Retention mechanism of fatty alcohol ethoxylates in reversed-phase liquid chromatography

Fatty alcohol ethoxylates (FAEs) are widely used nonionic surfactants that have distributions in both alkyl and poly(ethylene oxide) (PEO) chain length. Generally, two-dimensional liquid chromatography technique is required for the complete characterization of both distributions. By selecting a proper stationary and mobile phase condition, however, we can obtain fully resolved chromatograms of a FAE sample (Brij 30) with respect to both alkyl and PEO chain length by using a single reversed-phase C18 column and aqueous acetonitrile mobile phase. FAEs show a peculiar reversed-phase liquid chromatography (RPLC) retention behavior with an aqueous-organic mobile phase, the retention mechanism of which has not been fully elucidated. For a fixed alkyl chain length, FAEs with higher-molecular-mass PEO block elutes first and the van't Hoff plot of the retention factor shows a curvature. The unique retention behavior can be understood from the opposite thermodynamic characteristics associated with RPLC retention of PEO block and alkyl chain: the sorption process of PEO to the non-polar stationary phase shows deltaH(o) > 0 and deltaS(o) > 0 while the alkyl chain shows deltaH(o) < 0 and deltaS(o) < 0 in contrast. The relative magnitude of the two contributions can change the elution order of the FAE. Therefore the often found, inverted elution order of FAEs (the early elution of FAEs with longer PEO block) is due to the positive enthalpic interaction of PEO blocks, which is a characteristic of the hydrophobic interaction. And the curvature of the van't Hoff plots was analyzed assuming the temperature dependent thermodynamic variables.     

硅胶色谱柱的亲水作用保留机理及其影响因素

亲水作用色谱（HILIC）是替代反相色谱（RPLC）分离强极性及亲水性化合物的另一色谱模式,其分离机理与RPLC有很大不同,具有和RPLC互补的选择性。在HILIC模式中,采用正相色谱（NPLC）中的极性固定相及含高浓度有机溶剂（通常为乙腈）的水溶液为流动相。硅胶是开发最早、研究最为深入及应用最为广泛的HILIC固定相,本文介绍了硅胶色谱柱的HILIC保留机理,详细概述了操作条件如硅胶柱类型、流动相组成及柱温对HILIC分离的影响,并对硅胶填料色谱柱的HILIC模式的发展方向与应用前景进行了展望。     

2-芳基丙酸类手性药物色谱拆分的热力学研究

以0.5%和1.0%(体积分数)正丙醇-50 mmol/L磷酸盐缓冲液(pH 6.41)为流动相,温度74~313 K,在Chiral-AGP柱手性固定相上,考察了萘普生和布洛芬对映体在手性柱上的保留和分离行为。在实验范围内,温度升高对分离不利,随着温度的升高,对映体的保留时间、分离度和选择性因子都减少;萘普生对映体的分离度均比布洛芬大;流动相含1.0％正丙醇时,萘普生对映体和布洛芬对映体达到完全分离应控制的最高温度分别为298和288 K。用ln k对1/T作图得到的Van’t Hoff曲线都具有良好的     

Effects of temperature on retention of chiral compounds on a ristocetin A chiral stationary phase

The isocratic retention of enantiomers of chiral analytes, i.e. tryptophan, 1,2,3,4-tetrahydroisoquinoline and gamma-butyrolac tone analogs, was studied on a ristocetin A chiral stationary phase at different temperatures and with different mobile phase compositions, using the reversed-phase, polar-organic and normal-phase modes. By variation of the both mobile phase composition and the temperature, baseline separations could be achieved for these enantiomers. The retention factors and selectivity factors for the enantiomers of all investigated compounds decreased with increasing temperature. The natural logarithms of the retention factors (ln k) of the investigated compounds depended linearly on the inverse of temperature (1/T). van't Hoff plots afforded thermodynamic parameters, such as the apparent change in enthalpy (deltaH(o)), the apparent change in entropy (deltaS(o)) and the apparent change in Gibbs free energy (deltaG(o) ) for the transfer of analyte from the mobile to the stationary phase. The thermodynamic parameters (deltaH(o), deltaS(o) and deltaG(o)) were calculated in order to promote an understanding of the thermodynamic driving forces for retention in this chromatographic system.     

