RESUMEN
High performance anion exchange chromatography (HPAEC) with pulsed amperometric detection (PAD) was optimized for the analysis of oligosaccharides derived from the extracellular matrix component hyaluronan. Using this sensitive approach, the separation of oligosaccharides consisting of two (molecular weight ca. 0.8 kDa) up to 25-30 (molecular weight: ca. 9.5-11.4 kDa) disaccharide moieties was possible. Standard oligosaccharides (comprising 2-4 repetitive disaccharides) were detectable at very low amounts of 0.2-0.3 pmol (20-30 nM). Including 10 min of column equilibration, a complex mixture of low molecular weight hyaluronan can be analyzed within 40 min. The HPAEC method was successfully applied to the study of the size-dependency of both the action of bovine testicular hyaluronidase (BTH) and the precipitation of hyaluronan by cetyltrimethylammonium bromide (CTAB), a physicochemical reaction often used for the determination of hyaluronan and hyaluronidase activity.
Asunto(s)
Cromatografía por Intercambio Iónico/métodos , Ácido Hialurónico/análisis , Oligosacáridos/análisis , Animales , Bovinos , Cetrimonio , Compuestos de Cetrimonio , Electroforesis Capilar , Ácido Hialurónico/metabolismo , Hialuronoglucosaminidasa/metabolismo , Concentración de Iones de Hidrógeno , Límite de Detección , Modelos Lineales , Masculino , Oligosacáridos/metabolismo , Reproducibilidad de los Resultados , Acetato de Sodio , Hidróxido de Sodio , TemperaturaRESUMEN
A novel approach has been developed for the separation of organotin species with capillary electrophoresis hyphenated to time-of-flight mass spectrometry. It has been applied to the development of a method for the determination and speciation of organotin compounds namely, dibutyltin (DBT), tributyltin (TBT), diphenyltin (DPT) and triphenyltin (TPT) in water samples. Experiments were made with a special laboratory constructed CE instrument. A non-aqueous buffer system compatible with TOF-MS has been developed using ammonium acetate-acetic acid (50 mM and 1 M) in acetonitrile: methanol (80:20). The total analysis time is less than 3 min for these compounds under the conditions developed. The method has been applied successfully to the determination of these compounds in river water samples. Detection limits of the CE-TOF-MS method were between 1 and 8×10(-7) M, and between 2 and 11×10(-9) M (0.46 to 3.2 µg L(-1)) when used in conjunction with solid phase extraction. The short analysis time as well as good sensitivity and selectivity make it a useful approach for the fast screening of organotin compounds.
Asunto(s)
Agua Dulce/química , Compuestos Orgánicos de Estaño/aislamiento & purificación , Compuestos de Trialquiltina/aislamiento & purificación , Contaminantes Químicos del Agua/aislamiento & purificación , Electroforesis Capilar/métodos , Humanos , Límite de Detección , Extracción en Fase Sólida , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Factores de TiempoRESUMEN
We present an experimental approach to conducting fast capillary electrophoresis-mass spectrometry (CE-MS) measurements of very small samples in the nanoliter range. This is achieved by injecting sample very efficiently into a CE-MS system. Injection efficiency represents the ratio of injected sample to the amount of sample needed for carrying out the injection process (v/v). In order to increase this injection efficiency from typical values of 10(-3) to 10(-7), the concept of capillary batch injection is used to build an automated, small-footprint injection device for CE-MS. This device is capable of running true multi-sample measurement series, using minimal sample volumes and delivering an injection efficiency of up to 100 %. It is compatible with both aqueous and non-aqueous background electrolytes. As an additional benefit, CE-MS separations of a catecholamine model system in capillaries of 15 cm length under conditions of high electric field strength could be accomplished in 20 s with high separation efficiency. This report details design and specifications of the injection device and shows optimal parameter choices for injections with both high injection efficiency and high separation efficiency. Furthermore, a procedure is presented to coat the tip of a fused silica capillary with a silicone elastomer which acts as a seal between two capillaries.
RESUMEN
Fast capillary electrophoresis (CE) hyphenated to time-of-flight mass spectrometry (TOF-MS) of four organoarsenic species (glycerol oxoarsenosugar, sulfate oxoarsenosugar, arsenobetaine, arsenocholine) are presented using short length CE capillaries under high electric field strengths of up to 1.3 kV cm(-1) with small inner diameter (ID). The separation of arsenosugars by CE is demonstrated for the first time. An aqueous formic acid solution was employed as the background electrolyte (BGE) for the separation. Various acid concentrations were evaluated for their influence on migration times, separation efficiency as well as with regard to controlling the charge of the arsenic species. A 0.1 M formic acid/ammonium formate buffer (pH 2.8) proved to be suitable for the separation of the four species. A non-aqueous BGE was tested as an alternative buffer system for fast speciation analysis. Separation of arsenobetaine and arsenocholine could even be achieved within 10 s by pressure-assisted CE. Application of the optimized method for the analysis of extracts of a seagrass and a Wakame algae sample as well as the brown algae homogenate reference material IAEA-140/TM revealed a clear signal for the glycerol arsenosugar.
Asunto(s)
Arsénico/análisis , Electroforesis Capilar/métodos , Espectrometría de Masa por Ionización de Electrospray/métodos , Arsénico/clasificación , Tampones (Química) , PresiónRESUMEN
Fast capillary electrophoresis-mass spectrometry measurements under counter-electroosmotic analyte migration conditions are presented. Efficient separations of a homologous series of six hyaluronan oligosaccharides (comprising 1-6 hyalobiuronic acid moieties) could be completed in 65 s. Separations were achieved in short-length fused silica capillaries under high electric field strengths of up to 1.25 kV·cm(-1). Capillary inner diameters ranging from 5 to 50 µm were investigated, resulting in an optimal value of 15 µm. The influence of capillary dimensions and buffer composition on separation efficiency and sensitivity are discussed. Optimal separations were achieved using a 28 cm × 15 µm capillary, a separation high voltage of 35 kV, a background electrolyte of 25 mM ammonium acetate adjusted to pH 8.5, and negative ionization mode. The optimized method was successfully applied to a bovine testicular hyaluronidase digest of hyaluronan. Only minimal sample pretreatment for protein-containing samples is required. The simple manual injection procedure and fast separations allow for a sample throughput of 35 samples per hour.
Asunto(s)
Ácido Hialurónico/química , Oligosacáridos/análisis , Animales , Bovinos , Electroósmosis , Electroforesis Capilar , Ácido Hialurónico/metabolismo , Hialuronoglucosaminidasa/química , Hialuronoglucosaminidasa/metabolismo , Masculino , Espectrometría de Masas , Oligosacáridos/metabolismo , Testículo/enzimología , Factores de TiempoRESUMEN
Fast electrophoretic separations in fused silica capillaries (CE) coupled to time-of-flight mass spectrometry (TOF-MS) are presented. CE separations of the model analytes (epinephrine, norepinephrine, dopamine, histidine, and isoproterenol) under conditions of high electric field strengths of up to 1.25 kV cm(-1) are completed in 20 s. Coupling of CE with TOF-MS is accomplished using a coaxial sheath liquid electrospray ionization interface. The influence of parameters inherent to the interface and their effects, including suction pressure and dilution, are discussed. In addition to standard capillaries of 75 and 50 µm inner diameter (ID), separations in capillaries with IDs of 25, 15, and 5 µm have been successfully applied to this setup. The analytical performance is compared over this range of capillary dimensions, and both advantages and disadvantages are discussed.