毛细管离子色谱分析系统研究进展

离子色谱是目前分析离子型样品的成熟技术,其水相工作介质的特点相对于液相色谱更适合于生物样品的分析。毛细管离子色谱(CIC)由于其节省样品的特点在生物分析方面的优势更为明显。CIC大体上可分为开管型、填充柱型和整体柱型3种类型。本文以填充型CIC几大关键部件技术发展为主线,综述了近年来CIC的研究进展。     

Recent developments in ion chromatography

This paper summarizes how ion chromatography is now a multimode technique suitable for solving analytical problems in all areas of interest. Current and more recent applications will be overviewed within the new trends.     

Vitamin U-bonded stationary phase in capillary ion chromatography

A vitamin U-bonded stationary phase was prepared and the retention behavior of inorganic anions was examined using ion chromatography. Inorganic anions were retained on the vitamin U-bonded stationary phase under acidic as well as neutral eluent conditions in the ion-exchange mode. The elution order of the examined anions under neutral eluent conditions was nearly the same as that observed in common ion exchange mode, while the elution order observed under acidic eluent conditions was completely different from that observed in common ion exchange mode. The retention of the analyte anions under the neutral eluent conditions was due to the sulfonium groups of the vitamin U, while protonated primary amino groups caused retention of the analyte anions with different selectivity under acidic conditions. The retention factor of the analyte anions increased with decreasing eluent concentration under both eluent conditions. The present system was applied to the determination of bromide and nitrate contained in seawater.     

