3-Iodoacetylaminobenzanthrone as a fluorescent derivatizing reagent for thiols in high-performance liquid chromatography

A new thiol-reactive derivatizing reagent, 3-iodoacetylaminobenzanthrone (IAB) has been developed for thiol analysis in liquid chromatography. In aqueous methanol containing 15 mM pH 8.3 H₃BO₃-KCl-Na₂CO₃ buffer, IAB reacted with thiols at 35 °C for 15 min. The derivatives of IAB with glutathione (GSH), cysteine (Cys), homocysteine (Hcy) and N-acetylcysteine (Nac) were well separated on a C₁₈ column with the mobile phase of methanol-water (50:50, v/v) containing 15 mM pH 2.7 H₃cit-Na₂HPO₄ buffer. At λ_ex/λ_em=420/540 nm, the detection limits were 20, 20, 55 and 40 fmol (1, 1, 2.3 and 2 nM), respectively, with a signal-to-noise ratio of 3. Owing to the preferential selectivity of iodoacetamidyl moiety to SH group, amino acids, aliphatic amines, phenol and alcohols had no obvious interference with the determination. The proposed method has been applied to the determination of thiols in human blood with recoveries of 98.5-105.3%.     

高效液相色谱法测定胭脂红酸的研究

研究了以反相C18柱,V(甲醇)∶V(乙腈)∶V(pH 2.8 H3PO4)=12∶8∶80混合溶液为流动相,对胭脂红酸进行高效液相色谱分析的方法。胭脂红酸在2.5~40μg/mL范围内的吸光度线性相关系数达0.9998,方法回收率在98.2%~101.1%,相对标准偏差为0.78%~1.2%,已用于样品中胭脂红酸的测定。     

芯片液相色谱技术进展

微型化是现代分析仪器发展的重要趋势.微型化液相色谱仪器在提供与常规尺度液相色谱相同甚至更高分离效率的同时,可以有效减少溶剂和样品的消耗;在液相色谱-质谱联用中,低流速进样可以有效提高质谱离子源的离子化效率,提高质谱检测效率;对于极微量样品的分离,微型化的液相色谱可以有效减少样品稀释;液相色谱的微型化还有利于液相色谱仪器...     

原位衍生化技术在液相色谱和液相色谱-质谱联用分析中的应用

衍生化是将待分析物转化为更适合的物质形式以便于分析的有效手段。原位衍生化技术作为一种常用的柱前衍生化方法,可以在样品基质中同时完成分析物的萃取和衍生化,具有高效、灵敏和选择性好的优点。原位衍生化结合其他前处理技术广泛用于胺类、醛酮类、醇类、酚类、羧酸和巯基化合物的分析中,在生物、药物、食品、环境、化妆品分析等领域有广泛的应用。该文概述了原位衍生化的反应类型和代表性衍生试剂,综述了原位衍生化技术在液相色谱和液相色谱-质谱联用分析中的应用,并展望其发展趋势。     

Homogeneous liquid–liquid extraction combined with high performance liquid chromatography–fluorescence detection for determination of polycyclic aromatic hydrocarbons in vegetables

In this article, homogeneous liquid–liquid extraction (HOLLE), combined with HPLC-fluorescence detector (HPLC-FLD), has been developed for the extraction and determination of polycyclic aromatic hydrocarbons (PAHs) in vegetables. ACN was used as extraction solvent for the extraction of target analytes from vegetables. When the previous extraction process was over, the ACN extract was transferred to the water-immiscible organic phase, tetrachloroethane, used as extraction solvent in HOLLE procedures. Under the optimum conditions, repeatability was carried out by spiking PAHs at concentration level of 12.5 μg/kg, the RSDs varied between 1.1 and 8.5% (n = 3). The LODs, based on S/N of 3, ranged from 0.025 to 0.25 μg/kg. Relative recoveries of PAHs from cucumber and long crooked squash samples were in the range of 72.4–104.9% and 65.5–119.3%, respectively. Compared with the conventional extraction method, the proposed method has the advantage of being quick, easy to operate, and having low consumption of organic solvent.     

