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Rapid Separation (rapid + separation)
Selected AbstractsRapid separation of protein isoforms by capillary zone electrophoresis with new dynamic coatingsELECTROPHORESIS, Issue 11 2005William W. P. Chang Abstract Many cellular functions are regulated through protein isoforms. Changes in the expression level or regulatory dysfunctions of isoforms often lead to developmental or pathological disorders. Isoforms are traditionally analyzed using techniques such as gel- or capillary-based isoelectric focusing. However, with proper electroosmotic flow (EOF) control, isoforms with small pI differences can also be analyzed using capillary zone electrophoresis (CZE). Here we demonstrate the ability to quickly resolve isoforms of three model proteins (bovine serum albumin, transferrin, ,1 -antitrypsin) in capillaries coated with novel dynamic coatings. The coatings allow reproducible EOF modulation in the cathodal direction to a level of 10 -9 m2V -1s -1. They also appear to inhibit protein adsorption to the capillary wall, making the isoform separations highly reproducible both in peak areas and apparent mobility. Isoforms of transferrin and ,1 -antitrypsin have been implicated in several human diseases. By coupling the CZE isoform separation with standard affinity capture assays, it may be possible to develop a cost-effective analytical platform for clinical diagnostics. [source] Preparation and characterization of a molecularly imprinted monolithic column for pressure-assisted CEC separation of nitroimidazole drugsELECTROPHORESIS, Issue 16 2010Sulan Liao Abstract A polymethacrylate-based molecularly imprinted monolithic column bearing mixed functional monomers, using non-covalent imprinting approach, was designed for the rapid separation of nitroimidazole compounds. The new monolithic column has been prepared via simple in situ polymerization of 2-hydroxyethyl methacrylate, dimethylaminoethyl methacrylate and ethylene dimethacrylate, using (S)-ornidazole ((S)-ONZ) as template in a binary porogenic mixture consisting of toluene and dodecanol. The composition of the polymerization mixture was systematically altered and optimized by altering the amount of monomers as well as the composition of the porogenic solvent. The column performance was evaluated in pressure-assisted CEC mode. Separation conditions such as pH, voltage, amount of organic modifier and salt concentration were studied. The optimized monolithic column resulted in excellent separation of a group of structurally related nitroimidazole drugs within 10,min in isocratic elution condition. Column efficiencies of 99,000, 80,000, 103,000, 60,000 and 99,000,plates/m were obtained for metronidazole, secnidazole, ronidazole, tinidazole and dimetridazole, respectively. Parallel experiments were carried out using molecularly imprinted and non-imprinted capillary columns. The separation might be the result of combined effects including hydrophobic, hydrogen bonding and the imprinting cavities on the (S)-ONZ-imprinted monolithic column. [source] Development of a new method for analysis of Sudan dyes by pressurized CEC with amperometric detectionELECTROPHORESIS, Issue 11 2007Shaofeng Liu Abstract A new analytical method, pressurized CEC (pCEC) with amperometric detection (AD) using 1.5,,m RP nonporous silica packed columns has been developed for the rapid separation and determination of four Sudan dyes in hot chilli. The influence of several experimental parameters on the retention behavior has been investigated. The electrochemical oxidation of Sudans I,IV separated by pCEC can be reliably monitored with a carbon electrode at +0.95,V (vs. Ag/AgCl). Fast and efficient separation of the analytes was achieved within 7,min by pCEC under the optimum conditions with an ACN/water (95:5%) mobile phase containing formic acid (pH,4.3), 5% acetone and 0.002% triethylamine using a separation voltage of 12,kV. The detection limits for four Sudan dyes ranged from 8.0,×,10,7 to 1.2,×,10,6,mol/L. To evaluate the feasibility and reliability of this method, the proposed pCEC-AD method was further demonstrated with hot chilli samples spiked with Sudan dyes. [source] Rapid detection of Staphylococcus aureus by a combination of monoclonal antibody-coated latex and capillary electrophoresisELECTROPHORESIS, Issue 9 2006Peng Gao Abstract The rapid detection of pathogenic bacteria is extremely important in biotechnology and clinical diagnosis. CE has been utilized in the field of bacterial analysis for many years, but to some extent, simultaneous separation and identification of certain microbes from complex samples by CE coupled with UV detector is still a challenge. In this paper, we propose a new strategy for rapid separation and identification of Staphylococcus aureus (S.,aureus) in bacterial mixtures by means of specific mAb-coated latex coupled with CZE. An appropriate set of conditions that selectively isolated S.,aureus from the microorganisms Escherichia coli, Pseudomonas aeruginosa, and Klebsiella pneumoniae were established. S.,aureus could be differentiated