高效液相色谱同时测定芦荟中8种蒽醌类物质的含量

建立了高效液相色谱(HPLC)-二级阵列检测器(DAD)同时测定芦荟中8种蒽醌类物质(芦荟苷B、芦荟苷A、大黄素-8-O-葡萄糖苷、芦荟大黄素、大黄酸、大黄素、大黄酚、大黄素甲醚)的方法。样品经5 mL 90%甲醇在30℃超声提取20 min后,以甲醇-乙腈-0.05%磷酸溶液为流动相梯度洗脱,经Eclipse XDB-C_(18)柱分离,多波长同时检测。8种蒽醌类待测物在14 min内获得满意的分离。方法的检出限(S/N=3)为0.55~1.02μg/g,样品平均加标回收率为90.3%~107.8%,RSD为4.8%。     

毛细管整体柱的制备技术及其应用进展

毛细管整体柱是以其制备相对简单无需烧结塞子,渗透性好,柱效高,低柱压等优点,成为目前备受关注的液相色谱固定相.它具有较好的重现性,可进行快速分离,已被应用于毛细管电色谱(CEC)和微柱高效液相色谱(μ-HPLC).本文主要介绍近几年毛细管整体柱的制备技术及其应用概况.     

高效液相色谱法快速测定大黄中大黄素,大黄酸和芦荟大黄素的研究

研究了高效液相色谱法测定大黄中大黄素、大黄酸和芦荟大黄素。大黄中大黄素、大黄酸和芦荟大黄素用氯仿加热回流提取,提取液蒸干溶剂,以甲醇溶解定容待测。以安捷仑ZORBAX Stable Bound(4.6 mm×50 mm,1.8μm)色谱柱为固定相,质量分数0.1%的H3PO4和甲醇为流动相,梯度洗脱(0 min:VH3PO4∶V甲醇=27∶73;1 min:100%甲醇);在该色谱条件下,大黄素、大黄酸和芦荟大黄素在2.0 min内可达到基线分离;用紫外二极管矩阵检测器检测。方法标准回收率为99.4%~102%,相对标准偏差为1.5%~1.8%。可用于大黄中大黄素、大黄酸和芦荟大黄素的快速分析检测。     

毛细管电色谱分析苯氧羧酸类药残的研究

毛细管电色谱(CEC)是近年发展起来的一种高效、快速微柱分离方法,主要分为填充毛细管电色谱,开管毛细管电色谱和整体式毛细管电色谱.开管毛细管电色谱无柱塞和填料,不易产生气泡,且无涡流扩散,能获得较高柱效,其电渗流流速比填充柱大60%,适用于快速分析,具有良好的应用前景.     

梯度加压毛细管电色谱分离蛋白质

 以1.5 μm无孔硅胶颗粒(non-porous silica,NPS)为固定相,采用电压和压力联合驱动流动相,用反相梯度加压毛细管电色谱(p-CEC)在7.5 min内实现了核糖核酸酶A、细胞色素C、溶菌酶和肌红蛋白等4种蛋白质的快速、高效的分离。比较了梯度加压毛细管电色谱和微柱液相色谱(μ-HPLC)分离蛋白质的结果,同时考察了固定相、离子对试剂三氟醋酸(TFA)浓度和电压等条件对梯度加压毛细管电色谱分离蛋白质的影响。结果表明,梯度p-CEC可以通过调节电压精细调节带电溶质的保留,提高分离选择性,缩短分离时间,得到较高的柱效。该方法在蛋白质分离分析及蛋白质组学的研究中具有很大的应用潜力，为高效快速地分离蛋白质开辟了新的途径。     

强阳离子交换毛细管液相色谱-反相加压毛细管电色谱二维系统的构建及其在中药黄柏提取物分离中的应用

加压毛细管电色谱(pCEC)具有电泳和液相色谱的双重分离机理,其柱效高、选择性强、分辨率高和分离速度快并可进行梯度洗脱。我们在此基础上加入离子交换色谱模式,构建了强阳离子交换-反相加压毛细管液相色谱(micro strong cation exchange liquid chromatography/reversed phase pressurized capillary electrochromatography, μ-SCXLC/RP-pCEC)二维系统,并对中药黄柏的提取物进行了优化分离。第一维μ-SCXLC采用线性盐梯度分离,样品被切割成11个馏分洗脱收集后进入第二维,第二维脱盐后,采用RP-pCEC进行分离分析,梯度洗脱。以中药黄柏提取物为样品,此二维系统的分辨率和峰容量都较一维系统有很大提高,理论峰容量可达900左右,证明构建的二维体系非常适合复杂样品的分离分析。     

