体积排阻色谱和激光光散射对大豆蛋白热诱导聚集体的研究

采用体积排阻色谱(SEC-HPLC)和激光光散射(LLS)研究了不同浓度和离子强度下大豆蛋白热诱导聚集体的分子量分布和粒径分布。在离子强度为0时,SEC-HPLC的结果表明,热处理后的蛋白溶液主要由3部分组成,即聚集体、中间体和未聚集部分。聚集体部分随着浓度增加而逐渐增加;LLS的结果表明:体系有不均一的粒径分布,且浓度增加时体系的平均粒径增加。上述样品在较高离子强度下加热时,SEC-HPLC和LLS的结果都证明溶液中的中间体部分逐渐消失。因此,控制浓度和离子强度可以制备不同性质的大豆蛋白聚集体。     

凝胶过滤色谱-多角度激光散射法测定琥珀酰明胶自由氨基修饰度

琥珀酰明胶作为血浆代用品的临床疗效与其分子量分布和修饰度密切相关。本研究利用高效凝胶过滤色谱-多角度激光散射法(HPSEC-MALLS)并结合2,4,6-三硝基苯磺酸(TNBS)衍生法,探索建立一种测定琥珀酰明胶修饰度的方法。实验利用TNBS将琥珀酰明胶衍生,通过HPSEC-MALLS测定琥珀酰明胶的分子量变化,计算出琥珀酰明胶的修饰度。实验以人血清白蛋白为模型进行了方法验证,结果表明:该方法能较好地表征人血清白蛋白在TNBS衍生过程中的分子量变化,实验值与根据其氨基酸组成计算值一致。实验在HPSEC-MALLS测定琥珀酰明胶及TNBS衍生后琥珀酰明胶分子量及分布的基础上,利用凝胶过滤色谱分子量-保留时间拟合曲线,计算得到琥珀酰明胶的自由氨基修饰度为38%。结果表明利用高效凝胶过滤色谱-多角度激光散射法结合2,4,6-三硝基苯磺酸衍生法测定琥珀酰明胶的修饰度是可行的。     

Characterization of Nylons by Gel Permeation Chromatography and Low Angle Laser Light Scattering in 2,2,2-Trifluoroethanol

Abstract

A GPC method was developed for the analysis of several commercial nylons in trifluoroethanol + 0.05M LiBr using a styrene-divinylbenzene column custom-packed by Jordi Associates.

A broad molecular weight standard method was developed by interfacing GPC with LALLS to give the absolute molecular weight for each data point or elution volume along the chromatographic peak from a nylon sample of known molecular weight. The integrity of the interface was verified by static LALLS measurements; no loss or adsorption of solute was found in the chromatographic system.

A strong ionic effect was observed for nylon samples and the method to alleviate it was described. The molecular weights and distribution of the following nylons were determined by this method: nylon 6, nylon 4,6, and nylon 6,6. Much higher than quoted molecular weights were obtained for nylons when polymethyl methacrylates and ethylene glycols were used as standards; this necessitated the use of this broad molecular weight method for column calibration. The ambient operating conditions offered several advantages over the conventional m-cresol solvent which required operation at higher than 100 C in order to reduce the viscosity, possessed a strong odor, and occasionally resulted in degradation of polyamides.     

Continuous Viscometric Detection in Size Exclusion Chromatography

Abstract

Continuous viscometric detection is based on the measurement of pressure drop in an on-line small capillary tube in which chromatographic eluents flow at constant flow rate. This detector is always coupled with a concentration detector (usually refractometer) and located before it to avoid back pressure in the refractometer. In order to obtain reliable information for polymer samples, it is generally necessary to connect these two detectors to a computer which performs data acquisition and treatment.

First, we discuss the problem of shape, geometry and dimensions of the viscometer. The typical characteristics are the result of a compromise between contradictory targets, mainly small internal volume, low shear rate and low pressure drop. It is shown that Poiseuille's laminar flow is only obtained when coiling radius of the measurement tube is greater than 6 cm, which is not the case inside the refractometer. Accordingly, two pressure transducers are necessary to eliminate pressure drop data coming from refractometer.

