Cation-exchange liquid chromatography of choline and acetylcholine on free shielded silanols of silica-based reversed-phase stationary phases.

Free anionic functions present on the surface of reversed-phase packing materials were used for the selective cation-exchange preconcentration and separation of the neurotransmitters choline and acetylcholine from a biological matrix. The cation-exchange behaviour of different reversed-phase packing materials in the neat aqueous mobile phase, the properties of an end-capped column, the dependence of capacity factors and peak shape on the concentration of counter ions, ionic strength, pH and the addition of acetonitrile and optimum conditions for enzymatic conversion of solutes to hydrogen peroxide were studied. The studied reversed-phase columns exhibit better pH stability and longer lifetimes than normal silica-based cation exchangers. Acetylcholine is an effective and sensitive test sample for the measurement of adsorption on silica support. A large sample volume was injected onto a precolumn inserted instead of an injection valve and after injection the solutes were focused and separated on an analytical column with a mobile phase containing tetramethylammonium perchlorate as the counter ion.     

Reversed‐phase ion‐pair solid‐phase extraction and ion chromatography analysis of pyrrolidinium ionic liquid cations in environmental water samples

A method of reversed‐phase ion‐pair solid‐phase extraction combined with ion chromatography for determination of pyrrolidinium ionic liquid cations (N‐methyl‐N‐ethyl pyrrolidinium, N‐methyl‐N‐propyl pyrrolidinium, and N‐methyl‐N‐butyl pyrrolidinium) in water samples was developed in this study. First, ion‐pair reagent sodium heptanesulfonate was added to the water samples after static, centrifugation and filteration. Then, pyrrolidinium cations in the samples were enriched and purified by a reversed‐phase solid‐phase extraction column, and eluted from the column with methanol aqueous solution as eluent. Finally, the eluate collected was analyzed by ion chromatography. The separation and direct conductivity detection of these pyrrolidinium cations by ion‐exchange column using 1.0 mM methanesulfonic acid (in water)/acetonitrile (97:3, v:v) as mobile phase was achieved within 10 min. By using this method, pyrrolidinium cations in Songhua River and Hulan River were successfully extracted with the recoveries ranging from 74.2 to 97.1% and the enrichment factor assessed as 60. Pyrrolidinium cations with the concentration of 0.001?0.03 mg/L can be enriched and detected in the water samples. The developed method for the determination of pyrrolidinium ionic liquid cations in water samples is simple and reliable, which provides a reference for the study of the potential impact of ionic liquids on the environment.     

Distribution ratio, distribution constant and partition coefficient. Countercurrent chromatography retention of benzoic acid

There is some confusion in chromatography between terms such as solute distribution ratio, distribution constant and partition coefficient. These terms are very precisely defined in the field of liquid-liquid systems and liquid-liquid extraction as well as in the field of chromatography with sometimes conflicting definitions. Countercurrent chromatography (CCC) is a chromatographic technique in which the stationary phase is a support-free liquid. Since the mobile phase is also liquid, biphasic liquid systems are used. This work focuses on the exact meaning of the terms since there are consequences on experimental results. The retention volumes of solutes in CCC are linearly related to their distribution ratios. The partition coefficient that should be termed (IUPAC recommendation) distribution constant is linked to a single definite species. Using benzoic acid that can dimerize in heptane and ionize in aqueous phase and an 18 mL hydrodynamic CCC column, the role and relationships between parameters and the consequences on experimental peak position and shape are discussed. If the heptane/water distribution constant (marginally accepted to be called partition coefficient) of benzoic acid is 0.2 at 20 °C and can be tabulated in books, its CCC measured distribution ratio or distribution coefficient can change between zero (basic aqueous mobile phase) and more than 25 (acidic aqueous mobile phase and elevated concentration). Benzoic acid distribution ratio and partition coefficient coincide only when both dimerization and ionization are quenched, i.e. at very low concentration and pH 2. It is possible to quench dimerization adding butanol in the heptane/water system. However, butanol additions also affect the partition coefficient of benzoic acid greatly by increasing it.     

