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.     

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.     

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.     

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.     

微柱液相色谱的研究进展

介绍了微柱液相色谱（μ-LC）。从理论上简单讨论了μ-LC的柱特性、色谱洗脱效应和柱外效应等一系列问题,综述了μ-LC的柱技术的最新进展,讨论了μ-LC对仪器和附件的要求,特别是微流量输液和检测技术,还探讨了μ-LC与多维色谱、质谱等技术的联用。     

Concentration Effects in Size Exclusion Chromatography Under Equilibrium Stationary Conditions

Abstract

In size exclusion chromatography the elution volume increases with increasing concentration of injected polymer solutions. Several particular processes contribute to this concentration effect. Under model equilibrium stationary conditions, where the phases remain immobile, it is possible to eliminate dynamic phenomena connected with the higher viscosity of polymer solution in the chromatographic zone. The only factors operative here are the effect of changes in the effective size of macromolecules in solution with a change in concentration and the effect of the varying accessible pore volume with varying concentration. The ratio of these two contributions has been investigated both theoretically and experimentally. Theoretical calculations indicated that with both phenomena operating simultaneously, the elution volumes may increase or decrease with increasing concentration according to the given experimental conditions, as a result of the coupled influence of molecular parameters of the samples under study, of the thermodynamic quality of the solvent and of the distribution coefficient on the given porous material. Experimental results showed, however, that at least under stationary conditions secondary exclusion is probably operative to a less important degree.     

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.     

体积排阻色谱在蛋白质药物聚集体领域的应用

蛋白质聚集现象是生物制药行业面临的重大问题之一,对蛋白质药物有效性、安全性、质量可控性有很大影响。体积排阻色谱技术是蛋白质药物及其聚集体检测分析的标准技术,具有操作简单、分离条件温和、基本不破坏蛋白质药物结构等优点。但由于蛋白质与固定相存在非特异性相互作用,采用该法检测时存在洗脱延迟、色谱峰拖尾、基线漂移、蛋白质回收率低等问题。该文介绍了体积排阻色谱的分离原理及实际应用,并对该技术在蛋白质药物检测中存在的非特异性吸附问题及相关方法优化做了简要概述。同时列举了几种与体积排阻色谱互补的可用于蛋白质药物聚集体分析的技术。最后,对体积排阻色谱技术的发展前景进行了展望。     

Size Exclusion Chromatography Calibration Assessment Utilizing Coupled Molecular Weight Detectors

Abstract

This paper shows that the usual method of representing an SEC calibration curve by a single polynomial curve may often be inadequate with new high resolution columns. Data points wind about the fitted line. The significant magnitude and systematic nature of these deviations clearly appear when a plot of residuals is derived from the conventional calibration curve and expressed in terms of the percent error in molecular weight. The deviation of the calibration data from the fitted line was approximately 10% for the conventional molecular weight and intrinsic viscosity calibration curves. It became 20% for the universal calibration curve. LALLS and DV detectors were used together with the DRI detector to provide evidence that the calibration curve deviations were due to the column packings and not due to some other cause (e.g., vendor values of molecular weight). Use of a polynomial fit to a portion of the curve corresponding to the retention volume range of an unknown was used to show the significant improvement in results which occurred when the calibration variations were taken into account. At present, use of many individual narrow standards is necessary to elucidate the effect.     

Computer Assisted Data Acquisition and Analysis for Size Exclusion Chromatography

Abstract

Two programs, ACEACQ and ACEANN, provide data acquisition and analysis for Size Exclusion Chromatography (SEC). ACEACQ is used for data logging, real-time smoothing and data reduction by boxcar averaging. ACEACQ requires that traditional SEC hardware be coupled with a Hewlett Packard data logger and microcomputer. A Mettler electronic balance is connected to the computer in a novel fashion to continuously monitor the mass of solvent eluted during an experiment. ACEANN provides analysis for the data collected by ACEACQ. Calculations include molecular weight averages, molecular hydrodynamic diameter averages and column separation efficiency parameters. Significant reductions in analysis time have been realized along with improvements in accuracy of data acquisition, analysis and experimental reproducibility.     

Characterization of LC Stationary Phases by Inverse Size Exclusion Chromatography

Abstract

The most efficient use of a porous particulate stationary phase can only be made when the material in the column is fully characterized. While complete characterization includes both chemical and physical aspects, the macro structure provides the framework for the microscopic chemical interactions. The physical analysis is therefore critically important for a more complete calibration of the chromatographic effects.     