from the others by unique peaks in the electropherograms. The validity was also confirmed by LIF with antibodies specific to both the latex and the microbial cells. The LOD is as low as 9.0×105 colony forming unit/mL. We have also utilized this technology to identify S.,aureus in a stool sample coming from a healthy volunteer spiked successfully with S.,aureus. This CZE-UV technique can be applied to rapid diagnosis of enteritis caused by S.,aureus or other bacterial control-related fields needing rapid identification of target pathogens from microbial mixtures. In theory, this method is suitable for the detection of any bacterium as long as corresponding bacterium-specific antibody-coated latex is available. [source] High-throughput screening of kinase inhibitors by multiplex capillary electrophoresis with UV absorption detectionELECTROPHORESIS, Issue 1-2 2003Yan He Abstract Protein kinases play a major role in the transformation of cells and are often used as molecular targets for the new generation of anticancer drugs. We present a novel technique for high-throughput screening of inhibitors of protein kinases. The technique involves the use of multiplexed capillary electrophoresis (CE) for the rapid separation of the peptides, phosphopeptides, and various inhibitors. By means of UV detection, diversified peptides with native amino acid sequences and their phosphorylated counterparts can be directly analyzed without the need for radioactive or fluorescence labeling. The effects of different inhibitors and their IC50 value were determined using three different situations involving the use of a single purified kinase, two purified kinases, and crude cell extracts, respectively. The results suggest that multiplexed CE/UV may prove to be a straightforward and general approach for high-throughput screening of compound libraries to find potent and selective inhibitors of the various protein kinases. [source] Identification and determination of the saikosaponins in Radix bupleuri by accelerated solvent extraction combined with rapid-resolution LC-MSJOURNAL OF SEPARATION SCIENCE, JSS, Issue 13 2010Yun-Yun Yang Abstract A method based on accelerated solvent extraction combined with rapid-resolution LC,MS for efficient extraction, rapid separation, online identification and accurate determination of the saikosaponins (SSs) in Radix bupleuri (RB) was developed. The RB samples were extracted by accelerated solvent extraction using 70% aqueous ethanol v/v as solvent, at a temperature of 120°C and pressure of 100,bar, with 10,min of static extraction time and three extraction cycles. Rapid-resolution LC separation was performed by using a C18 column at gradient elution of water (containing 0.5% formic acid) and acetonitrile, and the major constituents were well separated within 20,min. A TOF-MS and an IT-MS were used for online identification of the major constituents, and 27 SSs were identified or tentatively identified. Five major bioactive SSs (SSa, SSc, SSd, 6,- O -acetyl-SSa and 6,- O -acetyl-SSd) with obvious peak areas and good resolution were chosen as benchmark substances, and a triple quadrupole MS operating in multiple-reaction monitoring mode was used for their quantitative analysis. A total of 16 RB samples from different regions of China were analyzed. The results indicated that the method was rapid, efficient, accurate and suitable for use in the quality control of RB. [source] Monitoring of mutarotation of monosaccharides by hydrophilic interaction chromatographyJOURNAL OF SEPARATION SCIENCE, JSS, Issue 6-7 2010í Pazourek Abstract Calibration based on the "single-point calibration method", a simple exponential transformation of the response function of an evaporative light scattering detector was improved and applied to analysis of selected saccharides under hydrophilic interaction chromatography mode (a polar phase LiChrospher100 DIOL, mobile phase acetonitrile/water). The improved approach to the calibration procedure yielded a calibration curve with an excellent linearity (quality coefficient <5%). This quantitative evaluation of chromatograms of D -galactose suggested that not only anomers but even pyranose and furanose forms of the anomers could be resolved , the resulting calculations of abundance of the anomeric form strongly correlated with data from the literature obtained mostly by NMR studies (analogous results were also obtained for D -arabinose, D -glucose, and D -mannose). Because of the rapid separation (retention time less than 10,min), the observed correlation enabled to monitor anomeric conversion (mutarotation) of monosaccharides. [source] Chemical fingerprint of commercial Radix Echinopsis and quantitative analysis of ,-terthienylJOURNAL OF SEPARATION SCIENCE, JSS, Issue 4-5 2010Xue Qiao Abstract New TLC, HPLC and LC/MS methods were developed for the rapid separation, characterization and quantitation of thiophenes in Radix Echinopsis, a herbal medicine, which has been used in China for long history. Nineteen commercial batches of this herb were analyzed by TLC and HPLC. Only five batches were derived