Liquid chromatography on unmodified and modified Spheron gels II. Barbiturates and sulfonamides

Summary The ethylene glycol methacrylate gel Spheron and ion-exchangers produced by the chemical modification of this gel (the cation exchanger Spheron S and the anion exchanger Spheron DEAE) can be used for reversedphase chromatography of barbiturates and sulfonamides. In addition to the hydrophobic effect, the unique selectivities of the functional groups of Spheron materials can be utilized for the chromatographic separation of these compounds, including those difficult to resolve on octadecyl silica.Principle author     

Liquid chromatography on unmodified and modified spheron gels I. Nucleosides,bases and related compounds

Summary The ethylene glycol methacrylate gel Spheron and ion exchangers produced by the chemical modification of this gel (such as the cation exchanger Spheron S and anion exchanger Spheron DEAE) are compared with octadecylsilica as column packing materials for reversedphase chromatography of nucleic acid constituents and related compounds. The different separation selectivities of the individual materials can be utilized for the chromatographic separation of these compounds.     

Analyses of phenolic compounds by microemulsion electrokinetic chromatography

In this study, a microemulsion electrokinetic chromatography (MEEKC) method was developed to analyze and detect 13 phenolic compounds (syringic acid, p-cumaric acid, vanillic acid, caffeic acid, gallic acid, 3,4-dihydroxybenzoic acid, 4-hydroxybenzoic acid, (+)-catechin, (-)-epigallocatechin, (-)-epicatechin gallate, (-)-epigallocatechin gallate, (-)-epicatechin, and (-)-gallocatechin), which are present in many plant-derived foods. The effects of cosurfactant, organic modifier, and oil were examined in order to optimize the separation of these phenolic compounds. The amounts of cosurfactant (cyclohexanol) and organic modifier (acetonitrile) were determined as the major influence on the separation selectivity, while the type of oil partially affected the separation resolution of the phenolic compounds. A highly efficient MEEKC separation method was achieved within 14 min by using a microemulsion solution of pH 2.0 containing 2.89% w/v SDS, 1.36% w/v heptane, 7.66% w/v cyclohexanol, and 2% w/v ACN. Furthermore, the present work could demonstrate that the nature of the oil phase has a significant influence on the separation selectivity of phenolic compounds.     

Liquid chromatography of phenolic compounds in natural water using on-line trace enrichment

Summary Two packing materials, C18 and PLRP-S, are studied for on-line trace enrichment of phenolic compounds in water. Various precolumns of different internal diameter are also tested and the addition of an ion-pair reagent to increase retention and thus, breakthrough volumes of phenolic compounds, is studied. Best results are obtained when a PLRP-S precolumn is coupled on-line with a C18 analytical column and DAD detector. Addition of TBA considerably increases breakthrough volumes. In contrast, when a C18 precolumn is used, breakthrough volumes are lower and it is impossible to determine TCP and PCP, under the experimental conditions used, because of interference of other nonpolar compounds in the samples. The performance of the system is evaluated with river and tap water and the preconcentration of 10 ml of sample in a PLRP-S precolumn involves a linear range between 1 g 1^–1 and 20 l^–1 and limits of determination between 0.5 g l^–1 and 1 g l^–1 are obtained.     

Determination of phenols in water by on-line solid-phase disk extraction and liquid chromatography with electrochemical detection

Poly(styrene-divinylbenzene) (PS-DVB) membrane extraction disks were used as sorbents for the on-line solid phase extraction of 13 phenols (nitro and chlorophenols) in river and tap waters. Determination was performed by liquid chromatography with electrochemical detection (LC-ED). An acetate buffer-acetonitrile-methanol mixture as mobile phase and amperometric detection at +1100 mV were used. High water volumes, up to 250 ml, can be preconcentrated without loss of phenols (recoveries between 80% and 100%) except for the more polar ones. Moreover, detection limits between 0.01 and 0.1 μg l⁻¹ in tap water and between 0.1 and 1.0 μg⁻¹ in river water were obtained. The method has been applied to the analysis of two river water samples.     

