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First Dimension (first + dimension)
Selected AbstractsCapillary sieving electrophoresis and micellar electrokinetic capillary chromatography produce highly correlated separation of tryptic digestsELECTROPHORESIS, Issue 14 2010Jane A. Dickerson Abstract We perform 2-D capillary electrophoresis on fluorescently labeled proteins and peptides. Capillary sieving electrophoresis (CSE) was performed in the first dimension and MEKC was performed in the second. A cellular homogenate was labeled with the fluorogenic reagent FQ and separated using the system. This homogenate generated a pair of ridges; the first had essentially constant migration time in the CSE dimension, while the second had essentially constant migration time in the MEKC dimension. In addition, a few spots were scattered through the electropherogram. The same homogenate was digested using trypsin, and then labeled and subjected to the 2-D separation. In this case, the two ridges observed from the original 2-D separation disappeared and were replaced by a set of spots that fell along the diagonal. Those spots were identified using a local-maximum algorithm and each was fit using a 2-D Gaussian surface by an unsupervised nonlinear least squares regression algorithm. The migration times of the tryptic digest components were highly correlated (r=0.862). When the slowest migrating components were eliminated from the analysis, the correlation coefficient improved to r=0.956. [source] Heart-cutting 2D-CE with on-line preconcentration for the chiral analysis of native amino acidsELECTROPHORESIS, Issue 6 2010Suzanne Anouti Abstract The use of transient moving chemical reaction boundary (tMCRB) was investigated for the on-line preconcentration of native amino acids in heart-cutting 2D-CE with multiple detection points using contactless conductivity detection. The tMCRB focusing was obtained by using ammonium formate (pH 8.56) as sample matrix and acetic acid (pH 2.3) as a BGE in the first dimension of the heart-cutting 2D-CE. Different experimental parameters such as the injected volume and the concentration in ammonium formate were optimized for improving the sensitivity of detection. A stacked fraction from the first dimension was selected, isolated in the capillary, and then separated in the second dimension in the presence of a chiral selector ((+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid). This on-line tMCRB preconcentration coupled with heart-cutting 2D-CE was applied with success to the chiral separation of D,L -phenylalanine, and D,L -threonine in a mixture of 22 native amino acids. The sample mixture was diluted in 0.8,M of ammonium formate, and injected at a concentration of 2.5,,M for each enantiomer with a volume corresponding to 10% of the total capillary volume. An LOD (S/N=3) of 2,,M was determined for L -threonine. [source] Proteome analysis of human liver tumor tissue by two-dimensional gel electrophoresis and matrixassisted laser desorption/ionization-mass spectrometry for identification of disease-related proteinsELECTROPHORESIS, Issue 24 2002Jina Kim Abstract Hepatocellular carcinoma (HCC) is a common malignancy worldwide and is a leading cause of death. To contribute to the development and improvement of molecular markers for diagnostics and prognostics and of therapeutic targets for the disease, we have largely expanded the currently available human liver tissue maps and studied the differential expression of proteins in normal and cancer tissues. Reference two-dimensional electrophoresis (2-DE) maps of human liver tumor tissue include labeled 2-DE images for total homogenate and soluble fraction separated on pH 3,10 gels, and also images for soluble fraction separated on pH 4,7 and pH 6,9 gels for a more detailed map. Proteins were separated in the first dimension by isoelectric focusing on immobilized pH gradient (IPG) strips, and by 7.5,17.5% gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels in the second dimension. Protein identification was done by peptide mass fingerprinting with delayed extraction-matrix assisted laser desorption/ionization-time of flight-mass spectrometry (DE-MALDI-TOF-MS). In total, 212 protein spots (117 spots in pH 4,7 map and 95 spots in pH 6,9) corresponding to 127 different polypeptide chains were identified. In the next step, we analyzed the differential protein expression of liver tumor samples, to find out candidates for liver cancer-associated proteins. Matched pairs of tissues from 11 liver cancer patients were analyzed for their 2-DE profiles. Protein expression was comparatively analyzed by use of image analysis software. Proteins whose expression levels were different by more than three-fold in at least 30% (four) of the patients were further analyzed. Numbers of protein spots overexpressed or underexpressed in tumor tissues as compared with nontumorous regions were 9 and 28, respectively. Among