Buffer Choice for Tuning the Selectivity in Reverse Phase Chromatography

Abstract

Retention behaviour of ionogenic species in high-performance liquid chromatography on reversed phase materials was studied, specifically dependence of buffer quality applied to mobile phases. The buffers' effect on retention of organic acids, amino acids and dipeptides is quantified by modelling capacity factors as a function of pH-values. At constant ionic strength, increasing capacity factors were observed going from phosphate to less polar citrate buffer, modification of accessible silanol groups of the stationary phase being responsible for this effect. Application of citrate buffer for separation of a seven-component mixture is demonstrated on the basis of a computerized search for optimum chromatographic performance. The evaluated factor levels (pH, methanol content and ionic strength) differ from those found using phosphate buffer-containing mobile phases.     

Potential Use of an Aminopropyl Stationary Phase in Hydrophilic Interaction Capillary Electrochromatography. Application to Tetracycline Antibiotics

We have demonstrated that the aminopropyl stationary phase (APS2) already used for analysis of polar compounds in hydrophilic interaction liquid chromatography can be successfully used in capillary electrochromatography. First, it was shown that the APS2 packing material affords a sustainable electroosmotic flow, the direction and magnitude of which depend on the nature of the electrolyte ions. A triple-layer model is proposed to describe this particular behaviour of the aminopropyl stationary phase. Finally, application to the separation of the tetracycline antibiotics used as test compounds in capillary electrochromatography is described and it is demonstrated that the final separation may be manipulated because of the electromigration and retention properties of the compounds, although migration times cannot be accurately predicted. Revised: 15 March and 6 July 2005     

Aminoglycoside analysis in food of animal origin with a zwitterionic stationary phase and liquid chromatography–tandem mass spectrometry

In this study, fourteen highly polar aminoglycoside (AGs) antibiotics were selected. Various stationary phases were tested, including Obelisc R, ZIC-HILIC, BEH amide and aminopropyl. The nature of the stationary phase, mobile phase (water or buffer solutions and acetonitrile), pH (percentage of formic acid), gradient conditions and injection solvents were systematically studied as relevant parameters for tuning retention selectivity and detectability of AGs in liquid chromatography electrospray tandem mass spectrometry (LC–(ESI)–MS/MS). Only the two zwitterionic phases (Obelisc R and ZIC-HILIC) achieved a proper chromatographic separation considering interferences due to the crosstalk effect in low resolution mass spectrometers. The water/acetonitrile mobile phase containing 1% formic acid used with Obelisc R provided more sensitivity than the highly concentrated buffered mobile phases required for ZIC-HILIC. A solid phase extraction (SPE) clean-up procedure with polymeric weak cation exchange (WCX) cartridges was optimized for honey, milk and liver samples. Different brands of cartridges and elution solvents were tested, and the Taurus WCX offered the best recovery rate with a buffer elution at pH 3. The final optimized method was validated in these matrices according to Decision 2002/657/EC. A monitoring campaign for sixty honey, milk and liver samples was carried out at the Food Authority Control in Geneva. The concentration of dihydrostreptomycin (DSTP) found in one ovine liver exceeded the established maximum residue levels (MRLs) within the European and Swiss legislations but it was compliant taking into account the validation data.     

Dual‐mode hydrophilic interaction normal phase and reversed phase liquid chromatography of polar compounds on a single column