Comprehensive two-dimensional liquid chromatography in the analysis of antioxidant phenolic compounds in wines and juices

A novel comprehensive two-dimensional liquid chromatographic (LC×LC) system was developed for the quantification of antioxidant phenolic compounds in wine and juice. The system allows parts of the sample that are well separated in the first column to pass directly to the detector after the first column, while the rest of the sample proceeds to the second column. The optimised LC×LC system employed a combination of two C18 columns, the latter column with an ion-pair reagent (tetrapentylammonium bromide). The relative standard deviations (RSD) for the retention times were better than 0.01% in the first dimension and on average 6.3% in the second. The RSD values of the peak volumes varied from 3% (protocatechuic acid) to 13% (caffeic acid, n = 3, 10 μg/ml).     

Optimum conditions for the separation of nucleobases and nucleosides on unmodified silica by HPLC

Summary The retention behaviour of nucleobases and nucleosides on unmodified silica with dichloromethane-methanol-water mixtures has been systematically investigated. The degree and order of retention can be varied over a wide range by changing the pH, the type and concentration of the acidic additives and by the methanol and water content of the mobile phase. The retention process cannot be considered as simple adsorption but rather as a very complex mixed distribution process of adsorption and absorption including the involvement of ion-pair formation. Further data on the effect of the type of silica (source of supply) on retention behaviour, column efficiency and column reproducibility are reported. The practical application of unmodified silica for the separation of nucleobases and nucleosides is demonstrated by the analysis of a hydrolysate of calf thymus DNA.     

Electrochemical detection of phenolic estrogenic compounds at carbon nanotube-modified electrodes

The use of a carbon nanotube-modified glassy carbon electrode (CNT-GCE) for the LC-EC detection of phenolic compounds with estrogenic activity is reported. Cyclic voltammograms for phenolic endocrine disruptors and estrogenic hormones showed, in general, an enhancement of their electrochemical oxidation responses at CNT-GCE attributable to the electrocatalytic effect caused by CNTs. Hydrodynamic voltammograms obtained under flow injection conditions lead to the selection of +700 mV as the potential value to be applied for the amperometric detection of the phenolic estrogenic compounds, this value being remarkably less positive than those reported in the literature using other electrode materials. Successive injections of these compounds demonstrated that no electrode surface fouling occurred. A mobile phase consisting of a 50:50 (v/v) acetonitrile:0.05 mol l⁻¹ phosphate buffer of pH 7.0 was selected for the chromatographic separation of mixtures of these compounds, with detection limits ranging between 98 and 340 nmol l⁻¹. Good recoveries were obtained in the analysis of underground well water and tap water samples spiked with some phenolic estrogenic compounds at a 14 nmol l⁻¹ concentration level.     

Structures and chromatographic characteristics of capsule-type silica gels coated with hydrophobic polymers in reversed-phase liquid chromatography

Summary Silicone polymer-coated silica gels modified with octadecyl and octyl groups (S/S-C₁₈, S/S-C₈), or “capsule-type silica gels” were developed as packing materials for reversed-phase liquid chromatography. They were obtained by coating the surface of totally porous silica gel with a homogeneous silicone polymer film, and thereafter modifying the coating polymer with octadecyl or octyl groups. Retaining the advantages of silica-based packings, they show strong resistance of alkali-like organic porous polymeric materials.     

Liquid chromatographic retention and separation of phenols and related aromatic compounds on reversed phase columns

Summary Isocratic column liquid chromatographic systems with UV absorbance detection at 280 nm have been developed for the separation of 29 phenolics and related compounds.The selectivity was investigated on silica-, carbon- and polymer-based separation columns for the separation of phenolic type of components. The effects of various acetonitrile/buffer mixtures, and pH of the mobile phase, and their impact on the retention of the phenols was assessed. Tables of retention times on the four columns for the 29 phenols with two different acetonitrile/buffer mixtures, together with the retention times at three pHs from 6.5 to 2.3 with varying levels of organic modifier on the LiChrospher RP 18 column are presented.As an application, the analysis of real river water samples from the Ebro river is described using a solid phase extraction step prior to injection into the chromatographic system.     