Comparing Dichromate Oxidation and Size Exclusion Chromatography of Dissolved Carbohydrates

Standardised methods are used to characterise the pulp or cellulosic material for its alkali resistance and alkali solubility. Today, pulp is usually characterised by its solubility in sodium hydroxide. The characterisation test was based on the treatment of pulp with sodium hydroxide solution of a defined concentration, according to a specified procedure. The dissolved organic matter was then determined by dichromate oxidation. The results obtained by size exclusion chromatography showed that both the amount and the molecular weight of the dissolved carbohydrates vary when investigating solubility and α-cellulose content of pulps. The results show further that S₁₈ values obtained by dichromate oxidation are overestimated when characterising pulps containing higher amounts of hemicellulose and/ or short chain cellulose, compared with results obtained by the SEC method. On the contrary, S₁₀ values are overestimated when determined by dichromate oxidation in higher alpha pulps.     

Size Exclusion Chromatography of Nonionic and Anionic Copolymers of Vinylpyrrolidone

Abstract

Size exclusion chromatography (SEC) of poly(vinylpyrrolidoneco-vinylacetate), PVPVA, and poly(vinylpyrrolidone-co-dimethylamincethylmethacrylate-co-vinylcaprolactan),PVPDMMAEMAVC, is evaluated in terms of resolution between polymer and solvent peaks using aqueous and nonaqueous mobile phases. A 1:1 (v/v) water/methanol, 0.1M LiNO₂ mobile phase is used with four Waters Ultrahydrogel columns with pore sizes of 120Å, 500Å, 1000Å, and 2000Å. DMF, 0.1M LiNO₃ mobile phase is used with three different two column sets. Two of the column sets are comprised of a mixed bed packing of poly(styrene-co-divinyl benzene), obtained from Shodex (KD80M), followed by either an Ultrahydrogel 120Å or a PLge1 100Å. The third two column set is a PLge1 10⁴Å followed by a PLgel 500Å. Any of the second columns in these sets improve separation between the trailing end of the polymer peak and the leading end of the solvent peak but the column set using an Ultrahydrogel 120Å yields a separation of these peaks whose valley is closer to the baseline. The aqueous mobile phase with the four column set yields a separation between the trailing end of the polymer peak and the leading end of the solvent peak whose valley is closest to the baseline. Recovery of PVPVA and PVPDMAEMAVC from the columns evaluated is 100%. Vinylpyrrolidone compositions of PVPVA ranging from 30 to 70 mole % were studied using both mobile phases. SEC of     

Size Exclusion Chromatography with Low Angle Laser Light Scattering Detection

Abstract

A review is given of the use of low angle laser light scattering (LALLS) detection in conjunction with size exclusion chromatography (SEC) to measure polymer molecular weight distributions without conventional SEC column calibration methods. A summary of light scattering theory is presented, and instrument configurations and principles of operation are described for two LALLS photometers. Also discussed are the overall performance of the SEC/LALLS technique and data from selected applications.     

High Performance Size Exclusion Chromatography Using Porous Glass Packing Materials

Abstract

Porous glass packing materials of average particle diameter 5 μm have been packed into a 7.2 mm i.d. x 25 cm column by viscousslurry packing parocedure. Average pore diameters of porous glasses were 170 Â, 500 Â, 1000 A, and 2000 A. The numbers of theoretical plates were between 7000 and 8000 per a column for porous glasses of pore diameters of 170, 500, and 1000 A, and 5000 for that of 2000 A. The retention volumes of narrow molecular weight-distribution polystyrene standards have been determined using tetrahydrofuran as mobile phase for the construction of calibration curves. Separations of polystyrene over molecular weight ranges of 1000 and 4,000,000 have been obtained by combining all four porous glass columns in series. Molecular weight averages of NBS 706 polystyrene have been measured and compared with the values determined with polystyrene gel columns. Both results were equivalent to the manufacturer's data. Porous glasses thus appear to be a useful packing materials for HPSEC.     

A New Packing for Aqueous Size Exclusion Chromatography Polyvinylpyrrolidone-Coated Silica

Abstract

Adsorption of polyvinylpyrrolidone on silica in aqueous medium is studied. Adsorption isotherms, thermogravimetry and elementary analysis show that a complete surface coverage is reached with large pore microbeads. The coating stability and its ability to prevent non-steric effects when used as packing in exclusion chromatography are demonstrated. Four different pore size packings were prepared for chromatographic evaluations. It is shown that water soluble polymers like polyethylene oxide, polyvinylalcohol, polyacrylamide and hydroxyethylcellulose can be chromatographied in pure water according to the steric exclusion mechanism.