注射用大黄粉针剂的高效液相色谱分析

采用反相高效液相色谱法考察注射用大黄粉针剂在不同梯度洗脱条件下的分离情况,优化的注射用大黄粉针剂高效液相色谱指纹图谱的分离条件是:色谱柱为Shim-pack CLC-ODS柱(6.0 mm i.d.×150 mm,5 μm);流动相为甲醇和0.1%(体积分数)磷酸溶液体系;检测波长254 nm;柱温40 ℃。在该色谱条件下,注射用大黄粉针剂各组分达到较佳分离,方法的稳定性、精密度、重现性好,为建立其指纹图谱奠定了基础。     

加压毛细管电色谱分离检测虎杖根中蒽醌类成分

建立了加压毛细管电色谱法(p CEC)检测大黄酸、大黄素、芦荟大黄素、大黄酚、大黄素甲醚5种蒽醌类成分的方法,并对虎杖根中蒽醌类的成分进行分析。该方法采用EP-100-20/45-3-C_(18)毛细管色谱柱(总长度45 cm,有效长度20 cm,直径为100μm,ODS填料3μm)进行分离,流动相为20 mmol/L Na H2PO4(pH 4.7)-乙腈(15∶85),流动相的总流速为0.04 m L/min,分离电压为+5 k V,紫外检测波长为254 nm。结果表明,5种蒽醌类成分的检出限(S/N=3)为0.60~2.54μg/m L,在3.57~162.68μg/m L范围内线性关系良好,相关系数均不小于0.998 2。将所建立的方法用于虎杖中蒽醌类成分的分析,取得良好的实验结果,在低、中、高3个加标浓度下的回收率为91.1%~101.2%,相对标准偏差(RSD)为0.03%~3.6%。     

光聚合整体式咖啡因印迹毛细管柱的制备及分离性能

分子印迹技术作为一种制备对目标分子具有专一识别能力的功能高分子的方法 ,近年来在化学化工、生物化学与生物技术的许多领域中得到广泛应用 [1～ 4 ] .分子印迹技术与微分离方法 (包括微柱液相色谱、毛细管电泳、毛细管电色谱和芯片分离等 )结合已引起人们极大的兴趣和关注[5,6] .毛细管柱是毛细管电色谱和微柱液相色谱的关键部件 ,目前普遍使用的是烷基键合硅胶微粒的填充柱 ,存在制备时须烧塞和填充两大困难 ,以及使用时易产生气泡和易折断等缺点 .将含被识别分子 (印迹分子 )、交联剂、溶剂、功能单体和引发剂的混合液注入毛细管 ,经光…     

丙二酰胺型大环多胺键合固定相在毛细管开管电色谱中的应用

毛细管电色谱 ( Capillary electrochromatography,简称 CEC)是近年发展起来的一种高效、快速的新型微柱分离方法 [1～ 4] .由于它在毛细管柱内填充液相色谱固定相或者在毛细管内壁键合固定相 ,且采用电渗流作为驱动力 ,因而兼有高效液相色谱和毛细管电泳的分离特点 ,已应用于复杂的药物分析 [2 ] .填充柱毛细管柱具有工艺要求高、容易产生气泡、焦耳热和价格昂贵等缺点 .开管柱电色谱( Open- tubular CEC,简称 OTCEC)是将固定相键合或涂覆在毛细管的内壁 ,避免了上述缺陷 .因此已引起高度重视 [3,4] .大环多胺的结构与冠醚类似 ,是一…     

Analysis of Five Pharmacologically Active Compounds from Rhodiola for Natural Product Drug Discovery with Capillary Electrophoresis

A rapid capillary electrophoresis method for the separation of five natural pharmacologically active compounds from extracted Rhodiola, namely salidroside, tyrosol, rhodionin, gallic acid and ethyl gallate has been developed. The separation of five natural pharmacologically active compounds was carried out in a fused-silica capillary with 14 mM boric acid, 30 mM SDS and 2.5% acetonitrile, adjusted to pH 10.7 with NaOH. Applied potential was 21kV. The temperature of the capillary was maintained at 25 °C by the instrument thermostating system, with the correlation coefficients of 0.9805–0.9989 for migration time, and relative standards of < 3.52% for peak areas. The established method is rapid and reproducible for the separation of five natural pharmacologically compounds from extracts of Rhodiola with satisfactory results.     