Cellulose dimethylphenylcarbamate-bonded carbon-clad zirconia for chiral separation in high performance liquid chromatography.

Porous zirconia particles are very robust material and have received considerable attention as a stationary phase support for HPLC. We prepared cellulose dimethylphenylcarbamate-bonded carbon-clad zirconia (CDMPCCZ) as a chiral stationary phase (CSP) for separation of enantiomers of a set of 14 racemic compounds in normal phase (NP) and reversed-phase (RP) liquid chromatography. Retention and enantioselectivity on CDMPCCZ were compared to those on CDMPC-coated zirconia (CDMPCZ) to see how the change in immobilization method of the chiral selector affects the retention and chiral selectivity. In NPLC, retention was longer and the number of resolved racemates was smaller on CDMPCCZ than on CDMPCZ. However, chiral selectivity factors for some resolved racemates were better on CDMPCCZ than on CDMPCZ. The longer retention on CDMPCCZ is likely due to strong, non-chiral discriminating interactions with the carbon layer on CDMPCZ. In RPLC only two racemates were resolved on CDMPCCZ, but retention times were shorter than, and resolutions were comparable to, those in NPLC, indicating a potential for improving chromatographic performance of the CDMPCCZ column in RPLC with optimized column preparation and separation conditions.     

Adsorption mechanism in reversed-phase liquid chromatography. Effect of the surface coverage of a monomeric C18-silica stationary phase

The effect of the bonding density of the octadecyl chains onto the same silica on the adsorption and retention properties of low molecular weight compounds (phenol, caffeine, and sodium 2-naphthalene sulfonate) was investigated. The same mobile phase (methanol:water, 20:80, v/v) and temperature (T = 298 K) were applied and two duplicate columns (A and B) from each batch of packing material (neat silica, simply endcapped or C1 phase, 0.42, 1.01, 2.03, and 3.15 micromol/m2 of C18 alkyl chains) were tested. Adsorption data of the three compounds were acquired by frontal analysis (FA) and the adsorption energy distributions (AEDs) were calculated using the expectation-maximization method. Results confirmed earlier findings in linear chromatography of a retention maximum at an intermediate bonding density. From a general point of view, the saturation capacity of the adsorbent tends to decrease with increasing bonding density, due to the vanishing space intercalated between the C18 bonded chains and to the decrease of the specific surface area of the stationary phase. The equilibrium constants are maximum for an intermediary bonding density (approximately 2 micromol/m2). An enthalpy-entropy compensation was found for the thermodynamic parameters of the isotherm data. Weak equilibrium constants (small deltaH) and high saturation capacities (large deltaS) were observed at low bonding densities, higher equilibrium constants and lower saturation capacities at high bonding densities, the combinations leading to similar apparent retention in RPLC. The use of a low surface coverage column is recommended for preparative purposes.     

反相高效液相色谱中溶质保留过程的热力学研究4: 焓熵补偿

依据液相色谱中溶质计量置换保留模型, 对溶质在反相液相色谱(RPLC)保留过程及其吸附、解吸附过程中的焓熵补偿进行了研究, 证实了在RPLC中焓熵补偿确实存在。从焓熵补偿的定义出发, 从理论上证明了溶质在保留过程中的焓熵补偿温度本质上为溶质保留值的收敛温度, 其数值为Z对1/T线性作图的斜率与截距之比。与惯常计算焓熵补偿温度的方法相比, 本文的方法所得补偿温度更为合理且不受流动相中强溶剂浓度变化的影响。     

亲水作用色谱填料的研究进展

亲水作用色谱是一种新型的色谱分离模式.此类色谱模式集反相色谱的经济廉价与正相色谱的优点于一体,有效补充了反相色谱的不足.简单介绍实验室中合成的新型亲水色谱固定相.     

