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161.
Thakur D Rejtar T Wang D Bones J Cha S Clodfelder-Miller B Richardson E Binns S Dahiya S Sgroi D Karger BL 《Journal of chromatography. A》2011,1218(45):8168-8174
Precise proteomic profiling of limited levels of disease tissue represents an extremely challenging task. Here, we present an effective and reproducible microproteomic workflow for sample sizes of only 10,000 cells that integrates selective sample procurement via laser capture microdissection (LCM), sample clean-up and protein level fractionation using short-range SDS-PAGE, followed by ultrasensitive LC-MS/MS analysis using a 10 μm i.d. porous layer open tubular (PLOT) column. With 10,000 LCM captured mouse hepatocytes for method development and performance assessment, only 10% of the in-gel digest, equivalent to ~1000 cells, was needed per LC-MS/MS analysis. The optimized workflow was applied to the differential proteomic analysis of 10,000 LCM collected primary and metastatic breast cancer cells from the same patient. More than 1100 proteins were identified from each injection with >1700 proteins identified from three LCM samples of 10,000 cells from the same patient (1123 with at least two unique peptides). Label free quantitation (spectral counting) was performed to identify differential protein expression between the primary and metastatic cell populations. Informatics analysis of the resulting data indicated that vesicular transport and extracellular remodeling processes were significantly altered between the two cell types. The ability to extract meaningful biological information from limited, but highly informative cell populations demonstrates the significant benefits of the described microproteomic workflow. 相似文献
162.
Tools for analyzing the phosphoproteome and other phosphorylated biomolecules: a review 总被引:1,自引:0,他引:1
Enrichment, separation and mass spectrometric analysis of biomolecules carrying a phosphate group plays an important role in current analytical chemistry. Application areas range from the preparative enrichment of phospholipids for biotechnological purposes and the separation and purification of plasmid DNA or mRNA to the specific preconcentration of phosphoproteins and -peptides to facilitate their later identification and characterization by mass spectrometry. Most of the recent improvements in this field were triggered by the need for phosphopeptide enrichment technology for the analysis of cellular protein phosphorylation events with the help of liquid chromatography–mass spectrometry. The high sensitivity of mass spectrometry and the possibility to combine this technique with different separation modes in liquid chromatography have made it the method of choice for proteome analysis. However, in the case of phosphoprotein analysis, the low abundance of the resulting phosphopeptides and their low quality fragment spectra interfere with the identification of phosphorylation events. Recent developments in phosphopeptide enrichment and fragmentation technologies successfully helped to overcome these limitations. In this review, we will focus on sample preparation techniques in the field of phosphoproteomics, but also highlight recent advancements for the analysis of other phosphorylated biomolecules. 相似文献
163.
A simple, low cost, fast and sensitive method is reported for the determination of the four endocrine disrupting chemicals (EDCs) 4-tert-butylphenol, 4-tert-octylphenol, bisphenol A and 17β-estradiol using pentafluoropyridine as the derivatizing reagent. These EDCs were determined by simultaneous extraction and derivatization in a solid phase analytical derivatization (SPAD) technique without the aid of any phase transfer catalyst (PTC) or an ion-pair mechanism. Recoveries of analytes as their tetrafluoropyridyl derivatives from water ranged from 71% for 4-tert-butylphenol to 106% for 17β-estradiol; from urine they ranged from 61% for 17β-estradiol to 91% for 4-tert-octylphenol; and from humic acids solution the ranged from 59% for 17β-estradiol to 104% for 4-tert-octylphenol in humic acid solutions. Calibration curves were constructed from a matrix of human male urine in the range 1-40 ng/mL and had coefficients of correlation greater than 0.99. For 4-tert-butylphenol, bisphenol A and 17β-estradiol the limits of quantitation were 5 ng/mL and for 4-tert-octylphenol it was 1 ng/mL. This method was applied to determine EDCs and detected 4-tert-octylphenol, bisphenol A and 17β-estradiol in concentrations comparable to those found in the literature. The method offers advantages in speed of analysis, reduced reagent and specificity of derivatization. 相似文献
164.
This study describes a new approach to cold-fiber solid-phase microextraction (CF-SPME) based on a combination of different extraction modes in the same extraction procedure. Also, the high quantity of water required to facilitate both the desorption of analytes from the matrix and their transport to the fiber coating is reported. The extraction mode was changed from the direct to the headspace mode in a single extraction while manipulating the extraction times and coating temperature to improve the extraction of compounds with different volatilities. Compounds with low volatility were better extracted in the direct mode, while the headspace mode was more appropriate for volatile compounds. Polycyclic aromatic hydrocarbons (PAHs) and phthalic acid esters (PEs) in sand or soil samples were used as model compounds and matrices in this study. The optimized conditions were: sample pH in the range of 4-7, addition of 12 mL of 194 g L(-1) aqueous NaCl solution in a 15 mL vial, and 80 min total extraction time with a sample temperature of 90°C (50 min in direct mode with coating at 90°C followed by 30 min in headspace mode with coating at 30°C). The proposed procedure was compared with conventional CF-SPME (with and without addition of water) and was found to be more effective for all the analytes, since it is capable of extracting both heavier and lighter compounds from soil samples in a single extraction procedure. The use of an excess of water and a combination of extraction modes in the same CF-SPME procedure are the main factors responsible for this enhancement. The proposed method was applied to the extraction of PAHs and PEs in spiked soil samples and excellent results were obtained for most of the compounds evaluated. 相似文献
165.
