Preparation of a mixed-mode hydrophilic interaction/anion-exchange polymeric monolithic stationary phase for capillary liquid chromatography of polar analytes

A novel cationic hydrophilic interaction monolithic stationary phase based on the copolymerization of 2-(methacryloyloxy)ethyltrimethylammonium methyl sulfate (META) and pentaerythritol triacrylate (PETA) in a binary porogenic solvent consisting of cyclohexanol/ethylene glycol was designed for performing capillary liquid chromatography. While META functioned as both the ion-exchange sites and polar ligand provider, the PETA, a trivinyl monomer, was introduced as cross-linker. The monolithic stationary phases with different properties were easily prepared by adjusting the amount of META in the polymerization solution as well as the composition of the porogenic solvent. The hydrophilicity of the monolith increased with increasing content of META in the polymerization mixture. A typical hydrophilic interaction chromatography mechanism was observed when the content of acetonitrile in the mobile phase was higher than 20%. The poly(META-co-PETA) monolith showed very good selectivity for neutral, basic and acidic polar analytes. For polar-charged analytes, both hydrophilic interaction and electrostatic interaction contributed to their retention. Peak tailing of basic compounds was avoided and the efficient separation of benzoic acid derivatives was obtained.     

Sweeping of neutral analytes via complexation with borate in capillary zone electrophoresis

Summary The sweeping concept is extended to capillary zone electrophoresis (CZE) separation of neutral solutes involving complexation with borate. Analogous to the pseudostationary phase in electrokinetic chromatography (EKC), the complexing agent (borate) serves as carrier for sweeping and separation in CZE. Therefore, similar to the retention factor in EKC, the focusing effect in the present system is directly related to the association constant between the analyte and complexing agent. Theoretical and some preliminary experimental studies gerenally suggest that the electrophoretic mobility of the complex and the concentration of the complexing agent affect the resulting length of narrowed zones. Moreover, sweeping using borate is affected by pH since borate complexation is pH dependent. From around 10 to 40-fold improvement in peak heights has been observed experimentally for some neutral test analytes (monosaccharides, catechols, and nucleosides)     

On-line multidimensional chromatography using packed capillary liquid chromatography and capillary gas chromatography

The introduction of selected fractions from a liquid chromatograph into a gas chromatograph has been described; however, analyses were performed by off-line experiments requiring collection and reinjection of the separate fractions or by on-line procedures where disadvantageously, only a fraction of the separated peak or a well resolved component in a mixture could be introduced into a gas chromatograph. This disadvantage is overcome by the apparatus and method described in this paper, which utilizes a multidimensional chromatography system employing a high efficiency, packed capillary LC column coupled on-line to a capillary gas chromatograph. The liquid chromatograph (so designed) can act as a highly efficient clean-up or chemical class fractionation step prior to introduction into the gas chromatograph, significantly reducing sample preparation times in many applications. Thus minor components in a complex matrix can be determined without prior sample clean-up, an example of which is the determination of polychlorinated biphenyls in a complex hydrocarbon matrix.     

Application of open tubular electrochromatography for acidic and basic analytes

Summary The separation of charged analytes by electrochromatography has been examined on porous-layer open tubular capillaries prepared using the sol gel method. An electroosmotic flow of about 2.10×10⁴ cm²V¹s¹ was obtained using between 10 and 30% acetonitrile in the mobile phase. Acidic diuretic drug compounds were successfully separated at high pH as were theN-alkylanilines in their basic and neutral forms. The limitation of open tubular columns was observed on separating some basic pharmaceutical drugs. These components showed severe peak tailing and were not resolved on a 20 μm i.d. porous silica layer open tubular column. Strongly acidic components could not be detected on these columns due to their higher counter electromobilities. The successful separation of neutral aryl alkyl ketones with an efficiency of 101,533 plates m¹ for butyrophenone was an indication of the improved phase ratio on this type of open tubular columns.     

Peak doubling in preparative liquid chromatography

Summary Peak doublets in LC are ascribed to longitudinal column zones of different permeability leading to a double front in the column.     

System peaks observed in capillary liquid chromatography with eluents containing triethylamine

Summary Triethylamine is often added to mobile phases in reversed-phase liquid chromatography for dynamic deactivation of free silanol groups of the stationary phase. It has been observed that eluents composed of methanol and triethylamine generate two system peaks in chromatograms obtained with LiChrosorb RP-select B stationary phase, whose retention times correspond to the dead time and to the retention time of triethylamine. It has been demonstrated that the system peaks can be positive or negative depending on the experimental conditions and may be incorrectly interpreted as peaks corresponding to sample components. An approach is outlined to unambiguous identify these system peaks in chromatograms of practical samples.     

