Simultaneous stacking of cationic and anionic compounds in single run capillary zone electrophoresis by two-end field amplified sample injection

In order to extend the application of field amplified sample injection (FASI) in high throughput analysis, a convenient and simple procedure, namely two-end field amplified sample injection (TE-FASI), was developed for the simultaneous stacking of cationic and anionic compounds in a single run capillary zone electrophoresis (CZE). Following the capillary-filling with a buffer of high conductivity, water plug was loaded into each end of the capillary; and two high-field strength zones were generated at both heads of the column when high voltage was applied. Therefore, under suppressed EOF cations and anions can be selectively FASI stacked at anode and cathode head, respectively. After separation, the stacked anions and cations are detected by a common detector placed in the center of the capillary. Under the optimized conditions, the limits of detection for the model cationic (matrine and oxymatrine) and anionic (5-sulfosalicylic acid) compounds were determined as 0.2, 0.2 and 0.06 ng/mL, respectively. Compared with non-stacking conditions, the sensitivities of these compounds were enhanced 1003-, 1330- and 1380-fold, respectively. The results of reproducibility, linearity and real sample analysis show that the proposed procedure is promising to be applied for the simultaneous quantification detection of trace cationic and anionic analytes.     

Fundamental studies on electrokinetic chromatography with PEGylated phospholipid micelles.

In this study, micelles prepared from distearoylphosphatidylethanolamine with covalently attached poly(ethylene) glycol) (PEG) of molecular weight 2000 (DSPE-PEG-2000) were employed in micellar electrokinetic chromatography (MEKC) as pseudostationary phases. Since DSPE-PEG-2000 contains long hydrophobic alkyl chains, an anionic phosphate group, and hydrophilic PEG chains, the prepared micelles are expected to provide a characteristic retention behavior for both neutral and ionic compounds. As a typical example, a baseline separation of phenol and 2-naphthol was successfully achieved by using the DSPE-PEG-2000 micelles as a background electrolyte for MEKC; such success clearly shows that the micelles can retain electrically neutral compounds. The MEKC separations of anionic and cationic compounds with a DSPE-PEG-2000 micellar solution and the enantioseparation of binaphthyl compounds with mixed micelles containing bile salt are also discussed.     

Simultaneous determination of artificial sweeteners, preservatives, caffeine, theobromine and theophylline in food and pharmaceutical preparations by ion chromatography.

A novel ion chromatographic method was proposed for the simultaneous determination of artificial sweeteners (sodium saccharin, aspartame, acesulfame-K), preservatives (benzoic acid, sorbic acid), caffeine, theobromine and theophylline. The separation was performed on an anion-exchange analytical column operated at 40 degrees C within 45 min by an isocratic elution with 5 mM aqueous NaH2PO4 (pH 8.20) solution containing 4% (v/v) acetonitrile as eluent, and the determination by wavelength-switching ultraviolet absorbance detection. The detection limits (signal-to-noise ratio 3:1) for all analytes were below the sub-microg/ml level. Under the experimental conditions, several organic acids, including citric acid, malic acid, tartaric acid and ascorbic acid, did not interfere with the determination. The method has been successfully applied to the analysis of various food and pharmaceutical preparations, and the average recoveries for real samples ranged from 85 to 104%. The levels of all analytes determined by this method were in good agreement with those obtained by the high-performance liquid chromatographic procedure. The results also indicated that ion chromatography would be possibly a beneficial alternative to conventional high-performance liquid chromatography for the separation and determination of these compounds.     

