catechine, (-)epicatechin-3-O-galloyl-(4beta-->2)-phloroglucinol, (-)epicatechin, and (-)epicatechin gallate are required for quantification of acid-catalysed phloroglucinol degradation products of procyanidins.     

Preparative isolation of polyphenolic compounds from Vitis vinifera by centrifugal partition chromatography

This study deals with a centrifugal partition chromatography developed for the separation of phenolic compounds from Vitis vinifera. EtOAc grape seed extracts were separated using the solvent system hexane-ethyl acetate-ethanol-water (1:8:2:7; v/v) in two fractions: one containing about 75% of flavanol monomers (catechin and epicatechin) corresponding to 18% of crude extract and another fraction B-type dimers (22% of crude extract). From the stalk extracts, we could separate stilbenoid compounds (resveratrol and its oligomers; 12% of crude extract) which were eluted in less than 30 min from flavanols (which required a few hours of additional elution). Using the same solvent system but in different ratios (4:5:3:3; v/v), we isolated the trans-resveratrol (7@1000; 90% purity).     

Determination of vitamin B12 in multivitamin tablets by multimode high-performance liquid chromatography

Quantitative determination of vitamin B₁₂ in B-complex tablets was performed by using multimode high-performance liquid chromatography. The multivitamin tablets (B₁, B₆ and B₁₂) were sonicated for 30 min in methanol–water (50:50, v/v) and diluted to appropriated volume with the same solvent. The resulting solution was filtered and the filtrate was analysed on a phenylpropanolamine bonded silica column (15 cm×4.6 mm I.D., 5 μm). The optimized mobile phase was 30 mM phosphate buffer (pH 3.00) containing 6% (v/v) acetonitrile at a flow-rate of 1 ml min⁻¹ and the detection was measured at 361 nm. The calibration graph prepared using standards was linear from 0.05 to 0.25 μg. The determination limit was 25 ng, the relative standard deviation was 0.47% and recovery from tablet solution was 100%. An analysis was completed in 5 min. The new method is simple, rapid and precise.     

Preparative isolation of procyanidins from grape seed extracts by high-speed counter-current chromatography

High-speed counter-current chromatography (HSCCC) has been applied to the separation of grape seed procyanidins. The isolation of dimeric to tetrameric procyanidins is achieved after removing the polymeric compounds by solvent precipitation. An additional clean-up by solid-phase extraction on polyamide improved the purities of the isolated compounds. The solvent systems ethyl acetate/2-propanol/water (40:1:40, v/v/v), ethyl acetate/2-propanol/water (20:1:20, v/v/v), and ethyl acetate/1-butanol/water (14:1:15, v/v/v) were successfully used for the fractionation. The combination of HPLC-MS, diode array detection, and NMR analysis, as well as phloroglucinolysis, confirmed the structures of the isolated compounds: B1 [EC-(4beta-->8)-C], B2 [EC-(4beta-->8)-EC], B3 [C-(4alpha-->8)-C], B4 [C-(4alpha-->8)-EC], B5 [EC-4beta-->6-EC], B7 [EC-(4beta-->8)-C], [ECG-(4beta-->8)-C], trimeric procyanidin C1 [EC-4beta-->8-EC-4beta-->8-EC], and the tetrameric procyanidin cinnamtannin A2 (where C: catechin, EC: epicatechin and ECG: epicatechin-3-O-gallate).     

Application of preparative high-speed counter-current chromatography for separation of methyl gallate from Acer truncatum Bunge

Preparative separation of methyl gallate in leaves extract of Acer truncatum Bunge was conducted using high-speed counter-current chromatography (HSCCC) with a solvent system composed of ethyl acetate-ethanol-water at volume ratios of 5:1:5 (v/v/v). In a single operation, 57.5 mg of methyl gallate was obtained from 120 mg of the extract. HPLC analyses of the counter-current chromatography (CCC) fraction revealed that the methyl gallate was having over 97% purity. Its structure was identified by 1H NMR and 13C NMR.     

Liquid chromatography of polymyxin B sulphate

A reversed-phase liquid chromatography method for analysis of polymyxin B sulphate is described. The method uses a YMC-Pack Pro, C₁₈, 5 μm, 250×4.6 mm I.D. column maintained at 30°C. The mobile phase comprises acetonitrile–sodium sulphate (0.7%, m/v)–phosphoric acid (6.8%, v/v dilution of 85%, m/m phosphoric acid)–water (22.25:50:5:22.75) at a flow-rate of 1.0 ml/min. Detection was by UV at 215 nm. The method is able to resolve polymyxin B₁, the major component, from more than thirty other components present in the complex. Robustness was evaluated by performing a full-factorial design experiment. The method showed good selectivity, repeatability, linearity and sensitivity.     

Separation and purification of baicalin and wogonoside from the Chinese medicinal plant Scutellaria baicalensis Georgi by high-speed counter-current chromatography

A preparative high-speed counter-current chromatography (HSCCC) method for isolation and purification of baicalin and wogonoside from the Chinese medicinal plant Scutellaria baicalensis Georgi (Huang-qin in Chinese) was successfully established by using ethyl acetate-methanol-1% acetic acid water (5:0.5:5, v/v) as the two-phase solvent system. The upper phase of ethyl acetate-methanol-1% acetic acid water (5:0.5:5, v/v) was used as the stationary phase of HSCCC. Baicalin (58.1 mg) and wogonoside (17.0mg) with the purity of 99.2 and 99.0%, respectively, were separated successfully in one-step separation from 120 mg of crude sample from S. baicalensi, Georgi. The structures of baicalin and wogonoside were identified by 1H NMR and 13C NMR.     

