COMPOSITION DETERMINATION OF BINARY POLYMER MIXTURES BY SIZE EXCLUSION CHROMATOGRAPHY WITH LIGHT SCATTERING DETECTION

Base on the principle of absolute quantification of size exclusion chromatography （SEC）, a light scattering （LS） detector coupled with a concentration detector （refractive index detector） is utilized to determine the compositions of complicated binary mixtures. A theoretical analysis predicts that the response factors for both LS and RI detectors are linear functions with the composition of any specified polymer mixtures in the binary polymer mixtures. Two pairs of complicated binary mixtures were used to test the theory mentioned in the present paper, and the experimental results show an excellent accordance with the theory.

Preparation and Application of Silica-based Perfusion Packing of Size Exclusion Chromatography for Quantitation of Polyacrylamide in Enhanced Oil Recovery Systems

The synthesis and characterization of a novel macroporous silica derived size exclusion chromatography （SEC） packing for quantitative analysis of high molecular weight （MW） polyacrylamide （PAM） are presented. Using this packing, a fast, sensitive and reproducible approach for quantitation of super high-MW PAM in demanding enhanced oil recovery （EOR） waters was developed and the effect of synthesis parameters on the properties of resultant materials was investigated. These parameters include salt addition, reaction temperature and duration, activation condition of functional groups on the silica surface, as well as the reaction cycles required for optimal silica modification. Moreover, SEC analysis conditions, such as mobile phase composition, flow rate, detection and sample preparation, were also explored and an optimal analysis protocol was developed. Under this optimized SEC analysis conditions, the synthesized macroporous materials proved satisfactory for quantification of PAM with average MW up to 22 million Daltons. An SEC analysis required less than few minutes with a detection limit of 1 ng, a linear response range of 0.1 to 75 mg/L with squared R value of 0.99 and reproducibility better than 9.2% RSD （relative standard deviation）. The analysis of PAM in highly saline oilfield production water containing interfering high MW polymeric surfactants indicated the recovery ranges from 92.5% to 110.1% for 1.0 mg/L PAM and 94.2% to 103.8% for 50 mg/L PAM solution. This study presented for the ftrst time that the reliable quantization of high MW PAM in highly demanding EOR waters can be achieved by SEC.

Molecular Weight Distribution of Dissolved Organic Matter in Lake Hongfeng Determined by High Performance Size Exclusion Chromatography (HPSEC) With On-Line UV-Vis Absorbance and Fluorescence Detection

The molecular weight distribution (MWD) of dissolved organic matter (DOM) in lake waters from Lake Hongfeng was examined using high performance size exclusion chromatography (HPSEC) with UV-vis absorbance and fluorescence detection. The elution curves obtained by absorbance and fluorescence techniques expressed similar patterns, with the exception of diminishing of large fraction and the peaks behind several seconds in fluorescence chromatograms. According to its molecular weight (MW), DOM in water samples is divided into several fractions: large ({>3.5} kDa); medium-large ({3.5}-{2.0} kDa); medium ({2.0}-{1.0} kDa) and small ({<1.0} kDa). The average molecular weight was calculated using the elution curve detected by UV-vis absorbance and fluorescence detection techniques. The results showed that the weight-average molecular weight (Mw) and number-average molecular weight (Mn) calculated by UV-vis absorbance techniques range from 1750 to 2050 Dalton and from 1450 to 1850 Dalton, respectively. And the Mw and Mn obtained by fluorescence detection are lower by 50 to 400 Dalton. As a reference, the molecular weight of Fluka humic acid (FHA) is larger than that of water samples by about 200 Dalton. The average molecular weight of DOM for water samples collected in March and July was compared. The results revealed that the molecular weight is lower for water samples obtained in July than that obtained in March, indicating the ambient environment has an influence on the molecular weight, including photo-degradation and biological activity.

Lysozyme refolding at high concentration by dilution and size—exclusion chromatography

This study of renaturation by dilution and size exclusion chromatogra phy (SEC) addition of urea to improve yield as well as the initial and final pro tein concentrations showed that although urea decreased the rate of lysozyme ref o lding, it could suppress protein aggregation to sustain the pathway of correct r efolding at high protein concentration; and that there existed an optimum urea c oncentration in renaturation buffer. Under the above conditions, lysozyme was su ccessfully refolded from initial concentration of up to 40 mg/mL by dilution and 100 mg/mL by SEC, with the yield of the former being more than 40% and that of the latter being 34.8%. Especially, under the condition of 30 min interval time, i.e. τ>2(t_R2 -t_R1 ), the efficiency was increased by 25% and the renaturation buffe r could be recycled for SEC refolding in continuous operation of downstream proc ess.

