Retention mechanism of peptides on a stationary phase embedded with a quaternary ammonium group: A liquid chromatography study

We investigated the mechanisms involved in the retention of various peptides on a stationary phase embedded with a quaternary ammonium group (BS C23), by high-performance liquid chromatography. This was compared with peptide retention on a conventional reversed-phase C18 (RP C18) column under isocratic conditions, to understand better the various mechanisms involved. Chromatographic characterization of the two stationary phases with “model” compounds showed that BS C23 is less hydrophobic than RP C18 and induces electrostatic interaction (attraction or repulsion) with ionized compounds. If reversed-phase partitioning was the predominant retention phenomenon, for both stationary phases, the retention mechanisms in BS C23 provided different selectivity to that of RP C18. Electrostatic attraction or repulsion was clearly observed between peptides and the permanent positively charged group embedded in BS C23 depending on the pH. For most of the peptides, a weak anion-exchange mechanism was observed on the quaternary ammonium-embedded stationary phase if mobile phases at neutral pH and low ionic strengths were employed.     

高效液相色谱手性固定相法拆分阿折地平对映体

建立了阿折地平对映体的高效液相色谱拆分方法。采用Chiralpak AD-H (250 mm×4.6 mm, 5.0 μm, Daicel公司)手性色谱柱在正相条件下直接拆分阿折地平对映体,考察了固定相种类、流动相组成及柱温等对阿折地平对映体分离的影响。确定了最佳的拆分条件: 流动相为正己烷-异丙醇(90:10, v/v),流速为0.8 mL/min,检测波长为254 nm;柱温为20 ℃;在此条件下阿折地平对映体的分离度为3.3。该法简单快速,重现性好。     

Retention mechanism divergence of a mixed mode stationary phase for high performance liquid chromatography

Mixed mode stationary phases utilize secondary retention mechanisms to add a dimensionality to the surface of high performance liquid chromatography (HPLC) adsorbents. This approach was used by several authors to improve the separation performance of single dimension separations. We explored the magnitude of these secondary interactions by performing an off-line two-dimensional (2D)-HPLC separation with a Scherzo SM-C18 column of a β-lactoglobulin tryptic digest with a mobile phase pH of 7 in the first dimension and 2 in the second. Mechanism divergence was determined using the peak capacity and a geometric approach to factor analysis, to measure the correlation. This separation was repeated with a C18 stationary phase as a control. It was found that the C18 column had a correlation coefficient of 0.784, smaller than the mixed mode column, 0.884. This indicated that the retention mechanisms of the C18 column were more divergent under these two pH environments than the mixed mode column. However, the SM-C18 still provided alternative selectivity of the peptides to that of the C18 and could be considered as a good alternative for further 2D-HPLC separations.     

直链淀粉手性固定相高效液相色谱法拆分比索洛尔对映体

采用直链淀粉手性固定相高效液相色谱法在正相条件下直接拆分了比索洛尔对映异构体。分别以异丙醇、乙醇为有机改性剂,考察了流动相的组成与配比、流速及柱温等因素对比索洛尔对映体分离的影响。确定了比索洛尔对映体的最佳拆分条件:流动相正己烷-乙醇-二乙胺(体积比为88∶12∶0.1),流速0.6 mL/min,检测波长270 nm,柱温20 ℃。该方法可快捷、简便地拆分比索洛尔对映体。     

High performance liquid chromatographic separation of dipeptide and tripeptide enantiomers using a chiral crown ether stationary phase

A chiral stationary phase (CSP) based on (-)-(18-crown-6)-2,3,11,12-tetracarboxylic acid was evaluated for the direct resolution of the enantiomers of dipeptides and tripeptides. The type and concentration of the acid and the methanol content were optimized with regard to retention time and resolution using Ala-Phe as model peptide. A mobile phase consisting of 10 mM sulfuric acid in 70% aqueous methanol was applied to the separation of a set of 16 structurally diverse dipeptides and tripeptides. Generally, the configuration of the amino acid at the N-terminus determined the enantiomer elution order. With a few exceptions the LL- and LD-enantiomers interacted stronger with the CSP compared to the corresponding DD- or DL-enantiomers. The experimental conditions also allowed the simultaneous separation of all four stereoisomers of Ala-Phe. Addition of ammonium sulfate generally reduced retention times and enantiomer resolution. Addition of triethylamine as modifier led to an overall increase of the retention times while the resolution did not show a general trend, increasing in the case of Ala-Ala but decreasing in the case of Ala-Phe.     

