多维高效液相色谱技术在蛋白质分离研究中的进展

蛋白质组学出现之后,多维高效液相色谱(multidimensional HPLC,MD-HPLC)系统以其快速、高效、自动化程度高以及容易与质谱等其他技术联用等优势而成为蛋白质组学相关分析技术中研究应用的热点。本文主要以本实验室在蛋白质组学研究中的技术进展为主线,介绍了多维高效液相色谱技术的发展,包括经典的“bottom-up”技术和“top-down”式的多维高效液相色谱技术路线,以及为了提高系统的分离通量而自行设计搭建的阵列式多维高效液相色谱平台,这些技术路线在蛋白质组学研究中有着极大的潜在应用价值。     

复杂生物体系中蛋白质高效分离分析技术的新进展

继人类基因组计划完成之后,作为一种新的研究策略,蛋白质组学在生命科学研究中发挥着愈来愈重要的作用。由于生物体系的复杂性和多样性,使得分离效率高、灵敏度高、通量高和动态范围宽的分离分析技术平台的研究和应用已成为蛋白质组学研究的重点和热点之一。着重介绍了近年来应用日益广泛的多维色谱预分离、毛细管液相色谱-质谱联用、毛细管电泳及其与质谱联用等高效分离分析技术在复杂生物体系的蛋白质分析中的最新进展。引用相关文献40篇。     

多维液相色谱分离技术及其在蛋白质组研究中的应用

对近年来多维液相色谱技术及其在蛋白质组学研究中的应用进行了系统综述。详细描述了由不同液相色谱模式构建的多维液相色谱系统,并介绍了其在蛋白质组表达谱、翻译后修饰、定量等方面的应用。此外,还对多维液相色谱的发展趋势和前景进行了展望。     

蛋白质/多肽的微流控二维芯片电泳*

在微流控芯片上构建多维分离系统,为蛋白质组学研究提供了一个有发展前景的高效分离分析技术平台。本文介绍了二维芯片电泳系统耦联模式选取及正交性评价的方法;综述了针对蛋白质/多肽分离分析的各种耦联模式微流控二维芯片电泳分析系统,如胶束电动力学色谱（MEKC）与毛细管区带电泳（CZE）,开管电色谱（OECE）与CZE,等电聚焦（IEF）与CZE, IEF与SDS毛细管凝胶电泳（CGE）, SDS-CGE与MEKC等。特别对二维电泳芯片切换接口的类型进行了分类,探讨了用于微流控二维芯片电泳系统的检测技术,并展望了微流控二维电泳芯片在蛋白质组学研究中的应用前景和发展方向。     

二维色谱技术及其在中药领域中的应用

二维色谱是近年发展起来的多维色谱分离技术,因其高分辨率、高峰容量、高灵敏度等优点,在复杂样品的分离分析中发挥了巨大作用．中药是一个复杂的化学体系,包含多种类化学成分,因此,分析和阐明中药中的化学成分是中药现代研究的一个关键问题．二维色谱技术在中药等复杂体系的应用中显示了重要的应用价值,也具有广阔的发展前景．本文对二维色谱的分类、主要部件以及在中药领域中的应用进行综述,重点介绍二维气相色谱和二维液相色谱在中药研究中的应用．     

多维立体分离分析技术面临的挑战性问题及对应策略

高效液相色谱(HPLC)、毛细管电泳(CE)等高效分离技术的发展为蛋白质组、天然产物、环境样品等复杂体系的分离分析提供了新的手段。然而一维分离模式所能提供的分辨率和峰容量仍十分有限,难以满足人们对复杂样品进行高效分离与高灵敏检测的要求。多维分离是近年来发展起来的一种新型复合分离技术,与一维分离模式相比,这种技术可以极大地提高峰容量,便捷地调整分离选择性,因此已成为近期分析化学领域的重要研究热点。多维液相分离方法可以采用不同的HPLC模式(如排阻色谱、离子交换色谱、亲和色谱、反相色谱等)和CE模式(如毛细管区带…     

色谱技术在蛋白质组学研究中的应用引言

蛋白质组是指一个有机体的基因组所表达的全部蛋白质。蛋白质组学是研究有机体蛋白质的组成及其变化规律的科学。蛋白质组成的高度复杂性和随时间、空间变化的特点,对蛋白质组的研究技术和方法提出了巨大挑战。色谱作为现代分离科学的核心技术之一,在蛋白质组研究中发挥了重要作用。我们可以通过对组织、细胞或体液中成千上万种蛋白质/多肽的色谱预分离,降低样本的复杂程度,提高蛋白质的鉴定率;我们可以通过亲和色谱对翻译后修饰的蛋白质/多肽进行特异性富集分离,去除非修饰的蛋白质/多肽,实现修饰蛋白的成功鉴定;我们还可以通过色谱 质谱联用技术,获得蛋白质/多肽在色谱分离中的保留时间或峰面积,实现蛋白质的规模化定量与鉴定等。 为了集中展示我国科学家在色谱技术及其在蛋白质组学研究中的应用方面所取得的成果,《色谱》杂志特此在2010年第2期编辑出版了“色谱技术在蛋白质组学研究中的应用”专栏。我们邀请了在该领域具有突出成绩或学术造诣的部分专家、学者撰写了相关的学术论文和综述。希望通过这些文章所介绍的工作,为进一步提高色谱技术在蛋白质组学研究中的应用水平,推动我国蛋白质组学的发展和取得创新性的研究成果作出贡献。     

