反相液相色谱对多肽的分离、纯化与制备

用反相高效液相色谱（RPLC）对两种化学合成多肽－－32肽和21肽进行了分离、纯化和制备。在用分析型RPLC色谱柱对多肽样品的制备过程中，对其进样量和洗脱梯度进行了选择。每次进样量为5mg，在最优化色谱条件下，用RPLC一步就可对21肽进行分离纯化，其纯度达到98.6%。而32肽由于样品组分更加复杂，RPLC一步纯化后其纯度仅有80％。通过对色谱分离条件的再次优化，对32肽进行二次分离纯化，纯度达杂，RPLC一步纯化后其纯度仅有80％。通过对色谱分离条件的再次优化，对32肽进行二次分离纯化，纯度达到96.4%。在其最优化条件下，通过多次样品收集和冷冻干燥，分别制备了高纯度的21肽和32肽各100mg。     

填充型纳流液相色谱柱技术的研究进展

纳流液相色谱是高效液相色谱微型化的重要形式,由于其具有溶剂消耗少、样品需求量小、检测灵敏度高以及与质谱联用兼容性好等特点,在生命科学、生物医药以及微纳分析等领域得到越来越广泛的应用。填充柱是最常用的纳流色谱柱类型,通常由球形色谱填料颗粒填充入毛细管内制成。样品组分是在色谱柱上实现分离,色谱柱的性能对色谱分析的选择性和分辨率至关重要,因此制备高质量的纳流色谱填充柱对获得高效的纳流液相色谱分离尤为关键。纳流色谱柱的制备包含两个重要技术环节：柱塞技术和填充工艺。近年来,随着柱塞与填充技术的进步,填充型纳流液相色谱柱的稳定性和分离性能得到了显著提升。本文对近十年来纳流液相色谱柱技术的研究进展,主要是柱塞技术和填充技术的研究和应用进展进行了概述,介绍了其在组学研究和生物制药领域中的应用,并对其发展前景进行了展望。     

胰岛素反相制备色谱的方法开发

以胰岛素反相制备色谱方法的开发和优化为目标,通过考察色谱保留参数、峰展宽及样品流出曲线的浓度分布等色谱参数,对流动相梯度、色谱填料、载样量等色谱条件进行了优化,并建立了胰岛素制备色谱峰参数的描述方法。结果表明,所建立的方法可快速筛选出最适于胰岛素分离的色谱条件(包括流动相梯度及分离填料),即流动相中的强洗脱溶剂(有机相)需采取缓梯度窄区间的变化条件,筛选出的分离填料需具备峰向两侧展宽且展宽程度较小、样品最高浓度居中分布的特点。将方法用于实际胰岛素粗品的纯化制备,获得了杂质去除效果好、胰岛素纯度高的产品。该法为胰岛素反相色谱纯化制备方法的快速建立提供了指导,具有较强的实用价值,同时为发展大分子化合物的制备色谱方法提供了参考。     

一种食用香精香料中8种成分同时测定的方法

本发明公开了一种食用香精香料中8种成分同时测定的方法,包括以下步骤:萃取溶液的配制、标准溶液的配制、样品溶液的制备、超高效液相色谱–串联质谱分析以及含量的计算。本发明通过对前处理条件、液相色谱条件和质谱条件的优化,建立了一种简便、快速、高通量的测定方法,本发明具有操作简单、灵敏度高、回收率好、重现性好且检测时间短、溶剂消耗小、检测通量高、检测效率高的优点。     

黄芪中黄酮类组分的高效液相色谱分离条件的快速优化

以黄芪为研究对象,对黄芪中乙酸乙酯部位的化学成分进行了高效液相色谱分析并对其色谱操作条件进行了快速优化。根据几个线性梯度下的保留时间来计算各组分的保留参数,然后利用重叠分辨图法确定其最佳分析条件。在选定的最佳条件下各组分分离情况良好。利用梯度保留时间计算保留参数比较方便快速,并可以有效地避免以往等度线性回归法遇到的峰识别问题。该方法更适用于实际复杂样品色谱分析条件的优化。     

