Simultaneous analysis of cationic, anionic, and neutral compounds using monolithic CEC columns

A new capillary electrochromatography (CEC) column for the simultaneous analysis of cationic, neutral, and anionic compounds using CEC-ESI-MS is described. Three different silica monolith columns were prepared by changing the poly(ethylene glycol) (PEG) contents for comparison of the separation property of these columns. Different separation programs were used for the simultaneous separation of different charged compounds under the same conditions. The column prepared with 80 mg of PEG separated typical compounds within 15 min using 1 M formic acid as the electrolyte. The analytes migrated in the order of cationic, neutral, and anionic compounds, which means that the migration order was mainly determined by the electrophoresis. The hydrodynamic flow by pressure from the inlet side was significant for a stable analysis to be achieved. The effect of the composition of the sheath liquid was also examined. All analytes (14 amino acids, thiourea, urea, citric acid, and ATP) were detectable when 1% acetic acid in 50% (v/v) methanol was used as the sheath liquid.     

Qualitative and quantitative study of nitrogen-containing compounds in heavy gas oil using comprehensive two-dimensional gas chromatography with nitrogen phosphorus detection

In this work, a GCxGC-nitrogen-phosphorus detection (NPD) methodology was developed to separate and quantitate nitrogen-containing compounds in Brazilian heavy gas oil. First, the NPD performance was improved in order to achieve best GCxGC performance. The geometry of this detector was also evaluated. The use of an extended jet improved significantly the peak shape. The GCxGC separation was studied using both first and second dimension columns with different internal diameters. The use of a thicker film in both dimensions provided better performance. LODs of 0.16-8.49 pg of individual compounds were achieved. Two different extraction techniques of the neutral and basic nitrogen-containing compounds were also evaluated. The method using ion-exchange resins to separate neutral and basic nitrogen-containing compounds was more efficient than the method using modified silica. As an example, the amounts (microg/g) of each class reported were: indole (2.77), alkyl carbazoles ranging from C(0) to C(6+) (1.467), alkyl benzocarbazoles from C(0) to C(4+) (793), alkyl quinolines (31.2) and alkyl benzoquinolines (21.6) were quantitated.     

Repeatability and reproducibility of retention data and band profiles on reversed-phase liquid chromatography columns. III. Results obtained with Kromasil C18 columns.

The reproducibility of the retention data and the band profiles was investigated with Kromasil C18 columns (silica-based monomeric type reversed-phase packing material). High precision data were obtained and statistically compared among five columns from the same batch (column-to-column reproducibility) and six columns, one from each of six different batches (batch-to-batch reproducibility). These data were acquired under five different sets of chromatographic conditions, for a group of 30 neutral, acidic and basic compounds selected as probes following an experimental protocol previously described. Data characterizing the retention time, the retention factor, the separation factor, the column efficiency and the peak asymmetry for the different probe compounds are reported. Factors describing the silica surface interaction with the selected probe compounds, such as the hydrophobic interaction selectivity, the steric selectivity, and the separation factors of basic compounds at different pH values were also determined. The influence of the underlying silica on these data and correlations between the chromatographic and physico-chemical properties of the different batches are discussed.     

Separation of basic, acidic and neutral compounds by capillary electrochromatography using uncharged monolithic capillary columns modified with anionic and cationic surfactants

A mode of capillary electrochromatography (CEC), based on the dynamical adsorption of surfactants on the uncharged monolithic stationary phases has been developed. The monolithic stationary phase, obtained by the in situ polymerization of butyl methacrylate with ethylene dimethacrylate, was dynamically modified with an ionic surfactant such as the long-chain quaternary ammonium salt of cetyltrimethylammonium bromide (CTAB) and long-chain sodium sulfate of sodium dodecyl sulfate (SDS). The ionic surfactant was adsorbed on the surface of polymeric monolith by hydrophobic interaction, and the ionic groups used to generate the electroosmotic flow (EOF). The electroosmotic mobility through these capillary columns increased with increasing the content of ionic surfactants in the mobile phase. In this way, the synthesis of the monolithic stationary phase with binary monomers can be controlled more easily than that with ternary monomers, one of which should be an ionic monomer to generate EOF. Furthermore, it is more convenient to change the direction and magnitude of EOF by changing the concentration of cationic or anionic surfactants in this system. An efficiency of monolithic capillary columns with more than 140000 plates per meter for neutral compounds has been obtained, and the relative standard deviations observed for to and retention factors of neutral solutes were about 0.22% and less than 0.56% for ten consecutive runs, respectively. Effects of mobile phase composition on the EOF of the column and the retention values of the neutral solutes were investigated. Simultaneous separation of basic, neutral and acidic compounds has been achieved.     