from correct species. The other 14 batches were identified as its adulterating species, Radix Rhapontici, as they did not contain characteristic thiophenes of Echinopsis species. By LC/MS, 16 thiophenes were identified from the methanol extracts of Radix Echinopsis and Radix Rhapontici according to their fragmentation behaviors in MS/MS. In addition, a fully validated HPLC method was established to determine the contents of ,-terthienyl in Radix Echinopsis. The contents ranged from 2.7 to 5.2,mg/g for five tested samples. The methods in this paper were simple and reliable, and could be used for the comprehensive quality control of Radix Echinopsis. [source] Evaluation of use of a very short polar microbore column segment in high-speed gas chromatography analysisJOURNAL OF SEPARATION SCIENCE, JSS, Issue 14 2008Peter Quinto Tranchida Abstract Very fast GC analyses are commonly carried out by using 10 m×0.1 mm id capillaries. In order to achieve rapid elution times (1,3 min), the latter are operated under suboptimum conditions. The present research is focused on the evaluation of use of a 0.1 mm id polar column segment (2 m), operated under near-to-optimum conditions, in very fast GC analysis. The results attained are compared with those derived from using a 10 m microbore column in very fast GC experiments. Prior to method development, the effects of gas velocity, temperature program rate, and sample amounts on analytical performance were evaluated. Following these preliminary applications, a complex lipidic sample, cod liver oil, was subjected to rapid separation (,2.1 min) on the 10 m capillary through the application of a 50°C/min temperature rate and a 130 cm/s gas velocity. The same matrix was analyzed on the 2 m capillary using the same temperature program rate and range, but with a close-to-ideal linear velocity. The results observed were of interest, as the separation was achieved in less time (1.45 min) with improved peak resolution. Finally, both methods were validated in terms of retention time and peak area repeatability, LOQ, and linearity. [source] High-performance liquid chromatographic separation of natural and synthetic desulphoglucosinolates and their chemical validation by UV, NMR and chemical ionisation-MS methodsPHYTOCHEMICAL ANALYSIS, Issue 4 2001Guy Kiddle Abstract Methods are described for the optimised extraction, desulphation and HPLC separation of desulphoglucosinolates. These methods provide rapid separation, identification and quantitative measurements of glucosinolates extracted from Brassica napus L and related crops, of unusual glucosinolates found in crucifer weed species, and also of synthetic alkylglucosinolates. The desulphoglucosinolates used in these studies were either chemically synthesised (at least one example from each major structural class), or purified from various plant sources. Validation of the identities of the desulphoglucosinolates was by comparison of retention times with standards, and by UV, 1H- and 13C-NMR and chemical ionisation MS analysis. A list of useful species, and the specific tissues, from which high concentrations of standards can be extracted is included. Copyright © 2001 John Wiley & Sons, Ltd. [source] High-speed separation and characterization of major constituents in Radix Paeoniae Rubra by fast high-performance liquid chromatography coupled with diode-array detection and time-of-flight mass spectrometryRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 1 2009E-Hu Liu A fast high-performance liquid chromatography (HPLC) method coupled with diode-array detection (DAD) and electrospray ionization time-of-flight mass spectrometry (ESI-TOFMS) has been developed for rapid separation and sensitive identification of major constituents in Radix Paeoniae Rubra (RPR). The total analysis time on a short column packed with 1.8-µm porous particles was about 20,min without a loss in resolution, six times faster than the performance of a conventional column analysis (115,min). The MS fragmentation behavior and structural characterization of major compounds in RPR were investigated here for the first time. The targets were rapidly screened from RPR matrix using a narrow mass window of 0.01,Da to restructure extracted ion chromatograms. Accurate mass measurements (less than 5,ppm error) for both the deprotonated molecule and characteristic fragment ions represent reliable identification criteria for these compounds in complex matrices with similar if not even better performance compared with tandem mass spectrometry. A total of 26 components were screened and identified in RPR including 11 monoterpene glycosides, 11 galloyl glucoses and 4 other phenolic compounds. From the point of time savings, resolving power, accurate mass measurement capability and full spectral sensitivity, the established fast HPLC/DAD/TOFMS method turns out to be a highly useful technique to identify constituents in complex herbal medicines. Copyright © 2008 John Wiley & Sons, Ltd. [source] Validation of a real-time monitoring method for aniline in freshwater by high-performance liquid chromatography on