Simultaneous analysis of basic, acidic and neutral compounds on an endcapped octadecylsilane silica-based monolith by pressure-assisted capillary electrochromatography

This simultaneous separation of basic, acidic and neutral analytes by pressure-assisted CEC (pCEC) using a hybrid (tetramethoxysilane and methyltrimethoxysilane) silica-based monolith, chemically modified with octadecyldimethylchlorosilane followed by endcapping with hexamethyldisilazane is described. The endcapping resulted in near Gaussian peaks for highly basic analytes such as nortriptyline without a significant loss in the EOF. The migration behaviour of analytes on this phase could be rationalised based on hydrophobicity, electrophoretic mobility and ion-exchange interactions. The high porosity of the monolith allowed manipulation of the linear velocity of mobile phases by the addition of varying amounts of pressure at the inlet to reduce analysis times and overcome the reversed migration of anionic species towards the detection window in cathodic EOF mode. The concomitant programmed application of pressure (2-4 bar) and voltage (27 kV) facilitated the simultaneous separation of four cationic, four neutral and two anionic compounds in 6 min with efficiencies ranging from 41 000 to 94 000, 57 000 to 77 000 and 180 000 to 210 000 theoretical plates/metre, respectively. The % RSD values of migration times and efficiencies in pCEC mode were less than 3.6 and 7.9%, respectively (n = 5).     

CE-ESI-MS of biological anions in plastic capillaries at high pH

In this study, the potential of poly(methylmethacrylate) (PMMA, Plexiglas) and polyether ether ketone (PEEK) tubing for CE-ESI-MS separations of anions at high pH values was examined. A set of model compounds of biological interest was used to investigate the main operational parameters for CE-ESI-MS, such as the sheath-flow interface design, the polarity of the ionization voltage, the use of ammonia-based separation electrolytes, and the sheath liquid composition. Optimum separations and detection sensitivities in negative ESI mode were obtained using a running electrolyte of 75 mM of ammonia at pH 11 and a sheath liquid of 60:40 v/v or 75:25 v/v isopropanol/water with 0.5% v/v of ammonia. At these experimental conditions, PMMA and PEEK capillaries show good hydrolytic stabilities and lower EOF values than fused-silica columns. Better separation resolutions were obtained with PMMA capillary, but this plastic rapidly swelled and bled because of its limited chemical resistance to the sheath liquid. PMMA columns equipped with a fused-silica tip were used for a safer exposure to the sheath liquid, but the inner surface of the fused-silica tips had limited stability at pH 11. On the other hand, good separations and reproducibility on migration times and peak areas were obtained using PEEK capillaries without capillary column deterioration.     

Capillary electrochromatography with zwitterionic stationary phase on the lysine-bonded poly(glycidyl methacrylate-co-ethylene dimethacrylate) monolithic capillary column

A polymer-based neutral monolithic capillary column was prepared by radical polymerization of glycidyl methacrylate and ethylene dimethacrylate in a 100 mum id fused-silica capillary, and the prepared monolithic column was subsequently modified based on a ring opening reaction of epoxide groups with 1 M lysine in solution (pH 8.0) at 75 degrees C for 10 h to produce a lysine chemically bonded stationary phases in capillary column. The ring opening reaction conditions were optimized so that the column could generate substantial EOF. Due to the zwitterionic functional groups of the lysine covalently bonded on the polymer monolithic rod, the prepared column can generate cathodic and anodic EOF by varying the pH values of running buffer during CEC separation. EOF reached the maximum of -2.0 x 10(-8) m2v(-1)s(-1) and 2.6 x 10(-8) m2v(-1)s(-1) with pH of the running buffer of 2.25 and 10, respectively. As a consequence, neutral compounds, ionic solutes such as phenols, aromatic acids, anilines, and basic pharmaceuticals were all successfully separated on the column by CEC. Hydrophobic interaction is responsible for separation of neutral analytes. In addition, the electrostatic and hydrophobic interaction and the electrophoretic migration play a significant role in separation of the ionic or ionizable analytes.     