Analysis of basic compounds at high pH values by reversed-phase liquid chromatography

Reversed phase high performance liquid chromatography (RPLC) is currently the method of choice for the analysis of basic compounds. However, with traditional silica materials, secondary interactions between the analyte and residual silanols produce peak tailing which can negatively affect resolution, sensitivity, and reproducibility. In order to reduce these secondary interactions, which comprise ion exchange, hydrogen bonding, and London forces interactions, chromatographic analyses can be carried out at low or high pH values where silanol groups and basic compounds are mostly uncharged. The chromatographic behaviour of a particular bidentate stationary phase, Zorbax Extend C18, was studied with a set of basic and neutral compounds. Thanks to a higher chemical stability than traditional silica based supports, analyses were carried out with a high pH mobile phase, which represents a good alternative to the acidic mobile phases generally used to reduce ion exchange interactions. The performance of this bidentate stationary phase was also compared with that of other supports and it was proved that it is advantageous to work with high pH mobile phases when analyzing basic compounds.     

固相萃取-液相色谱-串联质谱法同时测定尿液中9种多环芳烃代谢物

建立了固相萃取-液相色谱-串联质谱同时测定尿中2-羟基萘、1-羟基萘、2-羟基芴、3-羟基菲、1-羟基芘等9种多环芳烃代谢物的液相色谱-串联质谱测定方法。尿样中结合态的多环芳烃代谢物在β-葡萄糖苷酸酶-芳基硫酸酯酶缓冲液(pH 5.0)作用下,于37℃水浴中避光水解4 h后,以C18固相萃取小柱富集、净化,以甲醇洗脱,采用Waters Symmetry C18色谱柱,流动相为乙腈-0.2%氨水(72∶27,V/V)等度淋洗分离后进入质谱测定。在喷雾电压4 kV,毛细管温度300℃下,以3-羟基菲13C为内标,采用SRM模式负离子扫描方式测定,内标法定量。9种多环芳烃代谢物在尿中的线性范围为0.90～100μg/L;相关系数为0.9970～0.9990;回收率为79.0%～119.8%;相对标准偏差为4.3%～12.4%;检出限为0.04～0.90μg/L;结果表明,本方法可用于尿中9种多环芳烃代谢物的测定。     

Measurement of Retention Indexes on n-Alkane Scale in Reversed-Phase Liquid Chromatography and their Prediction from Molecular Structures

Abstract

A method was proposed in which the retention indexes (RI) defined on the n-alkane scale were measured by using 1-phenylalkane homologous series as reference compounds. Good agreement was observed between the RI values obtained with this method and those determined using n-alkanes as the reference. An attempt was made to predict the RIs based on the additivity for the constitutive atomic groups. The RIs were estimated with considerable accuracy by using increments of substituents themselves and positional increments between substituents. It was possible to distinguish methylnitroaniline isomers by comparison of the calculated RI values with the observed values.     

Liquid chromatography of polymers under limiting conditions of adsorption: III. Role of experimental variables

LC of polymers under limiting conditions of adsorption (LC LCA) is a novel method based on different mobility of (pore excluded) macromolecules compared to (pore permeating) solvent molecules. Polymer sample is injected in a solvent preventing its adsorption within the column. Eluent promotes sample adsorption. Under these conditions, macromolecules cannot leave its initial solvent and elute from the column independently of their molar mass. In contrast, a less interactive simultaneously injected polymer leaves its initial solvent zone and is eluted in the size exclusion mode. As a result, chemically different polymer species can be discriminated. The effect of selected experimental conditions was studied on the LC LCA behavior of poly(methyl methacrylate)s eluted from bare silica gel columns. The parameters were packing pore diameter, injected sample volume and concentration, as well as column temperature. The size independent elution was only little affected by the above parameters and LC LCA produced well-focused peaks. The LC LCA mechanism was operative even at a very large sample of both volume and concentration. This makes LC LCA a robust and user-friendly method, likely suitable also for characterization of minor components of polymer mixtures.     

