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。     

酪蛋白多肽的制备和色谱分离方法

为了得到低成本的多肽,本文利用胰蛋白酶对酪蛋白进行了充分的酶解。采用分析级反相高效液相色谱-电喷雾质谱联用技术(RP-HPLC/ESI-MS)分析了酶解产物各组分的组成,并通过改变流动相的梯度洗脱程序,优化了分析级色谱条件以充分分离相对含量较高的多肽组分;将优化的分析级色谱条件直接放大到制备级RP-HPLC中,在程序控制下通过紫外吸收信号结合ESI-MS信号共同引导实现了多肽的全自动化分离和收集。整个过程方便快捷,经过这样一个单一的分离步骤,得到了多个纯度较高的多肽。除此之外,本文还考察了流动相的酸碱性、柱上样量等因素对该体系制备级分离的影响,并对一次分离中分辨率不好的亲水性多肽混合物进行了二次分离,得到了多个新的多肽。本文建立的多肽制备方法为多肽和多肽材料的广泛应用提供了一种选择。     

多肽的反相梯度加压毛细管电色谱分离

以C18为固定相，采用电压和压力联合驱动流动相，研究反相加压毛细管电色谱分离多肽；考察了加压电色谱中，电压对带电和中性物质迁移的影响，实现了梯度加压毛细管电色谱分离6种多肽；结果表明，加压电色谱可以很好地抑制气泡形成，实验结果准确，重复性好；梯度加压毛细管电色谱在复杂样品的分离分析中，具有很大的潜力。     

反相液相色谱在蛋白质及多肽分离分析中的应用

反相液相色谱是一种以疏水作用为基础的色谱分离模式。由于它具有分辨率高、重复性好、回收率高等优点,在蛋白质及多肽的分离分析中得到了极为广泛的应用。本文简要介绍了反相液相色谱及其分离机理,对其在蛋白质和多肽研究中的应用如分离纯化、肽图分析、酶活测定、构象变化检测及疏水作用研究等作系统综述,并展望了反相液相色谱在这一领域的发展前景。     

逆流色谱技术分离蛋白质和多肽的研究进展

逆流色谱是一种连续高效的液-液分配色谱技术,具有进样量大、无不可逆吸附、回收率高和能有效保护样品生物活性等优点,在蛋白质和多肽的分离中具有独特的优势。本文综述了近年来几种新型的逆流色谱技术在蛋白质和多肽分离中的研究进展,并对该领域的未来发展进行了展望。     

高效液相色谱-质谱法测定牛Ⅰ型胶原含量

建立了一种基于高效液相色谱-质谱联用技术并通过特征多肽进行牛Ⅰ型胶原含量检测的方法。胶原海绵样品经酶解处理后进行色谱分离,在电喷雾离子源正离子扫描及保留时间依赖的单离子监测模式(SIM)下测定,外标法定量。采用胰蛋白酶特异性酶解得到牛Ⅰ型胶原的1个特征多肽为GFSGLDGAK。该多肽在0.645～21.5μg/mL质量浓度范围内线性关系良好(r~2=0.999 4);方法的定量下限为6.45×10~(-4)μg/mL;在3.10、2.48、1.55μg/mL 3个加标水平下,平均回收率为98.2%～102%;峰面积的相对标准偏差(n=6)为0.80%。胶原海绵样品经均匀性、稳定性和定值检测,确保了方法有效性,验证了该产品质量,且测得牛Ⅰ型胶原含量为(0.926±0.014) mg/mg。该方法简便快速,重现性好、稳定性高,可有效地对牛Ⅰ型胶原进行定量。     

多肽色谱分离后的柱后荧光衍生检测

高效液相色谱现在已成为多肽分离分析中分辨率最高的手段。色谱流出液中多肽的检测,目前最常用的是利用远紫外区的光吸收测定(205-220nm)。紫外吸收测定法简便易行,对样品没有破坏作用,但对移动相有严格的要求。使得在生物活性蛋白分离中所常用的一些缓冲液系统都难以应用。     