porous graphitic carbon/electrospray ionization tandem mass spectrometryRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 14 2004Raphaël Delépée Aniline is an anthropogenic organic compound widely used in polymer, rubber, pharmaceutical and dye industries but also used in biodegradability assays of chemical compounds as a positive biodegradation standard. By the two approaches, the rapid determination of aniline is necessary because of the high toxicity of aniline on hemoglobin. A high-performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS) method for the determination of aniline in water is described here. This method, using benzylamine as internal standard, was validated. No time-consuming sample preparation was needed. A rapid separation (7,min between two chromatographic runs) of aniline and benzylamine was performed on a Hypercarb porous graphitic carbon column using a gradient of methanol and 100,mM formic acid. The obtained limits of detection and quantification were 10 and 1,ng/mL, respectively. The response for aniline was quadratic. We show that this problem could be circumvented by showing that the [calculated concentration,=,f(introduced concentration)] function was linear. The linearity range was 10,1000,ng/mL. An example of an application consisting of an aniline 42-day degradation kinetic in water was demonstrated. Copyright © 2004 John Wiley & Sons, Ltd. [source] Selective Extraction of Free Astaxanthin from Haematococcus Culture Using a Tandem Organic Solvent SystemBIOTECHNOLOGY PROGRESS, Issue 4 2007Chang Duk Kang A novel tandem solvent process of dodecane and methanol was developed for the selective extraction of free astaxanthin from red encysted Haematococcus culture. The process consists of dodecane extraction for astaxanthin mixture from the culture (stage 1) and methanol extraction for free astaxanthin from the dodecane extract (stage 2). In the first stage, astaxanthin mixture was directly extracted to dodecane from the culture broth without cell harvest process, followed by a rapid separation of the dodecane extract and the culture medium containing cell debris by simple settling. In the second stage, free astaxanthin was selectively collected to methanol from the dodecane extract, accompanied with saponification of astaxanthin-esters by the addition of NaOH to methanol. During saponification, use of the optimum NaOH concentration (0.02 M) and low temperature (4 °C) reaction minimized the degradation of free astaxanthin, resulting in a total recovery yield of free astaxanthin of over 85%. The free-astaxanthin-containing methanol extract was also simply separated from dodecane by gravity settling, after which the astaxanthin-free dodecane was effectively recycled to the first stage, yielding a stable extractability of astaxanthin mixture during repeated extraction. Our results indicate the potential of the proposed tandem solvent process as an alternative extraction technology for the high-value antioxidant Haematococcus astaxanthin. [source] Preparation of Immobilized Metal Affinity Chromatographic Packings by Immobilization of Carboxymethylated Asparate (CM-Asp) Based on Monodisperse Hydrophilic Non-porous Beads and Their ApplicationCHINESE JOURNAL OF CHEMISTRY, Issue 7 2010Bolin Gong Abstract The hydrophilic immobilized metal affinity chromatographic packing was prepared by immobilization of carboxymethylated asparate (CM-Asp) as chelating ligand and Ni2+ as center ion on the base of monodispersed, 3.0 µm non-porous monodisperse poly(glycidylmethacrylate- co -ethylenedimethacrylate) (PGMA/EDMA) particles. The retention behavior of proteins and the effect of pH on the retention in the range from 4.0 to 9.0 were investigated on both the naked and metal ion chelated columns. Four proteins were quickly separated in 2.0 min with linear gradient elution at a flow rate of 3.0 mL·min,1 by using the synthesized Ni2+ -CM-Asp-PGMA/EDMA packings. The separation time was shorter than other immobilized metal affinity chromatography reported in the literature. The Ni2+ -CM-Asp-PGMA/EDMA column was further investigated for the rapid separation and purification of copper-zinc superoxide dismutase (Cu,Zn-SOD) from the blood of pig in 3.0 min with only one step. The results obtained were satisfactory. [source] Preparation of Medium Cation Exchange Stationary Phase of Polymeric Matrix and Their Chromatographic PropertiesCHINESE JOURNAL OF CHEMISTRY, Issue 1 2007Gang Chen Abstract Based on the monodisperse poly(glycidyl methacrylate-co-ethylenedimethacrylate) beads (PGMA/EDMA) with macropore as a medium, a new hydrophilic medium cation exchange (MCX) stationary phase for HPLC was synthesized by a new chemically modified method. The stationary phase was evaluated with the property of ion exchange, separability, reproducibility, hydrophilicity, effect of salt concentration, salt types, column loading and pH on the separation and retention of proteins in detail. It was found that it follows ion exchange chromatographic (IEC) retention mechanism. The measured bioactivity recovery for lysozyme was (96±5)%. The dynamic