亲水作用色谱固定相的发展及应用

亲水作用色谱作为一种高效液相色谱技术,在强极性和离子型化合物如氨基酸、碳水化合物和多肽等的分离分析中发挥着重要作用。作为色谱技术的核心,亲水作用色谱材料的发展直接影响着色谱分离的选择性和分离效率,制约着亲水作用色谱的应用和推广。目前商品化和学术报道的亲水作用色谱材料种类繁多,键合相结构丰富。本文从色谱材料结构出发,综述了近5年内基于硅胶基质的亲水作用色谱固定相,包括纯硅胶、氨基、氰基、二醇基、酰胺型、聚（琥珀酰亚胺）型、糖型和两性离子型键合相的发展及其在极性药物、蛋白质组学、代谢组学等方面的典型应用。同时简要介绍了近年来亲水作用色谱的色谱评价研究。     

生物制药研究中的多维色谱

对全二维气相色谱(GC×GC)、全二维液相色谱(HPLC×HPLC)、多维毛细管电泳等多维分离技术在生物制药研究中的应用进行了综述,其中对作者所在研究组在全二维气相色谱应用于中药及固相萃取-液相色谱联用分析系统等方面的工作做了重点介绍。由所综述的生物制药研究得出结论：多维分离方法以其高分辨、快速、自动化等特点已经在生物制药领域显示出它的巨大优势,并将发挥更大的作用。     

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.     

High‐performance liquid chromatography separation of unsaturated organic compounds by a monolithic silica column embedded with silver nanoparticles

The optimization of a porous structure to ensure good separation performances is always a significant issue in high‐performance liquid chromatography column design. Recently we reported the homogeneous embedment of Ag nanoparticles in periodic mesoporous silica monolith and the application of such Ag nanoparticles embedded silica monolith for the high‐performance liquid chromatography separation of polyaromatic hydrocarbons. However, the separation performance remains to be improved and the retention mechanism as compared with the Ag ion high‐performance liquid chromatography technique still needs to be clarified. In this research, Ag nanoparticles were introduced into a macro/mesoporous silica monolith with optimized pore parameters for high‐performance liquid chromatography separations. Baseline separation of benzene, naphthalene, anthracene, and pyrene was achieved with the theoretical plate number for analyte naphthalene as 36 000 m^?1. Its separation function was further extended to cis/trans isomers of aromatic compounds where cis/trans stilbenes were chosen as a benchmark. Good separation of cis/trans‐stilbene with separation factor as 7 and theoretical plate number as 76 000 m^?1 for cis‐stilbene was obtained. The trans isomer, however, is retained more strongly, which contradicts the long‐ established retention rule of Ag ion chromatography. Such behavior of Ag nanoparticles embedded in a silica column can be attributed to the differences in the molecular geometric configuration of cis/trans stilbenes.     

Off‐line comprehensive two‐dimensional reversed‐phase countercurrent chromatography with high‐performance liquid chromatography: Orthogonality in separation of Polygonum cuspidatum Sieb. et Zucc

Off‐line comprehensive two‐dimensional reversed‐phase countercurrent chromatography with high‐performance liquid chromatography was investigated in separation of crude ethanol extract from traditional Chinese medicinal herb Polygonum cuspidatum Sieb. et Zucc. Two‐dimensional contour plots for countercurrent chromatography with high‐performance liquid chromatography was obtained after comprehensive separation was completed. Total peak capacity was evaluated and approximately 810 peaks were obtained through a comprehensive two‐dimensional separation. A highly orthogonality of 52.23% and a large separation space occupancy of 88.86% were achieved. Meanwhile, it was found that several components could be well separated by countercurrent chromatography while they could not be separated by high‐performance liquid chromatography, and vice versa, which further indicated the orthogonality of the two separation methods. The off‐line comprehensive two‐dimensional countercurrent chromatography with high‐performance liquid chromatography provided a promising and powerful method for separation of complex natural products.     

Recent progress of enantioseparation under scale production (2014–2019)

Very different from enantiorecognition at an analytical level, preparative enantioseparation at scale production has to confront many problems. In recent years, various separation techniques have been developed with much progress including high‐performance liquid chromatography, liquid–liquid extraction, membrane separation, enantioselective solid‐phase absorption, and enantioselective precipitation. Among them, in order to achieve high enantiomerical purity of the enantiomers after separation, two approaches are commonly carried out. One is that continuous separation is necessary when one‐step separation cannot have satisfactory efficiency. Another is that most of the research groups focus on the construction of novel chiral environment with much more rigid structures of the selectors. Combined with these two aspects, we mainly introduce current topics, trends, strategies related to enantioseparation under large scale.     

