Capillary electrochromatographic separation of ionizable compounds with a molecular imprinted monolithic cationic exchange column

A polymer-based monolithic capillary column imprinted with 4-aminopyridine (4-AP) was prepared by a thermally-initiated polymerization process; and its performance as a capillary electrochromatographic medium was evaluated in separating 4-AP and 2-AP isomers. The effects of experimental parameters, such as pH value and ionic strength of the buffer, the acetonitrile content in the mobile phase, and the applied voltage, on the resolution of these isomers had been carefully investigated. It was found that in the retention process there were interplays of multiple mechanisms of ion-exchange, molecular imprinting, and electrophoresis. These mechanisms allowed more sophisticated control of experimental parameters in the separation of ionizable compounds.

Separation of peptides on mixed mode of reversed-phase and ion-exchange capillary electrochromatography with a monolithic column

The mixed mode of reversed phase (RP) and strong canon-exchange (SCX) capillary electrochromatography (CEC) based on a monolithic capillary column has been developed. The capillary monolithic column was prepared by in situ copolymerization of 2-(sulfooxy)ethyl methacrylate (SEMA) and ethylene dimethacrylate (EDMA) in the presence of porogens. The sulfate group provided by the monomer SEMA on the monolithic bed is used for the generation of the electroosmotic flow (EOF) from the anode to the cathode, but at the same time serves as a SCX stationary phase. A mixed-mode (RP/SCX) mechanism for separation of peptides was observed in the monolithic column, comprising hydrophobic and electrostatic interaction as well as electrophoretic migration at a low pH value of mobile phase. A column efficiency of more than 280000 plates/m for the unretained compound has been obtained on the prepared monoliths. The relative standard deviations observed for to and retention factors of peptides were about 0.32% and less than 0.71% for ten consecutive runs, respectively. Effects of mobile phase compositions on the EOF of the monolithic column and on the separation of peptides were investigated. The selectivity on separation of peptides in the monolithic capillary column could be easily manipulated by varying the mobile phase composition.

Determination of peptides and amino acids from wool and beer with sensitive fluorescent reagent 2-(9-carbazole)-ethyl chloroformate by reverse phase high-performance liquid chromotography and liquid chromotography mass spectrometry

A new method for the sensitive determination of amino acids and peptides using the tagging reagent 2-(9-carbazole)-ethyl chloroformate (CEOC) with fluorescence (FL) detection has been developed. Identification of derivatives was carried out by liquid chromotography mass spectrometry. The chromophore in the 2-(9-fluorenyl)-ethyl chloroformate (FMOC) reagent was replaced by carbazole, which resulted in a sensitive fluorescence lerivatizing agent CEOC. CEOC can easily and quickly label peptides and amino acids. Derivatives are stable enough to be efficiently analyzed by high-performance liquid chromatography. Studies on derivatization demonstrate excellent derivative yields over the pH range 8.8-10.0. Maximal yields close to 100% are observed with three- to fourfold molar reagent excess. Derivatives exhibit strong fluorescence and allow direct injection of the reaction mixture with no significant disturbance from the major fluorescent reagent degradation by-products, such as 2(9-carbazole)-ethanol and bis-(2-(9-carbazole)-ethyl) carbonate. In addition, the detection responses for CEOC derivatives are compared to those obtained with FMOC. The ratios AC(CEOC)/AC(FMOC) = 1.00-1.82 for fluorescence (FL) response and AC'(CEOC)/AC'(FMOC) = 1.00-1.21 for ultraviolet (UV) response are observed (here, AC and AC' are, respectively, FL and UV F response). Separation of the derivatized peptides and amino acids has been optimized on a Hypersil BDS C18 column. Excellent linear responses are observed. This method was used successfully to analyze protein hydrolysates from wool and from direct-derivatized beer. (C) 2003 Elsevier Science (USA). All rights reserved.

Capillary electrochromatography for separation of peptides driven with electrophoretic mobility on monolithic column

A mode of capillary electrochromatography for separation of ionic compounds driven by electrophoretic mobility on a neutrally hydrophobic monolithic column was developed. The monolithic column was prepared from the in situ copolymerization of lauryl methacrylate and ethylene dimethacrylate to form a C-12 hydrophobic stationary phase. It was found that EOF in this hydrophobic monolithic column was very poor, even the pH value of mobile phase at 8.0. The peptides at acidic buffer were separated on the basis of their differences in electrophoretic mobility and hydrophobic interaction with the stationary phase; therefore, different separation selectivity can be obtained in CEC from that in capillary zone electrophoresis (CZE). Separation of peptides has been realized with high column efficiency (up to 150 000 plates/meter) and good reproducibility (migration time with RSD < 0.5%), and all of the peptides, including some basic peptides, showed good peak symmetry. Effects of the mobile phase compositions on the retention of peptides at low pH have been investigated in a hydrophobic capillary monolithic column. The significant difference in selectivity of peptides in CZE and CEC has been observed. Some peptide isomers that cannot be separated by CZE have been successfully separated on the capillary monolithic column in this mode with the same buffer used.

Direct determination of sialic acids in serum by capillary electrophoresis with UV detection

A novel method for the determination of N-acetylneuraminic acid (NANA) and N-glycolylneuraminic acid (NGNA) has been developed using high-performance capillary electrophoresis with UV detection at 195 nm, without pre or post-column derivatisation. The acids were separated in a 50-cm, fused-silica capillary (50 mu m i.d, 45.5-cm effective length) with Na2B4O7-Na2HPO4 buffer. The detection limit for NANA is a concentration of 9.6 x 10(-6) M or, in terms of mass: 3.879 x 10(-14) mol (39 fmol). This method is applicable to determination of NANA in normal human serum. The results were also compared with those of the colorimetrie method.

