Retention behaviour of imidazolium ionic liquid cations on 1.7 μm ethylene bridged hybrid silica column using acetonitrile-rich and water-rich mobile phases

The potential of 1.7 μm ethylene bridged hybrid silica phase was investigated for the separation of twelve imidazolium-based ionic liquid cations. U-shaped retention profile was observed for all solutes with an increase in retention at both low and high acetonitrile content. Chromatographic behaviour of imidazolium cations in both hydrophilic interaction chromatography (HILIC) and per aqueous liquid chromatography (PALC) modes was studied by varying key parameters such as buffer concentration and pH, acid additive, organic modifier and column temperature. Experimental data provided some evidences that under PALC conditions cationic solutes are retained predominantly by mixed hydrophobic/ion-exchange interactions. In the HILIC mode, both partitioning and ion-exchange interactions are responsible for the retention of solutes. Compared to PALC, HILIC provided significantly higher efficiencies with less or even no peak tailing, better separation selectivity and greater resistance to overload. In PALC mode gradient elution was required to achieve adequate retentivity of all solutes but selectivity was not sufficient to distinguish between solutes with very similar hydrophobicity. In contrast, under HILIC conditions twelve solutes were almost completely resolved in less than 4 min by using isocratic elution. Summarizing, it could be concluded that ethylene bridged hybrid silica column providing a dual retention mechanism offers the possibility of selecting between the two retention modes with opposite separation selectivity, just by changing the composition of the mobile phase.     

A weak cation-exchange phase for the separation of biogenic amines by open tubular liquid chromatography

Summary The preparation and performance of a weak cation-exchange stationary phase for Open Tubular Liquid Chromatography (OT-LC) was investigated. The stationary phase was prepared in 5.4 μm I.D. fused silica capillaries byin situ photopolymerization of a mixture of silicon acrylate and acrylic acid. The influence of pH, counter ion concentration and organic modifier concentration of the mobile phase on the retention was studied with catecholamines as test solutes using LIF detection. Other biological amines like amino acids, small peptides and nucleic acid derivatives could be separated on this stationary phase as well. The kinetic performance of the stationary phase was studied with several cations and neutral solutes.     

羧基甜菜碱型亲水作用色谱柱的制备及性能研究

以甲基丙烯酰氧乙基二甲基乙酸铵(CBMA)为功能单体,利用表面引发原子转移自由基聚合(Surface-initiated atom transfer radical polymerization, SI-ATRP)技术,将CBMA接枝到硅胶表面,得到接枝聚合物CBMA的亲水作用色谱固定相(Silica-CBMA).通过改变SI-ATRP反应体系中单体的浓度,制备了3种不同接枝量的亲水作用色谱固定相.考察了Silica-CBMA固定相对有机酸类化合物的分离性能以及流动相中pH值、盐浓度、水含量等因素对溶质保留行为的影响.结果表明,在亲水作用色谱模式下,Silica-CBMA固定相对有机酸类化合物的分离是离子交换作用与亲水作用的混合色谱模式.流动相中盐浓度增大,溶质保留减弱,符合离子交换作用特征;固定相和溶质的离子化程度受流动相pH值影响较大,pH值增大,溶质保留增强;而溶质的保留时间随流动相水含量增加而降低则是典型的亲水作用色谱特征.使用自制Silica-CBMA柱,建立了芦丁片中维生素C、芦丁含量的亲水作用色谱测定方法,操作方法简单,为强极性样品的分离测定提供了新方法.     

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.     

Retention properties of internal-surface reversed-phase silica packing and recovery of drugs from human plasma

The properties of internal-surface reversed-phase silica packings developed for use in high-performance liquid chromatography, were investigated with regard to the effects of mobile phase conditions (pH, ionic strength, nature and concentration of organic modifiers, ion-pairing agent and surfactant) on the retention of some selected compounds having different polarities. The results indicated that pi-electron interactions play the main role in solute retention, although it is weaker than that on phenylsilica supports, and that weak cation-exchange properties exert a secondary effect on the retention of ionic solutes. The ion-pairing effect was found to be markedly weakened in mobile phases containing an anionic counter ion. In contrast, the addition of a surfactant (SDS) caused a marked increase in the retention of certain drugs at low pH and expanded the pH range of the mobile phase applicable to plasma samples. Based on these findings, the separation and recovery of several drugs (probenecid, lidocaine, cefpiramide and cefaclor) from human plasma were investigated, with emphasis on their protein bindings. The contour chromatogram of human plasma is also demonstrated in relation to the selection of the detection wavelength.     

Study of influence of additives of tyloxapol on the chromatographic characteristics of the model compounds: the comparative characterization of micellar mobile phases of tyloxapol and Triton X‐100

The chromatographic behavior of model compounds of biomedical significance (organic acids, amino acids, drugs) was investigated using mobile phases modified with tyloxapol. The influence of factors such as concentration of tyloxapol, content of organic modifier and pH of mobile phase on the retention factor of solutes was studied. The results were compared with the data obtained by elution with mobile phases containing Triton X‐100 additives, since units of Triton X‐100 are repeated in the structure of tyloxapol. Divergence in chromatographic behavior of model compounds was explained by the difference in physico‐chemical characteristics (microviscosity, polarity, critical micelle concentration, shape of micelles, etc.) of tyloxapol and Triton X‐100 micelles. Copyright © 2009 John Wiley & Sons, Ltd.     