Adopting a stationary phase convention circumvents problematic definition of the boundary between the stationary and the mobile phase in the liquid chromatography, resulting in thermodynamically consistent and reproducible chromatographic data. Three stationary phase definition conventions provide different retention data, but equal selectivity: (i) the complete solid phase moiety; (ii) the solid porous part carrying the active interaction centers; (iii) the volume of the inner column pores. The selective uptake of water from the bulk aqueous‐organic mobile phase significantly affects the volume and the properties of polar stationary phases. Some polar stationary phases provide dual‐mode retention mechanism in aqueous‐organic mobile phases, reversed‐phase in the water‐rich range, and normal‐phase at high concentrations of the organic solvent in water. The linear solvation energy relationship model characterizes the structural contributions of the non‐selective and selective polar interactions both in the water‐rich and organic solvent‐rich mobile phases. The inner‐pore convention provides a single hold‐up volume value for the retention prediction on the dual‐mode columns over the full mobile phase range. Using the dual‐mode monolithic polymethacrylate zwitterionic micro‐columns alternatively in each mode in the first dimension of two‐dimensional liquid chromatography, in combination with a short reversed‐phase column in the second dimension, provides enhanced sample information.     

Effect of mobile phase pH on the retention of nucleotides on different stationary phases for high-performance liquid chromatography

The main aim of the present investigation was to study the retention and separation of eight nucleotides with the use of seven stationary phases in RP HPLC mode. Two octadecyl columns were used; however, aminopropyl, alkylamide, cholesterol, alkyl-phosphate, and phenyl were also studied. Special attention was paid to the mobile phase buffer pH, since it appears that this factor is very influential in the case of chromatographic separation of nucleotides. The retention of nucleotides was greater for mobile phase pH?4.0 in comparison with pH?7.0 (except for octadecyl and phenyl packing). This is a consequence of protonization of polar groups present on the stationary phase surface. It was proved that aminopropyl, alkyl phosphate, alkylamide packing materials are not suitable for the resolution of nucleotides. On the other hand, cholesterol and phenyl stationary phases are alternatives for conventional octadecyl phases. Application of cholesterol packing allows separation of small, polar nucleotides, which is impossible to achieve in the case of octadecyl phase. Moreover, a phenyl support allows separation of nucleotides in a shorter time in comparison with octadecyl packing.     

Separation of polar mushroom toxins by mixed-mode hydrophilic and ionic interaction liquid chromatography-electrospray ionization-mass spectrometry

Reversed-phase liquid chromatography (RPLC) is commonly used to analyze nonvolatile contaminants and naturally occurring toxins in foods. However, polar compounds, such as hydrophilic polypeptides and quaternary ammonium salts, are often not satisfactorily separated by RPLC and present a challenge for analytical scientists. In this study, hydrophilic interaction liquid chromatography (HILIC), on an amide-based stationary phase in combination with electrospray ionization (ESI) tandem mass spectrometry (MS-MS), is successfully employed to simultaneously separate polar mushroom toxins, including amanitins and phallotoxins, which are cyclic oligopeptides and muscarine, a quaternary ammonium compound, in mushrooms. The sensitivity of different ionization modes is studied, and the positive ionization mode is found to provide a more sensitive and effective tool for the unambiguous identification of the concerned polar toxins because of their characteristic fragmentation patterns. The properties of the mobile phase are also found to have significant impacts on the separation. At a high acetonitrile (ACN) concentration, hydrophilic interaction dominates, and all analytes under study demonstrate a much higher affinity with the stationary phase. The addition of methanol (MeOH) as a modifier could further enhance the HILIC separation for amanitins, phallotoxins, and muscarine. Valley-to-valley separation is achieved upon the optimatizatiqn of the mobile phase (comprising of ACN, MeOH, and ammonium formate buffer at pH approximately 3.5) and the solvent gradient. HILIC coupled with ESI-MS-MS is demonstrated to be a novel technique for the simultaneous separation and confirmatory analysis of the concerned polar toxins by providing an environment of solubility and retention that could not be achieved through the use of RPLC.     