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.     

Enantiomeric separations of cationic and neutral compounds by capillary electrochromatography with monolithic chiral stationary phases of beta-cyclodextrin-bonded negatively charged polyacrylamide gels

Enantiomeric separation by capillary electrochromatography with beta-cyclodextrin-bonded negatively charged polyacrylamide gels was examined. The columns used are capillaries filled with a negatively charged polyacrylamide gel, a so-called monolithic stationary phase, to which allyl carbamoylated beta-CD (AC-beta-CD) derivatives covalently bind. The capillary wall is activated first with a bifunctional reagent to make the resulting gel bind covalently to the inner surface of the fused-silica tubing. Enantiomeric separations of 15 cationic compounds were achieved using the above-mentioned columns and mobile phases of 200 mmol l(-1) Tris-300 mmol I(-1) boric acid buffer (pH 7.0 or 9.0) or 200 mmol l(-1) Tris-300 mmol l(-1) boric acid buffer (pH 7.0) containing an achiral crown ether (18-crown-6). Enantiomeric separations of two neutral compounds were also achieved using 200 mmol l(-1) Tris-300 mmol l(-1) boric acid buffer (pH 9.0) as a mobile phase. High efficiencies of up to 150,000 plates m(-1) were obtained. Both the within- and between-run reproducibilities of retention time and separation factor were good. The reproducibilities of retention time and separation factor for three different columns prepared from a different batch of monomers were acceptable. The gel-filled capillaries were stable for at least 3 months with intermittent use, utilizing the mobile phase of 200 mmol I(-1) Tris-300 mmol I(-1) boric acid buffer (pH 9.0).     

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.     

The hydrophilic interaction like properties of some reversed phase high performance liquid chromatography columns in the analysis of basic compounds

The hydrophilic interaction chromatography (HILIC) like properties of an ACE cyano (CN) HPLC column was studied for the separation of some basic compounds. Good separation of a test mix of basic compounds was obtained with a mobile phase consisting of acetonitrile/water (95:5) containing 3.25 mM ammonium acetate. The retention times of the basic compounds decreased with increased ionic strength or with increased water content in the mobile phase. When Trishydroxymethyl aminomethane (Tris) (pK(a) 8.0), which is a weaker amine than ammonia (pK(a) 9.3), was used as an additive in the mobile phase retention of the basic compounds was increased. The ACE CN column gave excellent peak shapes for all the basic compounds. The utility of the column for impurity profiling of two basic drugs was tested and some impurities in oxprenolol were characterised by interfacing with Fourier transform mass spectrometry. It was also observed that ACE butyl and ACE phenyl columns retained basic compounds when the columns were eluted with a mobile phase consisting of acetonitrile/water (95:5) containing 3.25 mM ammonium acetate.     

Preparation and evaluation of zirconia-coated silica monolith for capillary electrochromatography

Silica monoliths were fabricated inside fused-silica capillaries. Then the monolithic columns were coated with membrane-like zirconia. The zirconia-coated silica monoliths exhibited different EOF behavior comparing with that of bare silica monoliths. The magnitude and direction could be manipulated by changing the running buffers. Due to the amphoteric characteristic of zirconia, the silica monoliths with zirconia surface facilitate the separation of basic compounds. Aromatic amines and alkaloids were separated without obvious peak tailing. The zirconia surface was easily modified with octadecylphosphonic acid for the separation of neutral compounds. Column efficiency as high as 90,000 and 80,000 m⁻¹ was obtained for beberine and naphthalene, respectively. Furthermore, the zirconia coating increased the stability of the monolithic columns. Even after being exposed to severe condition, there was no apparently efficiency decrease for the test samples.     