The special effect of fluoride on the chromatography of acidic analytes on polybutadiene-coated zirconia

Summary The special effect of fluoride as a Lewis base additive in suppressing the ligand-exchange interactions for acidic analytes on polybutadiene-coated zirconia (PBD-ZrO₂) has been investigated. We found that fluoride is more effective than phosphate in improving the separation efficiency for strong acids. The improvement is attributed to that fluoride has a smaller size and a more flexible coordination chemistry towards zirconium centers than phosphate; consequently, fluoride can more effectively improve the kinetics of the ligand-exchange processes for strongly acidic analytes. We demonstrated that using a small amount of fluoride in combination with a larger quantity of phosphate is a practical way to improve the separation efficiency and resolution for acidic analytes. Some example separations are presented.     

Cross validation of capillary electrophoresis and high-performance liquid chromatography for cefotaxime and related impurities

Summary A micellar electrokinetic chromatography method is presented which permits separation of cefotaxime and its major related impurities. Separation was carried out at 15 kV, using 30 mM sodium dihydrogen phosphate adjusted to pH 7.2 with 5 M NaOH and which contained 165 mM sodium dodecylsulfate as electrolyte. Results obtained by capillary electrophoresis were in good agreement with those of high-performance liquid chromatography with respect to the level of the major known impurities, total impurity content and cefotaxime purity.     

Quantitative aspects of electrolysis in electromembrane extractions of acidic and basic analytes

Electrolysis is omnipresent in all electrochemical processes including electromembrane extraction (EME). The effects of electrolysis on quantitative aspects of EME were comprehensively evaluated for a set of acidic (substituted phenols) and basic (basic drugs) analytes. EMEs were carried out across supported liquid membranes formed by 1-ethyl-2-nitrobenzene at standard EME conditions, i.e., acidic analytes were extracted from alkaline into alkaline solutions and basic analytes were extracted from acidic into acidic solutions. Electric potential applied across the EME systems was 50 V and extraction recoveries of analytes as well as pH values of donor and acceptor solutions were determined after each EME. It has been proven that electrolysis plays a more significant role than has ever been thought before in EME. Electrolytically produced H⁺ and OH^– ions had a significant effect on pH values of acceptor solutions and variations of up to 8.5 pH units were obtained at standard EME conditions. pH values of donor solutions were affected only negligibly due to their significantly higher volumes. The observed variations in pH values of acceptor solutions had fatal consequences on quantitative EME results of weak and medium strong acidic/basic analytes. A direct relation was observed between the decrease in extraction recoveries of the analytes, their pK_a values and the acceptor solution pH values. Acceptor solutions consisting of high concentrations of weak bases or acids were thus proposed as suitable EME operational solutions since they efficiently eliminated the electrolytically induced pH variations, offered stable EME performances and were easily compatible with subsequent analytical methods.     

Suitability of automated capillary and micro liquid chromatography for routine determination of drugs in human plasma samples from clinical pharmacokinetic investigations

One of the most widely acclaimed features of capillary and microcolumn LC, in comparison with conventional HPLC, is the enormous increase in mass sensitivity. Nevertheless, application of capillary and micro LC in quantitative trace bioanalysis, characterized by weak analyte concentrations in complex matrices, can only be of any practical utility if large sample volumes can be injected onto the columns without affecting chromatographic resolution and efficiency. Two applications of large volume injection in a non-eluting solvent (on-column focusing) for the quantitative analysis of drugs in biological fluids on both capillary and micro chromatographic systems are presented: the first example deals with a new selective H₁-antihistaminic drug, mizolastine, the second one with a well known calcium antagonist, diltiazem, and its main metabolites. For both compounds, results obtained on micro and capillary LC in comparison with conventional HPLC are reported. The results demonstrate that when conventional HPLC methods are transformed into either micro or capillary LC techniques, they gain in sensitivity. By means of an on-column focusing technique, it is possible to increase the sensitivity 3–5 fold in comparison to conventional HPLC methods, but not 50–60 fold as obtained on synthetic drug solutions. Column robustness, handiness, reproducibility, and suitability of micro systems for routine bioanalysis are discussed for both capillary and micro LC columns, as well as limits of the technique in trace organic analysis problems.     

Generation of split-flow micro-gradients for capillary HPLC

Summary The generation of a micro-gradient for micro HPLC capillary columns is described. The system used is a modified and improved version of a split flow gradient elution device. Gradient tests have been performed and have shown good reproducibility under all conditions. The system has been successfully coupled with a particle beam LC-MS interface and the analysis of a mixture of twenty seven basic or neutral pesticides is reported.     

Use of high speed liquid chromatography (HSLC) in the pharmaceutical industry. Practical aspects and limitations

Summary High performance liquid chromatography (HPLC) is widely used in drug development and quality control. The main objective is to obtain dependable results in the shortest time in order to reduce the time taken to develop drugs and to ensure better quality of the final compound. High speed liquid chromatography (HSLC) helps us to reach this objective. In this paper pratical aspects of HSLC are discussed mainly in terms of instrument requirements and the practical needs of users. Comparison between HPLC and HSLC separations of pharmaceutical preparations are given and discussed. The contribution of HSLC to decrease solvent consumption and consequently analysis cost and its role in reducing environmental pollution, are also mentioned.     