Octyl-type monolithic columns of 530 microm i.d. for capillary liquid chromatography

A novel monolithic capillary column (530 microm i.d.) was prepared for capillary liquid chromatography (CLC) by in situ copolymerization of octyl methacrylate (MAOE) and ethylene dimethacrylate (EDMA) in the presence of a porogen solvent containing 1-propanol, 1,4-butanediol, and water with azobisisobutyronitrile as the initiator. The influences of the contents of the porogen solvent, EDMA and the various concentration ratios of 1-propanol to 1,4-butanediol in the polymerization mixture on the morphology, porosity, globule size, stability and column efficiency were investigated. The morphology and pore size distribution of monolithic capillary columns were characterized by SEM and mercury intrusion porosimetry, respectively. Chromatographic evaluations of the columns were performed under CLC mode. The results showed that good permeability and stability can be obtained under optimal experimental conditions. The separation results of some acid, neutral and basic analytes demonstrated the hydrophobicity and low affinity to basic analytes of the new column. Three metal ions, i.e. Mg(II), Zn(II) and Cd(II) were also separated under ion-pair mode on the new monolithic capillary column and the results were acceptable.     

Routine analysis of catecholamines and metabolites in urine by a liquid chromatographic column switching system

A reversed-phase liquid chromatographic technique including a column switching system has been adapted for the routine measurement of catecholamines and their metabolites (14 compounds) in urine. From 1 ml of urine all the compounds and the internal standards were obtained according to combined extraction procedures involving organic solvent, anionic and weakly cationic resins. Finally four extracts (catecholamines, methoxamines, acidic and neutral derivatives) had to be chromatographed throughout a wholly automated apparatus. For each run, the column switching system determined the analytical columns to be used to obtain the separation of the compounds from interferences due to other co-extracted endogenous substances, while the analysis times remained between 20 and 40 min. Such a system allowed the rapid clean-up of columns (in direct- and back-flush mode) carried out between two consecutive injections. By coupling on-line fluorimetric and electrochemical detections the specificity of the technique could be checked, since the ratio of the responses of both detectors was an index of the purity of the peaks. Finally the advanced automation of the equipment allowed weekly the evaluation of catecholamines and the whole range of their known metabolites in 36 urine samples.     

Dual-opposite injection electrokinetic chromatography for the unbiased, simultaneous separation of cationic and anionic compounds

The concept of dual opposite injection in capillary electrophoresis (DOI-CE) for the simultaneous separation, under conditions of suppressed electroosmotic flow, of anionic and cationic compounds with no bias in resolution and analysis time, is extended to a higher pH range in a zone electrophoresis mode (DOI-CZE). A new DOI-CE separation mode based on electrokinetic chromatography is also introduced (DOI-EKC). Whereas conventional CZE and DOI-CZE are limited to the separation of charged compounds with different electrophoretic mobilities, DOI-EKC is shown to be capable of separating compounds with the same or similar electrophoretic mobilities. In contrast to conventional EKC with charged pseudostationary phases that often interact too strongly with analytes of opposite charge, the neutral pseudostationary phases appropriate for DOI-EKC are simultaneously compatible with anionic and cationic compounds. This work describes two buffer additives that dynamically suppress electroosmotic flow (EOF) at a higher pH (6.5) than in a previous study (4.4), thus allowing DOI-CZE of several pharmaceutical bases and weakly acidic positional isomers. Several DOI-EKC systems based on nonionic (10 lauryl ether, Brij 35) or zwitterionic (SB-12, CAS U) micelles, or nonionic vesicles (Brij 30) are examined using a six-component test mixture that is difficult to separate by CZE or DOI-CZE. The effect of electromigration dispersion on peak shape and efficiency, and the effect of surfactant concentration on retention, selectivity, and efficiency are described.     

Enantioselective ion-exclusion chromatography on teicoplanin aglycone and (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid stationary phases

Ion-exclusion chromatography is a well established technique for the analysis of achiral ionic species, but it has rarely been applied to chiral analytes. In this paper enantioselective ion-exclusion separations were developed on two commercially available HPLC phases: Chirobiotic TAG, based on teicoplanin aglycone, and Opticrown RCA (+), based on (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid. Chirobiotic TAG columns have a carboxylic acid group on the chiral ligand, which can be partially ionized to exclude anionic analytes by ionic repulsion. Under acidic conditions Opticrown columns have a cationic sublayer generated from the aminopropyl base silica that excludes cationic analytes. Both columns demonstrate a large dependence of efficiency on flow-rate, with the highest efficiencies at 0.1 ml/min on a 4.6 mm inner diameter column.     