色谱研究的最新重要进展

细内径毛细管柱自由溶液用于分离宽范围DNA片段 凝胶电泳是分离DNA最高效的方法,但将凝胶注入微纳分离通道或细内径毛细管柱内存在很大的技术难度。为了克服这一技术难题,美国University of Oklahoma的Shaorong Liu和University of Texas at Arlington的P. K. Dasgupta等在无胶、无管壁改性和无外加电场的情况下,应用流体动力学原理在细内径毛细管柱上实现了宽范围 (75 bp ～106 kb)DNA片段的高效分离。他们还对分离后所采集的DNA片段的馏分进行了聚合酶链反应(PCR)扩增实验,发现采集的DNA片段保持了良好的扩增活性。(来源: J. Am. Chem. Soc., 2010, 132: 41－41) LC-MS/MS技术分离鉴定饮用水中的2,6-二氯-1,4-苯并醌 用化学试剂对饮用水进行杀毒处理是水处理中广泛采用的技术和方法,对于饮用水中致病菌的灭杀和防止流行病的发生具有重要作用。饮用水杀毒处理过程中的化学试剂有可能与水中的其他有机试剂发生反应,产生对人体具有毒副作用的副产物。加拿大University of Alberta的Feng Qin和Xingfang Li等发展了固相萃取样品预处理和LC-MS/MS的多级反应监测(MRM)的分离分析集成技术,对水中卤代醌的检测灵敏度达到3～8.7 ng/L。他们采用所发展的方法在饮用水中检测到由于化学试剂杀毒处理产生的副产物2,6-二氯-1,4-苯并醌,这是国际上第一次在饮用水中检测到2,6-二氯-1,4-苯并醌的报道。(来源: Angew. Chem. Int. Ed., 2010, 49: 790－792) 反相-反相二维液相色谱高效分离分析磷酸化肽的新方法 磷酸化肽的高效分离分析对磷酸化蛋白质组学研究具有重要的意义。多维色谱是实现磷酸化肽高效分离分析的最有效的途径。中国科学院大连化学物理研究所的叶明亮和邹汉法等发展了高正交性反相-反相二维液相色谱分离磷酸化肽的新方法。他们通过对分离流动相的优化,首先将富集的磷酸化肽混合物在高pH流动相下进行90 min的梯度分离,并在每1 min采集一个馏分,再将每间隔45 min的馏分混合(如1 min与46 min馏分混合,2 min与47 min馏分混合……),总计产生45个馏分。第一维分离的常规方法是每2 min采集一个馏分(0－2 min馏分,2－4 min馏分……), 90 min的梯度分离可采集45个馏分。在低pH流动相下采用毛细管反相液相色谱-质谱联用技术对第一维采集的馏分进行分离鉴定,发现第一维分离中将每间隔45 min的馏分进行混合的方式可以有效提高二维分离的正交性;与常规的每2 min采集一个馏分的方式相比较,磷酸化肽的鉴定能力提高了30%以上。他们将发展的方法应用于肝脏磷酸化蛋白质组的规模化分析,在将假阳性率控制在1%以下(即FDR＜1%)的条件下初步鉴定了8000个以上的磷酸化位点。(来源: Anal. Chem., 2010, 82: 53－56) 纳流通道分离阿升(Attoliter)级样品的飞升(Femtoliter)级液相色谱至今尚无良好的方法与手段对珍贵的极微量生物样品进行分离分析。日本University of Tokyo的Takehiko Kitamori等报道了飞升级液相色谱(fLC)的研究成果。他们在玻璃微芯片上制备了宽度和深度为几百纳米的纳流通道。该系统的尺寸只有常规液相色谱系统的1/1011左右,流动相的流量为亚pL/min,进样量为几百阿升。fLC无需分离固定相,可以分离电荷不同的化合物。fLC可以克服常规液相色谱分离固定相非均一性和涡流扩散的缺点。液相色谱尺寸的缩小不仅具有进样量小的优点,而且可有效提高分离效率。fLC可以应用于极微量样品(如单细胞样品)的高效分离分析。(来源: Anal. Chem., 2010, 82: 543－547) 全二维气相色谱-飞行时间质谱应用于化学武器前体痕量杂质的特征指纹分析 化学攻击作为潜在的恐怖活动正日益引起关注。美国University of Washington的Carlos G. Fraga等探讨了将样品分析和化学计量学相结合的方法用于化学攻击犯罪案件中法医鉴定的可行性。他们以一种有可能作为化学武器攻击的有毒试剂——甲基磷酸二甲酯(DMMP)作为模型化合物,以29种痕量杂质为检测对象,应用全二维气相色谱-飞行时间质谱(GC×GC-TOF)对6批DMMP样品中的痕量杂质进行了指纹分析。进一步使用平行因子法(PARAFAC)对重叠峰进行数学分辨处理获得清晰的谱图,并通过与谱图库中标准谱图的比对成功地鉴定了检测到的化合物。通过对每一对样品的数据进行统计分析,结果表明其中5批DMMP样品中的痕量杂质化合物的含量有些很相近,有些存在一定的差异。而有2批DMMP样品中的痕量杂质则具有完全相同的指纹谱图。运用非负矩阵因子分析发现,DMMP样品由于得分值不同可以分为5类,其中有2个来自同一供应商的同一批样品不能实现区分,这是由于它们的痕量杂质含量相同造成的,而其他4个样品由于来自于不同批次或不同供应商而得到了很好的区分。这一结果表明可以通过痕量杂质的指纹分析有效追踪DMMP产品的来源。此外,他们还发现不同公司的某些DMMP样品存在特异性的痕量杂质分布。因此所发展的方法有可能成为化学攻击犯罪案件中法医鉴定可采用的有效手段。(来源: Anal. Chem., 2010, 82: 689－698)     

Recent developments and emerging directions in ion chromatography

This review provides a selected overview of some of the significant developments that have occurred in ion chromatography (IC) over recent years, together with some views on the future directions that IC will follow. The topics covered in the review include new stationary phases (especially monolithic phases), miniaturized IC systems (capillary, microchip and low-pressure formats), enhanced peak capacity through the use of complex eluent profiles and associated computer tools for simulation and prediction of retention, hyphenated IC systems and their use for speciation studies and metallomics, and the rapidly increasing use of IC in bioanalysis.     

A simplified ion exchange bead-based KOH electrodialytic generator for capillary ion chromatography

A simple potassium hydroxide electrodialytic generator (EDG) with singe membrane configuration is described. In this setup, one cation exchange resin (CER) bead is used to fabricate the EDG in place of the common membrane sheet. The device is implemented simply in a commercial stainless steel (SS) Tee which serves as both the EDG cartridge and the cathodic electrode. The present EDG has much lower internal volume (∼0.16 (L), which is well suited with capillary ion chromatography system. The device has been tested up pressures to 3200 psi and could be directly deployed on the high-pressure side of the pump. The electrolysis gas can be effectively removed by a segment of PTFE tubing. In the tested range of 0-100 mM, the KOH concentration is generated linearly with the applied current being near-Faradaic efficiency. The device permits both isocratic and gradient operation with good reproducibility, as demonstrated by the analysis of anions.     