High throughput and rapid isolation of extracellular vesicles and exosomes with purity using size exclusion liquid chromatography

Extracellular vesicles(EVs)have emerged as potential biomarkers for diagnosing a range of diseases without invasive procedures.Extracellular vesicles also offer advantages compared to synthetic vesicles for delivery of various drugs;however,limitations in segregating EVs from other particles and soluble proteins have led to inconsistent EV retrieval rates with low levels of purity.Here,we report a new high-yield(88.47%)and rapid(<20 min)EV isolation method termed size exclusion–fast protein liquid chromatography(SE-FPLC).We show SE-FPLC can effectively isolate EVs from multiple sources including EVs derived from human and mouse cells and serum samples.The results indicate that SE-FPLC can successfully remove highly abundant protein contaminants such as albumin and lipoprotein complexes,which can represent a major hurdle in large scale isolation of EVs.The high-yield nature of SE-FPLC allows for easy industrial scaling up of EV production for various clinical utilities.SE-FPLC also enables analysis of small volumes of blood for use in point-of-care diagnostics in the clinic.Collectively,SE-FPLC offers many advantages over current EV isolation methods and offers rapid clinical translation.

Simultaneous determination of dissolved organic nitrogen, nitrite, nitrate and ammonia using size exclusion chromatography coupled with nitrogen detector

Accurate quantification of dissolved organic nitrogen(DON) has been a challenge due to the cumulative analytical errors in the conventional method via subtracting dissolved inorganic nitrogen species(DIN) from total dissolved nitrogen(TDN). Size exclusion chromatography coupled with an organic nitrogen detector(SEC-OND) has been developed as a direct method for quantification and characterization of DON. However, the applications of SECOND method still subject to poor separations between DON and DIN species and unsatisfied N recoveries of macromolecules. In this study, we packed a series of SEC columns with different lengths and resin materials for separation of different N species and designed an independent vacuum ultraviolet(VUV) oxidation device for complete oxidation converting N species to nitrate. To guarantee sufficient N recoveries, the operation conditions were optimized as oxidation time ≥ 30 min, injection mass(sample concentration × injection volume) < 1000 μL × mg-N/L for macromolecular proteins, and neutral p H mobile eluent. The dissolved O_(2)concentration in SEC mobile phase determined the upper limit of VUV oxidation at a specific oxidation time. Compared to conventional HW50S column(20 × 250 mm),HW40S column(20 × 350 mm) with mobile phase comprising of 1.5 g/L Na2HPO_(4)·2H_(2)O + 2.5g/L KH_(2)PO_(4)(p H = 6.85) could achieve a better separation of DON, nitrite, nitrate, and ammonia. When applied to river water, lake water, wastewater effluent, groundwater, and landfill leachate, the SEC-OND method could quantify DON as well as DIN species accurately and conveniently even the DIN/TDN ratio reached 0.98.

Studies on the refolding of the reduced-denatured insulin with size exclusion chromatography

The refolding of the reduced-denatured insulin from bovine pancreas was investigated with the size exclusion chromatography(SEC).It was shown that the reduced-denatured insulin originally denatured with 7.0 mol·L-1 guanidine hydrochloride(GuHCl)or 8.0 mol·L-1 urea could not be refolded with a non-oxidized mobile phase.Although the oxidized and reduced glutathione(GSSG and GSH)were employed in the oxidized mobile phase,the reduced-denatured insulin still could not be renatured.However,in the presence of 2.0 mol·L-1 urea in the oxidized mobile phase employed,the reduced-denatured insulin can be refolded with SEC,and the aggregation of denatured insulin can be diminished by urea.In addition,the disulfide exchange of reduced-denatured insulin also can be accelerated with GSSG/GSH in the oxidized mobile phase.The three disulfide bridges of insulin were formed correctly and the reduced-unfolded insulin can be renatured completely.The results were further tested with reversed-phase liquid chromatography(RPLC)and hydrophobic interaction chromatography(HIC).