In a second part, we show how to extract information from pressure variation data. By using concentration data, pure solvent pressure and sample pressure it is possible to calculate intrinsic viscosity extrapolated to zero concentration at each point of the chromatogram. By comparison with intrinsic viscosity of the polymer used for calibration, a correction of hydrodynamic volume according to Benoit's universal calibration leads to absolute molecular weights.

In addition, for a linear polymer, the knowledge of log [η] versus log M leads to the determination of Mark-Houwink relationship coefficients. For branched polymers, viscosity laws are curved and the comparison between the linear law corresponding to the linear equivalent polymer and the experimental law allows the determination of the g' branching parameter distribution.     

时间分辨激光光散射测量系统

自选设计，装配了一套时间分辨的激光光散射测量系统，该系统摄像机，录像机，计算机和自编的软件实现了一体化的数据采集，显示和分析过程，本文给出了该系统的应用实例，球晶的光散射和环氧树脂的固化反应诱导的相分离过程。     

Size Exclusion Chromatography of Quaternized Poly(vinylpyrrolidone-co-dimethyl Amino Ethyl Methacrylate) Copolymer (PVPDMAEMA): Absolute Molecular Weights and Molecular Weight Distributions by Low Angle Laser Light Scattering and Universal Calibration

Abstract

In 0.1M tris. pH 7 buffer containing 0.5M LiNO₃, PVPDMAEMA (which exists as a cationic polyelectrolyte) was found to elute based on hydrodynamic volume on Waters Ultrahydrogel columns. Within the exclusion limit of the column set an accurate weight average molecular weight of PVPDMAEMA can be obtained by universal calibration which is in very good agreement with the SEC/LALLS result.     

Size Exclusion Chromatography of Polyamides

Abstract

We report here a new chromatographic method for characterization of polyamides and polyether-block-amide copolymers by Size Exclusion Chromatography. First, it concerns absolute characterization of polyamide probes by classical light scattering and Ubbelohde viscometry measurements and the choice of narrow distribution standards to calibrate the GPC system. The GPC analysis of polyamides 6, 11, 12 and copolymers is based upon the dual detection refractometer/on-line continuous viscometer at 130°C using columns packed with cross-linked polystyrene gels, benzyl alcohol as mobile phase and polytetrahydrofuran standards for calibration. The main results are absolute average molecular weights and distribution curve, intrinsic viscosity and viscosity law. In addition, viscometric data produced evidence of long-chain branching occuring beyond a limiting molecular weight in some polyamide 12 samples.     

Overloading and Degradation Study in GPC Using Low Angle Laser Light Scattering Detector (LALLS)

Abstract

The unique macromolecular compression effect or delayed GPC elution of high M.W. material was observed using the GPC + LALLS technique for a polystyrene standard of M.W. 5.0 E5. Further overloading which resulted in nonlinear fractionation is also described. The degradation of a 4.2 E6 standard after elution was detected by LALLS with calculated M.W. of 1.0-1.1 E6 but not by GPC, as indicated by normal GPC retention time and the shape of the chromatogram. This degradation was further verified by reinjection of the collected fractions into the GPC which showed a bimodal distribution with calculated M.W. of ca. 1.0 E6 via GPC calibration and LALLS. The source of degradation was attributed to the 2 micron exit frit in the single Jordi mixed bed column. Similar degradation of 2.3 E6 standard to same M.W. of ca. 1.0 E6 was also observed. The use of these standards as GPC calibrants should not be affected by this “post column” degradation.     

Characterization of Branched Poly (vinyl Acetate) by GPC and Low Angle Laser Light Scattering Photometry

Abstract

Poly (vinyl acetate), PVAC, synthesized by bulk polymerization over a range of initiator concentrations ([AIBN] = 10^?5 to 4 × 10^?3 g-mole/1), temperatures (50°C, 60°C, 70°C, and 80°C) and conversion levels (3 to > 90%) were characterized using low angle laser light scattering (LALLS) photometry to measure M_w of the whole polymers. A number of these samples were characterized using GPC with a differential refractive index (DRI) and LALLS detector to measure the molecular weight distribution (weight fraction versus M_w). M_w for PVAC samples synthesized at suitably low initiator levels at various conversions were found to agree with classical light scattering measurements after Graessley.