The influence of the mobile phase pH and the stationary phase type on the selectivity tuning in high performance liquid chromatography nucleosides separation

Analysis of the modified nucleosides is particularly important in the medical area because of a possibility of cancerogenic processes studies. The aim of this work was to study the selectivity tuning of modified nucleosides through the investigations of interactions analyte (modified nucleoside) <==> stationary phase <==> mobile phase. A series of homemade stationary phases with different surface properties has been utilized. All of them contain various interaction sites such as: cholesterol (SG-CHOL); n-acylamide (SG-CHOL, SG-AP); aminopropyl (SG-CHOL, SG-AP, SG-NH2, SG-MIX); cyanopropyl, phenyl, octyl (SG-MIX), octadecyl (SG-MIX, SG-C18) and silanols localized on the silica gel surface of all packings. The attempt to predict the main interactions responsible for the retention between nucleosides and stationary phase ligands was done on the basis of the elemental analysis, and proportional part of an individual ligand bonded to silica surface results. In order to study the influence of different packing types on the analyzed nucleosides retention, the relationship between pH of the mobile phase buffer and the selectivity of a stationary phase was investigated.     

Evaporation of the liquid stationary phase in gas chromatography

Summary The theory of the evaporation of the liquid stationary phase is elaborated and experimentally verified. On the basis of this theory the role played by the losses in the amount of liquid phase present is quantitatively determined. General techniques are examined which minimize the losses; these techniques are based on saturating the incoming carrier gas with liquid phase vapours and raising the pressure of the carrier gas in the column, e.g. by connecting a capillary to the column outlet in order to offer resistance to the gas flow. The application of these techniques ensures stable performance of the gas chromatographic columns using a volatile liquid phase.     

纤维素衍生物手性固定相高效液相色谱法分离盐酸川丁特罗对映体

以纤维素三-(3,5-二甲基苯基氨基甲酸酯)为手性固定相(Lux Cellulose-1),建立了在正相色谱条件下直接分离盐酸川丁特罗对映体的高效液相色谱法。考察了乙醇、异丙醇等有机改性剂,三氟乙酸、二乙胺等流动相添加剂和柱温对对映体分离的影响。结果显示,酸性和碱性添加剂对对映体分离的影响最为显著: 添加二乙胺时两对映体无分离趋势;添加三氟乙酸时对映体保留强,且分离趋势明显;而同时添加三氟乙酸和二乙胺则两对映体分离显著改善,分离度可达4.0。优化后的色谱条件: 色谱柱为Lux Cellulose-1手性柱(250 mm×4.6 mm, 5 μm),流动相为正庚烷-乙醇-三氟乙酸-二乙胺(88:12:0.3:0.05, v/v/v/v),流速为1.0 mL/min,紫外检测波长为246 nm,柱温为25 ℃。该方法简便,快速,可用于左旋盐酸川丁特罗原料中右旋异构体杂质的检查。     

杂化硅胶整体材料研磨法制备混合型高效液相色谱固定相

以聚乙二醇（PEG）为致孔剂，四甲氧基硅烷（TMOS）和乙烯基三甲氧基硅烷（VTMS）为杂化硅胶前驱体，在乙酸催化作用下使硅烷发生水解，在尿素加热分解提供的碱性环境下水解的硅烷进一步缩聚得到杂化硅胶整体材料。将此整体材料用球磨机研磨，然后用三羟甲基氨基甲烷处理，并洗涤干燥得到粒径为3 μm左右的硅胶颗粒。探索了不同反应条件对硅胶颗粒的大小、比表面积和孔径、表面形貌和分散性的影响；当TMOS和VTMS体积比为3：1时可以得到孔径为7.5 nm和比表面积为245 m²/g的硅胶颗粒。通过对所制得的硅胶颗粒表面进行C18（十八烷基二甲基氯硅烷）键合修饰和巯基-烯点击反应，得到混合型高效液相色谱固定相。对此固定相的测试结果表明以上硅胶色谱填料的制备方法具有一定的实用性。     

