共查询到20条相似文献,搜索用时 15 毫秒
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Plasma disaturated phosphatidylcholine (DSPC) concentration has been implicated as a risk factor for atherosclerosis. However, suitable methods for the estimation of these compounds in plasma are not available. In this paper, a method for the estimation of DSPC using argentation thin-layer chromatography and high-performance liquid chromatography is described. It is quantitative for the measurement of individual and total DSPC species and is not dependent on fatty acid chain length. The method employs hydrolysis of total plasma phosphatidyl choline by phospholipase C, followed by benzoylation of the diacylglycerols. The benzoates are then fractionated on silver nitrate-impregnated silica gel thin-layer chromatography plates, and the disaturated species separated and quantitated by high-performance liquid chromatography. The method is sensitive and reproducible and allows many samples to be done at once. With this method, the amounts of DSPC were found to be significantly higher in a group of normolipidemic diabetic subjects, compared to age-matched controls. 相似文献
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Catechols are co-planar cis-diols known to form stable, isolable complexes with borate under weakly basic conditions. We exploited this chemistry and developed a boronate affinity chromatography for isolating catechol siderophores. The method was applied to the isolation of chrysobactin, enterobactin, and an unknown catechol siderophore produce by Erwinia carotovora subsp. carotovora W3C105. Yields of chrysobactin and enterobactin purified by boronate affinity chromatography were at least two-fold greater than those achieved through alternate methods. The unknown catechol produced by E. carotovora subsp. carotovora W3C105 was isolated by boronate affinity chromatography and shown to be identical to chrysobactin. Boronate affinity chromatography enabled separation of catechol from its rust-colored decomposition products, and simultaneous isolation of catechol and hydroxamate siderophores. Boronate affinity chromatography is a rapid and efficient method for purifying catechol siderophores from bacterial culture supernatants 相似文献
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L R Ferguson G W Rewcastle J M Lello P G Alley R N Seelye 《Analytical biochemistry》1984,143(2):325-332
A method has been developed for extraction and purification of the major bile acids in human feces, and for their quantitative estimation using high-pressure liquid chromatography. Freeze-dried fecal material was extracted with alkaline ethanol and, after removal of neutral steroids, was subjected to thin-layer chromatography, followed by reversed-phase C18 silica cartridge (Sep-Pak) purification. The mixture was further separated into free, and glyco and tauro conjugates by ion-exchange chromatography. Subsequent resolution of individual bile acids was accomplished by HPLC using a counterion pairing method. 相似文献
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N Jentoft 《Analytical biochemistry》1985,148(2):424-433
A method for analyzing the carbohydrate composition of glycoproteins and similar glycoconjugates by methanolysis followed by reverse-phase high-pressure liquid chromatography of the perbenzoylated methyl glycosides has been developed. As described, the method is capable of quantifying sugars in the 1- to 10-nmol range while further optimization of procedures may increase the usable sensitivity by a factor of 10 or greater. Improved yields of the sugar derivatives have been achieved by incorporating several modifications of the original methanolysis procedure. This, together with the use of high-pressure liquid chromatography rather than gas chromatography for separating the sugar derivatives, eliminates the need for empirically determined molar response ratios. 相似文献
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A method for the estimation of urinary testosterone 总被引:1,自引:0,他引:1
1. A method has been developed for the estimation of testosterone in human urine by using acid hydrolysis followed by a quantitative form of a modified Girard reaction that separates a ;conjugated-ketone' fraction from a urine extract; this is followed by column chromatography on alumina and paper chromatography. 2. Comparison of methods of estimation of testosterone in the final fraction shows that estimation by gas-liquid chromatography is more reproducible than by colorimetric methods applied to the same eluates from the paper chromatogram. 3. The mean recovery of testosterone by gas-liquid chromatography is 79.5%, and this method appears to be specific for testosterone. 4. The procedure is relatively rapid. Six determinations can be performed by one worker in 2 days. 5. Results of determinations on human urine are briefly presented. In general, they are similar to earlier estimates, but the maximal values are lower. 相似文献