Evaluation of different operation modes of high performance liquid chromatography for the analysis of complex mixtures of neutral oligosaccharides

Chromatographic methods based on different HPLC operation modes, reverse phase (RP), high performance anion exchange chromatography (HPAEC), graphitized carbon chromatography (GCC) and hydrophilic interaction liquid chromatography (HILIC), have been developed and compared for the analysis of complex mixtures of neutral oligosaccharides with functional properties. Whereas GCC gave the best chromatographic separation of isomeric oligosaccharides with the same molecular weight (R(s) values in the range 1.0-4.0 and 2.4-5.6 for tetra- and pentasaccharides, respectively), HILIC provided the best results for mixtures including oligosaccharides of different degrees of polymerization (R(s) values of maltooligosaccharides between 3.4 and 6.2). Validation of the HILIC LC-MS method proved its utility for the analysis of oligosaccharide mixtures with functional properties: relative standard deviations lower than 10%, LOD's and LOQ's in the range 12.7-130.2 ng mL(-1) and 39.3-402.2 ng mL(-1), respectively, and linearity up to 10-20 μg mL(-1). Quantitative data for fructooligosaccharides, gentiooligosaccharides and dextransucrase cellobiose acceptor oligosaccharides were obtained by using this method.     

A review on solid phase microextraction-high performance liquid chromatography as a novel tool for the analysis of toxic metal ions

This paper reviews the practical applications of solid phase microextraction-High performance liquid chromatography in the analysis of toxic metal species as these are important contaminants and are carcinogenic. Their determination in formulations, in feed and food, and in complex environmental matrices (e.g., waste water and industrial effluents) often requires analytical methods capable of high efficiency, unique selectivity, and high sensitivity. Solid phase microextraction (SPME) requires low solvent consumption and is quick in use. SPME is used for extraction and online desorption of analytes with the mobile phase of HPLC and subsequent detection by UV, ICP-MS or ESI-MS as detectors. Different SPME-HPLC methods are summarized in this article to demonstrate the usefulness of this technique for metallic species of As, Cr, Pb, Hg and Se.     

超高效液相色谱法检测土壤中的多环芳烃

采用二极管阵列(PDA)检测器,建立了超高效液相色谱(UPLC)定性定量分析土壤中16种多环芳烃(PAHs)的方法。并将该方法与传统高效液相色谱(HPLC)的分析性能进行了详细的比较。研究结果表明,采用UPLC法分析16种PAHs具有分析速度快(13.5 min)、检出限低(2～20 pg)、灵敏度高等优点。     

基于超高效液相色谱-八端口转子阀的微升级预分级系统的建立

复杂生物系统蛋白质组的深度覆盖鉴定得益于近年来快速发展的高效色谱分离和串联质谱分析技术。二维高效液相色谱（2-D HPLC）是实现复杂肽混合物正交分离的有效手段，但其缺点是运行时间长，通常要求肽上样量在毫克级别，且收集时的组分体积大，后续合并流程复杂，而有望替代其的StageTip技术，则由于有限的色谱分离梯度，难以达到充分的正交分离。该文探索了超高效液相色谱（UPLC）和八端口转子阀联用，作为高效、便捷肽段预分离和收集系统的可行性。研究结果显示，将UPLC的高pH反相色谱分馏与在线LC-MS/MS相结合，可以实现高pH和低pH条件下基于肽段不同色谱保留行为的正交互补分离，在蛋白质鉴定方面表现出优越的性能。应用该方法对人肝癌细胞系HepG2进行3次技术重复试验，重复试验间具有非常高的定量重现性（决定系数R²>0.95）。与传统StageTip方法相比，肽段鉴定数提高了23.52%。该分级方法灵活、稳定，能够针对较少的样品开展蛋白质组深度覆盖分析。     

High performance liquid chromatography - Quantitative nuclear magnetic resonance - High performance liquid chromatography for purity measurement of human insulin

Quantitative Nuclear Magnetic Resonance (qNMR) is a reliable quantitative spectroscopic technique, wherein the intensity of a resonance line is directly proportional to the number of resonant nucleus, and the absolute content of the compound can be determined, this means the inorganic stabilizer in the sample would not affect the result of qNMR. High performance liquid chromatography (HPLC) is a common analytical method with a high separation capacity. This study combined HPLC and qNMR, to measure the purity of Human Insulin (HI). It started from an original HI. The first step is purifying the original HI by HPLC to get a purified HI, with organic purity of 99.78%. The second step is assessing the absolute content of the purified HI by qNMR, and got 40.25%. The third step is measuring the purity of original HI by HPLC again, using the purified HI as the reference material. This method, called HPLC-qNMR-HPLC, is more accurate (84.12%?±?1.14%) than the traditional IDMS (isotope dilution mass spectrometry) method (86.6%?±?3.4%). This study expanded the application of qNMR to proteins with molecular weight of about 5800, and showed that this method can be widely used in measuring the purity of macromolecular proteins.     