高效液相色谱手性固定相法直接拆分外消旋雷诺嗪

在反相以及正相争件下，利用自制的涂敷型纤维素-三（3，5-二甲基苯基氨基甲酸酯）手性固定相直接拆分了外消旋雷诺嗪，并考察了不同流动相对手性拆分的影响，特别是醇类物质对拆分影响。结果表明，醇的立体结构、极性对雷诺嗪的手性拆分均有影响。实验结果显示无论在正相条件下还是在反相条件下，涂敷型纤维素-三（3，5-二甲基苯基氨基甲酸酯）手性固定相均可以很好的拆分外消旋体雷诺嗪。     

High performance liquid chromatography with cyclodextrin and calixarene macrocycle bonded silica stationary phases for separation of steroids

β-Cyclodextrin, p-tert-butyl-calix[8]arene and chloropropyl bonded silica stationary phases have been prepared and were applied at the same time to develop a chromatographic procedure to separate steroids. In order to select the best type of stationary phase for the analysis, similar preparation processes of the two kinds of macrocycle stationary phases with the same spacer were adopted respectively. The chromatographic behaviors and retention mechanisms of the two kinds of macrocycle stationary phases for steroids were systematically studied and compared with those of chloropropyl bonded silica and ODS. The effect of mobile phase variables, such as methanol content, pH value of buffer, ionic strength and buffer composition on chromatographic behaviors was investigated. The results showed that the retention mechanisms of the four stationary phases for steroids were obviously different, and excellent separation was achieved on β-cyclodextrin bonded silica stationary phase (β-CD-BS), as a consequence of the structure and the properties of the stationary phase. The retention process on β-CD-BS exhibited inclusion complexation, hydrogen-bonding and weak hydrophobic interaction, while for p-tert-butyl-calix[8]arene bonded silica stationary phase (CBS), π-π and hydrogen-bonding besides hydrophobic interaction played an important role.     

Selectivity optimization in green chromatography by gradient stationary phase optimized selectivity liquid chromatography

Stationary phase optimized selectivity liquid chromatography (SOSLC) is a promising technique to optimize the selectivity of a given separation by using a combination of different stationary phases. Previous work has shown that SOSLC offers excellent possibilities for method development, especially after the recent modification towards linear gradient SOSLC. The present work is aimed at developing and extending the SOSLC approach towards selectivity optimization and method development for green chromatography. Contrary to current LC practices, a green mobile phase (water/ethanol/formic acid) is hereby preselected and the composition of the stationary phase is optimized under a given gradient profile to obtain baseline resolution of all target solutes in the shortest possible analysis time. With the algorithm adapted to the high viscosity property of ethanol, the principle is illustrated with a fast, full baseline resolution for a randomly selected mixture composed of sulphonamides, xanthine alkaloids and steroids.     

Liquid chromatography and characterization of ether-functionalized imidazolium ionic liquids on mixed-mode reversed-phase/cation exchange stationary phase

A new series [C_nO_mmim]Cl of imidazolium cation-based ionic liquids (ILs), with an ether functional group on the alkyl side-chain, has been prepared. The possibility of analyzing the ionic liquids by high performance liquid chromatography (HPLC) was investigated on mixed-mode reversed/cation exchange stationary phase with the aqueous-acetonitrile mobile phase. Elution parameters, such as retention factor, selectivity and column efficiency, were studied as functions of mobile phase composition and pH. The ILs were characterized by elemental analysis, and infrared, UV and ¹H, ¹³C NMR spectroscopy.     

聚合离子液体毛细管气相色谱固定相的性能评价

离子液体作为毛细管气相色谱固定相的选择性和热稳定性是近年人们关注的课题。本文合成了1-烯基-3-苄基咪唑-二(三氟甲基磺酰)亚胺盐(VBIm-NTf2)离子液体并经毛细管柱内聚合制得了聚合离子液体PVBIm-NTf2色谱柱。与VBIm-NTf2色谱柱相比,PVBIm-NTf2色谱柱具有良好的色谱选择性和热稳定性。PVBIm-NTf2色谱柱对Grob试剂、醇混合物、酯混合物和苯系物等都表现了很好的分离能力,并且色谱峰峰形窄且对称。该聚合色谱柱在250 ℃下老化6 h后仍具有良好的分离能力和选择性。本文还测定了PVBIm-NTf2的Abraham溶剂化参数,解析了该固定相与溶质间的相互作用,相关研究国内尚未见报道。研究表明,对常规离子液体进行柱内聚合是改善常规离子液体气相色谱固定相的热稳定性和选择性的有效途径。     