Enantiomeric Separations of Acidic and Neutral Compounds by Capillary Electrochromatography with β‐Cyclodextrin‐Bonded Positively Charged Polyacrylamide Gels

The novel enantiomeric separation of acidic and neutral compounds by capillary electrochromatography with β‐cyclodextrin‐bonded positively charged polyacrylamide gels was examined. The columns used are capillaries filled with a positively charged polyacrylamide gel, a so‐called monolithic stationary phase, to which allyl carbamoylated β‐CD derivatives covalently bind. The capillary wall was activated first by bifunctional reagent to make the resulting gel bind covalently inside the fused‐silica tubing. Enantiomeric separations of sixteen acidic and two neutral compounds were achieved using the above‐mentioned columns and 200 mmol dm^–3 Tris–300 mmol dm^–3 boric acid buffer (pH 8.1) as a mobile phase. High efficiencies of up to 150 000 plates m^–1 were obtained for dansyl‐DL‐amino acids. The within‐run and between‐run reproducibilities of retention time and separation factor were examined for three dansyl‐DL‐amino acids and warfarin. The relative standard deviations of the within‐run and between‐run reproducibilities of retention time were less than 1.2 and 1.3% over the six injections, respectively. Those of the separation factor were less than 0.3 and 0.2%, respectively. The gel‐filled capillaries were stable for at least four months with intermittent use.     

Comparative Enantiomer-Resolving Ability of Coated and Covalently Immobilized Versions of Two Polysaccharide-Based Chiral Selectors in High-Performance Liquid Chromatography

The separation of enantiomers of over 175 randomly selected chiral acidic, basic, and neutral compounds was studied on 4 polysaccharide-based chiral columns made by coating or covalent attachment of cellulose 3,5-dichlorophenylcarbamate or amylose 3,5-dimethylphenylcarbamate on the surface of silica. Triscarbamate derivatives of cellulose or amylose were used for the preparation of coated-type columns, while in the case of covalently immobilized chiral stationary phases, the respective polysaccharides were not completely carbamoylated but only close to triscarbamates. It was found that this minimal difference in the chemical composition of the polysaccharide derivatives resulted in significantly different enantiomer-resolving ability for certain groups of chiral compounds while only marginally different for other chiral analytes. This potential difference between coated- and covalently immobilized versions of the “same” chiral selector must be considered in method development with these columns, as well as in method transfer between them.     

Retention pattern profiling of fungal metabolites on mixed-mode reversed-phase/weak anion exchange stationary phases in comparison to reversed-phase and weak anion exchange separation materials by liquid chromatography-electrospray ionisation-tandem mass spectrometry

Retention properties of 79 fungal metabolites (including neutral, acidic, basic, and amphoteric compounds) were evaluated on distinct mixed-mode reversed-phase/weak anion exchange (RP/WAX)-type stationary phases by liquid chromatography-electrospray ionisation-tandem mass spectrometry (LC-ESI-MS/MS) in gradient as well as in isocratic elution mode. The RP/WAX separation materials were prepared by functionalising thiol-modified silica with N-(10-undecenoyl)-3-aminoquinuclidine and N-(10-undecenoyl)-3-alpha-aminotropane, respectively. To evaluate complementarity in chromatographic selectivity the physico-chemically heterogeneous solute set was analysed also on a RP phase (C(18)), an amino-type WAX phase, and a commercially available RP/WAX-like mixed-mode phase. Analytes may interact with the RP/WAX ligands via (attractive/repulsive) ionic, RP-like hydrophobic, as well as hydrophilic (HILIC) retention mechanisms. Individual interactive increments were found to be basically controlled by the nature and amount of organic modifier, pH value of eluent, and ionic strength of buffer additives. It could be demonstrated that RP/WAX columns offer the potential to separate compounds by exploiting a combination of various chromatographic interaction modes, which is not accessible with conventional RP and WAX columns. Such multi-modal properties increase both versatility and degrees of freedom for adjustment of chromatographic selectivity. For example, highly polar mycotoxins such as moniliformin were well retained on RP/WAX-type phases without compromising RP-selectivity for neutral (e.g. aflatoxins) and most basic solutes (e.g. epimer separation of ergot alkaloids) under fully MS-compatible conditions like a hydro-organic eluent with acetonitrile as organic modifier and an acetic acid/ammonium acetate buffer. Flexibility of the employed mixed-mode separation materials may be of value particularly for LC-ESI-MS/MS-based bioanalytics involving analytes with widely varying physico-chemical properties or applications prone to matrix effects.     