基于双回路技术的二维色谱在线/离线接口设计与评价

双回路技术是一套机械传动组件和流路系统,具有自动进样和馏分收集的双重功能。本研究基于双回路技术设计了一种二维液相色谱在线/离线接口,用以有效连接两种不同分离模式构建分离系统,提高系统峰容量,并对其功能进行了评价。以4种芳香族化合物为分离对象,利用设计的接口连接1套液相色谱系统,实现了进样、馏分收集、再进样纯度分析的多重操作集成;连接2套液相色谱系统,采用强阳离子交换模式对5种蛋白质样品进行初步分离,收集难分离组分,并通过再次进样到反相色谱模式的分离系统中,组分得到良好分离。进一步基于此接口连接1套强阳离子交换系统和1套微柱反相色谱系统,对牛血清白蛋白酶解产物进行在线二维分离,以1 m AU为积分阈值,共识别292个色谱峰。新型在线/离线接口可灵活实现样品微量制备、难分离组分的精细拆分和复杂样品的二维分离,为二维色谱系统的构建及分离研究提供了有力工具。     

基于高压制备液相的多维色谱技术在中药分离纯化中的应用

中药物质基础复杂,对其活性成分的分离一直是中药研究的难题.基于高压制备液相的多维色谱系统在高压制备液相色谱的基础上,结合了多种分离技术,极大地提高了色谱系统的分离性能和分离效率,更有利于对物质基础复杂的中药样品进行分离纯化.本文介绍了基于高压制备液相系统的多维色谱系统的基本原理、分离模式以及关键技术,并综述了其在中药分离纯化中的应用.     

醛酮化合物的液相色谱保留行为的考察及其多元分离条件的优化

考察了大气污染物中醛酮化合物在甲醇／水、乙腈／水和四氢呋喃／水三个二元流动相体系中的液相色谱保留行为,并对醛酮化合物在各体系中的选择性差异进行了考察。在此基础上选择最佳的流动相体系,应用“均匀－重复设计优化法”对醛酮样品进行多元等度分离条件的优化,获得了醛酮样品最优的液相色谱分离条件。     

磁性氧化石墨烯的制备及其在环境水样中7种内分泌干扰物检测中的应用

制备了十二胺修饰的磁性氧化石墨烯纳米材料并将其用于磁固相萃取。在对磁固相萃取材料合成与萃取条件以及液相色谱分离等条件进行优化的基础上,建立了磁固相萃取与高效液相色谱-紫外检测对环境水样中7种内分泌干扰物的分析方法。该方法对雌酮、雌二醇、雌三醇、双酚A、17α-乙炔基雌二醇、己烷雌酚和雄烯二酮7种内分泌干扰物的检出限在0.10~0.23 nmol/L之间。将该方法分别应用于废水样品和湖水样品的加标回收试验,回收率在73.9%~112.9%和74.9%~114.7%之间。该方法操作简便,分析成本较低,可为环境水体中内分泌干扰物的分析提供技术支持。     

顶空液相微萃取/气相色谱-质谱对中药枳壳中有机挥发物的快速分析

建立了顶空液相微萃取/气相色谱-质谱联用测定中药枳壳中有机挥发物的方法.在顶空液相微萃取实验条件优化的基础上,确定了最佳实验条件:以正辛烷作为有机萃取剂,体积为2 μL,样品用量为0.3 g,液滴距离样品表面0.8 cm处,萃取8 min后直接进样.与固相微萃取/气相色谱-质谱法相比,顶空液相微萃取法定性了30种成分,固相微萃取法定性了24种成分,顶空液相微萃取法操作简单、快速,实验结果灵敏度更高,且萃取效率高,重复性好,可用于中药枳壳中有机挥发物的快速分析.     

Mass-directed fractionation and isolation of pharmaceutical compounds by packed-column supercritical fluid chromatography/mass spectrometry.

An automated packed-column semi-preparative supercritical fluid chromatography/mass spectrometry (SFC/MS) system incorporating mass-directed fraction collection has been designed and implemented as an alternative to preparative HPLC and preparative HPLC/MS (PrepLC/MS) for the purification of pharmaceutical compounds. The system incorporates a single quadrupole mass spectrometer and a supercritical fluid chromatograph. Separations were achieved using a binary solvent system consisting of carbon dioxide and methanol. Purification of SFC-separated compounds was achieved incorporating mass-directed fraction collection, enabling selective isolation of the target molecular weight compound and eliminating the collection of undesired compounds (e.g., by-products, excess starting materials, etc.). Cross contamination between fractions and recoveries of the system were investigated. Mass spectrometer ionization with basic mobile additives is discussed, and examples of preparative SFC/MS chiral separations are presented. Early experiences suggest SFC will be a powerful and complementary technique to HPLC for the purification of pharmaceutical compounds.     