An electronic device and a technique which optimizes the sensitivity and the signal-to-noise ratio of the LALLS photometer throughout the molecular weight distribution (MWD) of the GPC chromatogram were devised. These considerably simplify the operation of the LALLS for both offline and online operation with GPC.

Most importantly it was unambiguously shown that the commonly used universal calibration parameter (UCP) with GPC, [n]M_w, is incorrect for polymers with molecules having the same hydrodynamic volume but different molecular weights, i. e., those with only chain branching (LCB), copolymers with compositional drift, and polymer blends. The correct UCP was found to     

三检测体积排除色谱表征聚合物支化结构

以α-溴代异丁酸叔丁酯(t-BBiB)为引发剂,CuBr/联二吡啶为催化体系,二乙烯苯(DVB)为支化单体,采用原子转移自由基聚合反应(ATRP)经过一步加料方法和先臂后核方法分别合成不同支化结构类型的支化聚合物,同时以四官能度引发剂经ATRP合成臂数确定的星形聚合物为参照。采用三检测体积排除色谱(TD-SEC)得到Zimm相对支化因子g′与g和表征聚合物支化结构类型的指数因子ε(g′=gε)。结果表明:随支化结构类型的不同,支化结构指数因子ε表现出不同的分子量依赖特性。星状支化聚合物的ε平均值<1,而且随分子量的变化保持相对恒定;无规支化聚合物的ε平均值>1,而且其值随分子量的增加不断减小。因此,可以由三检测体积排除色谱进行分析确证聚合物的支化结构类型。     

Determination of Brassinolide Analogs by High-Performance Liquid Chromatography with Evaporative Light Scattering Detection

An efficient method based on high-performance liquid chromatography with evaporative light scattering detection was developed for the separation and determination of four brassinolide analogs [24-epibrassinolide, (22S, 23S)-24-epibrassinolide, 28-homobrassinolide, and (22S, 23S)-28-epihomobrassinolide] without prior derivatization. The optimized analysis was carried out on a C₁₈ reversed-phase column (150 mm × 4.60 mm, 3 µm) at 30°C using isocratic elution of acetonitrile and water (38:62, v/v). The drift tube temperature of the detector was 60°C and the auxiliary gas (nitrogen) pressure was 360 kPa. The regression equations revealed linear relationships (R² = 0.9984–0.9994) within the test ranges. The limits of detection and quantification were in the ranges of 0.12 to 0.17 µg and 0.24 to 0.33 µg, respectively. The fully validated method was applied to quantify the active ingredient content in technical material and formulations and provides an alternative approach for quality control.     

Characterization of Xylansulfate by Size Exclusion Chromatography

Abstract

A sulfated, low molecular weight beech xylan was fractionated preparatively on Sephadex G-50 into ten fractions with molecular weights from 17 000 to 1 800. The degree of sulfation, refractive index increment, molecular weight, and specific optical rotation of each fraction was determined. The fractions were then used to calibrate a Sephacryl S–200 gel column for the molecular weight distribution analysis of xylansulfate.

The molecular weights, M _W, obtained by SEC for samples of xylansulfate were in good agreement (within 4 ± 1%) to the values derived by low angle light scattering.     

Temperature effects in Microcolumn Size Exclusion Chromatography

Abstract

Effects of column temperature on the column efficiency, retention time and stability of analytes were studied in microcolumn size-exclusion chromatography. Larger theoretical plates were achieved at column temperatures betwen 70 and 100 °C. In the constant-flow mode the retention time of analytes decreased with increasing column temperature, which was due mainly to thermal expansion of the mobile phase. When the column temperature was around or higher than the critical temperature of the mobile phase, the retention times of the analytes observed under supercritical pressure conditions still indicated dominancy of the size-exclusion mechanism, while another retention mechanism was involved under subcritical pressure conditions. In the case of the analysis of saccharides, the column temperature should be lower than 100 °C because oligosaccharides were decomposed at higher temperatures.     