166.
Victor C. Yang Howard Bernstein Charles L. Cooney Robert Langer 《Applied biochemistry and biotechnology》1987,16(1):35-50
By a combination of hydroxylapatite chromatography and negative adsorption on QAE-Sephadex at pH 8.3, heparinase (E.C.4.2.2.7)
can be successfully isolated from all the other mucopolysaccharase contaminants present inFlavobacterium heparinum. Hydroxylapatite isolates heparinase primarily from chondroitinases, hyaluronidase, and most glycuronidases. QAE-Sephadex
chromatography at pH 8.3 further separates heparinase from heparitinases, sulfatases, and the remaining glycuronidases. The
heparinase preparation thus obtained contains no statistically significant levels of other contaminating mucopolysaccharases
except for heparitinases that are present at an apparent maximum level of 3.4%. Owing to the presence of a crossreaction of
heparinase on heparitin sulfate at conditions employed for the assay of heparitinase, the heparitinase level of 3.4% could
be misleading because of the action of heparinase on heparitin sulfate. Characterization of this heparinase preparation shows
that the enzyme has an optimum salt concentration of 0.08M NaCl, an optimum pH of 6.5, an activation energy of 5 kcal/mol,
and a Km of 7.95 x 10-6 M. These parameters are almost identical to those displayed by a homogeneous heparinase preparation. The method described
here is suitable for scale-up purposes using batch Chromatographic procedures. 相似文献
167.
We investigate the quality of solutions obtained from sample-average approximations to two-stage stochastic linear programs
with recourse. We use a recently developed software tool executing on a computational grid to solve many large instances of
these problems, allowing us to obtain high-quality solutions and to verify optimality and near-optimality of the computed
solutions in various ways.
Research supported by the Mathematical, Information, and Computational Sciences Division subprogram of the Office of Advanced
Scientific Computing Research, U.S. Department of Energy, under Contract W-31-109-Eng-38, and by the National Science Foundation
under Grant 9726385.
Research supported by the Mathematical, Information, and Computational Sciences Division subprogram of the Office of Advanced
Scientific Computing Research, U.S. Department of Energy, under Contract W-31-109-Eng-38, and by the National Science Foundation
under Grant DMS-0073770.
Research supported by the Mathematical, Information, and Computational Sciences Division subprogram of the Office of Advanced
Scientific Computing Research, U.S. Department of Energy, under Contract W-31-109-Eng-38, and by the National Science Foundation
under Grants 9726385 and 0082065. 相似文献
168.
Ricardo J.N Bettencourt da Silva Helena Figueiredo M.Filomena G.F.C Camões 《Analytica chimica acta》2003,477(2):169-185
A methodology for the estimation of sample processing and sub-sampling performance based on the comparison of the global method experimental dispersion of results with the uncertainty estimated from developed models for the subsequent analytical steps is presented. This approach is a valuable alternative to the evaluation of adequate experimental information using a classical ANOVA, since the significance of the sample processing and sub-sampling is evaluated with a higher number of degrees of freedom for the same number of experimental assays, due to the high number of degrees of freedom associated with the uncertainty estimated for the subsequent analytical steps from the combination of the involved sources of uncertainty.Considering the construction of a model to describe the performance of the analytical steps following sample processing and sub-sampling over a broad concentration range, the experimental assays involved at the evaluation of the sample processing and sub-sampling can be performed at any concentration meeting the previously validated range and several months after the development of that model once its adequacy has been proven over time.This approach, which also allows the construction of a detailed performance model for the global analytical method over a broad concentration range, was applied to the determination of pesticide residues in apples by gas-chromatography with electron capture detector.Considering that no information was available regarding the samples heterogeneity, sub-sampling performance was evaluated considering a sample representing the worst expectable homogeneity. This was accomplished by spiking just one out of the 10 halves of apples processed in each sample.The developed model for the performance of the analytical method was successfully and easily applied to routine analysis through an automated link between the information generated by the chromatograph software with a file containing the model. 相似文献
169.
乌鲁木齐市安宁渠区蔬菜中重金属的分布特征研究 总被引:7,自引:0,他引:7
对乌鲁木齐市郊安宁渠区在土壤调查的基础上,又进行了蔬菜中的重金属调查.并对蔬菜进行了污染评价.结果标明:土壤的环境元素组合与蔬菜的不同.土壤和蔬菜的重金属聚类分析结果也不同.按食品卫生标准进行评价,蔬菜中Cd、Pb的超标率最高,大白菜的超标率比四季豆和长豇豆更高.长豇豆对重金属的富集系数相对较小. 相似文献
170.
m相依样本均值的Bootstrap及其随机加权逼近的收敛速度 总被引:2,自引:0,他引:2
本文研究了m相依样本均值的Bootstrap及随机加权逼近问题,讨论了有关收敛速度。 相似文献