these 37 spots, 1 overexpressed and 15 underexpressed spots, corresponding to 11 proteins, were identified. The physiological significance of the differential expressions is discussed. [source] The development of an emotion model based on colour combinationsINTERNATIONAL JOURNAL OF CONSUMER STUDIES, Issue 2 2006Young-Jin Lee Abstract The purpose of this study was to develop an emotion model based on the colour combinations popularly used for interior coordination in Korea. To summarize, the emotion model had the following features: (1) It consisted of three axes named as ,soft,hard' (first dimension), ,light,heavy' (second dimension) and ,splendid,sober' (third dimension). (2) The emotion descriptors were categorized into nine emotion groups and matched with the representing colour combinations. (3) This emotion model had a one-to-multiplicity correspondence structure between the colour combination and the emotion descriptor, whereas most of the previously developed models included only one-to-one correspondence. (4) It was observed that the emotion variable only showed a relatively consistent tendency within the space of the emotion model as the difference in the tone of colour combinations increased or decreased. The emotion model developed in this study can be used as a basis for the determination of local consumers' emotion on colour combinations to support colour planning in the industrial design field relevant to interior coordination. [source] Orthogonality of silver-ion and non-aqueous reversed-phase HPLC/MS in the analysis of complex natural mixtures of triacylglycerolsJOURNAL OF SEPARATION SCIENCE, JSS, Issue 21 2009Michal Hol Abstract The goal of this work is the study of possibilities of two basic separation modes used in the analysis of complex triacylglycerol (TG) samples of plant oils and animal fats, i.e. non-aqueous reversed-phase (NARP) and silver-ion HPLC coupled with atmospheric pressure chemical ionization mass spectrometry (APCI-MS). The orthogonality of both separation modes is tested for complex TG mixtures containing fatty acids (FAs) with different acyl chain lengths, different number, positions and geometry of double bonds (DBs) and different regioisomeric positions of FAs on the glycerol skeleton. The retention in NARP mode is governed by the equivalent carbon number, while the retention in silver-ion chromatography increases with the increasing number of DBs with a clear differentiation between cis - and trans- FAs. Moreover, silver-ion mode enables at least the partial resolution of regioisomeric TG mixtures including cis -/trans -regioisomers, as illustrated on two examples of randomization mixtures. Off-line 2D coupling of both complementary modes (NARP in the first dimension and silver-ion in the second dimension) yields the superior chromatographic selectivity resulting in the highest number of identified TGs ever reported for studied samples. Off-line 2D chromatograms are processed with the home-made software providing various ways of data visualization. [source] Purification of alkaloids from Corydalis yanhusuo W. T. Wang using preparative 2-D HPLCJOURNAL OF SEPARATION SCIENCE, JSS, Issue 9 2009Jing Zhang Abstract Two-dimensional preparative multi-channel parallel high performance liquid chromatography was successfully applied for the first time to isolate and purify alkaloids from Corydalis yanhusuo. The experiments were performed in off-line mode using the same preparative chromatographic column with pH 3.5 in the first and pH 10.0 in the second separation dimension. In the preparative process, UV-triggered fraction collection was used in the first dimension while UV and MS-triggered collection were used in the second dimension for reasons of sensitivity and complementarity. Two pure compounds and nine fractions were obtained in the first dimension. Then two representative fractions were further purified in the second dimension and six pure compounds were obtained. The results demonstrated that this procedure is an effective approach for the preparative isolation and purification of alkaloids from Corydalis yanhusuo. Based on the different pH values of the mobile phase in this method, it is also suitable for the preparative isolation and purification of other compounds from TCMs which are sensitive to the pH of the solutions. Moreover, this method will be a promising tool for the purification of low content compounds from natural products. [source] Linear peak capacity of a comprehensive multi-dimensional separationJOURNAL OF SEPARATION SCIENCE, JSS, Issue 19 2008Leonid M. Blumberg Abstract In order to resolve (quantifiably and identifiably separate) the same number of peaks in the analysis of the same mixture yielding statistically uniform peak distribution, a comprehensive 2-D separation needs a two times larger peak capacity than a 1-D separation does. Each additional dimension further reduces the utilization of the