Modeling the retention mechanism for high‐performance liquid chromatography with a chiral ligand mobile phase and enantioseparation of mandelic acid derivatives

The chromatographic retention mechanism describing relationship between retention factor and concentration of Cu²⁺(l ‐phenylalanine)₂ using chiral ligand mobile phase was investigated and eight mandelic acid derivatives were enantioseparated by chiral ligand exchange chromatography. The relationship between retention factor and concentration of the Cu²⁺(l ‐phenylalanine)₂ complex was proven to be in conformity with chromatographic retention mechanism in which chiral discrimination occurred both in mobile and stationary phase. Different copper(II) salts, chiral ligands, organic modifier, pH of aqueous phase, and conventional temperature on retention behavior were optimized. Eight racemates were successfully enantioseparated on a common reversed‐phase column with an optimized mobile phase composed of 6 mmol/L of l ‐phenylalanine or N,N‐dimethyl‐l ‐phenylalanine and 3 mmol/Lof copper(II) acetate or copper(II) sulfate aqueous solution and methanol.     

高效液相色谱手性流动相法拆分甲状腺素对映体

运用高效液相色谱手性流动相法（ＨＰＬＣ－ＣＭＰ）对影响甲状腺素对映体（Ｄ－,Ｌ－Ｔ４）分离方法的因素：三乙胺（ＴＥＡ）浓度,流动相ｐＨ值,铜离子（Ｃｕ２＋）浓度,Ｌ－脯氨酸（Ｌ－ｐｒｏ）浓度,柱温以及流动相的流速进行了系统的研究。同时,考察了色谱方法分离Ｔ４对映体的线性关系,精密度和准确度。线性响应范围为０．６～３．２　ｎｍｏｌ　（Ｄ－,Ｌ－Ｔ４）,线性相关系数为ｒＤ－Ｔ４＝０．９９８０,ｒＬ－Ｔ４＝０．９９９０,日内和日间的精密度分别为ＲＳＤ＜２．３％（ｎ＝６）,ＲＳＤ＜３．１５％（ｎ＝５）。结果表明本实验所得的色谱条件较文献报道的优越,分离条件简单,重现性好。ＨＰＬＣ－ＣＭＰ法测定甲状腺素对映体其意义在于该方法可为定量测定药品及人体血液中Ｄ－,Ｌ－Ｔ４两种异构体,为治疗药物监测（ＴＤＭ）和药物不良反应监测（ＡＤＲｓ）提供了依据。     

流动相离子色谱法测定杂多酸中氯与磷酸根离子

探讨了用流动相离子色谱法测定杂多酸中C l-与PO43-的分析条件。通过在淋洗液中加入两性离子3-(N-吗啉)-1-丙磺酸(3-(N-morpholino)propanesu lfon ic ac id,MOPS),调节流动相pH值,找到一种pH 6.98的流动相,同时优化离子对试剂的配比,并加入无机添加剂和有机改进剂,能有效的抑制杂多酸的分解,较精确地测定其中所含阴离子。     

Reduction of silanophilic interactions in liquid chromatography with the use of ionic liquids

A suppression of silanophilic interactions by the selected ionic liquids added to the mobile phase in thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC) is reported. Acetonitrile was used as the eluent, alone or with various concentrations of water and phosphoric buffer pH 3. Selectivity of the normal (NP) and the reversed (RP) stationary phase material was examined using a series of proton-acceptor basic drugs analytes. The ionic liquids studied appeared to significantly affect analyte retention in NP-TLC, RP-TLC and RP-HPLC systems tested. Consequently, the increased separation selectivity was attained. Due to ionic liquid additives to eluent even analytes could be chromatographed, which were not eluted from the silica-based stationary phase materials with 100% of acetonitrile in the mobile phase. Addition of ionic liquid already in very small concentration (0.5%, v/v) could reduce the amount of acetonitrile used during the optimization of basic analytes separations in TLC and HPLC systems. Moreover, the influence of temperature on the separation of basic analytes was demonstrated and considered in practical HPLC method development.     