High-throughput preparative process utilizing three complementary chromatographic purification technologies

A high-throughput process was developed in which wells in plates generated from parallel synthesis are automatically channeled to an appropriate purification technique using analytical data as a guide. Samples are directed to either of three fundamentally different preparative techniques: HPLC with UV-triggered fraction collection, supercritical fluid chromatography (SFC) with UV-triggered fraction collection, or HPLC with MS-triggered fraction collection. Automated analysis of the analytical data identifies the product compound mass and creates work lists based on chromatographic properties exhibited in the data so that each preparative instrument cherry picks the appropriate list of samples to purify when a preparative-scale plate is loaded.     

A straightforward means of coupling preparative high-performance liquid chromatography and mass spectrometry

Flow splitting to a mass spectrometer is a common way of coupling a highly specific detector to preparative or semi-preparative high-performance liquid chromatography (HPLC) purification of combinatorial libraries, drug metabolites, and characterizable impurities. The sensitive mass spectrometer consumes only a small fraction of the analyte while providing online structure-specific detection, and its output can thus be used to trigger collection of the desired fraction. Coupling mass spectrometry to preparative HPLC is difficult due to the susceptibility of the detector to fouling under conditions of high analyte concentration or solute amount, or to changes in solvent composition. We report here on a device, the mass rate attenuator (MRA), which automatically produces split ratios over a range of 100:1 to 100 000:1 under programmable user control. The MRA is a flow-control device that periodically gates a small aliquot from one liquid stream into another. The design allows the user to set the frequency of the gating without interruption of the HPLC flow stream. The MRA also allows control of the volume of the aliquot that is transferred between the flow streams. This additional control, compared to passive splitting devices, facilitates optimization of the tubing connecting the separation, detection and collection events. We demonstrate that such optimization can reduce the volume of the collected fraction without compromising recovery, thus reducing the time spent in evaporating solvents to reclaim purified products.     

Phosphatidylcholine isolation from egg yolk phospholipids by high-performance liquid chromatography

Cell membrane components have been increasingly recognized as important biochemicals in the fields of biochemistry and pharmacy due to their relationship with metabolite transport in the cells. Among the components, phosphatidylcholine (PC) is considered a valuable biochemical, because it is difficult to commercialize. PC demand has been largely increased in the fields of the nutrient, cosmetic and pharmacy industries, and so the development of a preparative chromatography process is critical to supply a low-cost PC. In this study, we investigated the HPLC separation of phospholipid originated from egg yolk, which contains 80% (w/w) PC and 15% (w/w) phosphatidylethanolamine. Column temperature, mobile phase composition and its flow-rate and kinds of stationary phase were varied to understand the effectiveness of PC separation. For studying the relationship between recovery yield and sample loading amount in HPLC, we performed overloading experiments. In this way, we successfully separated PC with over 99% purity and with 98% yield with the following HPLC operating conditions; pure methanol as a mobile phase, 2.0 ml/min flow-rate and 1000 mg/ml feed concentration in a KR-100-10SIL column.     

Rapid purification and scale-up of honokiol and magnolol using high-capacity high-speed counter-current chromatography

In this paper, a rapid separation approach has been developed using high-capacity high-speed counter-current chromatography (high-capacity HSCCC) to isolate and purify honokiol and magnolol, which are the main bioactive constituents from Houpu. The optimization of the solvent selection process, sample loading volume and flow rate is systematically studied using analytical high-capacity HSCCC. The optimized parameters obtained rapidly at analytical scale were used for a 1000 x scale-up preparative run using pilot scale high-capacity HSCCC in a MAXI-DE centrifuge. A crude sample of 43 g was successfully separated and the fractions were analysed by high-performance liquid chromatography (HPLC). This large scale preparative single step run yielded 16.9 and 19.4 g of honokiol and magnolol with purities of 98.6 and 99.9%, in only 20 min. This is the first time that high-performance counter-current chromatography has been used to purify multiple gram grade bioactive compounds in less than 1h and at such high concentrations of final products (10.8 g/l for magnolol and 7.0 g/l for honokiol).     