peak capacity of comprehensive multi-dimensional (MD) separation by a factor of two per dimension. As a result, the same peak capacity means different things for separations with different dimensionalities. This complicates the use of the peak capacity for comparison of the potential separation performance of the separations with different dimensionalities. To facilitate the comparison, a concept of a linear peak capacity has been proposed. The linear peak capacity of an MD separation is the peak capacity of a 1-D separation that, in the analysis of the same mixture, is statistically expected to resolve the same number of peaks as the MD separation is. There are other factors that differently affect the performance of the separations that have different dimensionalities. Peak capacity of a 2-D separation with a rectangular separation space is 27% larger than the product of the peak capacities of its first and second dimension. This advantage of a 2-D separation is essentially nullified by the fact that the peak capacity of the first dimension of an optimized 2-D separation cannot be higher than 80% of the peak capacity of its first dimension standing alone. All in all, the incremental peak capacity gained from addition of a second dimension will not exceed 50% of the peak capacity of the added second dimension. All results are valid for arbitrarily shaped (not necessarily Gaussian) peaks. [source] Characterization of biodiesel and biodiesel blends using comprehensive two-dimensional gas chromatographyJOURNAL OF SEPARATION SCIENCE, JSS, Issue 14 2008Warawut Tiyapongpattana Abstract In this work the development of a comprehensive 2-D GC flame ionization detection (GC×GC FID) method for biodiesel fuels is reported. This method is used for the analysis of fatty acid methyl esters (FAMEs) in both biodiesel (B100) and biodiesel blend (B5) samples. The separation of FAME was based on component boiling point in the first dimension and polarity in the second dimension by using a BPX5/BP20 column set to provide a measure of ,orthogonality' in the 2-D space. Here the columns are coupled with a cryogenic modulator operating in a novel temperature programmed mode (TM) whereby the cryotrap is progressively incremented in temperature as the oven temperature is increased. The final method employs eight cryotrap temperature settings. The developed GC×GC method is able to successfully characterize and identify both B100 and B5 FAME components, which are produced from a variety of vegetable oils, animal fats and waste cooking oils, with high precision. The method is capable of analysing FAME with carbon numbers C4,C24, and is particularly suitable to characterize various types of biodiesel, making it possible to differentiate the origin and type of FAME used in the biodiesel samples. [source] Determination and removal of impurities in 2-D LC-MS of peptidesJOURNAL OF SEPARATION SCIENCE, JSS, Issue 4 2006Albena Mihailova Abstract Problems occurring during operation of a 2-D LC-MS system for separation and identification of neuropeptides, such as contamination of the used salts and column bleed, are described. When using polysulfoethyl aspartamide, which is widely used as a strong cation exchange stationary phase in the first dimension, interfering peaks were observed in the second-dimension reversed-phase chromatograms. The observed peaks, found to be caused by column bleeding, had abundance above the threshold value and influenced the quality of the analyses. The origin of the peaks was verified and appropriate measures are proposed. Additionally, peaks caused by polyethylene glycols (PEGs), covering approximately 5 min of feasible chromatographic time in every fraction, were observed. The commercial ammonium formate salts used to prepare the first-dimension mobile phase were found to contain PEG impurities, and in subsequent work the salt solutions were prepared from formic acid and ammonia to avoid any additional contaminations. [source] Simple 2D-HPLC using a monolithic silica column for peptide separationJOURNAL OF SEPARATION SCIENCE, JSS, Issue 10-11 2004Hiroshi Kimura Abstract Separation of peptides by fast and simple two-dimensional (2D)-HPLC was studied using a monolithic silica column as a second-dimension (2nd-D) column. Every fraction from the first column, 5 cm long (2.1 mm ID) packed with polymer-based cation exchange beads, was subjected to separation in the 2nd-D using an octadecylsilylated (C18) monolithic silica column (4.6 mm ID, 2.5 cm). A capillary-type monolithic silica C18 column (0.1 mm ID, 10 cm) was also employed as a 2nd-D column with split flow/injection. Effluent of the first dimension (1st-D) was directly loaded into an injector loop of 2nd-D HPLC. UV and MS detection were successfully carried out at high linear velocity of mobile phase at 2nd-D using flow splitting for the 4.6 mm ID 2nd-D column, or with direct connection of the capillary column to the MS