1mm内径HPLC整体柱用于蛋白质的高效分离

介绍了1mm内径的聚苯乙烯-二乙烯基苯整体柱用于液相色谱反相梯度高效分离完整的蛋白质。文中详细研究了柱温、流速和梯度时间等对峰宽和峰容量的影响。在最优色谱条件下该柱高效地反相分离了核糖核酸酶A、肌红蛋白和碳酸酐酶的蛋白质混合物以及大肠杆菌提取蛋白。     

食品中罗丹明B的高效液相色谱荧光检测

目的:建立用固相萃取富集净化、高效液相色谱荧光检测器测定食品中禁用色素罗丹明B的方法.方法:样品中的罗丹明B用磷酸水/乙腈溶液提取,经C18固相萃取浓缩净化,用高效液相色谱荧光检测器进行测定.结果:罗丹明B在0.5～100 ng/mL浓度范围内线性相关系数为0.9999(p＜0.01),在1、2、5ng/g添加水平时的回收率为52.6%～82.5%,相对标准偏差为3.7%～12.1%(n=6),定量检出限为1.0 ng/g.结论:此法适用于食品中罗丹明B的测定,并具有快速、准确、灵敏等优点.     

反相高效液相色谱法测定龙眼中阿维菌素残留量

建立了利用反相高效液相色谱法(RP-HPLC)测定龙眼中阿维菌素残留量的方法.以乙腈为提取溶剂,用碱性氧化铝固相萃取小柱进行净化,经N-甲基咪唑和三氟乙酸酐衍生化后,用高效液相色谱法-荧光检测器(激发波长为365nm,发射波长为470nm)进行测定.在0.05 mg/L～4.0 mg/L浓度范围内,阿维菌素标准工作溶液的色谱峰高与质量浓度呈良好的线性关系,相关系数为0.9997,方法的添加回收率为88.0%～94.0%,相对标准偏差RSD在1.81%～3.55%之间,检出限为0.001mg/kg.实验证明,该方法简便、快速、准确,可用于龙眼中阿维菌素残留量的测定.     

液相色谱串联质谱检测鳗鱼中四种氟喹诺酮残留

建立了液相色谱/串联质谱法测定鳗鱼中恩诺沙星,环丙沙星,诺氟沙星,氧氟沙星的方法.对分离和检测这四种兽药残留的液相色谱条件、质谱条件、样品前处理方法等作了详细的研究.用乙腈萃取样品中的残留物,在氮气保护下将萃取液浓缩干,加流动相1.0 mL溶解残渣,正己烷液液萃取,弃上层液,下层溶液用作液相色谱分析荧光检测或质谱检测.在鳗鱼样品中添加四种残留物,添加浓度分别为0.01,0.050和0.100mg/kg,平均回收率(n=6)在70～84.3%之间,相对标准偏差为3.0%～6.3%.     

一种测量高浓度低体积蛋白样品ζ电势的新方法

从科学研究到基于蛋白质的制造业对蛋白质结晶、储存、结晶有着越来越浓厚的兴趣.因此,了解并控制溶液中蛋白质的性质是这些工作的基础.目前广泛应用于表征蛋白分散性和溶解性的强大工具是光散射,其中包括对尺寸和ζ电势测量的技术.本文介绍了一种在不进行样晶稀释的前提下,对高浓度、低体积蛋白样品进行ζ电势测定的元件,并考察了这一改进的意义.     

MALDI-TOF-MS研究糖蛋白核糖核酸酶B的一级结构

本文应用基质辅助激光解吸附电离飞行时间质谱 ( MALDI- TOF- MS)测定了糖蛋白核糖核酸酶 B的平均分子量及其糖基化程度。结合蛋白酶及内切糖苷酶酶切的方法 ,确证了核糖核酸酶 B的糖基化位点及糖苷键类型 ,测定值与理论值相符。为糖蛋白一级结构的直接质谱分析建立子快速、准确的方法。     