A novel method of prediction and optimization for preparative high-performance liquid chromatography separation

The preparation of components from a complex sample is a difficult task. The optimization of the separation and subsequent scale-up is usually carried out by trial and error. In this study, the relationship between retention parameters a and c for analytical and preparative separations was developed when the same solid adsorbent and mobile phase were used. The prediction and optimization of the preparative separation of a complex sample could be achieved by direct and simple conversion of the experimentally determined data from an analytical level. The novel method was successfully applied to optimize the separation of target compounds in traditional Chinese medicine (TCM).     

Optimization of the preparative separation of a chiral pharmaceutical intermediate by high performance liquid chromatography

The prediction of optimal conditions of the preparative HPLC separation of the enantiomers of a pharmaceutical intermediate was accomplished by employing analytical chromatographic data, i.e. sample injections at low concentrations. Various temperatures and mobile phase conditions were studied. It was assumed that the sample loadability of the stationary phase is constant for a constant value of the separation factor and different mobile phase conditions and temperatures. Using this assumption, possible production rates can be compared for different method conditions. Overloading experiments were carried out to verify that the procedure employed is adequate. It was found that the optimization approach used, changing the mobile phase composition and temperature to achieve the shortest cycle time while keeping the separation factor constant, could be applied to improve the production rate of the separation.     

High-throughput purification of combinatorial libraries I: a high-throughput purification system using an accelerated retention window approach

We have developed a high-throughput purification system to purify combinatorial libraries at a 50-100-mg scale with a throughput of 250 samples/instrument/day. We applied an accelerated retention window method to shorten the purification time and targeted one fraction per injection to simplify data tracking, lower QC workload, and simplify the postpurification processing. First, we determined the accurate retention time and peak height for all compounds using an eight-channel parallel LC/UV/MS system, and calculated the specific preparative HPLC conditions for individual compounds. The preparative HPLC conditions include the compound-specific gradient segment for individual compounds with a fixed gradient slope and the compound-specific UV or ELSD threshold for triggering a fraction collection device. A unique solvent composition or solvent strength was programmed for each compound in the preparative HPLC in order to elute all compounds at the same target time. Considering the possible deviation of the predicted retention time, a 1-min window around the target time was set to collect peaks above a threshold based on UV or ELSD detection. Dual column preparative instruments were used to maximize throughput. We have purified more than 500 000 druglike compounds using this system in the past 3 years. We report various components of this high-throughput purification system and some of our purification results.     

Orthogonal analytical screening for liquid chromatography-mass spectrometry method development and preparative scale-up

An analytical HPLC-MS screening methodology has been developed to improve preparative RP-HPLC-MS purifications in medicinal chemistry laboratories. Although several approaches have been previously described to optimize analytical separations, none of them met our needs for the optimization of preparative conditions. Our screening protocol is based on searching among several orthogonal conditions to find the optimum preparative separation. Five different buffer conditions, from low to high pH, two organic solvents, acetonitrile and methanol, and five stationary phases of different polarities and characteristics were used. The orthogonality of the system was demonstrated using both, a standard mixture and mixtures from synthesis. To carry out the screening one of the analytical "open access" HPLC-MS systems was modified to perform the analytical screening while maintaining the open-access functionality for synthesis reaction monitoring. A software tool for automated sample programming and data reporting was also developed.     

Automated post-collection concentration for purified preparative fractions via solid phase extraction

Recent advancements in preparative HPLC have improved and streamlined compound purification. However, fraction evaporation remains a bottleneck within the process. An alternative to fraction evaporation is to remove the water and reduce the overall volume of the collection by trapping the fraction onto a solid phase extraction (SPE) cartridge. This method (as opposed to analytical applications involving SPE) works by collecting and then diluting the fraction(s), passing the fraction(s) through a SPE, drying the SPE with nitrogen and ultimately eluting the concentrated fraction(s) in a small amount of 100% organic solvent. An appreciable breakthrough is not observed using this method. In addition, recovery from the SPE for the tested compounds rosmarinic acid and carvacrol, two naturally occurring antioxidants in oregano, was found to be 95-98% for a 100mg injection via preparative HPLC purification at 50mg/compound.