New benchmark for basic and neutral nitrogen compounds speciation in middle distillates using comprehensive two-dimensional gas chromatography

This paper reports an analytical method for the comprehensive two-dimensional gas chromatography (GC x GC) separation and identification of nitrogen compounds (N-compounds) in middle distillates according to their types (basicity). For the evaluation of the best chromatographic conditions, a non-polar x polar approach was chosen. The impact of the second dimension (stationary phase and column length) on the separation of basic and neutral N-compounds was evaluated by mean of two-dimensional resolution. This study revealed that the implementation of polar secondary column having free electron pairs improves drastically the separation of N-compounds. Indeed, the presence of permanent dipole-permanent dipole interactions between neutral N-compounds and the stationary phase was enlightened. The comparison of two different nitrogen chemiluminescence detectors (NCD) was also evaluated for GC x GC selective monitoring of N-compounds. Owing to higher resolution power and enhanced sensitivity achieved using developed chromatographic and detection conditions, it was possible to identify univocally and to quantitate N-compounds (i) by class of compounds and (ii), within a class, by carbon number. Finally, quantitative comparison of GC x GC-NCD with conventional gas chromatography illustrates the benefits of GC x GC leading to an excellent correlation with results obtained by American Society for Testing Materials (ASTM) methods for the determination of basic/neutral nitrogen ratio in diesel samples.     

Cationic double-chained surfactant as pseudostationary phase in micellar electrokinetic capillary chromatography for drug separations

The cationic double-chained surfactant didodecyldimethylammonium bromide (DDAB) was used as pseudostationary phase (PSP) in micellar electrokinetic capillary chromatography (MEKC). Its performance on the three kinds of drugs, i.e., basic, acidic, and neutral drugs, was systematically investigated. Nicotine, cotinine, caffeine, lidocaine, and procaine were selected as the model basic drugs. Good baseline separation and high efficiency were obtained under the optimal separation condition that consisted of 50mM phosphate (pH 4.0) and 0.08 mM DDAB. Three basic phenylenediamine isomers can also be well separated with DDAB in buffer. In addition, DDAB can form cationic bilayer on the capillary wall, thus the wall adsorption of basic analytes was greatly suppressed. Compared with commonly used CTAB, the separation of basic drugs was significantly improved with a much lower amount of DDAB present in the buffer. The DDAB-involved MEKC also showed superiority to CTAB upon the separation of acidic drugs, amoxicillin and ampicillin. In the case of neutral compounds, a good separation of resorcinol, 1-naphthol and 2-naphthol was achieved with 0.1mM DDAB and 30% (v/v) acetonitrile (ACN) present in buffer. Hence, it was concluded that the double-chained cationic surfactant DDAB can be a good substitute for traditional single-chained surfactant CTAB in MEKC.     

Repeatability and reproducibility of retention data and band profiles on six batches of monolithic columns

Chromatographic data were acquired for eight different mixtures, under five different sets of experimental conditions, for a total of 30 neutral, acidic and basic test compounds, on a series of six Chromolith Performance columns from Merck. These columns are made of a C₁₈ chemically bonded silica monolith. Each column belonged to a different production batch, so the data reported here characterize their batch-to-batch reproducibility. The parameters studied in this work were the retention times, the retention and separation factors, the hydrophobic and the steric selectivities, the column efficiencies, and the tailing factors for all 30 compounds.     

Repeatability and reproducibility of retention data and band profiles on reversed-phase liquid chromatography columns. V. Results obtained with Vydac 218TP C18 columns

The general goal of this work is to investigate the precision of chromatographic data and to determine which properties of chromatographic columns influence this factor. Chromatographic data were acquired under five different sets of experimental conditions for 30 neutral, acidic and basic test compounds on columns packed with Vydac 218TP C18, a polymeric, wide-pore silica-based stationary phase. Five columns packed with samples from the same batch of this packing material were used to measure the column-to-column reproducibility and six columns packed with material from six different batches to measure the batch-to-batch reproducibility. The parameters studied were the retention time, the retention and separation factors, the hydrophobic and the steric selectivities, the column efficiency, and the tailing factor.     

HPLC characterization of phenolics in lignocellulosic materials

Summary The application of HPLC with an electrochemical detector for the determination of phenolics in lignocellulosic materials is reported. The separation of phenolic acids and aldehydes (gallic acid, p-hydroxybenzoic acid, vanillic acid, p-coumaric acid, syringic acid, ferulic acid, vanillin, syringaldehyde and p-hydroxybenzaldehyde) on two different columns (reversed phase C6 and styrene-divinylbenzene PLRP-S) is shown. Chromatograms of phenolic compounds in neutral, basic and oxidative extracts of wheat straw treated with NaOH and white rot fungusStropharia rugosoannulata are reported along with quantitative results.     