Microcomputer Simulation of Elution Behaviour for Dicarboxylic Acids in Ion－Exclusion Chromatography

he present paper covers a model involving Donnan memhrane equilibrium andadsorption eluilibrium to describe the retention process for diprotic organic acids inion-exclusion chromatography. On this basis the microcomputer simulation of elu-tion behaviour for dicarboxylic acids was investigated. Influences of eluent acidityand sample concentration on the retention value were studied. The theoreticallypredicted results are in reasonable agreement with the experimental data.

Size Exclusion Mechanism, Suspension Flow through Porous Medium

A lot of investigations have been done in order to understand the mechanisms of the transport of particulate suspension flow through porous medium. In general, Deep Bed Filtration studies have been conducted to analyse the mechanism involved in the processes of capturing and retaining particles occurs throughout the entire depth of the filter and not just on the filter surface. In this study, the deep bed filtration mechanism and the several mechanisms for the capture of suspended particles are explained then the size exclusion mechanism has been focused (particle capture from the suspension by the rock by the size exclusion). The effects of particle flux reduction and pore space inaccessibility due to selective flow of different size particles will be included in the model for deep bed filtration. The equations for particle and pore size distributions have been derived. The model proposed is a generalization of stochastic Sharma-Yortsos equations. Analytical solution for low concentration is obtained for any particle and pore size distributions. As we will see, the averaged macro scale solutions significantly differ from the classical deep bed filtration model.

Mathematical modeling of salt-gradient ion-exchange simulated moving bed chromatography for protein separations

The salt-gradient operation mode used in ion-exchange simulated moving bed chromatography (SMBC) can improve the efficiency of protein separations. A detailed model that takes into account any kind of adsorption/ion-exchange equilibrium, salt gradient, size exclusion, mass transfer resistance, and port periodic switching mechanism, was developed to simulate the complex dynamics. The model predictions were verified by the experimental data on upward and downward gradients for protein separations reported in the literature. All design and operating parameters (number, configuration, length and diameter of columns, particle size, switching period, flow rates of feed, raffinate, desorbent and extract, protein concentrations in feed, different salt concentrations in desorbent and feed) can be chosen correctly by numerical simulation. This model can facilitate the design, operation, optimization, control and scale-up of salt-gradient ion-exchange SMBC for protein separations.

Continuous size fractionation of magnetic nanoparticles by using simulated moving bed chromatography

The size fractionation of magnetic nanoparticles is a technical problem,which until today can only be solved with great effort.Nevertheless,there is an important demand for nanoparticles with sharp size distributions,for example for medical technology or sensor technology.Using magnetic chromatography,we show a promising method for fractionation of magnetic nanoparticles with respect to their size and/or magnetic properties.This was achieved by passing magnetic nanoparticles through a packed bed of fine steel spheres with which they interact magnetically because single domain ferro-/ferrimagnetic nanoparticles show a spontaneous magnetization.Since the strength of this interaction is related to particle size,the principle is suitable for size fractionation.This concept was transferred into a continuous process in this work using a so-called simulated moving bed chromatography.Applying a suspension of magnetic nanoparticles within a size range from 20 to 120 nm,the process showed a separation sharpness of up to 0.52 with recovery rates of 100%.The continuous feed stream of magnetic nanoparticles could be fractionated with a space-time-yield of up to 5 mg/(L∙min).Due to the easy scalability of continuous chromatography,the process is a promising approach for the efficient fractionation of industrially relevant amounts of magnetic nanoparticles.

Determination of Uric Acid in Human Urine by Ion-exclusion Chromatography with UV Detection Using Pure Water as Mobile Phase

A simple, rapid and accurate ion-exclusion chromatographic method coupled with a UV detector for the determination of uric acid in human urine samples has been developed. The separation was carried out on an ion-exclusion column using only pure water as mobile phase. The detection wavelength was 254 nm and urine sample was injected directly without any pretreatment. Furthermore, the retention behavior of uric acid on the ion-exclusion column was researched when pure water and 1 mmol·L-1 HCI were used as mobile phase, respectively. The stability of uric acid was also further investigated within 28 days, In this method, the linear range of the calibration curve for uric acid was 0.25--100 mg·L-1, and the detection limit calculated at S/N=3 was 0.02mg·L-1 The proposed ion-exclusion chromatographic method has been used for the determination of uric acid in human urine.