Method development approaches for capillary ion electrophoresis

Capillary ion electrophoresis (CIE) is a capillary electrophoretic technique optimized for rapid determination of low-molecular-mass inorganic and organic ions. CIE predominantly employs indirect UV detection since the majority of the analytes lack specific chromophores. Described are three methods for detection and electrolyte optimization. The first method discussed approaches for optimizing sensitivity, selectivity and peak confirmation using a chromate electrolyte and selected detection wavelengths. Peak confirmation is aided by using both direct detection of analytes. The second and third methods involve an unattended electrolyte development approach for instruments that only provide fresh electrolyte on the injection side of the capillary. The electrolyte composition is changed in both the injection side vial and in capillary before each sample injection while leaving the receiving side electrolyte vial constant at the initial electrolyte composition. In one mode, the concentration of the electroosmotic flow (EOF) modifier used to induce anodic flow is varied while keeping the background electrolyte composition constant. In a second experiment, the background electrolyte co-ion is sequentially changed from high mobility to low mobility while keeping the EOF modifier concentration constant. The end effect is to achieve a broad range of controlled peak symmetry for analytes in a simple matrix. The results are compared to separations obtained when the injection side and receiving side electrolytes are manually matched.     

The role of column technology in capillary gas chromatography

After having witnessed, over a period of more than 20 years, the extremely difficult early development of capillary gas chromatography, the growth of scientific understandign, and finally its sudden blossoming into a commonly accepted technique, the author is confronted with a dichotomy. On the one hand, there are the dramatic advances in the fields of column preparation, and of sampling techniques. Implementation of these advances should logically result in correspondingly drastic progress in the average quality of application work in drastic progress in the average quality of application work in capillary gas chromatography. On the other hand, however, such progress is lacking. The greater the practical potential, the smaller the fractionm of that potential which is practically exploited. The reason is that most users interpret te recent advances as exempting them from their obligation to know and think about the many details of practical work, and to attempt standardized work with standard columns and techniques. Standardization of routine work is beneficial, provided the standards are optimized-a condition which all too often remains unfulfilled. Research work, performed in a realm of ever new and unknown conditions, is of poor quality when done by standardized means. Since the most critical part of optimization is column selection, the capillary column must be mastered as a familiar tool in the hands of the investigator. This is hardly possible in the absence of any direct contact with column technology.     

Comprehensive ultra-high pressure capillary liquid chromatography/ion mobility spectrometry

Summary An ultra-high pressure liquid chromatograph was interfaced to a moderately high-resolution nanoelectrospray ionization ion mobility spectrometer operating at ambient temperature and pressure to achieve fast multidimensional separations. The potential of using ion mobility spectrometry as the second dimension in a comprehensive arrangement with liquid chromatography to offer improved qualitative information for compounds under specific operating conditions, is discussed. Separation and detection of selected benzodiazepines and triazine herbicides are demonstrated. Composite peak capacities of 39 and 33 for benzodiazepine and triazine herbicide mixtures, respectively, were achieved in less than 75 s using a 16.5 cm×50 μm internal diameter fused silica capillary liquid chromatographic column packed with 1.5 μm diameter ODS-bonded silica particles.     

Multidimensional gas chromatography with capillary flow technology and LTM-GC

2-D GC is a logical and cost effective extension to 1-D GC for improving the separation resolution, selectivity, and peak capacity of an analytical system. The advent of electronic pressure control systems that are accurate to the third decimal place, combined with recently innovated chromatographic devices such as capillary flow technology, has eliminated many deficiencies encountered in current conventional 2-D GC by making the technique reliable and simple to implement in both production and research analytical facilities. Low thermal mass GC (LTM-GC) was successfully integrated with capillary flow technology to further enhance overall 2-D GC chromatographic system performance by providing not only faster throughput via rapid heating and cooling, but independent temperature control for each dimension to maximize separation power. As an example, despite the enhanced peak capacity obtained from conventional 2-D GC, alkyl naphthalene isomers such as 2,3-dimethyl and 1,4-dimethyl naphthalene coeluted. These two critical compounds were well resolved (R = 5.2) using 2-D GC with LTM-GC with a similar analytical time. This paper demonstrates the benefits of combining capillary flow technology with LTM-GC to provide major enhancements to conventional 2-D GC. The synergy of these techniques is highlighted with practical industrial applications.     