Pilot‐scale ion‐exchange centrifugal partition chromatography: Purification of sinalbin from white mustard seeds

The purification of p‐hydroxybenzylglucosinolate (sinalbin) on a multigram scale from a crude aqueous extract of white mustard seeds (Sinapis alba var. concerta) was successfully achieved by scaling up a strong ion‐exchange centrifugal partition chromatography (SIXCPC) laboratory procedure. Thus, the one‐step sinalbin purification was performed with 2.35 g of crude extract in ?170 min (830 mg/h) up to 70.3 g in ?160 min (26.3 g/h) by switching from a 200 mL laboratory scale column to a 5.7 L pilot‐scale column. The required biphasic solvent system contained ethyl acetate, n‐butanol, and water in 3:2:5 v/v/v proportions, Aliquat 336® (trioctylmethyl ammonium chloride) was added to the organic stationary phase (80 mM) and acted as ion‐exchanger. Potassium iodide in the aqueous mobile phase (80 mM) was used as sinalbin displacer. The 28.5 mass scale factor arose from the increase in mobile phase flow‐rate (from 2 to 50 mL/min), from the higher mass of injected white mustard seed extract (from 12 to 350 g), and from the calculated productivity (from 830 mg to 26.3 g). These results demonstrate that industry scale production of glucosinolates is easily performed by SIXCPC, thus providing pure reference standards for pharmacology studies.     

Enthalpic Partition-Assisted Size Exclusion Chromatography: 1. Principle of Method

A novel high performance liquid chromatographic (HPLC) method viz. “enthalpic partition assisted size exclusion chromatography” deliberately combines entropic and enthalpic partition mechanisms. It enables separation of homopolymers according to their molar mass with increased selectivity, as well as discrimination of polymer species differing in their nature/composition. Enthalpic partition of macromolecules takes place between the mobile phase and the stationary “liquid” of a different chemical nature, which is immobilized within pores of an appropriate carrier (a bonded phase). The extent of enthalpic partition depends on the accessibility of bonded phase for macromolecules and on the difference of polymer solubility in the mobile phase and in the solvated bonded phase. The enthalpic partition in favor of column packing arises from better solubility of polymer solutes in the solvated stationary phase compared to the mobile phase. Macromolecules are “pushed” into the solvated stationary phase and their retention volumes (V_R) increase. In the area of high molar masses, the extent of enthalpic partition as rule raises with the increasing size of macromolecules. However, under properly chosen experimental conditions the enthalpic partition may rapidly diminish with the sample molar mass (M), likely due to the solubility changes and/or due to partial exclusion of macromolecules from the pores. As result, the corresponding retention volumes sharply drop within a narrow range of M with the increasing size of macromolecules. This results in the log M vs. V_R dependences, which resemble in their form that for size exclusion chromatography but are much more flat indicating highly selective separations of homopolymers according to their molar masses. In this way, enthalpic partition “assists” entropic partition (size exclusion). Polymer species, which do not undergo enthalpic partition, elute from the HPLC column in the conventional size exclusion mode and can be discriminated from the partitioning species. Enthalpic partition assisted size exclusion chromatography can be utilized in separation and characterization of various homopolymers, and polymer blends.     

稠环芳烃在烷基膦酸改性锆镁复合氧化物固定相上的反相液相色谱保留行为

 以稠环芳烃为探针,考察了烷基膦酸改性锆镁复合氧化物材料的反相色谱性能。研究了稠环芳烃类化合物的结构与其保留值的关系,比较了烷基膦酸改性锆镁复合氧化物固定相和十八烷基键合硅胶ZorbaxODS对稠环芳烃异构体的选择性,并对可能的保留机理进行了讨论。以甲醇-水(体积比为75∶25)为流动相,在烷基膦酸改性锆镁复合氧化物固定相上分离了8种稠环芳烃类化合物。     

High-performance liquid chromatography on silica modified with temperature-responsive polymers

Summary The separation selectivity of temperature-responsive poly(N-isopropylacrylamide)-modified silica as a packing material for high performance liquid chromatography was investigated with steroids, alkaloids, and substituted anilines as solutes. The elution profiles of the solutes depended on the temperature of the column and the methanol content of the mobile phase, indicating that the separation selectivity could be controlled by the column temperature or the mobile phase composition.     