高效液相色谱荧光检测法测定植物性食品中阿维菌素类药物

建立了植物性食品中阿维菌素类药物(阿维菌素、伊维菌素、多拉菌素、甲氨基阿维菌素苯甲酸盐和乙酰胺基阿维菌素)残留量的高效液相色谱测定方法。用乙腈提取溶剂样品中的目标物,碱性氧化铝固相萃取小柱和炭黑氨基柱净化,经N-甲基咪唑和三氟乙酸酐衍生化后,用高效液相色谱-荧光检测器在激发波长为365nm,发射波长为475nm处测定。在添加浓度4.0～100μg/kg范围内,平均添加回收率在78.5%～99.2%之间,相对标准偏差在0.56%～7.25%之间,检出限为1.0μg/kg。方法简便、快速、准确。在0.004mg/L～2.0mg/L范围内,阿维菌素类药物标准工作溶液的色谱峰面积与质量浓度成良好的线形关系,相关系数为0.9995。     

基于表面开孔光纤的集成式亚硝酸盐微流荧光传感器

利用中空悬挂芯光纤研制了一种将荧光猝灭反应区建立在空心光纤内部的光纤集成荧光在线微流传感器。利用CO2激光器在光纤表面刻蚀微孔,使得试剂可由微孔注入光纤内部并混合形成稳定的微流。在悬挂芯光纤纤芯倏逝场的激发下,指示剂分子产生荧光,所产生的荧光被耦合到纤芯内部并在出射端被检测。文中利用光纤内部的荧光猝灭反应实验确定了亚硝酸盐溶液的浓度。结果显示:微流可在短时间通过光纤,传感器能以较快的速度检测溶液浓度。另外,当亚硝酸盐溶液的浓度为0.1~2.6mmol/L时,荧光猝灭程度与溶液浓度呈较好的线性关系,结果证明了该集成式光纤内微流控传感器方案用于微量荧光检测的可行性。     

凝胶渗透色谱的应用及进展

凝胶渗透色谱(GPC)是基于体积排阻的分离机理,通过具有分子筛性质的固定相,进行分离的。它广泛应用于农药残留的分析、不同相对分子质量物质的分离、相对分子质量的测定、在生化制药中以及蛋白质复性的研究。就这些方面进行了介绍,综述了凝胶渗透色谱在农药残留分析前处理中的应用进展并进行了展望。     

凝胶渗透色谱-高效液相色谱法测定食用菌中呋喃丹农药残留

建立了凝胶渗透色谱净化高效液相色谱法检测食用菌类中呋喃丹农药残留量的方法。样品经乙腈提取,氮气吹干,环己烷-乙酸乙酯(1∶1,V/V)溶液溶解后,凝胶渗透色谱进行净化,C18反相高效液相色谱柱分离后,采用液相色谱荧光检测器进行测定,外标法定量。呋喃丹在0.2~5.0ng/mL质量浓度范围内呈良好线性关系,相关系数r大于0.999,测定低限为0.01mg/kg。对空白食用菌样品进行3个浓度水平0.5、1.0和2.0mg/kg的加标回收试验,呋喃丹的加标回收率为78.3%~97.6%,相对标准偏差(RSD)为3.26%~11.69%。本实验所建立的方法简单,灵敏度高,适用于食用菌样品中的呋喃丹农药残留的分析检测。     

Atomic force microscopy studies of intercalation-induced changes in plasmid DNA tertiary structure

Structural transitions in the tertiary structure of plasmid DNA have been investigated using atomic force microscopy. Changes in superhelical stress were induced by ethidium bromide intercalation, and conformational effects monitored by recording topographic images from DNA complexes of various ethidium bromide : base pair stoichiometry. Significant changes in the tertiary structure of individual DNA molecules were observed with increasing ethidium bromide concentration. The first distinct conformational transition was from a predominantly relaxed structure to one consisting solely of toroidal supercoils. A further increase in ethidium bromide concentration resulted in the formation of regions of plectonemic supercoiling. The ratio of plectonemic : toroidal supercoiling gradually increased until an extremely tightly interwound structure of solely plectonemic supercoiling was finally adopted. The toroidal form of supercoiling observed in this study is unusual as both atomic force microscopy and electron microscopy techniques have previously shown that plectonemic supercoiling is the predominant form adopted by plasmid DNA.     