基于聚倍半硅氧烷微球的亲水/反相混合模式色谱填料的制备与评价

亲水/反相混合模式色谱应用广泛,但pH使用范围有限,不利于碱性药物的分离。该工作利用巯基-烯基点击化学合成了单分散多孔的半胱氨酸改性乙烯基功能化聚甲基倍半硅氧烷(C-V-PMSQ)微球。元素分析表明半胱氨酸成功键合在微球表面。C-V-PMSQ微球为介孔结构,单分散性好且具有优良的化学稳定性。以几种常见的核苷和核酸碱基作为测试样品,考察其色谱保留行为,溶质的保留因子随流动相中水相含量的变化呈现典型的U型曲线,表明C-V-PMSQ固定相具有亲水/反相的双重保留特征。使用该固定相可以分离苯的同系物及一系列亲水性与疏水性化合物。另外在高碱性流动相条件下利用亲水和反相模式成功分离了中药苦参中的3种主要活性成分,表明它在分离碱性药物方面具有较大的优势。     

Chiral discrimination of phenoxypropionic acid herbicides on teicoplanin phase: Effect of mobile phase modifier

Summary The chiral recgonition mechanism for a series of phenoxypropionic acid herbicides was investigated in reversed phase liquid chromatography (RPLC) on a teicoplanin stationary phase over a wide range of column temperatures. Thermodynamic constants, of the transfer of an enantiomer from the mobile to the teicoplanin stationary phases were determined. The van't Hoff plots for all solutes had a break at a critical temperature T^* showing a variation in the enantiomer retention mechanism due to a change in the conformational state of the teicoplanin, structure. Additionally, enthalpy-entropy compensation confirmed both the change in enantiomer interaction mechanism observed for regions T<T^* and T>T^* and the independence of this mechanism from (i) herbicide molecular structure,s i.e. the position of the chloro group on the phenol ring and (ii) the carbon absolute configuration. Moreover, the increasing enantioselectivity with increasing methanol fraction in the mobile phase was attributed to restriction of the solute association in the teicoplain, stationary phase, leading to favorable stereoselective interactions. This behavior was used to optimize chromatographic conditions for separation of herbicide enantiomers on teicoplanin.     

Chiral separability of boron cluster species studied by screening approaches utilizing polysaccharide‐based chiral stationary phases

In this study, the screening steps of chiral separation strategies with polysaccharide‐based chiral stationary phases were applied on boron cluster compounds in normal‐phase liquid chromatography (NPLC) and polar organic solvents chromatography (POSC). Since the screening steps were initially developed to analyze organic compounds, their applicability for boron clusters was investigated. Overall, the screening steps in NPLC were applicable for the separation of zwitterions, while for anions mostly no elution was observed. A hypothesis for the latter behavior is precipitation of anions in the nonpolar mobile phases. Ten out of 11 compounds could be partially or baseline separated on the NPLC screening systems. In POSC, all zwitterions were separated on at least one of the screening systems, with an overall lower retention as in NPLC. Anions were detected but not separated in the majority of the experiments. Also their retention on the chiral stationary phases was very limited. This study showed that the chiral discrimination potential of chemically modified polysaccharides is meaningful for chiral separations of structurally chiral boron cluster species, but needs further systematic research, in which recognition mechanisms should be further explored. In addition, some unusual peaks also indicated that conditions with a high separation efficiency must first be searched for some of the tested systems. Copyright © 2014 John Wiley & Sons, Ltd.     

蛋白质在合成阳离子交换剂上的色谱特性研究

用国产材料按间接法合成了螯合型弱阳离子交换剂,详细研究了合成填料的色谱性能,并与商品柱的分离效能进行了比较;在宽温度范围内研究了蛋白质在弱阳离子交换系统中的色谱热力学,测定了蛋白质在色谱过程变性时的热力学参数 (△H0和△S0) 和补偿温度β,提出用标准熵变△S0判断蛋白质的构象变化和用△H0与△S0的补偿关系鉴定蛋白质各变体在色谱系统保留机理的同一性。考察了螯合型弱阳离子交换剂与金属离子的作用,研究了蛋白质在金属螯合色谱中的保留机理。     