Approach to hydrophilic interaction chromatography column selection: Application to neurotransmitters analysis

This paper presents the comparison between 12 hydrophilic interaction liquid chromatography columns that are commercially available. The main factors influencing the retention and selectivity toward 12 neurotransmitters for these different HILIC systems have been studied. For additional information, the retention data have been analyzed statistically by factor analysis. Principal component analyses (PCA) were calculated to evidence different separation behaviour between the stationary phases, based on the retention data measured for three compound classes: anionic acidic compounds, cationic basic compounds and zwitterionic amino acids. Finally, a generic procedure is suggested for optimization of HILIC analyses, depending on the ionization state of the analytes.     

Retention Mechanism Studies of Selected Amino Acids and Vitamin B6 on HILIC Columns with Evaporative Light Scattering Detection

The goal of the study was to investigate separation mechanism of selected “essential” amino acids (leucine, isoleucine, threonine, tryptophan, proline, and glycine) and vitamin B6 in hydrophilic interaction liquid chromatography (HILIC) with the evaporative light scattering detection. Chromatographic measurements were made on three different HILIC columns: amide-silica (TSK-gel Amide-80), amino-silica (TSK-gel NH₂-100), and cross-linked diol (Luna HILIC). The retention behaviour of the analytes was investigated as a function of different binary hydro-organic mobile phases containing 10–90 % (v/v) acetonitrile. The compounds studied were separated under isocratic and gradient conditions. The best results of tested biologically active compounds separation were obtained on the TSK-gel NH₂-100 column. TSK-gel NH₂ column showed mixed HILIC–ion-exchange mechanism, the highest separation efficiency and better selectivity and resolution for tested analytes than the other studied column, especially at concentration of water in mobile phase lower than 30 % (v/v). Special attention was dedicated to the study of interactions among the stationary phase, mobile phase and the analytes.     

Rapid separation and sensitive detection method for beta-blockers by pressure-assisted capillary electrochromatography-electrospray ionization mass spectrometry

A new method for rapid separation and sensitive detection of beta-blockers by pressure-assisted capillary electrochromatography (pCEC) with electrospray ionization mass spectrometry (ESI-MS) using silica-based monolithic column was studied in this paper. The proposed method has been confirmed to be very powerful since the fast mass transfer property and good permeability of silica monolithic column was used in this pCEC-ESI-MS system. In this work, a silica monolithic column was prepared with sol-gel method for simultaneous fast separation of beta-blockers. Furthermore, in order to obtain the highly selective and sensitive result of pCEC-ESI-MS, both the CEC separation and MS detection parameters were optimized in detail. Under the optimized conditions, namely 80% acetonitrile and 20% 20 mmol/L ammonium acetate (pH 6.0) as the mobile phase, 20 kV and 8 bar as the separation voltage and the assisted pressure, isopropanol/water (1:1, v/v) containing 7.5 mmol/L acetic acid as the sheath liquid, and 3 microL/min as the flow rate of sheath liquid, seven beta-blockers were well separated within 11 min with detection limits in the range of 0.15-0.80 ng/mL (defined as S/N=3). The recoveries of spiked urine samples of these beta-blockers were between 86.3 and 103% with the RSDs lower than 8.0%. The real samples from some male volunteers were successfully analyzed and confirmed with the proposed method. Comparing with GC-MS or LC-MS, the new method has some superiority (such as fast analysis capacity and simple pretreatment) in clinical practice and doping control.     