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。     

A practicable strategy for enrichment and separation of four minor flavonoids including two isomers from barley seedlings by macroporous resin column chromatography,medium‐pressure LC,and high‐speed countercurrent chromatography

Separation of minor compounds especially with similar polarities and structures from complex samples is a challenging work. In the present study, an efficient method was successfully established by macroporous resin column chromatography, medium‐pressure liquid chromatography, and high‐speed countercurrent chromatography for separation of four minor flavonoids from barley seedlings. Macroporous resin column chromatography and medium‐pressure liquid chromatography were used for enrichment of these four flavonoids. High‐pressure liquid chromatography analysis showed the total content of these four flavonoids increased from 2.2% in the crude extract to 95.3% in the medium‐pressure liquid chromatography fraction. It was indicated that the combination of macroporous resin column chromatography and medium‐pressure liquid chromatography could be a practicable strategy for enrichment of minor compounds from complex sample. Then, high‐speed countercurrent chromatography was employed for separation of these four flavonoids using ethyl acetate/n‐butanol/water (0.1% glacial acetic acid) (4:1:5, v/v/v) as solvent system. As a result, four flavonoids including two isomers with purities higher than 98% were obtained. Interestingly, two flavonoids existing in one high‐pressure liquid chromatography peak were also successfully separated. All these indicated high‐speed countercurrent chromatography had great potential for separation of compounds with similar structures and polarities. This study provides a reference for efficient enrichment and separation of minor compounds from complex sample.     

用反相液相色谱分离多肽时峰容量与色谱柱长度关系的研究

在蛋白质组学研究中,多肽混合物的有效分离对蛋白质鉴定和蛋白质之间相互作用的研究起着决定性的影响。基于此,用反相液相色谱研究了在两个不同长度的色谱柱上分离多肽混合物时色谱柱长度与峰容量的关系,同时考察了梯度洗脱时间对峰容量和峰宽的影响。实验结果表明,色谱柱长度对峰容量有显著的影响,而延长梯度洗脱时间不仅可以增加峰容量,而且可以增加峰宽。这说明用毛细管液相色谱 串联质谱联用方法对多肽混合物进行分离鉴定时,采用较长的色谱柱和较长的梯度洗脱时间有利于对更多的多肽进行分析鉴定。     

Comparison of ultra high performance supercritical fluid chromatography,ultra high performance liquid chromatography,and gas chromatography for the separation of synthetic cathinones

A comparison of ultra high performance supercritical fluid chromatography, ultra high performance liquid chromatography, and gas chromatography for the separation of synthetic cathinones has been conducted. Nine different mixtures of bath salts were analyzed in this study. The three different chromatographic techniques were examined using a general set of controlled synthetic cathinones as well as a variety of other synthetic cathinones that exist as positional isomers. Overall 35 different synthetic cathinones were analyzed. A variety of column types and chromatographic modes were examined for developing each separation. For the ultra high performance supercritical fluid chromatography separations, analyses were performed using a series of Torus and Trefoil columns with either ammonium formate or ammonium hydroxide as additives, and methanol, ethanol or isopropanol organic solvents as modifiers. Ultra high performance liquid chromatographic separations were performed in both reversed phase and hydrophilic interaction chromatographic modes using SPP C18 and SPP HILIC columns. Gas chromatography separations were performed using an Elite‐5MS capillary column. The orthogonality of ultra high performance supercritical fluid chromatography, ultra high performance liquid chromatography, and gas chromatography was examined using principal component analysis. For the best overall separation of synthetic cathinones, the use of ultra high performance supercritical fluid chromatography in combination with gas chromatography is recommended.     

Application of two‐dimensional liquid chromatography in the separation of traditional Chinese medicine

Traditional Chinese medicines have been widely used to prevent and cure diseases for thousands of years. For the purpose of better understanding the extremely complicated traditional Chinese medicines, powerful separation techniques are essential. Two‐dimensional liquid chromatography has been proven to be more powerful for the separation of complex traditional Chinese medicines due to its enhanced peak capacity and resolution compared with one‐dimensional liquid chromatography. Enormous efforts have been made on the coupling of independent separation mechanisms to improve the resolving power for complex traditional Chinese medicine samples, including the development and introduction of novel stationary phases. This review aims to give an overview on the applications of two‐dimensional liquid chromatography in traditional Chinese medicine research since 2008, including comprehensive two‐dimensional liquid chromatography, heart‐cutting two‐dimensional liquid chromatography both in on‐line, and off‐line mode. Different couplings of separation modes were respectively discussed based on specific studies, with emphasis on the applications of novel stationary phases in the two‐dimensional liquid chromatography.