混合抗体的亲和色谱分离策略

乳腺生物反应器可以高效表达重组人单克隆抗体,但是目标产品与乳液原料中的牛抗体性质、结构非常类似,分离难度很大。本文对牛抗体和重组人抗体的种属差异进行了分析,并在此基础上制定了新型分离策略,采取Protein A亲和色谱和免疫亲和色谱来解决混合抗体的分离问题,并讨论了色谱洗脱模式对分离效果的影响。结果表明,Protein A亲和色谱结合梯度洗脱可以有效地纯化得到混合抗体,但是难以彻底分离重组人抗体和牛抗体;相比之下,使用Protein A亲和色谱结合置换色谱模式可以更加高效地分离混合抗体,最终可以得到纯度高达95%以上的重组人抗体,回收率可达95%以上。免疫亲和色谱同样可以有效地分离纯化重组单克隆抗体,且其通用性更强,可以应用于任何动物乳腺表达重组人抗体的分离纯化中。     

氨基酸组成相同序列不同的小分子多肽的液相色谱-质谱联用分析

多肽组学是近年来兴起的一门新型学科,质谱已成为多肽组学研究的强有力手段．然而,用于检测具有相同氨基酸组成但序列不同的多肽时,只能给出等同的分子离子峰,在多肽结构解析上受到一定限制．因此,发展色谱分离．质谱检测联用技术是分析具有相同氨基酸组成但序列不同的多肽的有效途径．本文建立了一种氨基酸组成相同序列不同的小分子多肽的反相液相色谱分离-电喷雾离子化质谱检测新方法．该方法采用高效液相色谱-质谱联用技术,以两种三肽Gly．Ser．Phe和Gly．Phe．Ser为模式样品对象,考察了小分子多肽在不同流动相组成、流动相添加剂及pH等条件下的液相色谱行为,并讨论其保留机理．研究结果表明,在最优化的实验条件下,该方法稳定性好,重现性高,为多肽组学研究中的多肽解析提供科学的分析方法．     

超高效液相色谱-质谱联用法监测人心房钠尿肽的化学合成

多肽作为神经递质、激素、受体等参与和调节许多重要的生命过程。随着对多肽结构与功能认识的深入,化学合成多肽在生物医药、生命分析等领域显示了重要价值。人心房钠尿肽（ANP）是一种由28个氨基酸组成的多肽类激素,其含量和代谢异常与心血管疾病、癌症等密切相关。针对ANP重要的生理功能,该文开展了ANP化学合成及其过程的监测与优化,设计了集固相合成线性直链多肽和液相氧化成环为一体的策略。基于高效液相色谱的分离性能和多级质谱的结构鉴定能力,建立了超高效液相色谱-串联质谱（UHPLC-MS/MS）分析新方法,对固相合成产物进行了分离分析,并对液相氧化方法进行了筛选和优化,最终分离纯化得到了目标多肽,为人心房钠尿肽的高效化学合成及氧化成环反应提供借鉴。     

离子色谱法分析金属离子的研究进展

综述了离子色谱法(IC)分析金属离子的研究进展,对目前应用于分析金属离子的阳离子交换IC、阴离子交换IC和螯合离子色谱进行了评述。阳离子交换IC是IC分析金属离子的主要形式,固定相为强酸(磺酸)型阳离子交换剂和弱酸(羧酸)型阳离子交换剂,结合适当的检测方法,阳离子交换IC可以测定碱金属、碱土金属、过渡金属、稀土离子、铵离子及低相对分子质量的有机胺类分子等。阴离子交换IC可以分析碱土金属、过渡金属、稀土离子等,对金属离子的分析具有更好的选择性,并可以实现金属离子和无机阴离子的同时测定。螯合离子色谱可以对复杂基体中的痕量金属离子进行测定。引用文献125篇。     

Simultaneous determination of positive and negative pharmaceutical counterions using mixed-mode chromatography coupled with charged aerosol detector

