On-Line Coupling of Thermal Field-Flow Fractionation with Low-Angle Laser Light Scattering

Abstract

The analysis of a polymethyl methacrylate sample dissolved in dimethylformamide is performed by using a low-angle laser light scattering photometer attached to a thermal field-flow fractionation channel and a differential refractometer. Relevant theoretical light scattering equations for flow-through operation are outlined. It is shown that the calibration curve of the separation system can be constructed in situ during the course of separation, without using any calibration standard. The average molecular weights as well as the molecular weight distribution curves of the polymer have been determined. The sensitivity of the light-scattering photometer has been measured, and it is compared to that of the differential refractometer in terms of signal-to-noise ratios. The various sources of errors in the molecular weight determination are discussed, and the potential of the coupling for physicochemical studies on the thermal diffusion of polymers is indicated. In spite of some inherent problems, this coupling is expected to have a very bright future if reliable low-angle light-scattering instruments can be made available at moderate prices.     

Second Virial Coefficients for Solutions of Polystyrene Mixtures Obtained With Low-Angle Laser Light Scattering

Low-angle laser light scattering measurements were performed on practically binary mixtures of standard polystyrenes dissolved in toluene. Second virial coefficients A₂ were obtained as a function of the composition of the mixed polymer. No solid evidence was found for the existence of maxima in A₂ vs composition. Existing theories predict a maximum. The possible existence of minima such as reported by Suzuki⁵ could not be confirmed with the present system.     

聚合物流动行为的光散射表征

简要地介绍了光散射原理及目前光散射技术在聚合物流变学的中的应用，归纳并分析了前人的一些主要工作成果。对于一些至今难以解释的实验现象提出了自己的一些认识，并对仍未有人涉足的振动力场下聚合物复杂流动光散射表征提出了新的设想。     

Biophysical Characterization of Adeno-Associated Virus Vectors Using Ion-Exchange Chromatography Coupled to Light Scattering Detectors

Ion-exchange chromatography coupled to light scattering detectors represents a fast and simple analytical method for the assessment of multiple critical quality attributes (CQA) in one single measurement. The determination of CQAs play a crucial role in Adeno-Associated Virus (AAV)-based gene therapies and their applications in humans. Today, several different analytical techniques, including size-exclusion chromatography (SEC), analytical ultracentrifugation (AUC), qPCR or ELISA, are commonly used to characterize the gene therapy product regarding capsid titer, packaging efficiency, vector genome integrity, aggregation content and other process-related impurities. However, no universal method for the simultaneous determination of multiple CQAs is currently available. Here, we present a novel robust ion-exchange chromatography method coupled to multi-angle light scattering detectors (IEC-MALS) for the comprehensive characterization of empty and filled AAVs concerning capsid titer, full-to-total ratio, absolute molar mass of the protein and nucleic acid, and the size and polydispersity without baseline-separation of both species prior to data analysis. We demonstrate that the developed IEC-MALS assay is applicable to different serotypes and can be used as an orthogonal method to other established analytical techniques.     

激光颗粒仪测定聚四氟乙烯细粒模压粉的平均粒径

运用激光粒度仪研究了聚四氟乙烯细粒模压粉的平均粒径和粒径分布的测定方法，通过试验确定了分散方法、分散剂用量、试样量、分散时间等影响因素，以建立稳定的测量体系。     

激光散射在生物高分子中的应用

本文综述了激光散射在生物高分子中的应用,着重以蛋白质为例分析了激光散射在表征生物高分子的分子量、尺寸、构象、缔合、聚集、结晶和分形等方面的重要作用,同时对它在生理分析上的应用作了简要介绍。     

High Performance Size Exclusion Chromatography of Polar Compounds in Heated and Non-Heated Fats

In this paper, separation of the polar compounds previously obtained from heated fats by column chromatography is proposed in order to increase knowledge of the alteration of frying fats. The analysis is carried out in 35 min by high performance size exclusion chromatography (HPSEC). The results demonstrate that the contribution of thermoxidative and hydrolytic degradations can be quantified. The method can also be applied to a better definition of non-heated fats.     