稠环芳烃在烷基膦酸改性锆镁复合氧化物固定相上的反相液相色谱保留行为

以稠环芳烃为探针 ,考察了烷基膦酸改性锆镁复合氧化物材料的反相色谱性能。研究了稠环芳烃类化合物的结构与其保留值的关系 ,比较了烷基膦酸改性锆镁复合氧化物固定相和十八烷基键合硅胶 Zorbax ODS对稠环芳烃异构体的选择性 ,并对可能的保留机理进行了讨论。以甲醇 -水 (体积比为 75∶ 2 5)为流动相 ,在烷基膦酸改性锆镁复合氧化物固定相上分离了 8种稠环芳烃类化合物     

利用后凝胶化反应制备低吸附性反相填料

在硅胶基质的反相填料 (如 C18、C8、C4 等 )上 ,蛋白质的变性 ,碱性分子峰展宽、拖尾甚至完全吸附等常被归因为硅羟基产生的次级效应 (Secondary Effect) [1～ 3] .我们认为 ,除填料的孔径效应外 [4 ] ,杂质离子与残余硅羟基的协同效应是导致此类问题的直接原因 ,如金属离子直接作用于溶质以及金属离子和硅羟基的远程作用力导致的溶质变性等 .为解决这一问题 ,可采用高反应性的新硅烷化试剂 ,以尽可能将硅羟基完全屏蔽 [5] ,或以封端试剂消除残余的硅羟基 .但是 ,用硅烷化试剂封端 (End-capping)在多数情况下并不能完全覆盖表面硅羟基 ,…     

单柱离子色谱法测定一价阳离子的流动相研究

对单柱离子色谱法测定一价阳离子的流动相进行了系统研究，阐述了一价阳离子的保留行为和电导检测行为与流动相之间的关系，分别对无机酸（硝酸）、有机酸（柠檬酸）和芳香碱（苯胺）为流动相测定一价阳离子进行了讨论，其中有机酸和无机酸是较为适宜的流动相。     

Determination of Uric Acid in Human Urine by Ion‐exclusion Chromatography with UV Detection Using Pure Water as Mobile Phase

A simple, rapid and accurate ion‐exclusion chromatographic method coupled with a UV detector for the determination of uric acid in human urine samples has been developed. The separation was carried out on an ion‐exclusion column using only pure water as mobile phase. The detection wavelength was 254 nm and urine sample was injected directly without any pretreatment. Furthermore, the retention behavior of uric acid on the ion‐exclusion column was researched when pure water and 1 mmol·L^?1 HCl were used as mobile phase, respectively. The stability of uric acid was also further investigated within 28 days. In this method, the linear range of the calibration curve for uric acid was 0.25–100 mg·L^?1, and the detection limit calculated at S/N=3 was 0.02 mg·L^?1. The proposed ion‐exclusion chromatographic method has been used for the determination of uric acid in human urine.     

The Nature of Aqueous Solutions: Insights into Multiple Facets of Chemistry and Biochemistry from Freezing‐Point Depressions

Contrary to current widely held beliefs, many concentrated aqueous solutions of electrolytes and nonelectrolytes behave ideally. For both, the same simple equation yields mole fractions of water that are equal to the theoretical activities of water. No empirical activity coefficients or ad hoc parameters are needed. Thermodynamic hydration numbers and the number of particles produced per mole of solute are found by searching freezing‐point depression measurements, as if asking the water, “How much available water solvent is left and how many solute particles are there?” The results answer questions currently under debate: Do solutes alter the nature of water outside their immediate surroundings? What is the number of ion pairs formed by various electrolytes and what affects extents of their formation? What are some factors that cause precipitation of proteins, latexes, and so forth from aqueous solutions upon addition of other solutes (Hofmeister series)? Which nonelectrolytes form aggregates in water and what are the implications? Why do different solutes affect viscosity differently? How do ion‐selective channels in cell membranes function at the molecular level?     