毛细管离子色谱法测定酒和饮料中阳离子

建立了毛细管离子色谱测定酒、饮料等样品中阳离子的分析方法。使用毛细管离子色谱柱IonPac CS12A(250 mm×0.4 mm, 8 μm),以甲基磺酸淋洗液发生器(EGC-MSA)产生18 mmol/L的甲基磺酸为流动相,进样量0.4 μL,在流速0.01 mL/min的条件下,采用自循环抑制电导检测的方法对啤酒、葡萄酒、白酒、果汁及奶茶等样品中的阳离子含量进行检测。结果表明,毛细管离子色谱法能满足阳离子含量的测定要求,系统稳定不易堵,在灵敏度方面优于常规离子色谱系统。该方法能够快速、准确地测定酒、饮料等样品中的5种阳离子(钠、铵、钾、镁和钙),回归方程的相关系数在0.9997以上,实际样品的加标回收率为95.2%～103.3%。该方法具有灵敏度高,操作简单,环境友好的特点。     

Recent advances in ion chromatography suppressor improve anion separation and detection

The Alltech DS-Plus suppressor improves and simplifies anion analysis by suppressor-based ion chromatography (IC). In addition to suppressing the mobile phase and enhancing the analyte signal, the DS-Plus suppressor removes carbonic acid from the suppressor effluent when carbonate-hydrogencarbonate mobile phases are used, reducing the water dip that interferes with early eluting peaks, increasing detection sensitivity, and enabling carbonate-hydrogencarbonate gradients. The ability to operate with carbonate-hydrogencarbonate gradients places exceptional separating power in the mobile phase, forgoing the need for expensive columns with special selectivity. Continuous operation and solid-phase chemistry eliminates the need for external regenerants, switching valves, and multiple operating modes.     

Recent developments in the high-performance chelation ion chromatography of trace metals

There have been a number of significant developments in the high-performance chelation ion chromatography (HPCIC) of trace metals in recent years. This review focuses on these developments, while giving important information on the fundamental parameters controlling the chelation sorption mechanism, including type of chelating group, stability constants, kinetics, and column temperature. The discussion pays particular attention to the types and properties of efficient chelating stationary phases which have been fabricated for certain groups of metals. The review also describes a number of major improvements in postcolumn reaction detection including the use of the latest reagents and noise reduction strategies to improve sensitivity and reduce LOD. In the final section, an indication of the applicability of HPCIC to a range of complex sample types is given with some key examples and chromatograms using the latest high-efficiency chelating phases.     

Fourier transform ion mobility spectrometry of barbiturates after capillary gas chromatography

This paper demonstrates a novel operating mode of an ion mobility detector (IMD) for obtaining both qualitative and quantitative data after capillary gas chromatographic separation of 5,5′-disubstituted barbiturates. Using a recently developed time dispersive Fourier transform method for ion mobility spectrometry, complete ion mobility spectra could be obtained for each component in the chromatogram. This type of spectra can be used for providing qualitative information on unknown compounds or for selecting the proper detector conditions needed when operating in the continuous mobility monitoring mode. In this study each of the five barbiturates investigated produced a Fourier transformed ion mobility spectrum containing one major product ion. When drift times corresponding to those of the product ions measured in the FT mode were monitored continuously, selective chromatographic detection of the barbiturates was achieved. In one case even isomers could be differentiated based on mobility characteristics.     

