RESUMEN
This investigation demonstrates the application of a new peak sharpening technique to improve the separation of difficult-to-resolve racemic mixtures in capillary electro-chromatography. Molecularly imprinted porous layer open tubular (MIP-PLOT) capillaries, prepared by a layer-on-layer polymerization approach with Z-l-Asp-OH as the template, were selected to validate the approach. SEM revealed that the polymer film thickness can be varied by changes in both the polymer composition and the layer-on-layer regime. Capillaries made with methacrylic acid as the functional monomer could not separate the Z-Asp-OH racemate, due to weak interactions between the MIP-PLOT material and the target analytes. In contrast, MIP-PLOT capillaries prepared with 4-vinylpyridine as the functional monomer resulted in increased ionic interactions with the target analytes. Separation of the enantiomers could be enhanced when a peak zone sharpening effect was exploited through the use of specific BGE compositions and by taking advantage of eigenpeak phenomena. In this manner, the position of a sharpening zone and the peak shape of the sample analytes could be fine-tuned, so that when the sharpening zone and the target analyte co-migrated the separation of the Z-l-Asp-OH enantiomer from its d-enantiomer in a racemic mixture could be achieved under overloading conditions.
Asunto(s)
Electrocromatografía Capilar/instrumentación , Electrocromatografía Capilar/métodos , Impresión Molecular , Polímeros , Asparagina/análogos & derivados , Asparagina/aislamiento & purificación , Electrocromatografía Capilar/normas , Diseño de Equipo , Piridinas , EstereoisomerismoRESUMEN
Vinylized iron oxide magnetic nanoparticles (VMNPs) were incorporated into polymethacrylate monolithic columns to develop novel stationary phases with enhanced separation performance. The VMNPs were dispersed in a polymerization mixture containing gycidyl methacrylate and ethylene glycol dimethacrylate as monomers, cyclohexanol and 1-dodecanol as porogens and azobisisobutyronitrile as initiator. The stability of the VMNPs in the polymerization mixture was investigated at several VMNP contents. Using short UV-polymerization times, polymeric beds with homogenously dispersed VMNPs were obtained. The novel stationary phases were characterized by scanning electron microscopy. The chromatographic performance of these hybrid monoliths was evaluated using alkyl benzenes and organophosphorous pesticides as test solutes. Using capillary electrochromatography, efficiencies up to 130,000 plates/m were achieved. The increase of the specific surface area of hybrid monoliths led to an increase in the retention of all the test analytes, and an enhancement of efficiency. The resulting hybrid monolithic columns exhibited satisfactory column to-column and batch-to-batch reproducibilities with RSDs values below 6%.
Asunto(s)
Electrocromatografía Capilar/instrumentación , Nanopartículas de Magnetita , Metacrilatos/química , Electrocromatografía Capilar/métodos , Electrocromatografía Capilar/normas , Microscopía Electrónica de Rastreo , Polímeros/química , Ácidos Polimetacrílicos/química , SolucionesRESUMEN
Duplex capillary columns with a packed and an open section are widely used in electrochromatography (CEC). The duplex column configuration leads to non-uniform voltage drop, electrical field distribution and separation performance. It also adds to the complexity in understanding and optimizing electrochromatographic process. In this study, we introduced a simplex column configuration based on single particle fritting technology. The new column configuration has an essentially uniform packed bed through the entire column length, with only 1mm length left unpacked serving as the optical detection window. The study shows that a simplex column has higher separation efficiency than a duplex column, especially at the high voltage range, due to the consistent distribution of electrical field over the column length. In comparison to the duplex column, the simplex column presented a lower flow rate at the same applied voltage, suggesting that an open section may support a higher speed than a packed section. In practice, the long and short ends of the simplex column could be used as independent CEC columns respectively. This "two-in-one" bi-functional column configuration provided extra flexibilities in selecting and optimizing electrochromatographic conditions.
Asunto(s)
Electrocromatografía Capilar/instrumentación , Electrocromatografía Capilar/normas , Reproducibilidad de los ResultadosRESUMEN
In our previous work we have described the synthesis, characterization, and optimization of the chromatographic efficiency of a highly crosslinked macroporous mixed-mode acrylamide-based monolithic stationary phase synthesized by in situ free radical copolymerization of cyclodextrin-solubilized N-adamantyl acrylamide, piperazinediacrylamide, methacrylamide and vinylsulfonic acid in aqueous medium in pre-treated fused silica capillaries of 100µm I.D. In the present work, we study with different classes of neutral analytes (with varied hydrophobicity) the impact of the type of retention mode (influenced by the type of analyte and the mobile phase composition) and the impact of the solute functionality on the chromatographic efficiency and peak symmetry with a monolith synthesized under optimized synthesis parameters. With this monolithic capillary high separation efficiencies (up to ca. 220,000m(-1)) are obtained for the separation of different analyte classes (alkylphenones, nitrotoluenes, and phenolic compounds with k=0.2-0.55) in the reversed-phase mode, in the normal-phase mode, and in the mixed mode. For neutral alkylanilines (k<0.25) plate numbers of about 300,000m(-1) are routinely reached in the reversed-phase elution mode. For phenolic solutes separated in a mixed mode there is a solute-specific influence on peak symmetry and chromatographic efficiency. With increasing efficiency of the monolith, axial diffusion becomes an important mechanism of band broadening. For those peaks, which do not show a significant asymmetry (asymmetry factor ≤1.05), it is confirmed that plate heights gained via the tangent method are equivalent to those gained via moment analysis.