Orthogonal test design for optimization of supercritical fluid extraction of daphnoretin, 7-methoxy-daphnoretin and 1,5-diphenyl-1-pentanone from Stellera chamaejasme L. and subsequent isolation by high-speed counter-current chromatography

Supercritical fluid extraction (SFE) of daphnoretin, 7-methoxy-daphnoretin and 1,5-diphenyl-1- pentanone from Stellera chamaejasme L. was performed. An orthogonal L9 (3)4 test design was applied to select the optimum extraction parameters including pressure, temperature, modifier and sample particle size on yield using an analytical-scale SFE system. The process was then scaled up by 100 times using a preparative SFE system under the optimized conditions of 25 MPa of pressure, 45 degrees C of temperature, 40-60 mesh of sample particle size and modified CO2 with 20% methanol. The yield of the crude extract from preparative SFE was 2.65%, which contained daphnoretin 25.2%, 7-methoxy-daphnoretin 22.8% and 1,5-diphenyl-1-pentanone 21.1%, respectively. Then the crude extract was successfully isolated and separated by preparative high-speed counter-current chromatography (HSCCC) with a two-phase solvent system composed of n-hexane-ethyl acetate-methanol-water (10:13:13:10, v/v) by increasing the flow-rate of the mobile phase stepwise from 1.0 to 2.0 ml/min after 90 min. The target compounds isolated and purified by HSCCC were analyzed by high-performance liquid chromatography (HPLC). The separation produced total of 69.2mg of daphnoretin at 99.2% purity, 63.4 mg of 7-methoxy-daphnoretin at 98.7% purity and 58.3 mg of 1,5-diphenyl-1-pentanone at 98.1% purity from 300 mg of the crude extract in one-step separation. The recoveries of daphnoretin, 7-methoxy-daphnoretin and 1,5-diphenyl-1-pentanone were 90.8, 91.5 and 90.4%, respectively, in HSCCC isolation step and the chemical structure identification was carried out by MS, 1H NMR and 13C NMR.     

Attomole catechins determination by capillary liquid chromatography with electrochemical detection.

Attomole quantities of catechins were determined by a capillary liquid chromatography system with electrochemical detection (CLC-ECD) and the system is applied to the determination of catechins in human plasma. The eight catechins: catechin (C), epicatechin (EC), gallocatechin (GC), epigallocatechin (EGC), catechin gallate (Cg), epicatechin gallate (ECg), gallocatechin gallate (GCg), and epigallocatechin gallate (EGCg), were separated within 10 min using a capillary column (0.2 mm i.d.) and a mobile phase of phosphoric acid (85%)-methanol-water (0.5:27.5:72.5, v/v/v), and were detected at +0.85 V vs. Ag/AgCl. Peak heights were found to be linearly related to the amount of catechins injected, from 200 amol to 500 fmol (r > 0.998). The detection limits of the catechins were 61 amol for EGC, 75 amol for EC, 54 amol for GC, 61 amol for C, 67 amol for GCg, 75 amol for EGCg, 75 amol for ECg and 89 amol for Cg (S/N = 3). Because the present method is highly sensitive and allows facile pretreatment for plasma sample, the time courses of concentrations of catechins (GCg, EC, EGCg, ECg, and Cg) and their conjugates in human plasma obtained from a 10 microl plasma sample after ingestion of green tea could be determined.     

Efficient new method for extraction and isolation of three flavonoids from Patrinia villosa Juss. by supercritical fluid extraction and high-speed counter-current chromatography

Supercritical fluid extraction (SFE) of orotinin, orotinin-5-methyl ether and licoagrochalcone B from Patrinia villosa was performed. The optimization of parameters including pressure, temperature, modifier and sample particle size on yield was carried out using an analytical-scale SFE system. The process was then scaled up by 100 times using a preparative SFE system under the optimized conditions of 25 MPa, 45 degrees C, a sample particle size 40-60 mesh and modified CO2 with 20% methanol. The yield of the preparative SFE was 2.82% (crude extract I) and the combined yield of orotinin, orotinin-5-methyl ether and licoagrochalcone B was 0.82 mg/g of dry sample mass. Then the crude extract I was re-dissolved in methanol and methanol soluble fraction (crude extract II, 0.17%) was obtained, which was successfully isolated and separated by a preparative high-speed counter-current chromatography (HSCCC) with a two-phase solvent system composed of n-hexane-ethyl acetate-methanol-water (5:6:6:6, v/v/v/v) by increasing the flow-rate of the mobile phase stepwise from 1.0 to 2.0 ml/min after 3 h. The target compounds isolated and purified by HSCCC were analyzed by high performance liquid chromatography. The separation produced total of 38.2 mg of orotinin at 99.2% purity, 19.8 mg of orotinin-5-methyl ether at 98.5% purity and 21.5 mg of licoagrochalcone B at 97.6% purity from 400 mg of the crude extract in a one-step separation. The recoveries of orotinin, orotinin-5-methyl ether and licoagrochalcone B were 91.1, 91.6 and 90.3%, respectively, and the chemical structure identification was carried out by UV, IR, MS, 1H NMR and 13C NMR.