interface. Two-minute fractionation in the 1st-D, 118-second loading, and 2-second injection by the 2nd-D injector, allowed one minute for gradient separation in the 2nd-D, resulting in a maximum peak capacity of about 700 within 40 min. The use of a capillary column in the 2nd-D led to less solvent consumption and better MS detectability compared to a larger-sized column. This kind of fast and simple 2D-HPLC utilizing monolithic silica columns will be useful for the separation of complex mixtures in a short time. [source] The evolution of comprehensive two-dimensional gas chromatography (GC×GC)JOURNAL OF SEPARATION SCIENCE, JSS, Issue 5-6 2004Tadeusz Górecki Abstract For a technology little over a decade old, comprehensive two-dimensional gas chromatography (GC×GC) has quickly reached the status of one of the most powerful analytical tools for volatile organic compounds. At the heart of any GC×GC system is an interface, which physically connects the primary and the secondary columns and acts to preserve the separation obtained in the first dimension (first column) while allowing additional separation in the second dimension. The paper presents a review of the technology, including fundamental principles of the technique, data processing and interpretation and a timeline of inventive contributions to interface design. In addition, applications of the technique are presented, with a more detailed discussion of selected examples. [source] Mobilizing the Law in China: "Informed Disenchantment" and the Development of Legal ConsciousnessLAW & SOCIETY REVIEW, Issue 4 2006Mary E. Gallagher This article critically examines the development of legal consciousness among legal aid plaintiffs in Shanghai. It is based on 16 months of research at a large legal aid center and in-depth interviews with 50 plaintiffs. Chinese legal aid plaintiffs come to the legal process with high expectations about the possibility of protecting their rights; however, they also have only a vague and imprecise knowledge of legal procedure and their actual codified rights. Through this process of legal mobilization, plaintiffs' legal consciousness changes in two separate dimensions: changes in one's feelings of efficacy and competency vis-à-vis the law, and changes in one's perception/evaluation of the legal system. Put another way, the first dimension is "How well can I work the law?" and the second is "How well does the law work?" In this study I observe positive changes in feelings of individual efficacy and competency that are combined with more negative evaluations/perceptions of the legal system in terms of its fairness and effectiveness. The positive feelings of efficacy and voice provided by the legal process encourage labor dispute plaintiffs in the post-dispute period to plan new lawsuits and to help friends and relatives with their legal problems. Disenchantment with the promises of the legal system does not lead to despondency, but to more critical, informed action. This study provides new evidence on the nature of China's developing legal system with a focus on the social response to the state-led "rule of law" project. [source] The design of a multi-dimensional LC-SPE-NMR system (LC2 -SPE-NMR) for complex mixture analysis,MAGNETIC RESONANCE IN CHEMISTRY, Issue 1 2006A. J. Alexander Abstract In this communication, we describe the design of an online multi-chromatographic approach to the routine NMR analyses of low-level components (,0.1%) in complex mixtures. The technique, termed LC2 -SPE-NMR, optimally combines multi-dimensional liquid chromatography with SPE technology for isolating, enriching and delivering trace analytes to the NMR probe. The fully automated LC2 -SPE-NMR system allows for maximal loading capacity (in the first, preparative LC dimension), close to optimal peak resolution (in the second, analytical LC dimension) and enhanced sample concentration (through SPE). Using this system, it is feasible to conveniently conduct a wide range of NMR experiments on, for example, drug impurities at the low microgram per milliliter level, even for components poorly resolved in the first dimension. Such a sensitivity gain significantly elevates the analytical power of online NMR technology in terms of the level at which substances of pharmaceutical significance can be structurally characterized. Copyright © 2006 John Wiley & Sons, Ltd. [source] Allelic variants of granule-bound starch synthase proteins in European bread wheat varietiesPLANT BREEDING, Issue 4 2000C. Marcoz-Ragot Abstract The composition of 324 European wheat cultivars were analysed at the three granule-bound starch synthase (GBSS I) loci. Protein separation was first made by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE. A specific two-dimensional (2D) electrophoresis (immobilized pH gradient × SDS-PAGE) using an Immobiline dry strip in the first dimension was developed to resolve the GBSS I proteins more clearly and