HPLC retention behavior on hydride-based stationary phases

Two stationary phases attached to a silica hydride surface, cholesterol and bidentate C18, are investigated with a number of pharmaceutically related compounds in order to illustrate the various retention mechanisms that are possible for these bonded materials. The test solutes range from hydrophilic to hydrophobic based on log P (octanol/water partition coefficient) and pKa values. The mobile phases consist of acidified (formic and perchloric acid) water/methanol or water/ACN mixtures. Of particular interest are the high organic content mobile phase compositions where the retention would increase if the bonded material was operating in the aqueous normal phase (ANP) mode. Plots of retention factor (k) versus mobile phase composition are used to elucidate the retention mechanism. A number of examples are presented where solutes are retained based on RP, ANP, or dual retention mechanisms. The silica hydride-based stationary phases can also retain compounds in the organic normal phase.     

Method development for separating organic carbonates by ion‐moderated high‐performance liquid chromatography

An ion‐moderated partition high‐performance liquid chromatography method was developed for the separation and identification of common organic carbonates. The separation of organic carbonates was achieved on an ion exclusion column with an exchangeable hydrogen ion. An isocratic, aqueous mobile phase was used for elution and detection was performed with a refractive index detector. The developed method was validated for specificity, linearity, limits of detection and quantification, precision and accuracy. All calibration curves showed excellent linear regression (R² > 0.9990) within the testing range. The limits of detection were 3.8–30.8 ppm for the analyzed carbonates. Improvements in the peak resolution of the chromatograms were achieved by decreasing the column temperature. Addition of the organic modifier, acetonitrile, to the eluent was found to have insignificant effects on the peak resolution. The developed method was demonstrated for analyzing organic carbonate components in the electrolyte system of a commercial lithium ion battery.     

阴离子型胶束液相色谱的溶质保留行为

以SDS阴离子表面活性剂作流动相,酸性、中性及两性药物为受试药物,运用三相平衡理论考察影响阴离子型胶束液相色谱（AMLC）溶质保留行为的几个因素。保留由溶质与胶束相及修饰后固定相的综合作用决定。有机调节剂正丙醇的加入改变了溶质从水相到固定相或到胶束相的平衡,保留取决于溶质疏水性和静电性间的平衡。此外对羟基苯甲酸酯类同系物的亲脂性与3种细菌最小抑菌浓度具有显著相关性,提示其抑菌机理主要取决于药物与生物膜的亲和性。     

Evaluation and application of liquid chromatographic columns coated with 'intelligent' ligands. II. Phospholipid column

The stationary phases of octadecylsilica (ODS) coated with phospholipid have been developed as a model of artificial lipid membranes for liquid chromatographic columns. An ODS column coated with phospholipid can be readily prepared by recycling a solution containing L-alpha-dipalmitoyl-phosphatidylcholine (DPPC) through an ODS column in a closed loop. DPPC becomes absorbed on the ODS surfaces by hydrophobic interaction between the acyl group of DPPC and the octadecyl group of the ODS surfaces. The DPPC column was usable when a mobile phase containing 30% (v/v) acetonitrile was delivered without detachment of the DPPC from the ODS surfaces. The retention behavior of ionic solutes on the DPPC column suggested that the retention was based on both ionic and electrostatic interactions between the solutes and the stationary phase. The retention factors on the DPPC column correlated well with the partition coefficients in liposome systems for alpha-adrenoceptor agonists and beta-blockers, indicating that the partition of solutes between the coated phase and buffer was similar to that in the liposome/water system. The DPPC column can be used in screening studies to predict the binding properties of drugs onto lipid membranes.     