Preparative isolation and purification of five compounds from the Chinese medicinal herb Polygonum cuspidatum Sieb. et Zucc by high-speed counter-current chromatography

High-speed counter-current chromatography (HSCCC) was applied to the separation and purification of five compounds from the Chinese medicinal herb Polygonum cuspidatum Sieb. et Zucc. The crude extracts from P. cuspidatum Sieb. et Zucc were treated with light petroleum-ethyl acetate-methanol-water (2:5:4:6, v/v). Sample 1 was obtained from the lower phase and sample 2 from the upper phase. The sample 1 was separated with light petroleum-ethyl acetate-water (1:5:5, v/v) and yielded 19.3mg of piceid, 17.6 mg of anthraglycoside B from 200mg of sample 1. The sample 2 was separated with light petroleum-ethyl acetate-methanol-water (3:5:4:6, v/v) and light petroleum-ethyl acetate-methanol-water (3:5:7:3, v/v) in a gradient elution and yielded 18.5mg of resveratrol, 35.3mg of emodin and 8.2mg of physcion from 220 mg of sample 2. The purity of each compound is over 95% as determined by HPLC. The chemical structures of these components were identified by (1)H NMR and (13)C NMR.     

Separation of Compounds in Traditional Chinese Medicines Using Comprehensive Two Dimensional Chromatography

Traditional Chinese medicine is an invaluable treasure of the Chinese nationalities. Thousands of natural species that are of pharmaceutical importance have been accumulated. Most of them are plants. Traditional Chinese medicines generally are of unusual complexity. Even a single medicinal material may contain hundreds of compounds. Since these compounds play cooperative roles pharmacologically, it is essential to analyze all compounds as a whole. It is also important for quality controls in each step of productions, such as raw material collection, processing, and manufacturing. A comprehensive two-dimensional separation system coupling capillary high performance liquid chromatography with fast capillary electrophoresis (μ-HPLC-CE) is developed to provide a powerful means to separate such complex samples. In the first dimensional separation, a home-packed micro-HPLC column was used to reduce the consultation of sample injection and avoid sample dilution. The second dimensional analysis was carried out by micellar electrokinetic chromatography(MEKC) considering the fact that most compounds in Chinese medicine are neutrals. In order to achieve high-speed separation^ theory of fast MEKC was extensively studied. Models of the fast MEKC migration behaviors were established. Relationships of theoretical plates vs. electric field strength and column length were derived. It is concluded that maintaining certain column length and applying high-voltage at the ends of the capillary simultaneously are the key points to achieving fast MEKC separation. A pulse-contacting interface was developed for the 2-D μ-HPLC-CE system. CE sampling was carried out in an instant contact of HPLC and CE columns in an optimized timing program. During the short contact period, a certain fraction of HPLC effluent was introduced into the CE capillary. Injection efficiency for such an interface was up to 81%. Relative standard deviation (RSD) of migration times and peak heights for 100 consecutive MEKC. separation were 3.0% and 1.8%, respectively. With this novel 2-D μ-HPLC-CE system, some traditional Chinese medicines were analyzed and hundreds of peaks were observed. For liquorice, over 110 components were fairly resolved. More than 250 peaks were obtained for a compound mixture of Cheng-Qi-Tang. The peak capacity of this novel comprehensive 2-D HPLC-CE system was estimated to be about 2400 in 80 min.     

Determination of two intact glucosinolates in vegetables and Chinese herbs

A reversed-phase HPLC method was developed for analyzing sinigrin and gluconasturtiin in six vegetable and two Chinese herb samples. A gradient program and mobile phases using methanol and 0.05% trifluoroacetic acid containing 20 mM ammonium acetate allowed sufficient retention and separation of the glucosinolates in the sample extracts. Quadrupole time-of-flight tandem mass spectrometry in negative ion electrospray ionization was used to analyze the fractions collected from the HPLC elution to confirm the identification of the glucosinolates. The levels of sinigrin and gluconasturtiin in the vegetables and Chinese herbs were determined by using an external calibration method. Concentrations of gluconasturtiin in the Chinese herbs were more than 15 times higher than those of sinigrin. Detection limits were 18 nmol g^–1 for sinigrin and 4 nmol g^–1 for gluconasturtiin when 50 g of fresh vegetable was analyzed.     