Prediction of peak shape in hydro-organic and micellar-organic liquid chromatography as a function of mobile phase composition

A simple model is proposed that relates the parameters describing the peak width with the retention time, which can be easily predicted as a function of mobile phase composition. This allows the further prediction of peak shape with global errors below 5%, using a modified Gaussian model with a parabolic variance. The model is useful in the optimisation of chromatographic resolution to assess an eventual overlapping of close peaks. The dependence of peak shape with mobile phase composition was studied for mobile phases containing acetonitrile in the presence and absence of micellised surfactant (micellar-organic and hydro-organic reversed-phase liquid chromatography, RPLC). In micellar RPLC, both modifiers (surfactant and acetonitrile) were observed to decrease or improve the efficiencies in the same percentage, at least in the studied concentration ranges. The study also revealed that the problem of achieving smaller efficiencies in this chromatographic mode, compared to hydro-organic RPLC, is not only related to the presence of surfactant covering the stationary phase, but also to the smaller concentration of organic solvent in the mobile phase.     

固相萃取-亲水作用色谱法测定废水中四环素类抗生素

建立了固相萃取(SPE)-亲水作用色谱法(HILIC)测定废水中金霉素(CTC)、强力霉素(DC)、四环素(TC)和土霉素(OTC) 4种四环素类抗生素(TCs)残留的新方法.水样经Oasis HLB固相萃取柱净化富集后, 采用以氨基色谱柱及高极性有机溶剂-水相缓冲溶液为流动相的亲水作用色谱法(HILIC)进行分析. 对流动相中缓冲溶液的类型和pH值、离子强度、 有机溶剂的浓度以及流速进行了优化, 确定了以V(乙腈)∶V(6.7 mmol/L柠檬酸铵缓冲溶液, pH 4.0)=85∶ 15混合液为流动相的最佳条件.本方法具有良好的线性关系(r> 0.999)和重现性(峰面积RSD<1.0%), 最低检出限(S/N=3)为12～30 μg/L, 4种四环素类药物添加水平在0.5～10 μg/L范围内的标准加入回收率为 90.6%～106.5%; 相对标准偏差为 2.5%～6.2%.本方法简便、准确、流动相离子强度低,适合于与质谱联用,用于屠宰场污水及医院污水等实际样品检测,结果满意.     

Separation of the catecholamine metabolites 4-hydroxy-3-methoxy- and 3-hydroxy-4-methoxymandelic acid by reversed-phase high performance liquid chromatography

The catecholamine metabolites 4-hydroxy-3-methoxy- and 3-hydroxy-4-methoxymandelic acid can be completely separated by reversed-phase high performance liquid chromatography with chemically bonded octadecylsilane as stationary phase and a citrate/ammonium phosphate buffer (pH 4.5) containing 8% methanol as mobile phase. The two isomers can be electrochemically detected and produce different hydrodynamic voltammograms.     

Investigation of polar stationary phases for the separation of sympathomimetic drugs with nano-liquid chromatography in hydrophilic interaction liquid chromatography mode

In this study, the retention and selectivity of a mixture of basic polar drugs were investigated in hydrophilic interaction chromatographic conditions (HILIC) using nano-liquid chromatography (nano-LC). Six sympathomimetic drugs including ephedrine, norephedrine, synephrine, epinephrine, norepinephrine and norphenylephrine were separated by changing experimental parameters such as stationary phase, acetonitrile (ACN) content, buffer pH and concentration, column temperature. Four polar stationary phases (i.e. cyano-, diol-, aminopropyl-silica and Luna HILIC, a cross-linked diol phase) were selected and packed into fused silica capillary columns of 100 μm internal diameter (i.d.). Among the four stationary phases investigated a complete separation of the all studied compounds was achieved with aminopropyl silica and Luna HILIC stationary phases only. Best chromatographic results were obtained employing a mobile phase composed by ACN/water (92/8, v/v) containing 10 mM ammonium formate buffer pH 3. The influence of the capillary temperature on the resolution of the polar basic drugs was investigated in the range between 10 and 50 °C. Linear correlation of ln k vs. 1/T was observed for all the columns; ΔH° values were negative with Luna HILIC and positive with aminopropyl- and diol-silica stationary phases, demonstrating that different mechanisms were involved in the separation.To compare the chromatographic performance of the different columns, Van Deemter curves were also investigated.     