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We have developed a method to isolate and enhance the detection of phosphopeptides using liquid chromatography (LC)/mass spectrometry on a tryptic-digested protein sample. The method uses an on-line two-dimensional chromatography approach that consists of strong cation exchange (SCX) followed by reversed-phase (RP) chromatography with mass spectrometric detection. At pH 2.6 or lower, tryptic phosphopeptides are not retained during the first-dimension SCX chromatography step. Thus the capture of these peptides in the flow-through by the second-dimension RP trap can dramatically reduce the complexity of the phosphopeptide chromatography, resulting in little or no suppression of the signal often caused by the coeluting nonphosphorylated peptides. The method provides higher phosphopeptide recovery and less nonspecific biding of acidic peptides than the commonly used enrichment methods, such as immobilized metal affinity chromatography. Since the widely adopted multidimensional LC strategy in shotgun proteomics uses a similar SCX-RP approach, the method can be adapted to detect and characterize phosphopeptides from a complex mixture in a single experiment. Limitations of the method are also discussed. 相似文献
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Summary Purification procedures for extracting and concentrating thermostable neutral proteinases using vacuum evaporation and ammonium sulfate precipitation, or adsorption chromatography on amberlite XAD-7 resin were compared. Adsorption chromatography proved to be the most effective method to concentrate, extract and partially purify the thermostable neutral proteases produced by Bacillus stearothermophilus NCIB 8924 and NRRL B-3880. Proteases can also be extracted from large volumes of culture media containing only weak proteolytic activity and a low protein concentration. Final purification of the thermostable neutral proteases was performed with an established affinity chromatography method. The method seems to be suitable for scaling up. 相似文献
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A semi-automated method for amino acid derivatization and analysis has been validated for use in analysis of protein biopharmaceuticals. The method includes protein hydrolysis, o-phthalaldehyde derivatization, and reversed-phase high-performance liquid chromatography analysis in a general-purpose UV-visible high-performance liquid chromatography system. Amino-acid derivatization is performed automatically by the high-performance liquid chromatography autosampler right before injection. The required validation parameters, i.e., specificity, linearity, accuracy, precision, limit of detection, and limit of quantification, were studied for bovine serum albumin and for a recombinant human Fab fragment. The method can be employed as an absolute quantification method for determination of extinction coefficients of recombinant proteins. 相似文献
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Joachim Stadler 《Analytical biochemistry》1976,74(1):62-72
A sensitive method (0.4 μg of hexoses routinely detectable) for quantitative determinations of sugars and amino sugars in biological material, particularly in membranes, is described. The method consists of a combination of thin-layer chromatography (tlc), gas-liquid chromatography (glc), and radiogas chromatography (rgc), using a highly thermostable phase (Silar 10 c) for the analysis of the specific alditol acetates. In this method, the losses incurred during hydrolysis and preparation for glc are assessed by comparison with the specific recoveries of added radiolabeled internal standards. 相似文献
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A versatile noninvasive method for adsorber quantification in batch and column chromatography based on the ionic capacity
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Within the Quality by Design (QbD) framework proposed by the International Conference on Harmonisation (ICH), high‐throughput process development (HTPD) and mechanistic modeling are of outstanding importance for future biopharmaceutical chromatography process development. In order to compare the data derived from different column scales or batch chromatographies, the amount of adsorber has to be quantified with the same noninvasive method. Similarly, an important requirement for the implementation of mechanistic modeling is the reliable determination of column characteristics such as the ionic capacity Λ for ion‐exchange chromatography with the same method at all scales and formats. We developed a method to determine the ionic capacity in column and batch chromatography, based on the adsorption/desorption of the natural, uv‐detectable amino acid histidine. In column chromatography, this method produces results comparable to those of classical acid?base titration. In contrast to acid?base titration, this method can be adapted to robotic batch chromatographic experiments. We are able to convert the adsorber volumes in batch chromatography to the equivalent volume of a compressed column. In a case study, we demonstrate that this method increases the quality of SMA parameters fitted to batch adsorption isotherms, and the capability to predict column breakthrough experiments. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:666–677, 2016 相似文献
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Summary An improved method for separating analogues of coenzyme F420 by isocratic reversed-phase high performance liquid chromatography is described. The method offers improved resolution, shorter chromatography runs and requires less complex apparatus. 相似文献
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Simultaneous analysis of beauvericin and moniliformin in fungal cultures and in cereal grain samples
Species ofFusarium subglutinans andFusarium proliferation have been found to produce two mycotoxins, beauvericin and moniliformin, under labolatory conditions as well as in infected