Polysiloxane-encapsulated stationary phase for reversed-phase high-performance liquid chromatography

Summary Silica beads of 6-μm average diameter were silanized with methylvinyldiethoxysilane and then subjected to encapsulation with poly(methylvinylsiloxane). The resulting product is a new stationary phase for reversed-phase high performance liquid chromatography (RP-HPLC) which has superior ability for the separation of polar, non-polar and basic compounds. The chromatographic peaks are symmetric. Its stability has been studied; after continuous use for three months the carbon content and chromatographic behaviour of the phase were unchanged. on to the silica surface to given an uniform organic film. Material prepared in this way has both good chromatographic behaviour and superior selectivity. Because contact of the silica matrix with the mobile phase is avoided, the alkali-resisting ability of the stationary phase is increased. The non-specific adsorption of alkaline solutes on to the silica surface is also avoided because of the complete coverage of surface silanol groups. Reports of stationary phases encapsulated with polystyrene [6], polybutadiene [I] and octadecylsiloxane polymers have recently appeared in the literature [3]. In this paper we report the encapsulation of poly-(methylvinylsiloxane) (analogous to the phase SE-31 often used in GC) on to a silica matrix previously modified with methylvinyldiethoxysilane. The resulting phase has superior performance in reversed-phase HPLC.     

High performance liquid chromatography on the chiral stationary phase (R,R)-DACH-DNB using carbon dioxide-based eluents

Fast and efficient separations of chiral stereolabile compounds were obtained at very low temperature on a π-acid chiral stationary phase (R,R-DACH-DNB) using carbon dioxide-based mobile phases containing alcoholic polar modifiers. Furthermore, efficient separations of the newly discovered spherical carbon cluster buckminsterfullerene (C₆₀) and the related higher fullerenes (C₇₀, etc.) have been performed on the same stationary phase using eluents based on either n-hexane or carbon dioxide.     

Hexylacrylate-based mixed-mode monolith,a stationary phase for the nano-HPLC separation of structurally related enkephalins

The potential of an in situ photopolymerized hexylacrylate-based monolithic stationary phase-bearing sulfonic acid groups was investigated by studying the chromatographic retention of small structurally related peptides (enkephalins) by nano-LC. Several retention mechanisms were highlighted. First, a reverse-phase chromatographic behavior toward neutral solutes due to hexylacrylate-moieties was demonstrated. Second, an evaluation of the influences of buffer pH suggested the involvement of a cation-exchange mechanism due to the presence of 2-acrylamido-2-methyl-1-propanesulfonic acid. This cation-exchange phenomenon was confirmed by the clear influence of Na⁺ concentration in the mobile phase on peptide retention.     

Selectivity optimization in liquid chromatography via stationary phase tuning

In striving for the best possible separation, the selectivity of stationary phases as an optimization parameter is often underestimated although there are many ways to influence this powerful tool. This review serves to provide an insight into the various ways of adapting the selectivity of a separation in liquid chromatography. Approaches via temperature and flow rate tuning are discussed as a basis followed by focusing on the stationary phase as the superior optimization parameter. Highly selective stationary phases hereby provide an advantage for groups of similar analytes. For more complex mixtures, separations can be improved using mixed-mode technologies where different retention mechanisms are combined. Serial coupling, mixed-bed columns, and stationary phase optimized selectivity liquid chromatography provide solutions to various degrees. Finally, the advantages of stationary phase tuning over adaption of mobile phase and/or temperature are presented in terms of optimum application range.     

一种混合模式反相/阴离子交换色谱固定相的制备与评价

通过在硅胶表面同时化学键合十八烷基和环氧基团,再用小分子叔胺进行环氧开环,制备得到表面带正电荷的反相模式固定相.该方法避免了反相模式固定相必要的封尾步骤,可在碱性样品分离中起到电荷屏蔽作用,消除因静电吸附而导致的峰拖尾现象,同时有利于提高固定相的耐水能力.该固定相表现出反相和亲水作用的双重保留机理,具有良好的运行稳定性...