Characterization of Standard Reference Material 1690 by Modulated Dynamic Light Scattering

ABSTRACT

We have measured the number average diameter (NAD) and the standard deviation of the diameter distribution (SDDD) of spherical particles from a sample of Standard Reference Material 1690 (SRM 1690) using the method of Modulated Dynamic Light Scattering to determine the diameter of single test particles by examining their Brownian motion as they sediment freely in dry air. Standard Reference Material 1690 is a water-suspension of 1 μm nominal diameter polystyrene spheres and has been characterized by National Institute of Standards and Technology (NIST) personnel as a primary reference standard for the calibration of particle-size-measuring instruments. We have obtained for SRM 1690 particles the values NAD = 0.9103 ± 0.0021 μm and SDDD = 0.0090 μm. The NIST-certified values for SRM 1690 are NAD = 0.895 ± 0.008 μm and SDDD = 0.0095 ± 0.0012 μm. The SDDD value from this work agrees with the NIST-certified value within the stated limits of uncertainty, but the NAD value is 1.7% larger than the NIST-certified value. We suggest that the discrepancy is due to the presence of nonspherical particles in the test suspensions measured by NIST.     

Characterization of Pore Size Distribution by Infrared Scattering in Highly Dense ZnS

A model based upon Mie theory was developed to calculate the amount of spectral infrared scattering caused by the presence of a porous second phase. Various in-line transmission curves were calculated and used to characterize the scattering effects of pore size and concentration. The in-line transmission from 2.5 to 10 fjim of ZnS samples hot-pressed at 137.8,172.3, and 206.7 MPa was measured using Fourier transform infrared spectrophotometry and compared with calculated transmission curves. Good agreement with measured results was obtained only when a size distribution effect was included. From the analysis, a bimodal distribution of pores was found to give the best agreement which duplicates the measured transmission.     

Intravesicular Genomic DNA Enriched by Size Exclusion Chromatography Can Enhance Lung Cancer Oncogene Mutation Detection Sensitivity

Extracellular vesicles (EVs) are cell-derived structures surrounded by a lipid bilayer that carry RNA and DNA as potential templates for molecular diagnostics, e.g., in cancer genotyping. While it has been established that DNA templates appear on the outside of EVs, no consensus exists on which nucleic acid species inside small EVs (<200 nm, sEVs) are sufficiently abundant and accessible for developing genotyping protocols. We investigated this by extracting total intravesicular nucleic acid content from sEVs isolated from the conditioned cell medium of the human NCI-H1975 cell line containing the epidermal growth factor (EGFR) gene mutation T790M as a model system for non-small cell lung cancer. We observed that mainly short genomic DNA (<35–100 bp) present in the sEVs served as a template. Using qEV size exclusion chromatography (SEC), significantly lower yield and higher purity of isolated sEV fractions were obtained as compared to exoEasy membrane affinity purification and ultracentrifugation. Nevertheless, we detected the EGFR T790M mutation in the sEVs’ lumen with similar sensitivity using digital PCR. When applying SEC-based sEV separation prior to cell-free DNA extraction on spiked human plasma samples, we found significantly higher mutant allele frequencies as compared to standard cell-free DNA extraction, which in part was due to co-purification of circulating tumor DNA. We conclude that intravesicular genomic DNA can be exploited next to ctDNA to enhance EGFR T790M mutation detection sensitivity by adding a fast and easy-to-use sEV separation method, such as SEC, upstream of standard clinical cell-free DNA workflows.     

小角激光光散射法测定纤维素硫酸钠的分子量

采用小角激光散射法测定了纤维素硫酸钠(NaCS)的分子量,考察了盐离子及其浓度对分子量测定的影响. 结果表明,取代度分别为0.28, 0.40, 0.73的3个样品的分子量分别为879.8, 593.5和226.9 kDa,盐离子浓度为0.05~0.075 mol/L时测定较准确,盐离子浓度为0~0.05 mol/L时光散射强度精密度较低,不利于分子量测定,粘度变化较大,表明此时NaCS分子处于膨胀伸展状态,不利于光散射法测定分子量.     