应用亲水作用色谱检测鸡蛋和肉中三聚氰胺及三聚氰酸二酰胺

应用亲水作用色谱(HILIC)对从市场上购买的鸡蛋和肉中的三聚氰胺及三聚氰胺降解产物三聚氰酸二酰胺进行了检测。采用的色谱柱为ZIC-HILIC柱,流动相为3 mmol/L磷酸二氢铵溶液(pH 6.9)-乙腈(20:80, v/v),流速为0.8 mL/min,检测波长为220 nm。在该体系下,三聚氰胺和三聚氰酸二酰胺的保留时间适中,与样品中的内源性物质有良好的分离。样品经0.1%磷酸提取,偏磷酸及乙腈沉淀蛋白质和糖类物质,以及P-SCX固相萃取柱净化。三聚氰胺和三聚氰酸二酰胺在0.4～40 mg/L范围内与峰面积呈良好的线性关系,样品定量限(按信噪比(S/N)不小于10计)为2 mg/kg,在2～10 mg/kg添加水平下的平均回收率为80%～105%,相对标准偏差小于10%。该方法具有良好的分离选择性,可用于鸡蛋和肉中三聚氰胺和三聚氰酸二酰胺的同时检测。     

多功能柱净化-高效液相色谱法检测谷物中的玉米赤霉烯酮

建立了玉米和小麦中玉米赤霉烯酮（ZEN）的多功能柱净化-高效液相色谱检测方法。样品经乙腈-水混合溶剂（V（乙腈）：V（水）=84：16）提取,通过多功能净化柱（MFC）进行一次性净化,以Symmetry^R C18柱为分离柱,甲醇-水（V（甲醇）：V（水）=68：32）为流动相进行高效液相色谱分离和检测。玉米赤霉烯酮的质量浓度在0．01～4．0μg／mL范围内呈良好线性,相关系数为0．9996。检出限为0．04μg／g,在0、04—5．0mg／kg添加范围内的回收率为87．5％～98．6％,相对标准偏差为1．5％～8．3％。     

高效液相色谱法测定环氧树脂粘接剂中的双酚A

建立了进出口建筑用环氧树脂粘接剂中双酚A的高效液相色谱-荧光检测法。对提取方法和色谱条件进行了研究,结果表明采用乙醇超声提取样品15min可获得良好的提取效果。以乙腈和水为流动相,采用梯度洗脱可消除样品的基质干扰。方法对环氧树脂粘接剂中双酚A的定量限(LOQ)为0.5 mg/kg,在2～4000 ng/mL范围内,线性相关系数为0.9996。在5个添加水平下回收率在89.1%～100.8%之间。     

Hollow fiber-based liquid phase microextraction combined with high-performance liquid chromatography for the analysis of gabapentin in biological samples

Three-phase hollow fiber microextraction technique combined with high performance liquid chromatography-ultra violet (HPLC-UV) was applied for the extraction and determination of gabapentin in biological fluids. Gabapentin (GBP) was derivatized with 1-fluoro-2,4-dinitrobenzene, as a UV absorbent agent in borate buffer (pH 8.2) before extraction. The derivative product of GBP was extracted from the 8.5 mL of acidic solution (source phase) into an organic phase (dihexyl ether) impregnated in the pores of a hollow fiber and finally back-extracted into 24 μL of the basic solution (pH 9.1) located inside the lumen of the hollow fiber (receiving phase). The extraction took place due to pH gradient between the inside and outside of the hollow fiber membrane. In order to achieve maximum extraction efficiency, different parameters affecting the extraction conditions were optimized. Under the optimized conditions, preconcentration factor of 95 and detection limit (LOD) of 0.2 μg L⁻¹ were obtained. The calibration graph was linear within the range of 0.6-5000 μg L⁻¹. Finally, the feasibility of the proposed method was successfully confirmed by extraction and determination of GBP in human urine and plasma samples in the range of microgram per liter and suitable results were obtained (RSDs < 6.3%).     