protein loading capacity of the synthesized MCX packings was 21.8 mg/g. Five proteins were almost completely separated within 6.0 min at a flow rate of 4 mL/min using the synthesized MCX resin. The MCX resin was also used for the rapid separation and purification of lysozyme from egg white with only one step. The purity and specific bioactivity of the purified lysozyme was found more than 95% and 70345 U/mg, respectively. [source] Rapid capillary electrophoresis time-of-flight mass spectrometry separations of peptides and proteins using a monoquaternarized piperazine compound (M7C4I) for capillary coatingsELECTROPHORESIS, Issue 8 2008Anisa Elhamili Abstract A monoquaternarized piperazine, 1-(4-iodobutyl) 4-aza-1-azoniabicyclo[2,2,2] octane iodide (M7C4I), has been evaluated as a surface derivatization reagent for CE in combination with TOF MS for the analysis of proteins, peptides, and protein digests. The M7C4I piperazine, at alkaline pH, forms a covalent bond via alkylation of the ionized silanols producing a cationic surface with a highly stable and reversed EOF. The obtained surface yields rapid separations (less than 5,min) of peptides and proteins at acidic pH with high separation efficiencies (up to 1.1×106 plates/m for peptides and up to 1.8×106 plates/m for proteins) and no observed bleeding of the coating reagent into the mass spectrometer. The simplicity of the coating procedure also enables fast (2,min) regeneration of the surface, if necessary. This is useful in the analysis of complex samples in order to prevent possible memory effects. The potential of using M7C4I-coated capillaries for MS analysis of complex samples is demonstrated by the separation of peptides, proteins, and protein digests. Even more, the spectacular thing in which large intact proteins with molecular masses over 0.5,MDa could be separated. The coating showed good ability to handle these large proteins with high efficiency and retained peak shape as demonstrated by separation of IgG1 (150,kDa) and thyroglobulin (669,kDa). [source] An optimized microchip electrophoresis system for mutation detection by tandem SSCP and heteroduplex analysis for p53,gene exons,5,9ELECTROPHORESIS, Issue 19 2006Christa N. Hestekin Abstract With the complete sequencing of the human genome, there is a growing need for rapid, highly sensitive genetic mutation detection methods suitable for clinical implementation. DNA-based diagnostics such as single-strand conformational polymorphism (SSCP) and heteroduplex analysis (HA) are commonly used in research laboratories to screen for mutations, but the slab gel electrophoresis (SGE) format is ill-suited for routine clinical use. The translation of these assays from SGE to microfluidic chips offers significant speed, cost, and sensitivity advantages; however, numerous parameters must be optimized to provide highly sensitive mutation detection. Here we present a methodical study of system parameters including polymer matrix, wall coating, analysis temperature, and electric field strengths on the effectiveness of mutation detection by tandem SSCP/HA for DNA samples from exons,5,9 of the p53 gene. The effects of polymer matrix concentration and average molar mass were studied for linear polyacrylamide (LPA) solutions. We determined that a matrix of 8%,w/v 600,kDa LPA provides the most reliable SSCP/HA mutation detection on chips. The inclusion of a small amount of the dynamic wall-coating polymer poly- N -hydroxyethylacrylamide in the matrix substantially improves the resolution of SSCP conformers and extends the coating lifetime. We investigated electrophoresis temperatures between 17 and 35°C and found that the lowest temperature accessible on our chip electrophoresis system gives the best condition for high sensitivity of the tandem SSCP/HA method, especially for the SSCP conformers. Finally, the use of electrical fields between 350 and 450,V/cm provided rapid separations (<10,min) with well-resolved DNA peaks for both SSCP and HA. [source] Hydrophilic interaction and reversed-phase ultra-performance liquid chromatography TOF-MS for metabonomic analysis of Zucker rat urineJOURNAL OF SEPARATION SCIENCE, JSS, Issue 9 2008Helen G. Gika Abstract Hydrophilic interaction chromatography (HILIC) provides a complementary technique to RP methods for the retention of polar analytes for LC-MS-based metabonomic studies. Combining the advantages of both RP and HILIC separations with the efficient and rapid separations obtained using sub-2 ,m particles via the recently introduced ultra-performance LC (UPLC) enables increased coverage of the metabolites present in biological samples to be achieved. Here an HILIC-UPLC-MS method was developed to provide metabolite profiles for urine samples obtained from male Zucker rats. The resulting data were compared with results obtained for the same samples by RP-UPLC-MS and demonstrated the complementary nature of the two separations with both methods enabling discrimination between the different sample types. Interestingly sample type differentiation was based on different markers. [source] | |||||||||||||