Asunto(s)
Acrilamida/química , Electrocromatografía Capilar/instrumentación , Electrocromatografía Capilar/normas , Cromatografía Liquida , Concentración de Iones de Hidrógeno , Interacciones Hidrofóbicas e Hidrofílicas , Dióxido de Silicio/químicaRESUMEN
Atmospheric pollution of anthropic origin is recognized as a major risk factor for health, in particular for respiratory and cardio-vascular systems. Among these pollutants, polycyclic aromatic hydrocarbons (PAHs) are placed on the list of US Environmental Protection Agency (EPA) as 'priority' pollutants and four of them are assigned as potential carcinogens by The International Agency for Research on Cancer (IARC). In the present work two capillary techniques-micellar electrokinetic chromatography (MEKC) and monolithic capillary electrochromatography (CEC)-were compared for the separation of eleven PAHs. Both techniques compared in the present work are fully compatible with every standard apparatus of capillary electrophoresis. For MEKC, enhancement of selectivity and decrease of the separation window of eleven PAHs were obtained with methanol:borate 25 mM (20/80, v/v) running buffer containing 10 mM of hydroxypropylated γ-cyclodextrins with low SDS content (25 mM). In case of CEC, two acrylate-based monolithic stationary phases (MSPs) were evaluated for their application in the separation of eleven PAHs. The best MSP based on butyl acrylate was compared with MEKC in terms of sample capacity, PAHs elution order, LOQ, efficiency and effect of pH. Influence of the hydrophobicity of mobile phase on the PAHs elution order was also studied.
Asunto(s)
Electrocromatografía Capilar/normas , Cromatografía Capilar Electrocinética Micelar/normas , Hidrocarburos Policíclicos Aromáticos/análisis , Electrocromatografía Capilar/métodos , Cromatografía Capilar Electrocinética Micelar/métodos , Hidrocarburos Policíclicos Aromáticos/químicaRESUMEN
Development of capillary electrochromatography (CEC) largely depends on column technology. The past ten years or so have seen a great number of CEC works performed on monolithic columns, due to simplicity and robustness in column fabrication. Monolithic columns eliminate the issue of column fritting, which conventionally made particle-packed capillary columns fragile and introduced nonuniformity to the chromatographic bed. The particulate packing material, however, is still a popular type of stationary phase widely used in CEC, as the rich library of HPLC packing material provides a wide range of choices for chromatographic separations performed in electrodriven mode. In this study, we investigated a purely physical fritting method, single particle fritting technology, to immobilize particulate chromatographic material inside capillary tube in a sinter-free manner to produce robust capillary columns. Single particle fritted columns present significantly improved column-to-column reproducibility (n=10) in peak efficiency, retention factor, peak area and asymmetry (%RSD=5.4, 7.7, 6.2 and 6.1, respectively, at 26 kV), enabling their practical application in high throughput parallel analysis using multiple columns.
Asunto(s)
Electrocromatografía Capilar/instrumentación , Electrocromatografía Capilar/métodos , Material Particulado/química , Electrocromatografía Capilar/normas , Microscopía Electrónica de Rastreo , Hidrocarburos Policíclicos Aromáticos/análisis , Reproducibilidad de los Resultados , Solventes/químicaRESUMEN
In Shotgun proteome analysis, where nano-flow is adopted to increase the sensitivity as well as extremely complicated samples such as proteolytic digest are inevitably confronted, monolithic capillary columns are widely used to improve the liquid chromatography separation performance. It is known that cartilage contains extensive amounts of extracellular matrix (ECM), in which collagens and aggrecans being the most abundant macromolecules. It is obvious that the high content of ECM components causes a challenge in the comprehensive proteome analysis of cartilage. In this study, a 7 cm x 150 microm i. d. phosphate strong cation exchange (SCX) monolithic capillary column was coupled with an 85 cm x 75 microm i. d. C12 reversed-phase monolithic capillary column for online two-dimensional separation of 20 microg tryptic digest of proteins extracted from human cartilage. After 14 salt steps fractionation and following gradient separation coupled with tandem mass spectrometry detection, finally 7 434 unique peptides, corresponding to 1 901 distinct proteins were positively identified. Then, the identified proteins were analyzed by Gene Ontology (GO), and it was found that most of the identified proteins were come from articular chondrocytes with low abundance, which is important for the researches of articular diseases.