正相模式/反相模式的二维液相色谱系统的构建与应用

以4.6mm×50 mm i. d.的Hypersil SiO2正相色谱柱为第一维,4.6mm×250 mm i. d.的Kromasil C18反相色谱柱为第二维,通过升高第二维色谱温度的方法增加两维流动相间互溶性的方法构建了定量环-阀切换接口的二维液相色谱系统(NPLC×RPLC)。根据有机溶剂的特征,在第一维正相色谱流动相中加入二氧六环;第二维反相色谱流动相中加入异丙醇,在改善流动相兼容性的同时,有效调整分离选择性。采用此系统对正天丸样品进行分离分析,达到1120的峰容量。     

Assessment by monte carlo simulation of thermodynamic correlation of retention times in dual-column temperature programmed comprehensive two-dimensional gas chromatography

The correlation of retention times in comprehensive two-dimensional gas chromatography caused by correlation of enthalpy and entropy changes between two stationary phases, methylsilicone and poly(ethylene glycol), was examined using commercial GC software and in-house Monte Carlo simulation. The enthalpy change, deltaH0, and entropy change, deltaS0, of 93 compounds were predicted from isothermal one-dimensional gas chromatograms predicted by the software. These values then were mimicked by Monte Carlo simulation, which removed the strong correlation of deltaH0 and modest correlation of deltaS0 between the two phases. Retention times in a comprehensive two-dimensional gas chromatogram (GC x GC) and in simulations of it were predicted for typical dual-capillary temperature-programmed conditions using the actual, correlated values of deltaH0 and deltaS0 and their uncorrelated Monte Carlo counterparts, respectively. The uncorrelated deltaH0 and deltaS0 values caused the retention-time range of the simulations' second dimension to expand substantially beyond that in the GC x GC. Other simulations were developed using a restricted range of uncorrelated deltaH0 and deltaS0 values to mimic more closely the retention-time range of the GC x GC's second dimension. The intervals between nearest neighbor retention-time coordinates were calculated in both the latter simulations and the GC x GC. The intervals were larger in the simulations than in the GC x GC because the former contained uncorrelated coordinates and the latter contained correlated ones, which clustered along or near the diagonal. The retention times in the first dimension of the GC x GC were Poisson distributed, as assessed by statistical-overlap theory. In contrast, the two-dimensional reduced retention-time coordinates in the GC x GC were not Poisson distributed, because retention times were correlated. However, the reduced retention-time coordinates in the simulations were Poisson distributed.     

Synthesis,chromatographic evaluation and hydrophilic interaction/reversed-phase mixed-mode behavior of a “Click β-cyclodextrin” stationary phase

A native β-cyclodextrin (β-CD) stationary phase was prepared by covalently bonding β-CD on silica particles via Huisgen [3 + 2] dipolar cycloaddition between the organic azide and terminal alkyne, the so-called Click chemistry. The resulting β-CD bonded silica (Click β-CD) was characterized by FT-IR, solid state ¹³C cross polarization/magic-angle spinning (CP/MAS) NMR and elemental analyses, which proved the successful immobilization of β-CD on the silica support with Click chemistry. The retentive properties of Click β-CD were investigated under hydrophilic interaction liquid chromatography (HILIC) mode in different mobile phase conditions with a set of polar compounds including nucleosides, organic acids and alkaloids. The effects of water content, concentration of the salt and pH of the buffer solution on the retention time were studied and the results demonstrated the typical retention behavior of HILIC on Click β-CD. Separation of very polar components, such as nucleosides and oligosaccharides, and chiral separation under HILIC mode were successfully achieved. In addition, Click β-CD was chromatographically evaluated with a set of flavone glycosides. The retention curves depending on the mobile phase of acetonitrile content were “U” curves, which is an indication of HILIC/RPLC mixed-mode retention behavior. The difference of the separation selectivity between HILIC and RPLC was described as orthogonality by using geometric approach and the orthogonality reached 69.4%. The mixed-mode HPLC properties and excellent orthogonality demonstrated the flexibility in HPLC methods development and great potential in two-dimensional liquid chromatography separation.     