ears. A method for simultaneous extraction, analysis and quantitation of both metabolites was elaborated. Recoveries were
85–97 % and 78–94 % for the first and the latter mycotoxin, respectively. Detection limit of beauvericin on high- performance
thin- layer chromatography plates (Merck 5633) after exposure to iodine vapours was 3 μg/g and by high- performance liquid
chromatography method 0.07 μg/g while moniliformin was analyzed at concentration level 1 μg/g by thin- layer chromatography
and 0.05 μg/g by high- performance liquid chromatography method. 相似文献
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Rat submandibular kallikrein was isolated in an 87% yield by a very quick and simple procedure involving hydrophobic interaction chromatography. Furthermore, that purification method was superior to both aprotinin-affinity chromatography and immunoaffinity chromatography for the purification of rat submandibular kallikrein. The kallikrein purified by hydrophobic interaction chromatography consisted of a number of isoenzymes. The major component of Mr 38,000 seen on SDS-gel electrophoresis was found to be the glycosylated kallikrein, whereas the minor component of Mr 26,000 represented the non-glycosylated enzyme. 相似文献
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Shinkichi Kamata Kenji Imura Akira Okada Yasunaru Kawashima Atsushi Yamatodani Takehiko Watanabe Hiroshi Wada 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》1982,231(2):291-299
A fluorometric method for the simultaneous analyses of phenethylamine, phenylethanolamine, tyramine and octopamine has been developed. The method involves ion-exchange chromatography, derivatization with fluorescamine, solvent extraction and then separation by thin-layer chromatography. The fluorescent spots are then quantitated by scanning. The detection limits of this method are about 10 pmoles for phenethylamine, phenylethanolamine and tyramine, and 20 pmoles for octopamine. The method was used for simultaneous analyses of putative neurotransmitter amines in whole rat brain. 相似文献
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A reliable high-performance liquid chromatography (HPLC) method is described for the separation of dimethylamino-azobenzenesulfonyl-amino acid (DABS-AA). The separation is accomplished by reversed-phase chromatography on a Hypersil ODS column (4.6×150 mm) withe a Hewlett-Packard liquid chromatography system. In addition to the developed sample and solvent preparation procedure, this precolumn modification method using dimethylaminoazobenzene sulfonyl chloride (DABS-CL) for amino acid analysis is proved reliable and sensitive. Five apolipoprotein B-100 tryptic peptides, two of them containing cysteine, were demonstrated as examples for the general application of this method in amino acid analysis. It is a useful method for analysis of cysteine- and cysteine-containing peptide and, furthermore, for determination of sulfhydryl and disulfide linkages of proteins. 相似文献
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蛋白的色谱复性及同时纯化 总被引:25,自引:2,他引:25
对近年来新发展的用液相色谱(LC)进行蛋白质复性及同时纯化的方法做了评述,详细介绍了蛋白质在4种液相色谱上的复性及同时纯化的方法、设备和影响因素,并对各自的优缺点进行了比较,为色谱法作为研究蛋白质折叠及用于基因工程生产治疗蛋白质的复性及同时纯化技术的进一步应用提供依据。 相似文献
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Dai X Fang X Zhang C Xu R Xu B 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2007,857(2):287-295
Uric acid is an important diagnostic marker of catabolism of the purine nucleosides, and accurate measurements of serum uric acid are necessary for proper diagnosis of gout or renal disease appearance. A candidate reference method involving isotope dilution coupled with liquid chromatography/mass spectrometry (LC/MS) has been described. An isotopically labeled internal standard, [1,3-(15)N(2)] uric acid, was added to serum, followed by equilibration and protein removal clean up to prepare samples for liquid chromatography/mass spectrometry electrospray ionization (LC/MS-ESI) analyses. (M-H)(-) ions at m/z 167 and 169 for uric acid and its labeled internal standard were monitored for LC/MS. The accuracy of the measurement was evaluated by a comparison of results of this candidate reference method on lyophilized human serum reference materials for uric acid (Standard Reference Materials SRM909b) with the certified values determined by gas chromatography/mass spectrometry reference methods and by a recovery study for the added uric acid. The method performed well against the established reference method of ion-exchange followed by derivatization isotope dilution (ID) gas chromatography mass spectrometry (ID-GC/MS). The results of this method for uric acid agreed well with the certified values and were within 0.10%. The amounts of uric acid recovered and added were in good agreement for the three concentrations. This method was applied to determine uric acid in samples of frozen serum pools. Excellent precision was obtained with within-set CVs of 0.08-0.18% and between-set CVs of 0.02-0.07% for LC/MS analyses. Liquid chromatography/tandem mass spectrometry electrospray ionization (LC/MS/MS-ESI) analysis was also performed. The LC/MS and LC/MS/MS results were in very good agreement (within 0.14%). This LC/MS method, which demonstrates good accuracy and precision, and is in the speed of analysis without the need for a derivatization stage, qualifies as a candidate reference method. This method can be used as an alternative reference method to provide an accuracy base to which the routine methods can be compared. 相似文献