激光散射法快速测定催化剂粒度分布

采用激光粒度仪快速分析催化剂的粒度,利用激光光束对样品池内的样品颗粒进行衍射,散射光的角度与颗粒的粒度成反比,散射光的强度与颗粒的浓度成正比,所有测量由计算机分析测量并计算。该方法分析速度极快,测量范围广,操作简单、快捷,可以很好的满足对各种催化剂、粉尘的粒度分析的需要。     

Order-Disorder Conformation Change of Succinoglycan in Aqueous Sodium Chloride as Studied by Static and Dynamic Light Scattering

Summary Static and dynamic light scattering measurements have been made on a sodium salt sample of succinoglycan in 0.01 M aqueous NaCl at different temperatures between 25 and 75°C where the polysaccharide undergoes a thermally induced change from an ordered (helical) to disordered conformation with raising temperature T. The weight-average molecular weight M _w, the z-average radius of gyration, and the hydrodynamic radius sharply decrease in a relatively narrow T range (around 55°C) in which the specific rotation was previously found to change sigmoidally with T. In particular, the value of M _w (4.55 × 10⁵) in the ordered state at 25°C is twice as large as that (2.27 × 10⁵) in the disordered state at 75°C, giving decisive evidence that the helical structure of the polysaccharide in aqueous NaCl is composed of paired chains. It is concluded that this structure is a double-stranded helix and breaks directly into two disordered chains with increasing T. Received: 6 July 2001 / Accepted: 24 July 2001     

Gradient Elution HPLC of Fats and Oils with Laser Light Scattering Detection

Gradient elution HPLC of oils and fats using reversed-phase HPLC separation systems with laser light-scattering detection are described. Potential advantages and shortcomings are discussed. Flexible solvent gradient programming permits an adaptation of the system to several types of applications. Using systems of this type the usual solubility problem with long-chain saturated triglycerides can easily be overcome. A useful test-mixture consisting of a series of saturated triglycerides two carbon numbers apart can be obtained by interesterification of hydrogenated palm kernel oil and hydrogenated palm oil. HPLC systems of this type will also permit the simultaneous investigation of diglycerides and triglycerides in oils.     

Polarized Light Scattering by Small Particles

All of the polarized light scattering properties of single particles and suspensions of particles are contained in the 16-element Mueller matrix. For single-particle studies the particle is electrostatically suspended, the light scattering functions are measured, and the particle is then extracted from the suspension chamber by placing it on the point of a needle. The particle is examined subsequently with an electron microscope to determine precisely its size and shape. Results will be presented for single polystyrene spheres, sphere multiplets, approximately cubic NaCl crystals, and cubes of MgO. Recent studies of suspensions have concentrated on oceanic hydrosols. Some results and general features of these hydrosols will be presented.     

HPLC-ELSD法测定低聚甘油的组成

建立了高效液相色谱-蒸发光散射检测器(HPLC-ELSD)测定低聚甘油组成的方法。以Econosphere Amino(250×4.6 mm,5μm)为色谱柱,乙腈-水(85∶15,V/V)为流动相,流速为1.0 mL/min,柱温为28℃,进样量为30μL,ELSD漂移管温度为32℃,载气流速为1.5 L/min。结果表明,在优化条件下,甘油、二聚甘油和三聚甘油浓度分别在100～300μg/mL、100～400μg/mL和200～600μg/mL范围内呈良好线性,相关系数分别为0.9979、0.9921和0.9969,定量下限分别为1.5、1.0和2.0μg,回收率为97%～103%,RSD<3.0%(n=6)。     

Separation and Quantification of Vegetable Oil Based Polyols by High Performance Liquid Chromatography with Evaporative Light Scattering Detection

Polyols with terminal primary alcohol functionalities were obtained from canola oil via an ozonolysis and hydrogenation process. A high performance liquid chromatography (HPLC) method with evaporative light scattering detection (ELSD) was developed for separating and quantifying the polyol products. Linear calibration curves were obtained for the mono-ol, diol and triol components with correlations (r ²) above 0.98. According to the standard curve, the content of mono-ol, diol and triol can be obtained from their HPLC-ELSD chromatograms.