A mixed-mode chromatography coupled with charged aerosol detector (CAD) method was developed in this work to simultaneously determine pharmaceutical counterions including both inorganic ions and organic ions in the forms of cations and anions. 25 commonly used pharmaceutical ions were studied and simultaneously separated within 20 min by this single method. A silica based mixed-mode column with reversed-phase/cation-exchange/anion-exchange modes was used. It provides reversed-phase, strong cation-exchange and weak anion-exchange properties at the same time. It also provides the HILIC behavior at high percentage of organic solvent. The effects of mobile-phase organic strength, buffer ions, ionic strength, pH and column temperature have been investigated to optimize the method as well as to understand the retention and separation mechanisms. Conventional HPLC system was used and no special chromatography system is needed. The presented method has been employed successfully for screening and quantitative analysis of counterions, unknown ionic impurities and salts in active pharmaceutical ingredients and in process control samples with excellent accuracy, precision and sensitivity. This method provides a simple, fast and generic approach to speed up pharmaceutical research and development process and enhance lab efficiency. The similar methodologies can be applied to other ion analysis.     

Two-dimensional strong cation-exchange liquid chromatography/reversed-phase pressurized capillary electrochromatography for separation of complex samples

A 2-D separation platform was constructed using micro strong cation-exchange liquid chromatography (μ-SCXLC) and reversed-phase pressurized capillary electrochromatography (RP-pCEC) for the analysis of complex samples. Samples were fractionated by the first-dimension μ-SCXLC with a linear solvent gradient and then injected into the second-dimension RP-pCEC for further separation. The μ-SCXLC/RP-pCEC 2-D system with three separation mechanisms, namely strong cation-exchange, reversed-phase chromatography and electrophoresis, provided high selectivity, high resolution and high peak capacity compared to one-dimensional chromatographic approaches. Separation effectiveness of this 2-D system was demonstrated by the analysis of different kinds of complex samples, such as traditional Chinese medicine Cortex Phellodendri, bovine serum albumin (BSA) tryptic digest and real serum tryptic digest. A theoretical peak capacity of approximately 1200 was achieved, which proves its promising potential for the separation and analysis of complex samples.     

离子排斥色谱结合光度检测的水质监测系统用于测定离子型营养物（英文）

A unified ion-exclusion chromatography(IEC) system for monitoring anionic and cationic nutrients like NH + 4,NO 2,NO 3,phosphate ion,silicate ion and HCO 3 was developed and applied to several environmental waters.The IEC system consisted of four IEC methodologies,including the IEC with ultraviolet(UV) detection at 210 nm for determining NH + 4 on anion-exchange separation column in OH form connected with anion-exchange UV-conversion column in I form in tandem,the IEC with UV-detection at 210 nm for determining simultaneously NO 2 and NO 3 on cation-exchange separation column in H + form,the IEC with UV-detection at 210 nm for determining HCO 3 on cation-exchange separation column in H + form connected with anion-exchange UV-conversion column in I form in tandem,and the IEC with visible-detection based on molybdenum-blue reaction for determining simultaneously silicate and phosphate ions on cation-exchange separation column in H + form.These IEC systems were combined through three manually-driven 6-port column selection valves to select each separation column to determine selectively the ionic nutrients.Using this sequential water quality monitoring system,the analytical performances such as calibration linearity,reproducibility,detection limit and recovery were also tested under the optimized chromatographic conditions.This novel water quality monitoring system has been applied successfully for the determination of the ionic eutrophication components in sub-urban river waters.     