Retention Mechanism Studies of Selected Amino Acids and Vitamin B6 on HILIC Columns with Evaporative Light Scattering Detection

The goal of the study was to investigate separation mechanism of selected “essential” amino acids (leucine, isoleucine, threonine, tryptophan, proline, and glycine) and vitamin B6 in hydrophilic interaction liquid chromatography (HILIC) with the evaporative light scattering detection. Chromatographic measurements were made on three different HILIC columns: amide-silica (TSK-gel Amide-80), amino-silica (TSK-gel NH₂-100), and cross-linked diol (Luna HILIC). The retention behaviour of the analytes was investigated as a function of different binary hydro-organic mobile phases containing 10–90 % (v/v) acetonitrile. The compounds studied were separated under isocratic and gradient conditions. The best results of tested biologically active compounds separation were obtained on the TSK-gel NH₂-100 column. TSK-gel NH₂ column showed mixed HILIC–ion-exchange mechanism, the highest separation efficiency and better selectivity and resolution for tested analytes than the other studied column, especially at concentration of water in mobile phase lower than 30 % (v/v). Special attention was dedicated to the study of interactions among the stationary phase, mobile phase and the analytes.

     

Routine Analysis of Catecholamines and Metabolites in Urine by a Liquid Chromatographic Column Switching System

Abstract

A reversed-phase liquid chromatographic technique including a column switching system has been adapted for the routine measurement of catecholamines and their metabolites (14 compounds) in urine. From 1 ml of urine all the compounds and the internal standards were obtained according to combined extraction procedures involving organic solvent, anionic and weakly cationic resins. Finally four extracts (catecholamines, methoxamines, acidic and neutral derivatives) had to be chromatographed throughout a wholly automated apparatus. For each run, the column switching system determined the analytical columns to be used to obtain the separation of the compounds from interferences due to other co-extracted endogenous substances, while the analysis times remained between 20 and 40 min. Such a system allowed the rapid clean-up of columns (in direct- and back-flush mode) carried out between two consecutive injections. By coupling on-line fluorimetric and electro-chemical detections the specificity of the technique could be checked, since the ratio of the responses of both detectors was an index of the purity of the peaks. Finally the advanced automation of the equipment allowed weekly the evaluation of catecholamines and the whole range of their known metabolites in 36 urine samples.     

Determination of trigonelline, nicotinic acid, and caffeine in Yunnan Arabica coffee by microwave-assisted extraction and HPLC with two columns in series

A simple, rapid method was developed for simultaneous extraction of trigonelline, nicotinic acid, and caffeine from coffee, and separation by two chromatographic columns in series. The trigonelline, nicotinic acid, and caffeine were extracted with microwave-assisted extraction (MAE). The optimal conditions selected were 3 min, 200 psi, and 120 degrees C. The chromatographic separation was performed with two columns in series, polyaromatic hydrocarbon C18 (250 x 4.6 mm id, 5 microm particle size) and Bondapak NH2 (300 x 3.9 mm id, 5 microm particle size). Isocratic elution was with 0.02 M phosphoric acid-methanol (70 + 30, v/v) mobile phase at a flow rate of 0.8 mL/min. Good recoveries and RSD values were found for all analytes in the matrix. The LOD of the three compounds was 0.02 mg/L, and the LOQ was 0.005% in the matrix. The concentrations of trigonelline, nicotinic acid, and caffeine in instant coffee, roasted coffee, and raw coffee (Yunnan Arabica coffee) were assessed by MAE and hot water extraction; the correlation coefficients between concentrations of the three compounds obtained were close to 1.     

Using subcritical/supercritical fluid chromatography to separate acidic,basic, and neutral compounds over an ionic liquid-functionalized stationary phase

We packed an ionic liquid (IL)-functionalized stationary phase – based on 1-octyl-3-propylimidazolium chloride covalently bounded to silica gel – into a 3.2 mm × 250 mm column for the simultaneous separation of acidic, basic, and neutral compounds using carbon dioxide subcritical/supercritical fluid chromatography (SFC), and examined the effects of the pressure, temperature, co-solvents, and additives on the retention behavior of the analytes. The model compounds tested for SFC separation are acetaminophen, metoprolol, fenoprofen, ibuprofen, naphthalene, and testosterone. The data indicate that hydrogen-bonding and hydrophobic interactions between the analytes and the IL-modified stationary phase seem to involve in the separation process. Simultaneous separation of acidic, basic, and neutral compounds via SFC was successful at a co-solvent content of 20% MeOH, a pressure of 110 bar, and a column temperature of 35 °C. The relative standard deviations of the retention times and peak areas at 50 ppm were all less than 4 and 8% (n = 6), respectively.     