反相高效液相三元流动相台阶梯度分离条件的快速优化方法

 根据溶质在柱内的迁移规律 ,建立了一种利用线性梯度实验快速获得溶质保留值方程系数 ,然后以串行响应函数为优化指标进行多台阶梯度分离条件优化的方法。与利用等度实验获得保留值方程的方法相比 ,该法可以大大缩短优化时间。通过该方法对芳香胺和衍生化氨基酸样品进行了分离 ,获得了满意的分离度 ,表明该方法的预测精度很好。     

Migration of neutral solutes by double stepwise gradient elution in capillary electrochromatography

Characteristics of electroosmotic flow (EOF) and the migration of neutral solutes under double stepwise gradient elution in capillary electrochromatography were studied systematically. EOF velocity proved to be the function of operation time changing with the introduction of the second mobile phase. Accordingly, the retention of components also changed. The migration of neutral solutes was studied under the following three situations; A, components eluted when the column was filled only with the first kind of mobile phase; B, solutes eluted still in the first kind of mobile phase while at that time two kinds of mobile phase coexisted in the column and C, samples eluted in the second kind of mobile phase. Equations to describe the retention times of components under these three kinds of conditions were deduced and applied to predict the retention times of 12 aromatic compounds. Relative errors between experimental and calculated values were below 5.0%, which proved the reliability of the equations. In addition, parameters that might affect the retention time of solutes, such as the transferring time of mobile phase vials, the capacity factors of components and EOF velocities two steps were studied systematically.     

Neutral octadecyl monolith for reversed phase capillary electrochromatography of a wide range of solutes

A neutral octadecyl monolithic (ODM) column for RP capillary electrochromatography (RP-CEC) has been developed. The ODM column was prepared by the in situ polymerization of octadecyl acrylate (ODA) as the monomer and trimethylolpropanetrimethacrylate (TRIM) as the crosslinker, in a ternary porogenic solvent containing cyclohexanol, ethylene glycol, and water. The ODM column exhibited cathodal EOF over a wide range of pH and ACN concentration in the mobile phase despite the fact that it was devoid of any fixed charges. It is believed that the EOF is due to the adsorption of ions from the mobile phase onto the surface of the monolith thus imparting to the neutral ODM column the zeta potential necessary to support the EOF required for mass transport across the monolithic column. Furthermore, the adsorption of mobile phase ions to the neutral monolith modulated solute retention and affected the separation selectivity. The wide applications of the neutral ODM column were demonstrated by its ability to separate a wide range of small and large solutes, both neutral and charged. While the separation of the neutral solutes was based on RP retention mechanism, the charged solutes were separated on the basis of their electrophoretic mobility and hydrophobic interaction with the C18 ligands of the stationary phase. As a typical result, the neutral monolithic column was able to separate peptides quite rapidly with a separation efficiency of nearly 200,000 plates/m, and this efficiency was exploited in tryptic peptide mapping of standard proteins, e. g., lysozyme and cytochrome C, by isocratic elution.     

Selectivity of some basic solutes on a poly(methyltetradecylsiloxane)-silica stationary phase

Complex analyses of polar compounds, especially basic ones, require more selective stationary phases. The present paper describes a stationary phase prepared by thermal immobilization of poly(methyltetradecylsiloxane) onto chromatographic silica (PMTDS-SiO(2)). This stationary phase presents hydrophobic and ion-exchange interactions that confer both high retention and unique selectivities for basic solutes. The influence of ion-exchange interactions is confirmed by the increase in retention factors of basic solutes when the mobile-phase pH changes from acidic to neutral and by the decrease in retention factors when the mobile-phase pH changes from neutral to alkaline. The ion-exchange properties of the stationary phase are enriched in neutral mobile phase (pH 7-7.5) using soft Lewis bases such as tricine and tris as buffers but are suppressed in both acidic (pH 2.5-6) and highly alkaline mobile phases (pH≤10). Increasing both temperature and flow rate permits more rapid separations while maintaining the selectivity. The stability of the stationary phase is evaluated with acid, neutral and alkaline mobile phases.     

Modelling of elution-extrusion counter-current chromatography using perfect replacement approach

Basing on the perfect replacement approach the equilibrium cell model is developed to describe the separation process in elution-extrusion counter-current chromatography (EECCC). As is known, EECCC consists of three steps: classical elution, sweeping elution, and extrusion. The perfect replacement approach means that during sweeping elution step, the mobile phase contained in the column moves and interacts with the "old" stationary phase in the same mode as during the classical elution step; the "new" and "old" stationary phases do not mix; and after the contacting with the mobile phase the concentration of solutes in the "old" stationary phase remains constant and this stationary phase volume is pushed ahead to the exit of the column. Equations are presented allowing the simulation of the chromatogram of solutes eluted from the column with the mobile phase during the elution period and the chromatogram of solutes pushed out of the column with the stationary phase during the extrusion period of EECCC. These equations can help to choose the optimal conditions for conducting elution-extrusion counter-current chromatography.     

