Fiber formation from liquid-crystalline precursors. II. Cellulose in N,N-dimethylacetamide-lithium chloride

Fibers were spun from isotropic and biphasic solutions of regenerated cellulose (DP = 290) in N, N-dimethylacetamide +7.8% LiCl using water as a coagulant. There is an increase in mechanical properties through the isotropic → anisotropic transition with moduli reaching 22 GPa. The problem with this system is that crystallization encroaches on the biphasic region, and a pure mesophase is never observed. However, owing to the slow nucleation rate of the crystals, a biphasic solution is stable for a long time and can be spun to yield high modulus and orientation. The best approach is to use small flow gradients (extrusion rate and pick-up ratio) and to allow long times for homogenization and nucleation of the mesophase.     

Concentration effect in hyaluronan analysis by size exclusion chromatography

Summary The concentration effect in size-exclusion chromatographic analysis of hyaluronans of various relative molecular masses (RMM) has been studied. A critical concentration has been found that is negatively dependent on the hyaluronan molecular mass; the higher the biopolymer molecular mass, the smaller the injected sample where the concentration effect should be taken into account for accurate evaluation of molecular mass distribution. At higher temperatures, however, the concentration effect diminishes significantly.     

Analysis of sodium polycarboxylates in detergents by size exclusion chromatography

Summary This paper describes the quantitative analysis and preparative isolation of sodium polycarboxylates in detergents by means of gel permeation chromatography. An analytical monitoring method separates the polymers from other low molecular detergent ingredients within 10 minutes. There is no separation of the various molecular weight polycarboxylate macromolecules themselves. They elute from the column as a single narrow peak at the exclusion volume. A second preparative gel filtration method allows isolation of polycarboxylates in amounts necessary for further characterization. Appropriate sample pretreatments and possible interferences are discussed.     

Characterization of large water-soluble macromolecules by size exclusion chromatography

A method for characterizing very large, water-soluble polymers by size exclusion chromotography (SEC) has been developed. Sephacryl S1000 packing material, a precision syringe pump, and an eluent pressure detector have been utilized to produce highly accurate chromatograms of polymers having molecular hydrodynamic diameters up to 250 nm. Previous SEC analysis has been limited to polymers having hydrodynamic diameters of less than 120 nm.     

Determination of total polyvinylpyrrolidone (PVP) in ophthalmic solutions by size exclusion chromatography with ultraviolet-visible detection

A straightforward size exclusion chromatography (SEC) method was developed and validated for the determination of total polivinylpyrrolidone (PVP) in ophthalmic solutions using the unusual combination of size exclusion chromatography (SEC), ultraviolet-visible detection and quantitation of an analyte peak that elutes in the total exclusion volume of the column. Samples of opthalmic solutions are diluted with water and injected onto a TSKgel G1000PW, 7.5 mm i.d. × 30 cm, 12 μm column at 50°C, with 80:20 0.1M sodium acetate-methanol mobile phase and UV detection at 220 nm. Validation was successful for a stability indicating pharmaceutical method, with parameters including specificity, accuracy, linearity, and precision within typical pharmaceutical acceptance criteria. A stress study with acid, base, peroxide, heat, and light indicates that there is no interference from drug, product, or excipients.     

Error introduced into determination of pore size distribution by size exclusion chromatography

Simultaneous use of large standard molecules and small particles of the product examined gives rise to errors in pore size determination by size exclusion chromatography. This error is calculated for packings of spherical particles, thus making corrections possible.     

Stochastic theory of size exclusion chromatography by the characteristic function approach

A general stochastic theory of size exclusion chromatography (SEC) able to account for size dependence on both pore ingress and egress processes, moving zone dispersion and pore size distribution, was developed. The relationship between stochastic-chromatographic and batch equilibrium conditions are discussed and the fundamental role of the 'ergodic' hypothesis in establishing a link between them is emphasized. SEC models are solved by means of the characteristic function method and chromatographic parameters like plate height, peak skewness and excess are derived. The peak shapes are obtained by numerical inversion of the characteristic function under the most general conditions of the exploited models. Separate size effects on pore ingress and pore egress processes are investigated and their effects on both retention selectivity and efficiency are clearly shown. The peak splitting phenomenon and peak tailing due to incomplete sample sorption near to the exclusion limit is discussed. An SEC model for columns with two types of pores is discussed and several effects on retention selectivity and efficiency coming from pore size differences and their relative abundance are singled out. The relevance of moving zone dispersion on separation is investigated. The present approach proves to be general and able to account for more complex SEC conditions such as continuous pore size distributions and mixed retention mechanism.     