Gradient thin-layer chromatography of plant extracts

Summary A modified Niederwieser chamber for stepwise gradient elution, consisting of a PTFE capillary to store the series of eluents and a horizontal glass sandwich chamber with a glass distributor was used for the chromatographic separation of complex plant extracts (Seboren, Hemorigen and Pectosol) used in therapy. Densitograms demonstrate markedly improved separations of the extracts in comparison to isocratic elution.Produced by Polish Reagents, Melgiewska 18, 20-234 Lublin, Poland     

高效液相色谱-四极杆飞行时间质谱检测中国杨树型蜂胶、巴西绿蜂胶和杨树胶中的酚类化合物及真伪鉴别

建立了用于检测中国杨树型蜂胶、巴西绿蜂胶和杨树胶中23种酚类化合物的高效液相色谱-四极杆飞行时间质谱（HPLC/Q-TOF MS）法，并进行了差异性分析。蜂胶和树胶样品用甲醇-水（1：1，v/v）溶解后，过0.45 μm有机相滤膜后进样。采用Agilent Eclipse Plus C₁₈色谱柱分离，以乙腈和0.1%（体积分数）甲酸水溶液为流动相进行梯度洗脱，电喷雾正离子模式全扫描方式检测，扫描范围为m/z 100~1000，外标法定量。结果表明，23种酚类化合物在10~200 μg/L范围内线性关系良好，相关系数均大于0.99。桔皮素和刺芒柄花素的检出限和定量限分别为0.2和1 μg/L，其他化合物的检出限和定量限分别为2和10 μg/L。在10、25和50 mg/kg 3个添加水平下，23种酚类化合物在3种样品基质中的提取回收率为70.2%~122.6%，RSD值均小于10%。水杨苷、肉桂酸、咖啡酸和香豆酸可以作为中国杨树型蜂胶掺假鉴别的特征化合物，咖啡酸、阿魏酸、白杨素、咖啡酸苯乙酯、松属素、高良姜素、香豆酸、异鼠李素、山柰素和阿替匹林C可以作为辨别中国杨树型蜂胶和巴西绿蜂胶的特征化合物。该结果对蜂胶产品的质量控制具有一定的参考意义。     

Characteristics of electroosmotic flow and migration of neutral solutes under stepwise gradient elution of capillary electrochromatography

A theoretical study on the velocity of electroosmotic flow (EOF) and the retention times of neutral solutes under multiple-step gradient of capillary electrochromatography (CEC) was carried out, focusing on that with three kinds of mobile phases. Through the model computations, the detaining time of the second kind of mobile phase in the column was proved to play an important role in affecting EOF. The variation speed of EOF was shown to be determined by the differences among dead times in different steps. In addition, the prediction of the retention times of 13 aromatic compounds under gradient mode was performed with the deduced equations. A relative error below 3.3% between the calculated and experimental values was obtained, which demonstrated the rationality of the theoretical deduction. Our study could not only improve the comprehension of stepwise gradient elution, but also be of significance for the further optimization of separation conditions in the analysis of complex samples.     

Effect of Temperature in micro-HPLC Separation of PAHs under Isocratic Conditions Using Octadecyl and Alkylamide Phases

The main difficulty in micro-HPLC separation is the manipulation with the composition of the mobile phase (degassing of solvents and a slow establishment of equilibrium) but the problem of elution can be solved by temperature optimalization. The effect of temperature in micro-HPLC separation of the 16 polycyclic aromatic hydrocarbons (PAHs) (mixture SRM 1647 of the US EPA) has been studied using conventional C₁₈ and new developed AP phase (an amide group localized in the hydrophobic ligands). All the investigations have been performed under isocratic conditions (binary hydroorganic mobile phase: acetonitrile/water). The results have shown that, in the case of AP phase, application of temperature gradient (from 298 to 303 K), enabled the attainment of complete 16 PAHs separation (especially of the first 4 solutes).     

Accuracy mass screening and identification of phenolic compounds from the five parts of Abutilon theophrasti Medic. by reverse-phase high-performance liquid chromatography-electrospray ionization-quadrupoles-time of flight-mass spectrometry

A rapid and resolutive reverse-phase high-performance liquid chromatography-electrospray ionization-quadrupoles-time of flight-mass spectrometry method was established for the screening and identification of the phenolic compounds in the 70% ethanolic extracts from the five parts (roots, stems, leaves, seeds, and exocarps) of Abutilon theophrasti Medic.. Separation and detection conditions were optimized by using a 22 mixing standard, which included phenolic acids, flavonoids and a naphthalene compound. Optimum LC separation was achieved on a C(18) analytical column (250 mm x 4.6 mm id, 5 μm) by gradient elution with water containing 0.1% v/v formic acid (pH 2.4) and acetonitrile as mobile phases, at a flow rate of 1.0 mL/min. The developed method was applied to the study on the constituents of A. theophrasti Medic., and 16 compounds were unequivocally identified with standards. Meanwhile, 37 constituents were tentatively identified by comparing with references. In addition, accurate molecular formulae were conjectured for unknown compounds. To our knowledge, little is known about how these compounds are distributed in A. theophrasti Medic.. Hence, it is clear that the comprehensive analysis of the phenolic compounds of A. theophrasti Medic. is helpful for the quality control and understanding the usage and function of the herb and its products.     

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.