A multifunctional dual membrane electrodialytic eluent generator for capillary ion chromatography

A multifunctional electrodialytic generator (EDG) for capillary ion chromatography (CIC) is described. The same device can generate acidic, basic or saline eluents. Two oppositely charged ion exchange beads are used to fabricate the EDG; the dual ion exchanger configuration ensures the production of gas-free eluent, obviating the need of a gas removal device used with single ion exchanger EDG's. The ability of the same EDG to produce different eluents is governed solely by the choice of the respective feed solutions; this is presently demonstrated by generation of diverse eluents such as Na₂CO₃/NaHCO₃, CH₃SO₃H, and KNO₃. The EDG is implemented simply in a commercial cross fitting and has been tested up pressures to 2000 psi. Under typical operating conditions, the zero current concentration (open circuit penetration, OCP) is negligible. The generated eluent concentration linearly increases with applied current with a slope that is essentially Faradaic. The device permits both isocratic and gradient operation with good reproducibility, as demonstrated by the analysis of anions using HCO₃⁻/CO₃²⁻ EDG.     

The importance of capillary electrophoresis,capillary electrochromatography,and ion chromatography in separations of inorganic ions

The importance of capillary electrophoresis (CE), capillary electrochromatography (CEC), and ion chromatography (IC) in inorganic ion analyses is outlined. Methods for improving the reliability of the CE measurements are briefly described. Selectivity optimization in CE analyses of inorganic cations and anions is discussed. Using the Peakmaster program, CE system peaks (system zones, eigenmobilites) and some important CE parameters, such as effective mobilities, electromigration dispersion, indirect UV, and direct conductivity signals, are predicted and compared with experimental analyses.     

Developments in suppressor technology for inorganic ion analysis by ion chromatography using conductivity detection

A review is presented detailing the development and use of suppression devices for the conductimetric detection of inorganic ions by ion chromatography (IC). An overview of the general response equation for conductivity detection is also given. Topics of discussion include the role and function of suppressors, the development of early suppressors including packed column and membrane devices from 1975 to 1990 and the subsequent progression towards present day commercially available suppressors and recent innovations. Post-suppression devices for signal enhancement are also discussed.     

Recent innovation in capillary electrokinetic chromatography with replaceable charged pseudostationary phases or additives

Recent developments of separation of neutral analytes in capillary systems with the mobile phase driven by the electroosmotic flow (EOF) and charged additives acting as a pseudostationary phase are reviewed. As pseudostationary phases a number of additives are used. Soluble polymers, either anionic or cationic, were applied as alternatives to micelles. Monomeric charged additives are also intended to form associates with the analytes, leading to selective retention and separation in a similar way as the polymeric pseudostationary phases. Dendrimers, spherical macromolecules with highly branched chains and charged terminal groups, are successfully applied for the separation of lipophilic analytes. Polymers with covalently stabilized structures are introduced in the form of permanent micelles and are therefore insensitive to the mobile phase composition, enlarging the applicability of micellar electrokinetic capillary chromatography (MEKC).     

Recent developments in capillary electrokinetic chromatography with replaceable charged pseudostationary phases or additives

Although electrochromatography in packed beds or monolithic columns has gained enormous interest, techniques based on charged pseudostationary phases like micelles are of high practical importance in electrically driven separation science. However, nonmicellar alternatives, e.g., using charged soluble polymers or smaller additives are still attractive, as they allow high concentrations of organic solvents, and their application is not limited by the critical micellar concentration. This review discusses the developments in the field of electrokinetic chromatography with these additives in the last three years, covering ionic polymeric pseudostationary phases, dendrimers and so-called micelle polymers, but also small molecules which implement separation selectivity due to their specific interaction with the analytes.     

Poly(ethylene oxide)-bonded stationary phase for capillary ion chromatography

Methyl-capped poly(ethylene oxide) moieties were chemically bonded to silica gel using an amine-reactive modification reagent and evaluated as the stationary phase for ion chromatography. In this work, primary amino groups of an aminopropylsilica packing material were reacted with methyl-PEO₁₂-NHS ester (succinimidyl-{[N-methyl]-dodecaethyleneglycol} ester) in phosphate buffer (pH 7.0) at room temperature. The prepared poly(ethylene oxide)-bonded stationary was evaluated for the separation of inorganic anions, and the retention behavior of inorganic anions on the prepared stationary phase was examined. The elution order of the investigated anions was the same as that observed in common ion chromatography. Both cations and anions of the eluent affected the retention of the analyte anions. Ion exchange was involved for the retention of analyte anions, although the present stationary phase does not possess any discrete ion-exchange sites. The stationary phase was applied to the separation of trace anions contained in tap water and a rock salt.