Successive defects asymmetric simple exclusion processes with particles of arbitrary size

This paper uses various mean-field approaches and the Monte Carlo simulation to calculate asymmetric simple exclusion processes with particles of arbitrary size in the successive defects system. In this system, the hopping probability p （p 〈 1） and the size d of particles are not constant, Through theoretical calculation and computer simulation, it obtains the exact theoretical results and finds that the theoretical results are in agreement with the computer simulation. These results are helpful in analysing the effect of traffic with different hopping probabilities p and sizes d of particle.

Analysis of Cordyceps by multi-column liquid chromatography

Cordyceps is a famous traditional Chinese medicine (TCM) that has been used in China for hundreds of years. In the present study a multi-column liquid chromatography (MC-LC) system was developed for the qualitative analysis of macromolecules and micromolecules in Cordyceps. The MC-LC system includes a size exclusion pre-column, a size exclusion column (SEC) and a reversed phase column (RP) which were controlled by column-switching valves. The sample was separated by the size exclusion pre-column into two fractions (macromolecules and micromolecules). These fractions were further separated on SEC and RP columns, respectively. A diode array detector (DAD) and a mass spectrometer (MS) were used to detect the components. This MC-LC method was utilized for analysis of Cordyceps samples. Two macromolecular peaks and 15 micromolecular peaks were found in Cordyceps, and 11 of the micromolecular peaks were identified as adenosine-5'-monophosphate (AMP), phenylalanine, uridine, hypoxanthine, inosine, guanine, guanosine, deoxyadenosine-5'-monophosphate (dAMP), adenosine, adenine and cordycepin (or its isomer). This method is useful for quality control of Cordyceps. (C) 2017 Chinese Pharmaceutical Association and Institute of Materia Medica, Chinese Academy of Medical Sciences. Production and hosting by Elsevier B.V.

Characterization of blueberry exosome-like nanoparticles and miRNAs with potential cross-kingdom human gene targets

Edible plant derived exosome-like nanoparticles(ELNs)have been shown to have multiple nutraceutical functions.However,the diversity of plant materials makes the plant derived ELN study challenging.More efforts are still needed to explore the feasible isolation methods of edible plant derived ELNs and the possible roles of food-derived ELNs in improving human health.In this study,a size exclusion chromatography based method was compared with the traditional ultracentrifugation method to isolate blueberry derived ELNs(B-ELNs),and the miRNA profile of B-ELNs was analyzed by high-throughput sequencing.A total of 36 miRNAs were found to be enriched in B-ELNs compared with berry tissue,and their potential cross-kingdom human gene targets were further predicted.Results showed that size exclusion chromatography was effective for B-ELN isolation.The most abundant miRNAs in B-ELNs mainly belonged to the miR166 family and miR396 family.Target gene prediction indicated that B-ELNs could potentially regulate pathways related to the human digestive system,immune system and infectious diseases.

Accuracy of Protein Size Estimates Based on Light Scattering Measurements

There are two types of light scattering measurements: static light scattering (SLS) and dynamic light scattering (DLS). The SLS method is used to estimate the molecular weight (MW) of particles by measuring the time-averaged intensity of light scattered by the particles, whereas the DLS method is used to estimate the diffusion coefficient of particles by observing the time-correlation of scattered light intensity. These techniques have recently been applied to the investigation of the aggregation, denaturation and folding, and complex formation of proteins in solution. However, the accuracy of protein size measurement by light scattering is poorly understood. In the present study, we carried out the size measurements of five globular proteins by SLS and DLS at a detection angle of 90。 and compared these data to measurements made by size exclusion chromatography (SEC). The difference (%) between the MW estimated from each method and the MW calculated from the amino acid sequence (namely the calibration residual error) was regarded as an index of measurement accuracy. The averaged calibration residual errors were 5.2 and 4.7 for SEC and SLS measurements, respectively. For the DLS measurements, the extrapolation of the apparent hydrodynamic radii to a protein concentration of zero may effectively eliminate the interparticle and hydrodynamic interactions and significantly reduced the averaged calibration residual error to 4.8%. Our results suggested that the size of globular proteins can be estimated using light scattering measurements with an accuracy equivalent to that of SEC.