Asunto(s)
Cartílago/química , Cromatografía Liquida/métodos , Proteoma/análisis , Proteómica/métodos , Espectrometría de Masas en Tándem , Electrocromatografía Capilar/instrumentación , Electrocromatografía Capilar/normas , Humanos , Proteoma/químicaRESUMEN
A biphasic monolithic capillary column with 10 cm segment of strong-cation exchange monolith and 65 cm segment of reversed-phase monolith was prepared within a single 100 microm i.d. capillary. Separation performance of this column was evaluated by a five-cycle online multidimensional separation of 10 microg tryptic digest of yeast proteins using nanoflow liquid chromatography coupled with tandem mass spectrometry, and it took 12 h for whole separation under the operating pressure only approximately 900 psi. Totally, 780 distinct proteins were positively identified through assignment of 2953 unique peptides at false-positive rate less than 1%. The good separation performance of this biphasic column was largely attributed to the good orthogonality of the strong-cation exchange monolith and reversed-phase monolith for multidimensional separation.
Asunto(s)
Electrocromatografía Capilar/instrumentación , Proteínas Fúngicas/aislamiento & purificación , Proteoma/análisis , Proteómica/instrumentación , Espectrometría de Masas en Tándem , Electrocromatografía Capilar/normas , Resinas de Intercambio de Catión , Cromatografía Liquida , Sistemas en LíneaRESUMEN
A pressurized CEC (pCEC) method with postcolumn detection cell had been developed for quantifying the lignans from Fructus schisandrae extracts. The effects of different experimental conditions, such as the ACN content of the mobile phase, the concentration and pH of the buffer, the applied voltage, and the supplementary pressure were studied. Five lignans (schisandrin, gomisin A, schisantherin C, deoxyschizandrin, schisandrin B) were baseline separated using a mobile phase of ACN-phosphate buffer (pH 5.4; 5 mM) (40:60 v/v) under -4 kV applied voltage. The method showed the satisfactory retention time and peak area repeatability. The calibration curves were linear in the range 50.0-1000.0 microg/mL for schisandrin, 20.0-500.0 microg/mL for gomisin A, 10.0-200.0 microg/mL for schisantherin C, 20.0-500.0 microg/mL for deoxyschizandrin, and 20.0-500.0 microg/mL for schisandrin B. The correlation coefficients were between 0.9978 and 0.9989. With this pCEC system, fingerprints of F. schisandrae were preliminarily established to distinguish two members S. chinensis (Turcz.) Baill. and S. sphenanthera Rehd. Et Wils. of F. schisandrae by characteristic peaks, and evaluate the quality of various sources of raw materials by determining the contents of the five lignans.
Asunto(s)
Electrocromatografía Capilar/métodos , Plantas Medicinales/química , Schisandra/química , Tampones (Química) , Electrocromatografía Capilar/instrumentación , Electrocromatografía Capilar/normas , Electroquímica , Diseño de Equipo , Concentración de Iones de Hidrógeno , Lignanos/análisis , Lignanos/química , Estructura Molecular , Presión , Control de CalidadRESUMEN
Silica-based monolithic column with high rigidity and good permeability was prepared by a modified sol-gel technology, characterized by shorter fabrication process and higher success rate. A macrocyclic antibiotics-based chiral monolithic column was successfully prepared by modifying the monolithic matrix with a homemade macrocyclic antibiotics--norvancomycin by adopting a single-step on-column derivatization process. The newly synthesized column was assessed for its enantioselectivity in chromatographic modes of reversed-phase and polar organic-phase. The effect of the mobile phase composition on the retention and enantioselectivity of the system was also investigated in detail. It was shown that beta-receptor blockers can be best resolved under the mobile phase composition of methanol-acetonitrile-triethyl-amine-acetic acid (80: 20: 0.1 : 0.1, v/v). Under reversed-phase conditions, a nearly linear increase of electroosmotic flow (EOF) was observed with the pH changing from 4.0 to 7.0, which shows that the predominant source of EOF is still resulting from the silica backbone while the chiral selector of norvancomycin contributes little. A number of racemic pharmaceuticals with different structures were separated in reversed-phase mode with different resolutions. It was shown experimentally that the column prepared in this way had broader enantioselectivity as well as better batch-to-batch reproducibility.