to confirm some results. Very low polymorphism was found. Among the 324 cultivars analysed, only one carried a Wx-A1 null allele (Wx-A1b) and none was found to have the Wx-2D null allele. As described in the literature the Wx-B1 locus was more polymorphic and the null allele was encountered in 11 cultivars. The use of 2D electrophoresis allowed us to find another type of variant which presented as having thicker band with same mobility as the Wx-D1 protein in SDS-PAGE. Twelve per cent of the cultivars analysed presented this band and could have been previously mistaken for cultivars carrying the Wx-B1 null allele. Indeed this band probably corresponded to the Wx-B1, or Wx-B1e allele overlapping with the Wx-D1a allele in SDS-PAGE. [source] Two-dimensional reference map for the basic proteome of the human dorsolateral prefrontal cortex (dlPFC) of the prefrontal lobe region of the brainPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 13 2010Ciara A. McManus Abstract We describe a 2-DE proteomic reference map containing 227 basic proteins in the dorsolateral prefrontal cortex region of the human brain. Proteins were separated in the first dimension on pH 6,11 IPG strips using paper-bridge loading and on 12% SDS-PAGE in the second dimension. Proteins were subsequently identified by MS and spectra were analyzed using an in-house proteomics data analysis platform, Proline. The 2-DE reference map is available via the UCD 2-DE Proteome Database (http://proteomics-portal.ucd.ie:8082) and can also be accessed via the WORLD-2DPAGE Portal (http://www.expasy.ch/world-2dpage/). The associated protein identification data have been submitted to the PRIDE database (accession numbers 10018,10033). Separation of proteins in the basic region resolves more membrane associated proteins relevant to the synaptic pathology central to many neurological disorders. The 2-DE reference map will aid with further characterisation of neurological disorders such as bipolar and schizophrenia. [source] Proteomic and functional alterations in brain mitochondria from Tg2576 mice occur before amyloid plaque depositionPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 4 2007Frank Gillardon Dr. Abstract Synaptic dysfunction is an early event in Alzheimer's disease patients and has also been detected in transgenic mouse models. In the present study, we analyzed proteomic changes in synaptosomal fractions from Tg2576 mice that overexpress mutant human amyloid precursor protein (K670N, M671L) and from their nontransgenic littermates. Cortical and hippocampal tissue was microdissected at the onset of cognitive impairment, but before deposition of amyloid plaques. Crude synaptosomal fractions were prepared by differential centrifugation, proteins were separated by 2-D DIGE and identified by MS/MS. Significant alterations were detected in mitochondrial heat shock protein 70 pointing to a mitochondrial stress response. Subsequently, synaptosomal versus nonsynaptic mitochondria were purified from Tg2576 mice brains by density gradient centrifugation. Mitochondrial proteins were separated by IEF or Blue-native gel electrophoresis in the first dimension and SDS-PAGE in the second dimension. Numerous changes in the protein subunit composition of the respiratory chain complexes I and III were identified. Levels of corresponding mRNAs remain unchanged as shown by Affymetrix oligonucleotide array analysis. Functional examination revealed impaired state 3 respiration and uncoupled respiration in brain mitochondria from young Tg2576 mice. By immunoblotting, amyloid-beta oligomers were detected in synaptosomal fractions from Tg2576 mice and reduced glucose metabolism was observed in Tg2576 mice brains by [14C]-2-deoxyglucose infusion. Taken together, we demonstrate alterations in the mitochondrial proteome and function that occur in Tg2576 mice brains before amyloid plaque deposition suggesting that mitochondria are early targets of amyloid-beta aggregates. [source] On-line 2D-LC-ESI/MS/MS determination of rifaximin in rat serumBIOMEDICAL CHROMATOGRAPHY, Issue 11 2009R. Nageswara Rao Abstract A highly sensitive and selective on-line two-dimensional reversed-phase liquid chromatography/electrospray ionization,tandem mass spectrometry (2D-LC-ESI/MS/MS) method was developed and validated to determine rifaximin in rat serum by direct injection. The 2D-LC-ESI/MS/MS system consisted of a restricted access media column for trapping proteins as the first dimension and a Waters C18 column as second dimension using 0.1% aqueous acetic acid:acetonitrile as mobile phase in a gradient elution mode. Rifampacin was used as an internal standard. The linear dynamic range was 0.5,10 ng/mL (r2 > 0.998). Acceptable precision and accuracy were obtained over the calibration range. The assay was successfully used in analysis of rat serum to support pharmacokinetic studies. Copyright © 2009 John Wiley & Sons, Ltd. [source] | |||||||||||||