新型苯基桥键色谱固定相的热力学性质

将制备的球形苯基桥键型杂化介孔色谱固定相与商品化的C18和苯基键合硅胶固定相对比,研究其热力学性质。以稠环芳烃为例,探讨了溶质在固定相和流动相之间的迁移焓变、迁移熵变等热力学参数的变化。结果表明,与两种商品化的固定相不同,实验制备的苯基桥键固定相不存在明显的焓-熵补偿效应,证实新型桥键固定相分离机理是疏水作用、π-π作用、包结作用等协同作用的结果。     

基于巯基-烯点击化学法的β-环糊精固定相多模式色谱保留行为研究

基于巯基硅胶与单取代-6A-烯丙氨基-β-环糊精的巯基-烯点击化学反应,制备了β-环糊精(Click TE-CD)共价键合固定相。元素分析结果表明β-环糊精被成功键合到硅胶表面。以黄酮苷类化合物为模型,考察了Click TE-CD固定相在亲水、反相和超临界流体色谱等分离模式下的色谱保留行为。黄酮苷类化合物保留时间随流动相中乙腈含量的变化呈现典型的U型曲线,表明Click TE-CD固定相具有亲水/反相的双重保留特性。应用几何学方法测得Click TE-CD固定相在反相/亲水、亲水/超临界、反相/超临界混合模式下的正交性分别为69.8%、50.8%、50.8%。对比复杂中药样品降香提取物在反相、亲水、超临界等模式下的分离情况,结果表明Click TE-CD固定相在分离中药复杂样品方面具有极大潜力,可以在一根色谱柱上通过分离模式的改变,实现二维液相色谱的分离。Click TE-CD固定相不同分离模式的分离性能和较好的正交性表明该固定相具有在液相色谱方法发展和二维液相色谱分离方面应用的潜力。     

糖类化合物在两性离子亲水作用色谱柱上的保留行为评价

糖类化合物因其极性强,在反相色谱模式下保留较弱,因此常用亲水作用色谱(HILIC)对其进行分离分析。本文以9种糖类化合物的混合物为研究对象,系统评价了其在Click TE-Cys亲水色谱柱上的保留行为,分别考察了流动相中有机相比例和盐浓度对其保留行为的影响。实验证明:9种糖类化合物按极性由小到大的顺序依次从Click TE-Cys色谱柱上被洗脱下来。随着有机相比例的增加,糖类化合物的保留增强;随着盐浓度的增加,除唾液酸外的糖类化合物的保留增强。用顶替-吸附液相相互作用模型模拟了糖类化合物在HILIC上的保留行为,采用保留方程ln k=a+blnC_B+cC_B描述HILIC的保留规律,对HILIC的保留值进行多元线性回归。结果表明糖类化合物在Click TE-Cys色谱柱上的保留行为符合HILIC的保留规律。     

Chromatographic selectivity triangles

2010 marked the 50th anniversary of the use of selectivity triangles to characterize chromatographic phases. Such plots ultimately identify and quantify the blend of intermolecular interactions that occur between solutes and solvents/phases. The first chromatographic triangle was proposed by Brown and applied to GC stationary phases. Snyder then developed the influential solvent selectivity triangle (SST) based on the gas-liquid partition data of Rohrschneider. The SST was combined with simplex experimental designs to optimize RPLC separations. Subsequent criticisms of the work revolved around the inaccurate predictions that resulted from the SST. These inaccuracies ultimately relate to the inability of the SST to account for the effects of water on the interaction ability of organic solvents. Other criticisms focused on the selection of the three probe solutes (ethanol, dioxane, and nitromethane) that were used to define the apices of the SST. Here, the concerns include the lack of explicit consideration of dispersion interactions and the fact that the three probes do not represent any single intermolecular interaction but rather reflect a blend of intermolecular interactions. The SST approach was modified for NPLC by redefining the triangle apices to reflect the localization, general adsorption, and basicity of NPLC mobile phase modifiers. Because water is generally absent in NPLC, the triangle approach leads to better predictions for NPLC than for RPLC. In subsequent modifications of selectivity triangles, Fu and Khaledi have created a micellar selectivity triangle (MST) based on linear solvation energy relationships (LSERs) and Zhang and Carr have used the Dolan-Snyder hydrophobic subtraction model to create RPLC column selectivity triangles. We end this review by highlighting more recent methods for comparing selectivities and by discussing a new 3D visualization tool for classifying chromatographic systems as having similar or different fundamental energetics of retention and hence having similar or different selectivities.