基于热裂解气相色谱-质谱技术的熟宣劣化评价研究初探

本研究通过测试不同人工模拟加速老化环境下宣纸样品的力学性能、pH值和热裂解产物,探究了熟宣劣化程度评价的新方法。结果表明:净皮单宣与熟宣的抗张指数、耐折度、pH值以及主要热裂解产物的含量存在明显的差异。热裂解气相色谱-质谱技术的样品需要量少、无需前处理,可有效实现对不同老化时间、不同老化方式的熟宣劣化程度的评价。实验中主要热裂解产物中左旋葡萄糖酮产率因随老化时间变化显著而可作为纸张酸化劣化的内在指示指标,湿度因素是影响左旋葡萄糖酮产率也即熟宣劣化的显著环境因素。

碱性蛋白酶酶解蛋白制备活性肽的质控研究

以胶原蛋白和大豆分离蛋白为主要研究对象,应用体积排阻色谱(SEC)结合多角度激光光散射(MALLS)及示差折光(RI)联用技术对碱性蛋白酶酶解蛋白制备肽段的过程进行了详细分析。在酶解过程中,不同来源的胶原蛋白(牛、鸡)和大豆分离蛋白在分子量分布、体系的分散度及粒径变化规律上表现出了典型的差异。结果表明,牛胶原蛋白在酶解开始时就迅速被降解产生了大量小分子肽段,鸡胶原蛋白相对于牛胶原蛋白在相同的酶解条件下更难产生小分子肽段,大豆分离蛋白随着酶的加入则是表现出了先聚集再降解的特性。SEC-MALLS技术可以有效把控蛋白酶解的过程以及体系中肽段分子量分布,该技术可以为活性肽产品的开发及品质检测提供依据。

高效液相色谱-质谱技术在蛋白质组学中的应用

蛋白质组学研究在生物医学领域发挥了重要作用,然而研究面临的主要难点在于其研究对象的复杂性和多样性。随着质谱技术的快速发展,高效液相色谱-质谱(HPLC-MS)分离分析复杂生物样品已经成为蛋白质组学研究的基础工具。蛋白质组学的研究从肽段分离,延伸到蛋白质和蛋白质复合物的分离,随着分析物的分子质量不断增大,其结构和组成复杂性也持续增加,分子特性也发生改变。面对不同的蛋白质组学研究对象,选择不同的分离模式、分离条件以及固定相参数是进行深度蛋白质组学研究的关键。本文综述了实验室常用的液相色谱分离模式,包括反相色谱(RPLC)、亲水相互作用色谱(HILIC)、疏水相互作用色谱(HIC)、离子交换色谱(IEC)和体积排阻色谱(SEC),以及其不同的组合模式与质谱联用在自下而上(bottom-up)分析、自上而下(top-down)分析、蛋白-蛋白相互作用分析中应用的研究。具体分析了色谱流动相与被分析对象之间的兼容性问题、色谱流动相与质谱兼容性问题,以及多维色谱中不同色谱模式之间流动相的兼容性问题。重点关注存在不兼容问题时研究者所提出的解决方案。此外,本文还评述了HPLC-MS结合样本前处理的方法在外泌体和单细胞蛋白质组学中的应用研究。总之,文章聚焦于近年来HPLC-MS技术在蛋白质组学中的研究进展,旨在为未来蛋白质组学领域的研究提供参考。

碧根果致敏原Car i 1的分离纯化及表征鉴定

目的分离纯化碧根果致敏原Car i 1,并对其结构进行表征鉴定。方法以新鲜碧根果果仁为原料,通过粉碎、脱脂、浸提、粗分级、凝胶过滤层析,对碧根果致敏原蛋白Car i 1进行分离纯化。结合十二烷基硫酸钠-聚丙烯酰胺凝胶电泳、液相色谱-串联质谱法和免疫印迹法3种方法对Cari1进行鉴定,并通过圆二色谱仪与紫外分光光度计表征其二、三级结构。结果本方法纯化获得碧根果致敏原Cari1,单轮制备量可达5 mg以上,且纯度大于95%,蛋白质高级结构未被破坏,能够被全部3名碧根果过敏患者的血清准确识别。结论该纯化方法技术路线简单、设备要求低且单次制备量高,总得率可达65%,操作便捷,为碧根果致敏原Car i 1的相关研究奠定了物质基础。