High-performance size exclusion chromatography of biological products

Summary High-performance size exclusion chromatography has been applied to the analysis and separation of a range of biological products — substances and preparations used in human medicine that cannot be completely defined by chemical and physical means. A variety of proteins and polysaccharides have been examined and results are described for a number of blood products, and polysaccharides of bacterial origin. This method is superior to conventional size esclusion for characterization of such substances and permits their isolation for further examination, without work-up, by biological and immunological techniques. The selection of appropriate column supports is considered. The limitations are considered of this procedure for studying the interaction between antithrombin III and heparin as the basis of a non-biological assay method.Presented at the 14th International Symposium on Chromatography London, September, 1982     

Study of the abnormal late co-elution phenomenon of low density polyethylene in size exclusion chromatography using high temperature size exclusion chromatography and high temperature asymmetrical flow field-flow fractionation

The elution behaviour of linear and branched polyethylene samples in SEC was studied. For the branched samples an abnormal late co-elution of large and small macromolecules manifests itself as an abnormal re-increase of the molar mass and the radius of gyration values detected with multi angle light scattering at high elution volumes in SEC. The late co-elution of small and large macromolecules cannot be explained by the SEC mechanism alone. The influence of several experimental parameters on the late co-elution was studied. It was found that the type of SEC column and the flow rate have a significant influence. The late eluting part of the sample was fractionated and separated by HT-SEC- and HT-AF4-MALS. The different results of both methods have been discussed with the aim to find possible explanations for the late elution. The experiments indicate that especially large branched structures show an increased tendency for the phenomenon.     

Pore structure characterization of organic-inorganic materials by inverse size exclusion chromatography

Summary In Inverse Size (or Steric) Exclusion Chromatography (ISEC) measurements, the investigated material is used as a stationary phase in a chromatographic column and the elution volumes of a series of standard solutes with different molecular size are measured. By an appropriate choice of mobile phase and type of standard solutes, specific (enthalpic) interactions between investigated material and solutes are eliminated so that the elution volumes depend on the porous structure of the column filling only. Then, a mathematical treatment of elution data can provide detailed information on both the macropores and the microporous structure of e.g. a swollen polymer gel in a polymer that is grafted onto silica. The basic principle of the evaluation of porosimetric information from chromatographic data is the assumption that the real porous structure of an investigated sample can be modelled as a collection of discrete pore fractions, each containing pores of different but uniform size and of simple geometrical shape. Problem then is to determine the combination of volumes of these fractions which yields the best agreement between computed and experimental values of the elution volumes of standard solutes. It is possible to perform the ISEC measurements either in an organic solvent or non-solvent (e.g. tetrahydrofuran or methanol) or in water, depending on the compatibility of the investigated material with the respective environment. For non-polar polymers, like copolymers of styrene and divinylbenzene, the use of tetrahydrofuran as the mobile phase with alkanes and polystyrenes as standard solutes has been recommended. Alternative ISEC investigation of the same material in an organic and an aqueous environment can provide additional information on its lipo- or hydrophilicity. This method has provided specific information, not obtainable by mercury porosimetry, when modifying silica by a coupling agent, polymerizing different monomers to different extents.     

Analysis of polyamides by size exclusion chromatography and laser light scattering

The molar mass analysis of polyamides is complicated due to the fact that only a limited range of solvents can be used and association and aggregation phenomena have to be screened by adding electrolytes to the mobile phase. Optimum SEC behaviour is obtained when hexafluoroisopropanol + 0.05 mol/L potassium trifluoroacetate is used as the mobile phase. The calibration of the SEC system can be conducted in different ways. While a calibration with narrow disperse polymethyl methacrylate standards does not yield accurate molar mass information, the quantification can be done using an “artificial” calibration curve. This calibration curve is obtained by correcting the PMMA calibration curve with polyamide molar mass data from light scattering. The resulting molar mass distributions for different types of polyamides are compared with molar masses that are determined by size exclusion chromatography with a light scattering detector and an excellent correlation is obtained.     

Direct determination of band broadening in size exclusion chromatography

A simple method to correct the measured extent of band broadening in size exclusion chromatography for the contribution of narrow (polydisperse) standards is presented. It is based on the assumptions that commercial polymer standards can be described by a Poisson distribution and the additivity of peak variances. Two sets of standards (polystyrene from two suppliers) were investigated under normal working conditions, i.e. a combination of four columns with different porosities and a flow rate of 1 ml/min. Furthermore, the polystyrene standards were used to determine the extent of band broadening for four additional combinations of columns (varying in their separation range and porosities) as a function of the elution volume. The assumption of a constant peak variance for band broadening turned out to be a (very) rough approximation for some combinations of columns, but all results taken together demonstrate that this assumption is not generally applicable. Qualitative agreement between theory and experiment was found with a rearranged van Deemter equation.     