Study of the retention behavior of small polar molecules on different types of stationary phases used in hydrophilic interaction liquid chromatography

The retention behavior of a large group of analytes (35) with varied properties (pK_a and logP) was studied on eight hydrophilic interaction LC columns with different surfaces, stationary phase chemistries, and types of particles. The acetonitrile content (5–95%), buffer concentration (0.5–200 mM), and pH of the mobile phase (3.8 and 6.8) were evaluated for their effects on the retention behavior. The type of stationary phase had a significant impact on the selectivity and retention time of the tested analytes. Completely different selectivity was observed on the aminopropyl stationary phase. In this study, the influence of the buffer concentration was similar for all tested columns, except for the aminopropyl stationary phase. Increasing the buffer concentration led to decreased retention times for the basic compounds and increased retention times for the acidic compounds, while the inverse behavior was observed on the aminopropyl stationary phase. The selectivity of the individual stationary phases was evaluated at pH 3.8 and 6.8. Much lower selectivity differences between the stationary phases were observed at pH 6.8 than pH 3.8. Bare silica stationary phases were used in the comparison of the particles (fused‐core and fully porous particles of 3 and 1.7 μm) and the columns provided by different manufacturers.     

Development and evaluation of new zwitterionic hydrophilic interaction liquid chromatography stationary phases based on 3-P,P-diphenylphosphonium-propylsulfonate

New zwitterionic stationary phases were synthesized by covalently bonding 3-P,P-diphenylphosphonium-propylsulfonate to silica gel. The resulting materials possess both a negatively charged sulfonate group and a positively charged quaternary phosphonium group, which means that there is no net charge over a wide pH range. The retention mechanism and chromatographic behavior of polar solutes under HILIC conditions were studied on these zwitterionic phases. Compared to the commercial ZIC-HILIC column and a bare silica gel stationary phase, the newly synthesized zwitterionic stationary phases provided greater retention, higher peak efficiency and better peak symmetry in the HILIC mode. The analytes examined included: β-blockers, nucleic acid bases and nucleosides, salicylic acid and its analogues, and water soluble vitamins. Factors, such as the type of organic modifiers, solvent composition, pH and the buffer concentration of the mobile phase, have been considered as potential variables for controlling the chromatographic retention of polar analytes.     

Chromatographic behavior of new deazapurine ribonucleosides in hydrophilic interaction liquid chromatography

The chromatographic behavior of new biogenic purine nucleosides in hydrophilic interaction liquid chromatography was examined on three different stationary phases, namely bare silica, and amide‐ and cyclofructan‐based stationary phases. The effects of buffer concentration, pH and acetonitrile‐to‐aqueous‐part ratio in the mobile phase on retention and peak shape were assessed. The retention coefficients and peak symmetry values substantially differed with respect to analytes´ structures, stationary phase properties and mobile phase composition. The bare silica column was unsuitable for these compounds under the chromatographic conditions tested due to very broad and asymmetrical peaks. Furthermore, the cyclofructan‐based stationary phase provided almost Gaussian peak shapes of all deazapurine nucleosides under most conditions tested. Therefore, the cyclofructan‐based stationary phase is the most suitable choice for the chromatographic analysis of nucleosides.     

Hydrophilic interaction liquid chromatography (HILIC)—a powerful separation technique

Hydrophilic interaction liquid chromatography (HILIC) provides an alternative approach to effectively separate small polar compounds on polar stationary phases. The purpose of this work was to review the options for the characterization of HILIC stationary phases and their applications for separations of polar compounds in complex matrices. The characteristics of the hydrophilic stationary phase may affect and in some cases limit the choices of mobile phase composition, ion strength or buffer pH value available, since mechanisms other than hydrophilic partitioning could potentially occur. Enhancing our understanding of retention behavior in HILIC increases the scope of possible applications of liquid chromatography. One interesting option may also be to use HILIC in orthogonal and/or two-dimensional separations. Bioapplications of HILIC systems are also presented.     