Indirect detection in liquid chromatography. II. Response models for reversed-phase non-ionic systems

Summary Uncharged solutes devoid of inherent detectable properties can be monitored and quantified in reversed-phase chromatographic systems by including a non-ionic detectable component (probe) in the eluent. Response models have been developed, which are based on the fact that the distribution of the solute and the probe are connected to each other by common interaction effects, such as competition for the binding surface. The direction and magnitude of the indirect response can be predicted quantitatively by means of expressions which clearly show the influence of the retention of the solute relative to the probe as well as the effect of the solid phase coverage by the probe. The relatively low response obtained in non-ionic systems may be due to a low loading of the probe on the solid phase and to a simultaneous distribution of the solute in complexed and uncomplexed forms, the two distribution modes giving opposite response patterns.     

Simultaneous determination of positive and negative pharmaceutical counterions using mixed-mode chromatography coupled with charged aerosol detector

A mixed-mode chromatography coupled with charged aerosol detector (CAD) method was developed in this work to simultaneously determine pharmaceutical counterions including both inorganic ions and organic ions in the forms of cations and anions. 25 commonly used pharmaceutical ions were studied and simultaneously separated within 20 min by this single method. A silica based mixed-mode column with reversed-phase/cation-exchange/anion-exchange modes was used. It provides reversed-phase, strong cation-exchange and weak anion-exchange properties at the same time. It also provides the HILIC behavior at high percentage of organic solvent. The effects of mobile-phase organic strength, buffer ions, ionic strength, pH and column temperature have been investigated to optimize the method as well as to understand the retention and separation mechanisms. Conventional HPLC system was used and no special chromatography system is needed. The presented method has been employed successfully for screening and quantitative analysis of counterions, unknown ionic impurities and salts in active pharmaceutical ingredients and in process control samples with excellent accuracy, precision and sensitivity. This method provides a simple, fast and generic approach to speed up pharmaceutical research and development process and enhance lab efficiency. The similar methodologies can be applied to other ion analysis.     

Determination of phenolic compounds in water samples by on-line solid-phase extraction—supercritical-fluid chromatography with diode-array detection

Summary The eleven Environmental Protection Agency (EPA) priority phenolic compounds have been determined by solid-phase extraction (SPE) coupled on-line to supercritical-fluid chromatography (SFC) with diodearray detection. The variables affecting chromatographic separation were optimized and the analytes were separated at 40 °C in two diol columns connected in series; a gradient of methanol, as modifier, and CO₂ was used as mobile phase. Under these conditions, all the compounds studied were separated to baseline in less than 13 min. PLRP-S and LiChrolut EN were tested as sorbents in a 10×3 mm i.d. laboratory-packed precolumn for solid-phase extraction. An ion-pair reagent, tetrabutylammonium bromide (TBA), was used in the extraction process to increase break-through volumes. The performance of the method was checked with tap and river waters and the pre-concentration of 20 mL of sample in a PLRP-S pre-column enabled phenolic compounds to be determined at low μg L⁻¹ levels with limits of detection ranging between 0.4 and 2 μg L⁻¹. The repeatability and reproducibility between days (n=3) for real samples spiked at 10 μg L⁻¹ were lower than 10%.     

Liquid chromatography of dextrans on porous silica beds

Kinetics, equilibrium isotherms and chromatography retention times for sorption of dextrans T-10, T-20, T-40, T-70, T-110, T-161, T-250 and T-500 on porous silica were measured at 25 degrees C. The Henry constant and retention factors for the dextrans were obtained. The values of the partition coefficient for the distribution of the dextrans between the bulk solution and the pore space were calculated within the framework of a pore volume filling model with consideration given to the ratio between the sizes of the macromolecular coils and the pore inlet. The measurements showed that this parameter depends on the structure of the sorbent and the molecular mass distribution of the dextran. The interaction of aqueous dextran solution with porous silica is characterized by the sieve effect. Large macromolecular coils of dextran T-161 cannot penetrate into the pore space of the silica sorbent with pore diameter 14 nm. The difference in Henry law constants calculated from adsorption and chromatographic data for dextrans T-70 and T-110 can be explained by the slow diffusion of dextran macromolecules into silica pores under chromatographic conditions.