磷酸根离子在阴离子交换树脂上的保留行为及其机理探讨

首次发现磷酸根离子在阴离子交换柱上以两个色谱峰流出。在研究磷酸根离子的保留行为的基础上,提出了Ｈ２ＰＯ－４在固定相中进一步离解的保留机理,即Ｈ２ＰＯ－４在与阴离子交换树脂交换基进行离子交换的过程中,由于树脂交换基和淋洗离子的电荷相互作用促使一部分Ｈ２ＰＯ－４进行第２级离解。由于Ｈ２ＰＯ－４和ＨＰＯ２－４在阴离子交换树脂上的保留值不同,导致磷酸根离子出现“双峰”。     

Chitosan-coated silica gel as a new support in high-performance liquid chromatography

Summary A new stationary phase was prepared by immobilizing the chitosan, a natural hydrophilic polymer, on microparticolate silica gel. The effect of the polarity, pH and ionic strength of the mobile phase has been studied in order to find optimal conditions for the separation of nucleotides and aminoacids. The influence of the properties of the mobile phase on the retention was examined, allowing to employ the chitosan-coated silica gel both to anion exchange and adsorption, depending on the pH and the polarity of the eluent used.     

Retention behaviour of an homologous series of oligodeoxythymidilic acids using reversed-phase ion-pair chromatography

Summary The retention behaviour of an homologous series of phosphorylated oligodeoxythymidylic acid (pd(T)_5–18) oligonucleotides was studied using reversed-phase ion-pair chromatography with isocratic elution conditions. The effects of temperature, pH, eluent ionic strength, percentage organic modifier, concentration and alkyl chain length of the ion-pairing reagent were investigated. The retention behaviour was generally explicable by current theoretical models of ion-pair chromatography. However, the marked effect of mobile phase pH on the retention of the oligonucleotides was unexpected, and this was ascribed to the presence of ionisable residual silanols on the surface of the reversed-phase packing material.     

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。     

流动相组成、浓度和pH对蛋白质在金属螯合柱上的保留特性的影响

 系统研究了流动相中盐的性质和浓度、溶液 pH以及竞争配体对蛋白质在金属螯合色谱中保留值的影响。导出了描述蛋白质在金属螯合色谱中保留特征的数学表达式 ,提出用洗脱强度指数ε表征盐溶液的洗脱能力。根据不同色谱条件下蛋白质的保留特性 ,发现蛋白质在金属螯合色谱中的保留是配位、静电和疏水的协同作用。对与蛋白质强结合的金属螯合柱 ,以配位作用为主 ,静电作用为辅 ;对弱结合的金属柱 ,以静电作用为主 ,配位作用为辅。在流动相中加入高浓度非成络盐 ,可增强蛋白质和固定相间的疏水作用。     

离子交换与离子排斥色谱柱串联体系中阳离子的多峰现象及其机理

研究了串联柱体系中阳离子的“多峰现象”。在阳离子交换柱后面接上阴离子分析用的离子排斥柱构成一个串联柱体系,当以酒石酸（ＴＡ）和吡啶二羧酸（ＰＤＣ）的混合溶液作淋洗液时,每一种阳离子同时出现３个色谱峰。这是因为从阳离子交换柱流出的阳离子与有络合作用的两种淋洗剂阴离子形成络合物,使流动相中淋洗剂阴离子浓度减少以及两种淋洗剂阴离子在离子排斥柱中被保留且保留值不同。     

Effect of the ionic composition of a mobile phase on the chromatographic retention of profen enantiomers on a chiral adsorbent with grafted eremomycin antibiotic

The effect of ionic composition of mobile phase on retention and chromatographic separation of optical isomers of several derivatives of 2-arylpropanoic acid on a chiral stationary phase Diaspher-Chiralsel-E with a grafted eremomycin antibiotic was studied. As was shown, the mechanism of retention differs from that for hydrophobic reverse-phase adsorbents, as it involves a substantial ion-exchange component that determines the nonmonotonic dependence of the retention factor and the enantioselectivity on eluent pH. The effect of the concentration of buffer salt in the eluent on the chromatography of this class of compounds is discussed.     