Comparative studies of Protein Glycosylation Using Isotope Labeling and Electrospray Ion Trap Mass Spectrometry

In the analysis of glycoprotein, glycoprotein was often digested with trypsin before MS analysis. Several causes including glycoprotein concentration, glycosylation site occupancy and glycan profile may account for the difference if the signal of a specific glycopeptide was found to be different. A strategy was proposed for comparative analyses of glycoprotein in which the change of glycoprotein concentration, the change of glycosylation site occupancy and the change of glycan profile could be differentiated. Comparative analysis was performed by stable isotope labeling using formaldehyde-d0 and formaldehyde-d2 along with cellulose microcrystalline enrichment and mass spectrometry analysis. The utility of the proposed strategy was demonstrated with ribonuclease B. The change of glycoprotein concentration was studied using samples prepared with different quantity of ribonuclease B. Level of glycosylation site occupancy was studied by mixing different ratios of ribonuclease B and ribonuclease A. The change of the site-specific glycan profile was studied by mixing ribonuclease B treated with α-mannosidase and intact ribonuclease B. ESI-MS analysis of glycopeptides was performed on a linear ion trap mass spectrometer. With the capability of MS2 and MS3 of the instrument, the site of glycosylation and the glycan sequence may also be obtained.     

A Site-Specific Proteomic Approach for Studying Protein S-nitrosylation

In the analysis of glycoprotein, glycoprotein was often digested with trypsin before MS analysis. Several causes including glycoprotein concentration, glycosylation site occupancy and glycan profile may account for the difference if the signal of a specific glycopeptide was found to be different. A strategy was proposed for comparative analyses of glycoprotein in which the change of glycoprotein concentration, the change of glycosylation site occupancy and the change of glycan profile could be differentiated. Comparative analysis was performed by stable isotope labeling using formaldehyde-d₀ and formaldehyde-d₂ along with cellulose microcrystalline enrichment and mass spectrometry analysis. The utility of the proposed strategy was demonstrated with ribonuclease B. The change of glycoprotein concentration was studied using samples prepared with different quantity of ribonuclease B. Level of glycosylation site occupancy was studied by mixing different ratios of ribonuclease B and ribonuclease A. The change of the site-specific glycan profile was studied by mixing ribonuclease B treated with α-mannosidase and intact ribonuclease B. ESI-MS analysis of glycopeptides was performed on a linear ion trap mass spectrometer. With the capability of MS² and MS³ of the instrument, the site of glycosylation and the glycan sequence may also be obtained.     

Heat capacity and latent heat measurements of CoMnSi using a microcalorimeter

A new method of utilizing a commercial silicon nitride membrane calorimeter to measure the latent heat at a first order phase transition is presented. The method is a direct measurement of the thermoelectric voltage jump induced by the latent heat, in a thermally isolated system ideally suited for single crystal and small microgram samples. We show that when combined with the ac calorimetry technique previously developed, the resultant thermal measurement capabilities are extremely powerful. We demonstrate the applicability of the combined method with measurements on a 100 microm size fragment of CoMnSi exhibiting a sizable magnetocaloric effect near room temperature, and obtain good agreement with previously reported values on bulk samples.     

The transition of mild to severe wear of ceramics

The transition of mild to severe wear of ceramics depends on the operating conditions (normal load, velocity and temperature) and material properties (like grain size, mechanical and thermal material properties). Adachi et al. [Wear 203-204 (1997) 291] introduced the transition of mild to severe wear of ceramics by defining a mechanical severity parameter based on the work of Hamilton [Proc. Inst. Mech. Eng. 197C (1983)] and a thermal severity parameter based on the work of Ashby et al. [Tribol. Trans. (34) (1991) 577]. Metselaar et al. [Wear 249 (2001) 962] improved the thermal severity parameter using the temperature model introduced by Bos [Frictional heating of tribological contacts, Ph.D. Thesis, University of Twente, 1995]. Better prediction of wear transition in the region where the transition is dominated by thermally induced wear was achieved. The combination of the mechanical severity parameter and the thermal severity parameter for Peclet number (Pe) higher than 2 is presented in this paper. This model is verified experimentally and gives an improved prediction of the mild to severe wear transition of ceramics.     