Effects of ionic liquid as additive and the pH of the mobile phase on the retention factors of amino benzoic acids in RP-HPLC

As an organic salt, ionic liquids are widely used as new solvent media. In this paper, three positional isomers, such as o-amino benzoic acid, m-amino benzoic acid, and p-amino benzoic acid are separated with four different ionic liquids as additives to the mobile phase using reversed-phase (RP) high-performance liquid chromatography (HPLC). Amino benzoic acids are biologically active substances; the p-isomer is present in a group of water-soluble vitamins and is widely known as a sunscreen agent. The ionic liquids used are 1-butyl-3-methylimidazolium tetrafluoroborate, 1-ethyl-3-methylimidazolium tetrafluoroborate, 1-ethyl-3-methylimidazolium methylsulfate, and 1-octyl-3-methylimidazolium methylsulfate. The effects of the length of the alkyl group on the imidazolium ring and its counterion, the concentrations of the ionic liquid, and the effect of the pH of the mobile phase on the retention factor of the amino benzoic acid isomers are studied. Separation with the ionic liquid in the eluent was better than the separation without the ionic liquid. The pH mainly affected the retention and elution order of the solutes in RP-HPLC.     

UPLC a Powerful Tool for the Separation of Imidazolium Ionic Liquid Cations

Ultra-performance liquid chromatography (UPLC) in reversed-phase (RP), ion pair (IP) and hydrophilic interaction chromatography (HILIC) has been investigated for the separation of imidazolium-based ionic liquid (IL) cations. Among the three stationary phases (i.e., C18, C8 and phenyl) studied under RP conditions the phenyl phase provided much stronger retention for the IL cations. Four acids (hydrochloric, methanesulfonic, perchloric and trifluoroacetic) as mobile phase additives were compared in light of their effects on the retention of IL cations. It was shown that the retention of all IL cations decreased upon acidification of the mobile phase, possibly due to suppression of residual silanol ionization. Very fast (~3 min) and efficient RP-UPLC separation of six cations was achieved by gradient elution with acetonitrile?Cwater mobile phase containing 2.5 mmol L^?1 perchloric acid. In IP-UPLC all solutes were well resolved in about 4 min by gradient elution with acetonitrile?Cwater mobile phase containing 1 mmol L^?1 sodium 1-octanesulfonate as ion pairing reagent. Finally, under HILIC conditions by using isocratic elution with acetonitrile?Cwater (85:15, v/v) mobile phase containing 5 mmol L^?1 ammonium formate (pH 3.2) the separation time was reduced to less than 2 min while maintaining excellent peak shapes and sufficient resolution. Compared to current LC systems UPLC allowed considerably faster separations with better peak shapes.     

Effects of amine modifiers on retention and peak shape in reversed-phase high-performance liquid chromatography

A systematic evaluation of the effects of 15 different amine modifiers on the retention and peak symmetry of three solutes, a primary, a secondary, and a tertiary amine, is presented. Using automated experimentation, mobile phase combinations for each modifier over a pH range of 2.5 to 8 were investigated. The effect of changing the sodium ion concentration of the mobile phase was also examined. The importance of hydrophobic, ion exchange, and hydrogen bonding interactions as mechanisms for retention and peak symmetry of positively charged solutes is discussed.     

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.     

溶质在两种反相色谱柱中的动力学色谱行为

研究了不同流速下4种溶质在两种不同反相色谱柱上的色谱图及它们的动力学色谱性质,进一步验证了溶质相对保留值(RRT)与流动相流速之间存在良好的双对数线性关系,并对溶质在流速改变时洗脱顺序发生改变的现象从动力学因素上进行了合理的解释;同时还发现色谱填料的孔径及拓扑学构型不同将导致溶质在其上具有不同的动力学色谱参数及动力学色谱行为。     

Enantiomeric separation of some pharmaceutical intermediates and reversal of elution orders by high-performance liquid chromatography using cellulose and amylose tris(3,5-dimethylphenylcarbamate) derivatives as stationary phases

Several pairs of enantiomers of pharmaceutical intermediates were separated by HPLC directly on cellulose and amylose tris(3,5-dimethylphenylcarbamate) derivatives (Chiralcel OD and Chiralpak AD) using hexane as mobile phase with 2-propanol or ethanol as modifier. The separation and elution order of the enantiomers on the two columns using different alcohol modifiers were compared. Reversal of the elution order of some enantiomeric pairs associated with increased retention of many of these solutes upon changing the mobile phase modifier from 2-propanol to ethanol was observed. The effect of structural variation of two pairs of enantiomers on their k' and separation factor alpha was noted. Chiralcel OD and Chiralpak AD columns provided different retention, separation and elution order of some of the enantiomeric pairs.     

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。