The analysis of pharmaceutical bases on a silica stationary phase by capillary electrochromatography using aqueous mobile phases

Summary The capillary electrochromatographic (CEC) separation of a range of pharmaceutical bases was investigated on a commercially available silica stationary phase using aqueous mobile phases. The effects of mobile phase composition, buffer pH, applied voltage, and buffer anion on the retention behaviour of these bases were studied. Promising chromatography was obtained at pH 7.8 but was later found to be irreproducible. However, successful and reproducible chromatography of the bases was achieved at pH 2.3. We have previously demonstrated that the addition of mobile phase additives such as TEA-phosphate at low pH values has resulted in excellent CEC analysis of bases on reversed-phase packing materials. The same approach was applied to the analysis of bases on the silica phase in order to improve peak shape. Excellent chromatography was obtained for the analysis of strong pharmaceutical bases such as benzylamine, nortriptyline and diphenhydramine. The experimental investigations have shown that the CEC separation of a range of pharmaceutical bases can routinely be achieved with excellent peak shapes and peak efficiencies as high as 320,000 plates m⁻¹.     

Simple and Rapid Determination of Denaturants in Alcohol Formulations by Hydrophilic Interaction Chromatography

A hydrophilic interaction chromatography (HILIC) technique has been developed and validated for determination of common denaturants (denatonium benzoate, crystal violet and methylene blue) in denaturated alcohol formulations. Among the three different polar stationary phases (i.e., aminopropyl, cyanoethyl and silica) studied the cyanoethyl phase provided much stronger retention for the organic cations. It was shown that high efficiencies were reached only with anionic ion-pairing reagent that reduces the interactions with the silanol groups. The anion ion-pairing strength under HILIC conditions was: acetate < formate < trifluoroacetate < perchlorate. This study also investigated the effect of various experimental factors on the retention of the cyanoethyl stationary phase, such as acetonitrile content, pH and ion-pairing anion concentration in the mobile phase. The separation of three denaturants was achieved in about 8 min with a mobile phase containing 60% (v/v) acetonitrile and 10 mmol L⁻¹ HClO4. The proposed method was validated and applied to the determination of danaturating agents in various Lithuanian denaturated alcohol formulations.     

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.     

Separation behavior of basic compounds on unbonded silicon oxynitride and silica high‐performance liquid chromatography stationary phases with reversed‐phase eluents

Unbonded silicon oxynitride and silica high‐performance liquid chromatography stationary phases have been evaluated and compared for the separation of basic compounds of differing molecular weight, pK_a, and log D using aqueous/organic mobile phases. The influences of percentage of organic modifier, buffer pH, and concentration in the mobile phase on base retention were investigated on unbonded silicon oxynitride and silica phases. The results confirmed that unbonded silicon oxynitride and silica phases demonstrated excellent separation performance for model basic compounds and both the unbonded phases examined possessed a hydrophobic/adsorption and ion‐exchange character. The silicon oxynitride stationary phase exhibited high hydrophilicity compared with silica with a reversed‐phase mobile phase. An ion‐exclusion‐type mechanism becomes predominant for the separation of three aimed bases on the silicon oxynitride column at pH 2.8. Different from silicon oxynitride stationary phase, no obvious change for the retention time of three model bases on silica stationary phase at pH 2.8 can be observed.     

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。     

亲水作用色谱在糖类化合物分析中的应用

糖类化合物的结构和组成分析对于探究糖的结构与功能的关系具有重要作用。亲水作用色谱(HILIC)对糖类等极性化合物具有良好的分离效果。该文介绍了适合糖类分析的常用HILIC柱固定相及其分离机制,论述了强洗脱溶剂比例、流动相p H值、缓冲盐浓度和色谱柱温度对HILIC分离效果的影响,并举例说明了HILIC法在单糖组成、糖胺聚糖二糖组成、寡糖聚合度、糖苷、糖脂、糖醇以及N-/O-糖链分析中的应用。