基于离子淌度质谱研究低浓度甘油对蛋白质结构的影响

甘油作为水溶液中蛋白质的稳定剂,研究其存在时蛋白质的结构变化对蛋白质相关研究和应用具有重要意义。本研究以牛血清白蛋白(BSA)为模型蛋白,采用离子淌度质谱(IM-MS)法和动态光散射法考察在近似生理条件下低浓度甘油对蛋白质结构的影响。在一定浓度范围内,甘油可引起BSA平均电荷和碰撞截面积(CCS)的增加,单个电荷状态也经历了CCS值的增加,还可导致BSA水化半径的增加,表明低浓度甘油可引起溶液中蛋白质结构松散。此外,含0.5%甘油的BSA去折叠的开始电压较高,且去折叠过渡发生在更宽的电压范围内,表明低浓度甘油可提高蛋白质的稳定性。本工作可为甘油稳定蛋白质的机制研究提供参考。     

镀锡银钎料扩散过渡区生长建模及数值分析

采用温度梯度法对镀锡银钎料进行热扩散处理,形成了扩散过渡区。借助金相显微镜（OM）、原子力显微镜（AFM）对扩散过渡区的厚度、表界面形貌进行分析研究,发现热扩散处理加快了Sn原子的扩散速度,使得Sn原子在银钎料中的浓度升高,扩散过渡区的厚度增加。以Sn原子在镀锡银钎料扩散过渡区的浓度变化为研究对象,由Fick定律建立了扩散过渡区总厚度的数学模型,借助Arrehenius方程对其参数进行数值分析;利用MATLAB软件和扩散过渡区厚度的试验数据对所建模型进行求解,得到了200℃、220℃镀锡银钎料扩散过渡区Sn原子生长的本构方程。试验结果证实所建本构方程在一定程度上可定量表征镀锡银钎料扩散过渡区Sn原子的生长规律。     

The ‘corpuscle’ stereological problem—re-evaluation using slab fragment volumes and application to the correction of DNA histograms from sections of spherical nuclei

A new category of stereological size distribution unfolding models is introduced. It is based on the use of the volumes of particle slab fragments, in addition to their profile dimensions. When spheres are cut by a slab of known (constant) thickness, an estimation of discrete sphere sizes from section data is then possible, as only one parent sphere solution exists for any slab fragment, given the latter's projection size and volume. The unfolding algorithm consists in sequentially testing a set of equations: only one of the solutions satisfies various constraints on bounds. A precise determination of the section thickness is required. Truncation parameters, instead of being troublesome inputs as in classical unfolding models, become valuable outputs. This model offers the first stereologically valid solution to the important problem of correcting DNA-amount histograms obtained from sectioned spherical nuclei. Under the (biologically reasonable) assumption that the nuclear volume is proportional to the DNA amount, it is possible to estimate the DNA concentration and, subsequently, compute discrete slab fragment volumes from corresponding DNA values. An application to Feulgen-stained rat liver sections is shown. Measurements of hepatocytic nuclear-profile areas and integrated optical densities are obtained by automated image analysis (IBAS), and the model is used to unfold the section-obtained DNA histogram. A maximum likelihood fitting of the final distribution with chi functions allows a parametric estimation of ploidy frequencies. This model can only be used for acceptably spherically shaped particles.     

Distribution-free estimation of sphere size distributions from slabs showing overprojection and truncation,with a review of previous methods

A distribution-free method is presented for unfolding the size distribution of an aggregate of disjoint opaque spherical particles in a translucent medium, from the circular profiles observed in the orthogonal projection of sections of a certain thickness through the medium. This situation pertains especially to light and transmission electron microscopy. The model accounts for overprojection (or ‘Holmes effect’) due to section thickness, and for two forms of truncation (or ‘missing profiles’ mechanisms), simultaneously. The profile data may be grouped into an arbitrary class system, chosen by the user, and the procedure can be programmed in a medium-size desk calculator. In order to incorporate the above features, however, we had to choose a finite-difference unfolding algorithm, the advantages and disadvantages of which are discussed early in the paper in the context of the main alternative unfolding procedures hitherto available. The performance of the method is tested against three sets of real data previously analysed by a maximum likelihood method (Keidinget al., 1972).     

固相萃取-气相色谱法鉴定水体中泄漏的油介质

建立了固相萃取-气相色谱法鉴定循环冷却水系统中泄漏油介质的方法,优化了萃取富集条件和色谱操作条件.实验证明,利用谱图分析和标准谱图比对的方法可以鉴定泄漏入水体中的油介质,及时发现泄漏源.