A Calibration Technique for Size Exclusion Chromatography

Abstract

A modified Weibull distribution function is shown to be useful in calibrating the molecular size separation capabilities of both rigid and swellable gel packing materials. Two parameters are used in this function which are related to a packing material's micropore volume distribution. The calibration curves of a set of different packing materials connected in series were predicted from the Weibull calibration functions obtained for each individual packing material.     

Quantitative Size Exclusion Chromatography: Assessing New Developments

Abstract

Methods of assessing new technology are achieving increased importance because rapid technological changes cause obsolescence of evaluations before they are completed. Users now must often evaluate the specific version of the new technology that they receive. Four major areas are used here to provide examples of assessment methods: high resolution columns, coupled concentration-molecular weight-differential viscometer detectors, flow rate monitoring using a thermal pulse flowmeter and determination of Mark-Houwink constants from polydisperse standards. Specific methods of assessment examined centre about error analysis and sensitivity analysis. Several of the methods use the conventional calibration curve. The idea of correction priority (i.e. thoroughly examining the most fundamental significant corrections first) is emphasized.     

Size Exclusion Chromatography of Poly(vinylpyrrolidone)

Abstract

Poly(vinylpyrrolidone) and poly(ethylene oxide) separate by hydrodynamic volume on Toyo Soda TSK-PW columns in a mixed solvent mobile phase of 50:50 (v/v) MeOH/H₂O containing 0.1M LiNO₃. From this separation a single universal calibration curve based on hydrodynamic volume [η]M can be obtained. Accurate weight average molecular weights of PVP were obtained by both SEC/LALLS and universal calibration showing good agreement between the two methods. SEC/LALLS overestimates the number average molecular weight for broad distribution polymers due largely to the lack of sensitivity of the LALLS detector to the low molecular weight portion of the polymers, while the universal calibration method slightly underestimates the number average molecular weight as compared to osmometric values.     

高效液相色谱-蒸发光散射检测法测定蛋黄卵磷脂的含量

建立了蛋黄磷脂中卵磷脂（即磷脂酰胆碱,PC)的高效液相色谱-蒸发光散射检测（HPLC-ELSD）的测定方法。以Nov a-Pak Silica 60A硅胶柱(3.9 mm i.d.×150 mm,4 μm)为分离柱,正己烷-异丙醇-3%冰醋酸水溶液(体积比为35∶65 ∶8)为流动相，等度洗脱,流速1.0 mL/min,柱温30 ℃。蒸发光散射检测器漂移管温度50 ℃,雾化气(空气)压力350 kPa。在上述条件下测得PC在0.16~1.61 g/L范围内线性关系良好(r2=0.9979),检测限为0.64 μg,方法的精密度为3.2%(n=5),回收率为98.2%~128.2%。将该方法用于实际样品的测定,获得了令人满意的结果。该方 法预处理简单,分析速度快,可用于蛋黄磷脂中卵磷脂的测定。     

Size Exclusion Chromatography of Polymers With Random Tetrafunctional Branching

Abstract

The behaviour of polydisperse branched copolymers of methyl methacrylate with a small amount of randomly situated tetrafunctional ethylenedimethacrylate units was investigated by means of size exclusion chromatography (SEC). A procedure has been suggested for the conversion of apparent values of molecular parameters of real polymer branched systems (M_{n, app}; M_{w, app} obtained from SEC data by calibration of the separation system using a linear polymer) into actual values. This was made possible by off-line coupling of SEC and viscometry. The branching was characterized by the weight average number of branching sites in the macro-molecule, m_w, and the branching index, y.     

Characterization of Polymer Architectures by Multidetection Size Exclusion Chromatography

Summary: This paper discusses the analysis of star-branched and dendritic polyisobutylenes (PIBs) using branching parameters based on radii of gyration R_g,z and hydrodynamic radii R_h,z. R_g,z and R_h,z were measured by Size Exclusion Chromatography equipped with a Wyatt Technology Viscostar viscometer (VIS), a Wyatt Optilab DSP RI detector, a Wyatt DAWN EOS 18 angle Multiangle Light Scattering (MALS) detector and a Wyatt Quasi-elastic Light Scattering QELS detector. Branching parameters were calculated from these measurements and compared with those computed for these architectures.