Comprehensive sample analysis using high performance liquid chromatography with multi-detection

Herein we assess the separation space offered by a liquid chromatography system with an optimised uni-dimensional separation for the determination of the key chemical entities in the highly complex matrix of a tobacco leaf extract. Multiple modes of detection, including UV–visible absorbance, chemiluminescence (acidic potassium permanganate, manganese(IV), and tris(2,2′-bipyridine)ruthenium(III)), mass spectrometry and DPPH radical scavenging were used in an attempt to systematically reduce the data complexity of the sample whilst obtaining a greater degree of molecule-specific information. A large amount of chemical data was obtained, but several limitations in the ability to assign detector responses to particular compounds, even with the aid of complementary detection systems, were observed. Thirty-three compounds were detected via MS on the tobacco extract and 12 out of 32 compounds gave a peak height ratio (PHR) greater than 0.33 on one or more detectors. This paper serves as a case study of these limitations, illustrating why multidimensional chromatography is an important consideration when developing a comprehensive chemical detection system.     

离子液体作高效液相色谱流动相添加剂分离测定芳香胺

建立了以离子液体作反相高效液相色谱流动相添加剂分离测定邻苯二胺、苯胺和对甲苯胺3种芳香胺的方法。实验以C18反相色谱柱为分离柱,采用紫外检测方法,考察了检测波长、甲醇含量、咪唑离子液体烷基链长度、离子液体溶液浓度等条件对分离和测定的影响,并与其它分离测定芳香胺的方法进行了比较。优化的色谱条件为:以甲醇/1-丁基-3-甲基咪唑四氟硼酸盐水溶液(3.0mmol/L,乙酸调节pH 3.5)=30/70(V/V)为流动相;检测波长254 nm;流速1.0mL/min;柱温30℃。在此条件下,3种芳香胺达到基线分离,在6.5 min之内分离完全;在1～40 mg/L范围内,线性回归方程的相关系数达到0.99以上;检出限为0.07～0.41 mg/L。将本方法应用于废水的测定,加标回收率在92.3%～96.7%之间,相对标准偏差小于3.5%。     

液相色谱梯度洗脱中的谱带压缩效应

谱带压缩效应是梯度洗脱区别于等度洗脱的重要特征。经典的范德姆特(van Deemter)理论塔板高度方程基于等度洗脱推导得到,因此不能对谱带压缩效应进行描述。在梯度洗脱中,保留因子(k)会随流动相组成(φ)的改变而发生变化,这就使得对梯度洗脱机理的研究要比等度洗脱复杂许多。该文对近10年来谱带压缩效应的研究进展,特别是溶剂强度模型(即描述ln k与φ关系的数学表达式)的非线性特征对谱带压缩因子(G)的影响进行了述评,指出为了更好地认识谱带压缩效应需要将这种非线性因素考虑在内。     

Light emitting diode induced chemiluminescence and its application as a detector for high performance liquid chromatography

Some categories of compounds, including quinones, coumarins, flavins, and xanthene dyes, were found to produce strong chemiluminescence (CL) signals with luminol in sample solution under the irradiation of light emitting diodes (LED) with proper wavelengths. Based on this phenomenon, a compact photochemical reactor was constructed to develop a novel LED induced CL detector for high performance liquid chromatography (HPLC). The effects of related parameters including LED wavelength, luminol concentration, flow rate, pH, and eluents of HPLC were investigated in detail. Under the optimized conditions, the limits of detections (LODs) were in the range of 0.2–80 ng mL⁻¹. The applications and accuracy of the proposed method were validated by analyzing food samples such as milk powder, beer, candy and beverage with satisfactory results.     

液相色谱塔板高度方程的统一形式

研究了色谱分离过程中物质的径向扩散和流动相发热对柱效能的影响。从热传导方程出发,运用色谱过程动力学原理推导了包括考虑流动相径向扩散、色谱柱发热影响的液相色谱塔板高度方程：

该方程概括了高效液相色谱（HPLC）、超高效液相色谱（UPLC）、毛细管电色谱（CEC）和消滞留层液相色谱（ESFLC）塔板高度与各种因素的关系。方程最后一项代表了径向扩散和柱发热对塔板高度的贡献。当流动相线速度较低且柱内径较细时,流动相摩擦生热和径向扩散对塔板高度的贡献趋近于零,塔板高度方程还原成Horvath和Lin的方程;当流动相线速度较高时,由于流动相摩擦生热,柱轴心与边缘温差增加,导致流动相线速度径向分布差异,使得柱效率降低。柱轴心与边缘的温差与流动相线速度平方成正比。该文指出,在流动相高线速度情况下,液相色谱的柱效率与柱内径密切相关,采用细内径柱有利于实现高速与高效率;过高的流动相线速度将导致色谱柱效率崩溃。