Extracolumn band broadening in capillary liquid chromatography

A commercially available capillary LC instrument was modified to investigate and control the contribution of different instrument components on extracolumn band broadening. Quantitative estimations of dispersion induced by several equipmental parts were carried out. Injection parameters could be optimized to achieve the theoretical value of 12 for a profile factor describing a rectangular sample profile. Additionally, an additive injector flow channel dependent dispersion effect was found. A practical approach for minimizing instrumental effects in capillary LC is suggested. The results were compared with those obtained with an HPLC instrument designed for conventional size columns.     

Acid-induced transient isotachophoretic stacking of basic drugs in co-electroosmotic flow capillary zone electrophoresis

Online sample concentration or stacking of basic drugs by transient isotachophoresis with the injection of an acid in co-electroosmotic flow capillary zone electrophoresis was studied experimentally and with computer simulation. The acid stacking strategy afforded an order of magnitude improvement in concentration sensitivity for model tricyclic antidepressant and β blocker drugs.     

Effect of column diameter on efficiency in capillary supercritical fluid chromatography

Fused silica capillary columns with internal diameters from 100 to 25 μm were coated with SE-54 and evaluated under supercritical fluid chromatographic conditions using carbon dioxide as mobile phase. Experimental results compared well with theoretical predictions. At ten times the optimum mobile phase velocity and for a capacity factor, k of 3, efficiencies of 2300 to 5600 plates m^?1 were obtained for column diameters of 100 to 25 μm, respectively.     

毛细管液相色谱法分离植物内源激素

建立了毛细管液相色谱法(CLC)同时分离测定4种植物内源激素的方法.采用硅胶基质ODS整体柱(27 cm×100 μm i.d.)作为分离柱,以带有光程为3 mm的光纤检测池的紫外检测器作为检测手段,在室温下以含10 mmol/L乙酸(pH 3.0)的V(甲醇):V(水)＝35:65为流动相,以0.6 μL/min的流速进行等度洗脱.采用溶剂梯度效应和扩展光程的检测池来提高检测灵敏度,该方法测定4种植物内源激素的检出限(S/N=3)在27.7～196.1 ng/mL之间,峰面积和保留时间的相对标准偏差(RSD)小于2.8%.方法已用于不同种类玉米样品的测定.     

Potential of microcolumn liquid chromatography and capillary electrophoresis with flame photometric detection for determination of polar phosphorus-containing pesticides

Summary The potential of microcolumn liquid chromatography (μlC) and capillary electrophoresis (CE) with on-line, flame photometric detection (FPD) in the P-selective mode has been studied for determination of polar P-containing pesticides, glyphosate and its main metabolite, aminomethylphosphonic acid (AMPA), ethephon, fosetyl-aluminium and acephate. Acephate was determined by reversed-phase μLLC-FPD using large-volume injections with peak compression, the other compounds were determined by μLC-FPD and CE-FPD using simple, large-volume injection procedures to obtain limits of detection of 7.5-500 ng·mL⁻¹ and 1.0 μ·mL⁻¹, respectively. The methods showed acceptable repeatability and robustness and were successfully applied for rapid and selective determination of pesticides in fruit, vegetable and water samples.     

Automated solid-phase extraction for determination of amodiaquine, chloroquine and metabolites in capillary blood on sampling paper by liquid chromatography

Summary A bioanalytical method using solid-phase extraction and high-performance liquid chromatography (LC) has been developed and validated for the determination of amodiaquine, chloroquine and their metabolites in 100 μL blood applied to sampling paper. The intra-assay precision for all analytes was <5% at 2000 nM, <7% at 750 nM and <10% at 100 nM. The interassay precision for all analytes was <4% at 2000 nM, <7% at 750 nM and <12% at 100 nM. The lower limit of quantitation was 100 nM for all analytes. The limit of detection was 30 nM in 100 μL venous blood applied to sampling paper.     

Siloxane/silarylene copolymers as stationary phases for capillary gas chromatography part I: Evaluation of silanol terminated dimethyl substituted polymers

The thermal stability of silicones can be improved on replacement of certain of the oxygen atoms in the polymer backbone by phenyl groups. Such a polymer has been synthesized and evaluated for use as stationary phase in fused silica capillary gas chromatography; the polymer was dimethyl substituted and silanol terminated. A selectivity was provided by the phenyl groups in the backbone. For comparative purposes, a silanol-terminated dimethylpolysiloxane has also been evaluated. Both stationary phases gave columns of highest separation efficiency and the supporting fused silica surface was deactivated by the stationary phases on thermal treatment. Further, low column bleeding was observed at the maximum temperature tested, 370°C. The phenyl-containing phase could be immobilized to 60% by heat treatment, but the pure dimethylpolysiloxane was 10% immobilized. The influence on immobilization of factors such as nature of the supporting surface, stationary phase silanol content, reaction temperature and atmosphere in the column during reaction has been studied.