Chromatographic evaluation of reversed-phase/anion-exchange/cation-exchange trimodal stationary phases prepared by electrostatically driven self-assembly process

This work describes chromatographic properties of reversed-phase/cation-exchange/anion-exchange trimodal stationary phases. These stationary phases were based on high-purity porous spherical silica particles coated with nano-polymer beads using an electrostatically driven self-assembly process. The inner-pore area of the material was modified covalently with an organic layer that provided both reversed-phase and anion-exchange properties while the outer surface was coated with nano-sized polymer beads with strong cation-exchange characteristics. This design ensured spatial separation of the anion-exchange and the cation-exchange regions, and allowed reversed-phase, anion-exchange and cation-exchange retention mechanisms to function simultaneously. Chromatographic evaluation of ions and small molecules suggested that retention of ionic analytes was influenced by the ionic strength, pH, and mobile phase organic solvent content, and governed by both ion-exchange and hydrophobic interactions. Meanwhile, neutral analytes were retained by hydrophobic interaction and was mainly affected by mobile phase organic solvent content. Depending on the specific application, selectivity could be optimized by adjusting the anion-exchange/cation-exchange capacity ratio (selectivity), which was achieved experimentally by using porous silica particles with different surface areas.     

锆胶基质阳离子色谱柱填料的制备

以10μm锆胶微球包覆聚苯乙烯制成强酸、弱酸型的阳离子色谱柱填料,具有良好的色谱性能。使用D ionex-120型双柱离子色谱仪,较好地分离碱金属、碱土金属离子及铵离子,比较了不同交换基团对溶质保留的影响。由于锆胶基质具有高的化学稳定性和机械强度,用作离子色谱柱填料有良好的应用前景。     

Simultaneous separation of inorganic anions and cations by using anion-exchange and cation-exchange columns connected in tandem in ion chromatography

Inorganic anions and cations in environmental waters were determined by ion chromatography. Stationary and mobile phases were examined for the simultaneous separation of both anions and cations. Cations detection by UV detection requires a mobile phase with a UV absorbing additive, which indirectly visualizes cations as negative peaks. Simultaneous separation of anions and cations were achieved when using an eluent that consists of inorganic acid with weak basic amino acid as additives. It was convenient to separate both anions and cations by coupling anion-exchange and cation-exchange columns in tandem. The order of the separation columns connected affected the elution profiles. When the eluent comprises of multiple anions and a single cation, the anion-exchange column should be connected in the upper stream, whereas when the eluent comprises multiple cations and a single anion, the cation-exchange column should be connected in the upper stream. Use of switching valves also allowed simultaneous separation of anions and cations in a single chromatographic run. In the present work, operating conditions were optimized for the simultaneous separation of anions and cations.     

Behaviour of selenium and tellurium species and their determination by ion chromatography

Summary An ion chromatographic method has been developed for the separation of Te (IV) and Se(IV) in hydrochloric acid mobile phases; the method has been used to determine tellurium in a high-purity non-stoichiometric semiconducting ZnCdTe-based material. Different cation-exchange columns (IonPac CS2, CS3, CS10), a mixed bed ion-exchange column (IonPac CS5), a multi-mode cation-exchange column (OmniPac PCX-500), anion-exchange columns (IonPac AS4, AS4A, AS5, AS5A, AS10, AS11) and a multi-mode anion-exchange column (OmniPac PAX-500) were evaluated for ion chromatographic separation of Se and Te and to study the chemical forms in which the analytes were eluted. The chromatographic data obtained enabled the calculation of both the sign and the chaarge of the eluting species.     

氨基酸分析方法的研究进展

对目前应用于氨基酸分析的主要方法作了较详细评述。这些方法包括柱后衍生高效阳离子交换色谱法、柱前衍生反相高效液相色谱法、高效阴离子交换色谱-积分脉冲安培检测法等。引用文献73篇。     

Peak parking technique for the simultaneous determination of anions and cations

An ion chromatography method is described for the simultaneous determination of anions (Cl^–, NO₃^–, and SO₄^2–) and cations (Na⁺, NH₄⁺, K⁺, Mg²⁺, and Ca²⁺) using a single pump, a single eluent and a single detector. An anion-exchange column modified with chondroitin sulfate C facilitated the elution of the above three anions using 5 mM tartaric acid as the eluent in isocratic mode, whereas the same eluent facilitated the separation of the above five cations on a commercially-available cation-exchange column. The separation columns were connected in series via two six-port switching valves, so the required cation-exchange or anion-exchange separation could be carried out by selecting the appropriate positions for the switching valves. The separations were completed in 30 min.