基于反相高效液相色谱法构建 QSRR 模型测定萘类及蒽醌类化合物的正辛醇-水分配系数

正辛醇-水分配系数(Kow )是评价药物毒性、活性及跨膜转运等的重要参数,但直接测定法实验过程复杂。本研究采用反相高效液相色谱(RP-HPLC)法,以甲醇-水为流动相,以29种已知 Kow的酸性和中性苯系物及萘类、醌类衍生物为模型化合物,以保留时间两点校正法( DP-RTC)校正保留时间,并由 Snyder-Soczewinski方程得100%水相保留因子(kw ),建立了表观正辛醇-水分配系数 Kow＂与 kw 的定量关系(Quantita-tive structure-retention relationship, QSRR)模型,并对模型进行了内、外部验证。结果表明,不同 pH 下的 QSRR模型线性相关性 R2=0.974~0.976,内部验证( R2cv =0.970~0.973)和外部验证结果(6种验证化合物,1.4%≤相对误差(RE)≤7.9%)令人满意,与考虑了分子结构参数后建立的线性溶剂化能模型(LSER)相比无差异。将建立的 QSRR 模型应用于11种萘类和蒽醌类化合物的 Kow测定,并与软件计算值、摇瓶法实验值比较,结果表明,本方法准确性更高,且简单快捷,可用于快速准确预测复杂混合物体系中组分的 Kow。     

Multisyringe chromatography (MSC) using a monolithic column for the determination of sulphonated azo dyes

A methodology based on multisyringe chromatography with a monolithic column was developed to determine three sulphonated azo textile dyes: Acid Yellow 23, Acid Yellow 9 and Acid Red 97. An ion pair reagent was needed because of the low affinity between the monolithic column and the anionic dyes. The proposed analytical system is simple, versatile and low-cost and has great flexibility in manifold configuration.The method was optimized through experimentation based on experimental design methodology. For this purpose two blocks of full factorial 2³ were done sequentially. In the first experimental plan, the factors studied were: the % of acetonitrile in organic phase, the % of H₂O in the mobile phase and the kind of ion pair reagent. In this stage, a simple configuration was used which has only one syringe for the mobile phase.After the first experimentation, we added a second syringe with a second mobile phase to the multisyringe module and performed a second full factorial 2³. The factors studied in this case were: the % of acetonitrile in the second mobile phase, the pH and the concentration of ion pair reagent in both mobile phases. After this design, the optimal conditions were selected for obtaining a good resolution between the peaks of yellow dyes (1.47) and the elution of red dye in less than 8 min.The methodology was validated by spiking different amounts of each dye in real water samples, specifically, tap water, well water and water from a biological wastewater lagoon.     

有机酸和有机酸盐的静电离子色谱研究

 以涂敷了两性表面活性剂胆汁酸盐的十八烷基键合硅胶为固定相 ,以去离子水为流动相 ,选用电导检测器 ,研究了有机酸及其盐的同时分离 ;探讨了草酸钠、柠檬酸、苯甲酸钠溶液的色谱分离条件及分离机制 ,并应用于饮料中的苯甲酸钠和柠檬酸的定量测定。实验表明 ,这种以纯水为流动相的色谱过程 ,不仅具有快速简单、检测背景干扰小、重现性好等特点 ,而且有效地克服了二次环境污染 ,是一种对环境友好的分析方法。     

Ion-pair chromatography of acidic drug metabolites and endogenic compounds.

Liquid-liquid chromatographic systems based on ion-pair partition with silica microparticles as the support for the stationary phase have been used for the separation of anionic compounds of biochemical and pharmacological interest. A high separating efficiency can be obtained with both aqueous and organic mobile phases and the retention is easily regulated by the nature and the concentration of the quaternary ammonium counter ion, present in the aqueous phase. The influence of the composition of the liquid phases on the selectivity and separating efficiency has been studied, as well as equilibration methods and the stability of the systems. Examples are given of separations of sulphonamides, barbiturates, glucuronic and sulphuric acid conjugates of steroidal compounds and phenols glycine conjugates of carboxylic acids (hippuric, nicotinuric and salicyluric acid) and anionic metabolites of biogenic amines (indoleacetic, benzoic, mandelic and phenylacetic acid derivatives).