Determining the molar mass of a plasma substitute succinylated gelatin by size exclusion chromatography-multi-angle laser light scattering,sedimentation equilibrium and conventional size exclusion chromatography

The clinical effectiveness of succinylated gelatin as a plasma substitute depends strongly on its molar mass, determined conventionally by size exclusion chromatography (SEC). This study evaluates different SEC calibration standards in comparison with two independent "absolute" methods for determining the weight average molar mass (M(w)) of a succinylated gelatin sample. SEC calibrated using succinylated gelatin fractions correlated well with size exclusion chromatography-multi-angle laser light scattering (SEC-MALLS) and sedimentation equilibrium whereas SEC calibrated with unmodified gelatin, sodium polystyrene sulfonates or pullulans overestimated M(w) by over 20%. Universal calibration was equivocal. The problems associated with the preparation of succinylated gelatin fractions suggest that an absolute method such as SEC-MALLS may be a more suitable choice for determining the M(w) in succinylated gelatins.     

High performance size exclusion chromatography of polyamic acid

Molecular weight studies of polyamic acids are complicated by polyelectrolyte effects. Although early work on the molecular weight determination of polyamic acids employed salts to suppress this polyelectrolyte effect, recent publications reveal that such salts can cause precipitation of polyamic acids from solution, however, a mobile phase consisting of 0.03M LiBr/0.03M H₃PO₄/1% vol THF in dimethyl formamide was reported to successfully suppress the noted polyelectrolyte effect without causing the precipitation of polyamic acid and give satisfactory size exclusion chromatograms using columns packed with “macro-porous” glasses. However, the development time was long (ca. 4 h) and considering the lability of the polyamic acids, the results must be viewed with skepticism. We find that use of a similar “mixed” mobile phase with Zorbax® PSM-Bimodal columns using size exclusion chromatography in the high performance mode (HPSEC) provides well resolved and reproducible chromatograms and molecular weight data with development times of < 15 min. Aside from the avoidance of long development times, which can lead to questionable results, this procedure permits the facile routine analysis of polyamic acid on a daily basis. The procedure has great utility as an analytical tool to support fundamental studies of polyamic acid chemistry and two examples of this application are given.     

Interlaced size exclusion liquid chromatography of monoclonal antibodies

Size exclusion chromatography is a widely performed analysis of monoclonal antibodies, primarily used to monitor the levels of higher weight molecular species such as aggregates. Owing to the subtleties of these separation mechanisms and frequently observed partial resolutions of components in these separations, many common methods for increasing the method throughput are not practical as they trade off resolution for speed. Short columns, high flow rates and smaller particles are examples of these approaches. In this paper a practical method is demonstrated for injecting samples onto the column in rapid succession and gating the detection window to monitor the elution of each sample individually. At any given instant approximately two samples are eluting through the column. By co-ordinating the injection and detection time windows the samples can be kept discrete and significant throughput enhancements achieved, up to nearly 2-fold improvements are demonstrated. A rudimentary theory is development to show that the throughput improvements can be predicted to approximation by simple column characteristics. Experimental results for a series of monoclonal antibodies demonstrate the equivalency of the method to a conventional injection approach, the throughput increase, and the robustness of the method.     

Plate-model applied on concentration effects in size exclusion chromatography

Abstract

A universal procedure of modeling chromatography separation, based on the plate model, has been developed. On each plate, the equilibrium between the analyte in the mobile and stationary phases is established. This equilibrium may be described by the concentration-dependent partition coefficient. The establishment of the equilibrium on each plate is followed by the displacement of the analyte in the mobile phase by one plate, establishment of new equilibrium, etc. The result is a series of elution curves with positions of maxima dependent on injected mass of the polymer. The procedure has been tested on modeling concentration effects on the basis of dependences of the partition coefficient on local concentrations on each plate. By comparison with the experiment, the slope of the concentration dependence of the partition coefficient of polystyrene in benzene was estimated. It is in qualitative agreement with the literature data obtained by a computer simulation of chromatography separation of polymers in good solvents.     

Study of polymer complexes by size exclusion chromatography coupled with light scattering in combination with fluorescence spectroscopy

Poly(methyl methacrylate) stereocomplexes prepared at different concentration in dilute tetrahydrofuran solutions were studied by size exclusion chromatography coupled with refractive and light scattering detectors in combination with fluorescence spectroscopy. A considerable increase in segment density due to complexation compared with free poly(methyl methacrylate) chain was only slightly affected by the polymer concentration in solution where stereocomplexes were formed. At polymer concentrations up to 3×10⁻³ g cm⁻³, an increase in non‐uniformity of polymer complex molecular weight and size and a shift to higher values of both were observed. In semidilute solutions (at c > 3×10⁻³ g cm⁻³) stereocomplexes virtually did not become heavier and larger.     

Investigation and interpretation of band broadening in size exclusion chromatography

Study of Band Broadening occurring in Size Exclusion Chromatography (SEC) is reported using very narrow PS standards obtained and characterised by Temperature Gradient Interaction Chromatography (TGIC). Chromatograms are fitted by Exponentially Modified Gaussian functions (EMG) and mapping of band broadening is obtained for different column sets. Interpretation of the skewing of the chromatograms is proposed with a new model using Brownian motion properties inside the pores. That explains why band broadening and tailing become so important near total exclusion volume.