Separation of positional isomers by the use of coupled shape-selective stationary phase columns

The successful separation of 2- and 3-methyl-substituted positional isomers of butanol, butyl acetate, and butanoic acid and its ethyl ester, is reported. These compounds are of interest in the study of wine flavour, however the separation of the 2- and 3-methyl isomers may present problems, and more so in the presence of the wine matrix components, when single capillary column gas chromatography (GC) is used. The strategy to achieve separation was based on the use of shape-selective cyclodextrin derivative (CDD) capillary columns (commonly referred to as chiral columns). These columns provide simultaneous resolution of the enantiomeric pairs of the 2-methyl isomers, and at the same time the ability to separate the 3-methyl isomer from the 2-methyl is achieved in all but the case of the ( S)-2- and 3-methylbutanol. The advantages of using shape-selective columns to perform this study is demonstrated, with coupling of two CDD columns giving improved separations of these compounds. Although these compounds are relatively volatile, cryogenic modulated comprehensive two-dimensional GC was shown to provide good pulsed peak profiles with chiral separation in the first dimension when a thicker film trapping column segment was employed. The components of interest were well separated from other wine matrix components.     

High-efficiency peptide analysis on monolithic multimode capillary columns: Pressure-assisted capillary electrochromatography/capillary electrophoresis coupled to UV and electrospray ionization-mass spectrometry

High-efficiency peptide analysis using multimode pressure-assisted capillary electrochromatography/capillary electrophoresis (pCEC/pCE) monolithic polymeric columns and the separation of model peptide mixtures and protein digests by isocratic and gradient elution under an applied electric field with UV and electrospray ionization-mass spectrometry (ESI-MS) detection is demonstrated. Capillary multipurpose columns were prepared in silanized fused-silica capillaries of 50, 75, and 100 microm inner diameters by thermally induced in situ copolymerization of methacrylic monomers in the presence of n-propanol and formamide as porogens and azobisisobutyronitrile as initiator. N-Ethylbutylamine was used to modify the chromatographic surface of the monolith from neutral to cationic. Monolithic columns were termed as multipurpose or multimode columns because they showed mixed modes of separation mechanisms under different conditions. Anion-exchange separation ability in the liquid chromatography (LC) mode can be determined by the cationic chromatographic surface of the monolith. At acidic pH and high voltage across the column, the monolithic stationary phase provided conditions for predominantly capillary electrophoretic migration of peptides. At basic pH and electric field across the column, enhanced chromatographic retention of peptides on monolithic capillary column made CEC mechanisms of migration responsible for separation. The role of pressure, ionic strength, pH, and organic content of the mobile phase on chromatographic performance was investigated. High efficiencies (exceeding 300 000 plates/m) of the monolithic columns for peptide separations are shown using volatile and nonvolatile, acidic and basic buffers. Good reproducibility and robustness of isocratic and gradient elution pressure-assisted CEC/CE separations were achieved for both UV and ESI-MS detection. Manipulation of the electric field and gradient conditions allowed high-throughput analysis of complex peptide mixtures. A simple design of sheathless electrospray emitter provided effective and robust low dead volume interfacing of monolithic multimode columns with ESI-MS. Gradient elution pressure-assisted mixed-mode separation CE/CEC-ESI-MS mass fingerprinting and data-dependent pCE/pCEC-ESI-MS/MS analysis of a bovine serum albumin (BSA) tryptic digest in less than 5 min yielding high sequence coverage (73%) demonstrated the potential of the method.     

胺型键合固定相分离碱性物质的影响因素考察

 以某些碱性物质为探测溶质 ,中性物质乙苯为参照溶质 ,详尽考察了检测温度、缓冲液中阳离子浓度、流动相的流速分别在流动相的 pH值为 3 0和 7 0时对胺型键合固定相分离碱性物质的影响。研究结果表明 ,随着温度的升高 ,碱性物质的保留时间先下降较快 ,然后趋于平缓 ,而柱效和峰形分别得到了提高和改善 ;缓冲液中阳离子浓度对碱性物质的保留行为有较大的影响 ;碱性物质的最佳流速较中性物质小。由于胺型键合固定相中的疏水烷基链中引入了极性官能团氨基 ,因此碱性物质在该固定相上的行为比在传统的固定相上更为复杂。