Investigation of degradation mechanisms by portable Raman spectroscopy and thermodynamic speciation: the wall painting of Santa María de Lemoniz (Basque Country, North of Spain)

To characterise the polymeric properties of processed lignins, a new method has been developed using hydrophobic interaction chromatography (HIC). This method separates the lignin polymers into fractions based on differences in hydrophobicity using low pressure liquid chromatography (LPLC). The hydrophobic column material consists of monodisperse polystyrene/divinylbenzene beads. An elution gradient was prepared monitoring the electrolyte concentration and pH. Citric acid buffer, containing ammonium sulphate that promotes adsorption to the column material, was used as mobile phase in a step-wise gradient together with ethanol (20/80% (v/v) ethanol/water, pH 12) and isopropanol (40/60% (v/v) isopropanol/water, pH 12). Depending on eluent composition, the degree of elution was 94% or higher. With the HIC method developed, lignosulphonates and kraft lignins were separated into seven distinctive peaks according to hydrophobicity.     

非抑制型离子色谱法测定碱金属、铵离子和烷基胺

 采用非抑制型离子色谱法、以氨磺酸作流动相分离测定了Li+ 、Na+ 、NH+ 4、K+ 、甲胺、乙胺和正丙胺等 7种物质 ,研究了氨磺酸流动相浓度对溶质保留值的影响。结果发现 ,随着流动相浓度的增大 ,溶质保留值减小 ;适宜的流动相浓度为 4 0mmol/L。以 4 0mmol/L氨磺酸为流动相测得上述 7种物质的检出限及工作曲线的线性回归方程。方法应用于自来水试样的分析 ,结果良好。     

Prediction of the chromatographic behaviour for a series of diuretic compounds

Summary The proportion of organic modifier and the pH of the acetonitrile-water mixtures used as mobile phases were optimized in order to separate a group of diuretic compounds covering a wide range of physyco-chemical properties. The Linear Solvation Energy Relationship (LSER) formalism based either on the multiparameter π^*, β and α scales or the single solvent polarity parameterE _T ^N , have been used to predict their chromatographic behaviour as a function of the percentage of acetonitrile in the eluent. Moreover, correlation established between retention and pH of the aqueous-organic mobile phases have been used to predict the chromatographic behaviour of the diuretic compounds studied as a function of the eluent pH. Linear correlation between a function of the eluent pH. Linear correlation between the chromatographic retention and theE _T ^N polarity parameter of mobile phases containing different percentages of organic modifier has been obtained Based on the knowledge of the acid-base dissociation constant the relation between retention and mobile phase pH has also been linearized. These relationship allowed an important reduction of the experimental retention data needed for developing a given separation and a great improvement in chromatographic optimization schemes.     

NAD-硅胶亲和色谱固定相的合成和在核苷酸及其碱基分析中的应用

ＮＡＤ－硅胶亲和色谱固定相的合成和在核苷酸及其碱基分析中的应用于世林,苗凤琴,杨朝霞,冯茹（北京化工大学应用化学系,北京,１０００２９）关键词烟酰胺腺嘌呤二核苷酸（ＮＡＤ）,亲和色谱,核苷酸烟酰胺腺嘌呤二核苷酸（ＮＡＤ,辅酶Ⅰ）是多种脱氢酶的辅酶,通...     

Effect of the eluent pH and acidic modifiers in high-performance liquid chromatography retention of basic analytes

The retention of ionogenic bases in liquid chromatography is strongly dependent upon the pH of the mobile phase. Chromatographic behavior of a series of substituted aniline and pyridine basic compounds has been studied on C18 bonded silica using acetonitrile-water (10:90) as the eluent with different pHs and at various concentrations of the acidic modifier counter anions. The effect of different acidic modifiers on solute retention over a pH range from 1.3 to 8.6 was studied. Ionized basic compounds showed increased retention with a decrease of the mobile phase pH. This increase in retention was attributed to the interaction with counter anions of the acidic modifiers. The increase in retention is dependent on the nature of the counter anion and its concentration in the mobile phase. It was shown that altering the concentration of counter anion of the acidic modifier allows the optimization of the selectivity between basic compounds as well as for neutral and acidic compounds.     