Protein A immobilized monolithic capillary column for affinity chromatography

Monolithic capillary columns for affinity chromatography were prepared by an in situ polymerization procedure using glycidyl methacrylate (GMA) as a monomer and trimethylolpropane trimethacrylate (TRIM) and ethylene dimethacrylate (EDMA) as cross-linkers, respectively. Scanning electron microscopy was applied to characterize the morphology of the end of monolithic capillary and mercury intrusion porosimetry to characterize the polymer rod prepared within the confines of a stainless steel column with 50 mm x 4.6 mm i.d. under the same polymerization condition. Obvious differences in the porous properties between the TRIM- and EDMA-based monoliths could be observed. Moreover, the mechanical stability of these two monolithic capillary columns was compared by testing the reproducibility of the column performance. The rod prepared with GMA and TRIM proved to be mechanically more stable than that prepared with GMA and EDMA. Protein A was immobilized on the monolithic rod for affinity chromatography and the experiments were performed on a capillary electrophoresis instrument, using its pressure system as the driving force. Non-specific adsorption was not observed on the TRIM-based affinity column, as proved with bovine serum albumin (BSA) as a test protein. The affinity column prepared with GMA and TRIM was then applied to determine the hIgG concentration in human serum. The correlative coefficient of the calibration curve reached 0.9942. The amount of adsorbed hIgG was unaffected by the flow rate of the loading buffer, which makes this method suitable for fast determination of biomacromolecules in microliter samples. (C) 2002 Elsevier Science B.V All rights reserved.

Modeling the effect of ozone loss on photobleaching of chromophoric dissolved organic matter in the St. Lawrence estuary

Temporal trends in total ozone for the St. Lawrence estuary were estimated from ground-based measurements at the NOAA/CMDL station in Caribou, Maine. Linear regression analysis showed that from 1979 to 1999 total ozone has decreased by about 3.3% per decade on an annual basis and ≤6.2% per decade on a monthly basis relative to unperturbed (pre-CFC) levels. The influence of increased ultraviolet-B (280–320 nm) radiation associated with ozone depletion on water column photochemical processes was evaluated by modeling the photobleaching of chromophoric dissolved organic material (CDOM). Linear regression analysis showed small (<0.5% per decade), but statistically significant upward trends in maximum noontime photobleaching rates. Most notably, positive trends in relative rates for May, June, and July, when maximum absolute rates are expected, were predicted. A global model based on TOMS ozone data revealed increases in photobleaching of ≤3% per decade at high latitudes in the Southern Hemisphere. Radiation amplification factors for increases in photochemically weighted UV (280–400 nm) in response to ozone depletion were estimated at 0.1 and 0.08 for photobleaching of CDOM absorbance at 300 and 350 nm, respectively. Application of the laboratory-based model to conditions that more closely resembled those in situ were variable with both overestimation and underestimation of measured rates. The differences between modeled rates and observed rates under quasi-natural conditions were as large or larger than the predicted increases due to ozone depletion. These comparisons suggest that biological activity and mixing play an important, but as yet ill-defined, role in modifying photochemical processes.

Grafting acrylic acid onto polypropylene by reactive extrusion with pre-irradiated PP as initiator

In this paper, the modification of polypropylene (PP) with acrylic acid (AA) by reactive extrusion using pre-irradiated PP (rPP) as initiator was investigated. It was found the relatively high graft degree (Gd) and slight degradation of modified PP was obtained when 20 wt% rPP was used. This result can be explained in terms of effective concentration of free radicals.

Preparation and Characterization of Melt Grafting 2-acrylamido-2-methyl-1-propanesulfonic Acid onto Pre-Irradiated Linear Low Density Polyethylene

Linear low density polyethylene (LLDPE) was functionalized with 2-acrylamido-2-methyl-1-propanesulfonic acid (AMPS) by using -ray pre-irradiation in air in a twin-screw extruder. Fourier-transformed infrared spectroscopy (FT-IR) and electron spectroscopy for chemical analysis (ESCA) were used to characterize the formation of LLDPE-g-AMPS copolymers. The content of AMPS in LLDPE-g-AMPS was determined by using element analysis instrument. The effects of concentrations of monomer, reaction temperature and pre-irradiation dose on degree of grafting were investigated. The critical surface tension of LLDPE-g-AMPS was measured by using contact angle method. The influences of the degree of grafting on crystallization properties were studied by using DSC. Compared with neat LLDPE, the crystallization temperature increased about 4C, and crystallinity decreased with increasing degree of grafting. Crystallization rates of grafted LLDPE were faster than that of plain LLDPE at the same crystallization temperature.

Determination of benzhexol hydrochloride by capillary zone electrophoresis with an end-column electrochemiluminescence detection

A capillary zone electrophoresis with end-column electrochemiluminescence (ECL) detector was described for the determination of benzhexol hydrochloride. The detection was based on the tris(2,2'-bypyridine)ruthenium(II) [Ru(bpy)(3)(2+)] ECL reaction with the analyte. Electrophoresis was performed using a 25 mum i.d. uncoated capillary. 10 mM sodium phosphate buffer (pH=8.0) was used as the running buffer. The solution in the detection cell was 80 mM sodium phosphate (pH=8.0) and 5 mM)21 Ru(bpy)(3)(2+). A linear calibration curve of three-orders of magnitude was obtained (with a correlation coefficient of > 0.999) from 1.0X10(-8) to 1.0X10(-5) M and the limit of detection was 6.7 X 10(-9) M (S/N= 3). This just provides an easy and sensitive method to determine the active ingredient in pharmaceutical formulations.