厚朴酚键合硅胶液相色谱固定相的制备、表征与性能评价

通过γ-巯丙基三甲氧基硅烷(KH-590)的作用, 将具有抗菌功能的中草药厚朴的主要药用成分厚朴酚键合在硅胶表面上, 制备了厚朴酚键合硅胶液相色谱固定相. 采用红外光谱、元素分析和热重分析对该固定相进行了表征. 以苯同系物、5种吡啶、6种苯胺和8种芳香羧酸类化合物为溶质探针, 初步考察了该新型固定相的基本色谱性能, 研究了其对这些化合物的保留机理. 结果表明, 该固定相的反相色谱性能类似于十八烷基键合硅胶固定相(ODS), 分离原理与疏水性作用有关; 另外, 该固定相包含有别于疏水性作用的氢键作用、π-π电荷转移作用和偶极-偶极等作用, 多种作用力使其在分离某些可电离的碱性和酸性化合物时表现出更好的选择性和分离效果. 厚朴酚配体的多种作用位点对快速分离极性芳香化合物有重要贡献.     

Properties of two amide‐based hydrophilic interaction liquid chromatography columns

Hydrophilic interaction liquid chromatography is a separation technique suitable for the separation of moderately and highly polar compounds. Various stationary phases (SPs) for hydrophilic interaction liquid chromatography are commercially available. While the SPs based on the same type of ligand are available from different providers, they can display a distinct retention characteristics and separation selectivity. The current work is focused on characterization and comparison of the separation systems of two amide‐based HPLC columns from two producers, i.e. XBridge Amide column and TSK gel Amide‐80 column. Several characterization procedures (tests) were used to investigate the differences between these columns. The chromatographic behavior of selected analytes indicates that multimodal interactions are responsible for retention and separation on these columns. Multiple testing approaches were used in order to reveal subtle differences between the SPs. Both amide‐based columns showed certain differences in retention, selectivity, and plate counts. Based on the tests used in this study, we conclude that the investigated columns provide a different degree of H‐bonding interactions.     

Mixed-mode reversed-phase and ion-exchange monolithic columns for micro-HPLC

This paper describes the fabrication of RP/ion-exchange mixed-mode monolithic materials for capillary LC. Following deactivation of the capillary surface with 3-(trimethoxysilyl)propyl methacrylate (gamma-MAPS), monoliths were formed by copolymerisation of pentaerythritol diacrylate monostearate (PEDAS), 2-sulphoethyl methacrylate (SEMA) with/without ethylene glycol dimethacrylate (EDMA) within 100 microm id capillaries. In order to investigate the porous properties of the monoliths prepared in our laboratory, mercury intrusion porosimetry, SEM and micro-HPLC were used to measure the monolithic structures. The monolithic columns prepared without EDMA showed bad mechanical stability at high pressure, which is undesirable for micro-HPLC applications. However, it was observed that the small amount (5% w/w) of EDMA clearly improved the mechanical stability of the monoliths. In order to evaluate their application for micro-HPLC, a range of neutral, acidic and basic compounds was separated with these capillaries and satisfactory separations were obtained. In order to further investigate the separation mechanism of these monolithic columns, comparative studies were carried out on the poly(PEDAS-co-SEMA) monolithic column and two other monoliths, poly(PEDAS) and poly(PEDAS-co-2-(methacryloyloxy)ethyl-trimethylammonium methylsulphate (METAM)). As expected, different selectivities were observed for the separation of basic compounds on all three monolithic columns using the same separation conditions. The mobile phase pH also showed clear influence on the retention time of basic compounds. This could be explained by ion-exchange interaction between positively charged analytes and the negatively charged sulphate group.