RESUMEN
The preparation of capillary microfluidic reactor with co-immobilized trypsin and chymotrypsin with the use of a low-cost commercially available enzymatic reagent (containing these proteases) as well as the evaluation of its usefulness in proteomic research were presented. The monolithic copolymer synthesized from glycidyl methacrylate (GMA) and ethylene glycol dimethacrylate (EDMA) was used as a support. Firstly, the polymerization conditions were optimized and the monolithic bed was synthesized in the fused silica capillary modified with 3-(trimethoxysilyl)propyl methacrylate (γ-MAPS). The polymer containing epoxy groups was then modified with 1,6-diaminohexane, followed by the attachment of glutaraldehyde and immobilization of enzymes. The efficiency of the prepared monolithic Immobilized Enzyme Microreactor (µ-IMER) with regard to trypsin activity was evaluated using the low-molecular mass compound (Nα-benzoyl-l-arginine ethyl ester, BAEE). The activities of both enzymes were investigated using a macromolecular protein (human transferrin, Tf) as a substrate. In the case of BAEE, the reaction product was separated from the substrate using the capillary liquid chromatography and the efficiency of the reaction was determined by the peak area of the substrate. The hydrolysis products of transferrin were analyzed with MALDI-TOF which allows for the verification of the prepared enzymatic system applicability in the field of proteomic research.
Asunto(s)
Quimotripsina/química , Enzimas Inmovilizadas/metabolismo , Proteómica/métodos , Tripsina/química , Arginina/análogos & derivados , Arginina/química , Reactores Biológicos , Cromatografía Liquida , Compuestos Epoxi/química , Humanos , Metacrilatos/química , Compuestos de Organosilicio/química , Polimerizacion , Proteínas/metabolismo , Silanos/química , Dióxido de Silicio/químicaRESUMEN
This paper presents a second part of our research devoted to cholesterol-based polymeric monolithic stationary phase. The obtained capillary columns were successfully used for separations of alkylbenzenes, steroid hormones and polycyclic aromatic hydrocarbons during isocratic or gradient elutions. The columns showed excellent thermal stability. Increasing the temperature resulted in decrease of the retention factors and peak widths, but selectivity and efficient separations have been retained in the studied temperature range of 20 to100°C. Additionally, the van't Hoff model presented a non-linear relationship of lnk versus 1/T plots, which is likely the result of liquid crystal properties of cholesterol. The studied capillary monolithic columns showed extremely fast re-equilibration after gradient elution and found to be stable under such conditions as: fast flow rate, high acetonitrile content in the mobile phase (no swelling) and high temperature.
Asunto(s)
Colesterol/química , Acetonitrilos , Cromatografía Liquida/instrumentación , Cromatografía Liquida/métodos , Hormonas Esteroides Gonadales/análisis , Hidrocarburos Policíclicos Aromáticos/análisis , Polímeros/química , TemperaturaRESUMEN
A novel, cholesterol-based polymeric monolithic stationary phase for capillary liquid chromatography, was prepared by thermally initiated in-situ polymerization. Cholesteryl methacrylate (CholMA) was used as a functional monomer and trimethylolpropane trimethacrylate (TRIM) was a cross-linker, while azobisisobutyronitrile (AIBN) was an initiator. Isooctane and toluene were chosen as "poor" and "good" solvent, respectively, as constituents of the porogen solvent. Isocratic elutions of alkylbenzenes and separation of the testing mixture of o-terphenyl and triphenylene were conducted for all of the monoliths to assess their hydrophobicity and planar selectivity characteristic for cholesterol-based stationary phases. The synthesized columns demonstrated efficiency exceeding N=10,000 plates and a plate height of ca. H=30 µm. Column preparation was found to be highly reproducible; the relative standard deviation (RSD) values (n=3) for day-to-day and column-to-column were less than 4.08 and 2.02%, respectively, based on retention factor of alkylbenzenes.
Asunto(s)
Colesterol/química , Cromatografía Liquida/instrumentación , Capilares , Cromatografía Liquida/métodos , Interacciones Hidrofóbicas e Hidrofílicas , Metacrilatos/química , Polímeros/química , Porosidad , Solventes/químicaRESUMEN
Monolithic molecularly imprinted polymers extraction columns have been prepared in fused-silica capillaries by UV or thermal polymerization in a two-step process. First, a poly-(trimethylolpropane trimethacrylate) (polyTRIM) core monolith was synthesized either by UV or thermal polymerization. Then it was grafted with the mixture of methacrylic acid (MAA) as a functional monomer, ethylene dimethacrylate (EDMA) as a cross-linking agent, 5,7-dimethoxycoumarin (DMC) as an aflatoxin-mimicking template, toluene as a porogen solvent and 2,2-azobis-(2-methylpropionitrile) (AIBN) as an initiator of the polymerization reaction. Different thermal condition of the photografting and different concentrations of the grafting mixture were tested during polymerization. The extraction capillary columns were evaluated in the terms of their hydrodynamic and chromatographic properties. Retention coefficients for aflatoxin B1 and DMC were used for assessment of the selectivity and imprinting factor. The obtained results indicate that the temperature of photografting and concentration of the grafting mixture are key parameters that determine the quality of the prepared MIPs. From the MIP columns characterized by the highest permeability the column of the highest imprinting factor was applied for isolation of aflatoxins B1, B2, G1 and G2 from the model aqueous sample followed by on-line chromatographic separation. The process was performed using a micro-MISPE-microLC-LIF system of a novel design, which allowed for detection of the eluates from the sample preparation part as well as from the chromatographic separation.
Asunto(s)
Aflatoxinas/aislamiento & purificación , Cumarinas/química , Metacrilatos/química , Impresión Molecular , Polímeros/química , Cromatografía Liquida/instrumentación , Cromatografía Liquida/métodos , Reactivos de Enlaces Cruzados/química , Permeabilidad , Polimerizacion , Polímeros/síntesis química , Dióxido de Silicio , Agua/químicaRESUMEN
Breath is a rich mixture containing numerous volatile organic compounds at trace amounts (ppbv-pptv level) such as: hydrocarbons, alcohols, ketones, aldehydes, esters or heterocycles. The presence of some of them depends on health status. Therefore, breath analysis might be useful for clinical diagnostics, therapy monitoring and control of metabolic or biochemical cell cycle products. This Review presents an update on the latest developments in breath analysis applied to diagnosing different diseases with the help of high-quality equipment. Efforts were made to fully and accurately describe traditional and modern techniques used to determine the components of breath. The techniques were compared in terms of design, function and also detection limit of different volatile organic compounds. GC with different detectors, MS, optical sensor and laser spectroscopic detection techniques are also discussed.