Behavior of peptides and computer-assisted optimization of peptides separations in a normal-phase thin-layer chromatography system with and without the addition of ionic liquid in the eluent

The addition of an ionic liquid into the mobile phase appeared to be useful in optimization of chromatographic separation of peptides. Different behavior of peptides in thin-layer chromatography (TLC) was observed after addition of 1-ethyl-3-methylimidazolium tetra fluoroborate to the eluent in comparison to the system without the ionic liquid. Nonlinear dependence of the retention coefficient, R(M), of peptides on the volume percentage of acetonitrile in the eluent was found in normal-phase TLC with and without immidazolium tetra fluoroborate in the mobile phase. In general, R(M) increased with increasing concentration of acetonitrile. In TLC systems without the ionic liquid, R(M) can be described well with a quadratic function. On the other hand, in a TLC system with an ionic liquid as the additive to the mobile phase, the retention behavior is better described with a third-degree polynomial function. The potential usefulness of ionic liquids for optimization of separation of peptides was demonstrated. Optimization of the separation conditions was supported by a commercially available computer program.     

Anion separations for liquid chromatography using propylpyridinium silica as the stationary phase

This work describes the characterization and potential applications of a silica-based anion-exchange phase prepared by a two-step modification process that incorporates a propylpyridinium group. The effects of pH and eluent concentration on anion separation were examined using 150 mm × 3.9 mm HPLC columns packed with the new phase. The mobile phase pH values ranged from 3.8 to 6.6 using phthalic acid/Tris solutions. The best separation was achieved using 2.5 mmol L⁻¹ phthalate/2.4 mmol L⁻¹ Tris solution at pH 4.2 as mobile phase with non-suppressed conductivity detection. The new stationary phase was used for the separation of some inorganic and organic anions showing good resolution. The stability of the silica-based anion exchange phase was also evaluated.Analytical curves, for concentrations ranging from 0.25 to 10 mg L⁻¹ for the inorganic anions chloride, nitrite, bromide and nitrate, showed good linear correlations (r > 0.998). The method was tested with certified rainwater samples. The measured and certified values were in good agreement, indicating that the new phase holds significant promise for the analysis of these anions in environmental samples.     

Interconversion of gradient and isocratic retention data in reversed-phase liquid chromatography: Effect of the uptake of eluent modifier on the retention of analytes

The experimental technique of mass spectrometric tracer pulse chromatography was used to study the effect of the sorption of eluent components by a C₁₈-bonded silica RPLC packing on the retention of a series of test analytes during isocratic and gradient elution experiments. The analytes of interest were a substituted phenol, a substituted nitroaniline, an anti-malaria drug, tetrahydrofuran, and methanol. The eluent used was a mixture of acetonitrile and water. The solutes and isotopically labeled eluent components were injected at fixed time intervals during each gradient run. The mass specific detector allowed the assignment of individual analyte peaks even when there was overlap in the chromatograms from successive injections. Thus, the retention time of each analyte could be determined as a function of gradient slope and initial eluent composition at the time of each injection. Experimental gradient retention time data were then compared with the calculated results from two theoretical models. The first model assumed the velocity of the mobile phase and eluent were equal. The second and most realistic model assumed the velocity of the eluent was less than the velocity of the mobile phase due to the uptake of eluent by the stationary phase. Gradient retention times predicted by the two models were reasonably accurate with the sorption model giving slightly more accurate values. Inverse calculations, i.e., calculation of isocratic retention factors from gradient elution data were also carried out with very similar results. That is, the model allowing for the uptake of eluent was slightly more accurate than the model assuming no eluent-stationary phase interaction.     

Determination of Amino Acid Enantiomers in Pharmaceuticals by Reversed-Phase High-Performance Liquid Chromatography

Precolumn derivatization with the reagent o-phthalic aldehyde/N-acetyl-L-cysteine (OPA/NAC) was used for the determination of amino acid enantiomers by reversed-phase high-performance liquid chromatography. The influence of the composition and pH of the eluent on the separation of the resulting derivatives was studied with the example of four amino acids. It was found that the highest selectivity and efficiency of the separation of OPA/NAC derivatives of amino acids is attained with the use of the eluent methanol–0.01 M Na₂HPO₄ (pH 6.0). The optimum composition of the mobile phase and conditions of the gradient elution were selected for the separation of a mixture of 20 amino acid derivatives. A procedure was developed for the determination of amino acid enantiomers in parenteral nutrition preparations. The procedure was used for the determination of D-isomers of arginine, alanine, methionine, phenylalanine, and leucine in the preparation Polyamine.