Gisela Berth

The degree of acetylation of chitosans and its effect on the chain conformation in aqueous solution

The average degree of acetylation (DA) of chitosans from several sources as well as the distribution of the DA across the molecular weight distribution was measured by titration with poly(styrene sulphonate) as counter polyanion and toluidine blue as metachromatic indicator. The method was shown to be useful within a broad range of DAs. There was no compositional heterogeneity for more than 90% of the population in any sample. Parallel macromolecular characterisation by static light scattering and viscometry standing alone or connected with gel permeation chromatography (GPC) revealed no measurable effect of the DA on the chain conformation where the DA varied between a few percent and ∼60%. Relationships between intrinsic viscosity and radius of gyration with molecular mass were established. The data when considered with previous work suggest no evident differences between chitosans from different sources and no significant effect of the ionic strength between ∼0.1 and 0.3 M on the conformation.

Physico-chemical characterization of chitosans varying in degree of acetylation

Abstract Three commercial chitosans varying in average degree of acetylation (DA) were characterized in terms of average molar massesMn,Mw, the second virial coefficientB and radius of gyrationRgz, and intrinsic viscosities by membrane osmometry, static light scattering, and capillary viscometry in acetate buffer of pH 4.5. To obtain satisfactory light scattering data, the method used for clarification was of great importance. Combined ultracentrifugation and membrane filtration was found to be the method of choice. Using the model for wormlike chains with excluded volume and a logarithmic molar mass distribution, the persistence lengthLp was determined asLp = 6 nm. No concentration-dependent association was observed for the low concentrations studied. Samples were also fractionated on Sepharose CL-2B for subsequent light scattering and viscosity measurements to establish relationships betweenMwand [η] andRg, respectively. These relationships were found to be independent of the DA for the bulk of molecularly dispersed chitosan. Model calculations aimed at checking the consistency of data led, as to be expected, to an increasing excluded volume effect with increasing molar mass and steadily increasing polydispersities of the fractions with increasing elution volume. The levelling off of the [η]-Mw andRg-Mg relations for fractions of increasingly high molar mass was assigned to chemical heterogeneity of samples in terms of DA distributions.

The molecular weight distribution and conformation of citrus pectins in solution studied by hydrodynamics

Abstract The molecular weight distribution of a citrus pectin has been analysed by a combined approach using gel-permeation chromatography with low-speed sedimentation equilibrium. (1) A pectin preparation from citrus fruit was fractionated on Sepharose CL-2B/Sepharose CL-4B. (2) Weight average molecular weights of the fractions were determined by low speed sedimentation equilibrium in multichannel cells. (3) An absolute calibration for the column for this material was thereby defined. (4) The (lognormal) molecular weight distribution thus obtained is consistent with a weight average of (90000±10000) g/mol, obtained separately on unfractionated material, and consistent with a distribution obtained on the same material but using light scattering as the molecular weight probe. The conformation of the pectin fractions in solution was studied in terms of: (1) the Wales-Van Holde parameter, k s [η] ; (2) Mark-Houwink-Kuhn-Sakurada plots of sedimentation coefficient and intrinsic viscosity data versus molecular weight; (3) rod models and (4) worm-like-coil models. The sedimentation data is consistent with a rod model (or a worm-like-coil with a large persistence length) with mass per unit length ∼ 430 g mol−1 nm−1. The intrinsic viscosity data is also consistent with a rod model but shows some anomalous features which may be suggestive of worm-like-coil behaviour at higher molecular weight, although it is not possible to fit this data with a realistic value of the mass per unit length.

Studies on the heterogeneity of citrus pectin by gel permeation chromatography on Sepharose 2 B/Sepharose 4 B

Abstract Commercial high methoxyl citrus pectin was purified by ultracentrifugation or ion exchange chromatography prior to GPC on Sepharose 2 B/Sepharose 4 B. The molecular weights by light scattering and the intrinsic viscosities of the fractions were determined. The effects of the purification procedures on the heterogeneity in shape are discussed by combining the Mark-Houwink plots with the universal calibration plot and the elution profile, respectively. Pectin fractions prepared by GPC are investigated in the same manner. GPC cannot differentiate between lower molecular weight extended coils and more compact high molecular weight molecules of increased polymer density. They are eluted together within a broad elution volume range.

The determination of the molecular weight distribution of pectins by calibrated GPC Part I. Calibration by light scattering and membrane osmometry

Abstract Commercial high methoxyl citrus pectin was fractionated on Sepharose 2 B/Sepharose 4 B. Fractions were used to calibrate GPC by light scattering and membrane osmometry. The angular dependence of light scattering of the fractions was interpreted in terms of a two-component system giving reliable M w / M n ratios for the major molecularly dispersed component which forms coils of considerable stiffness in solution. A minor particulate spherical component was identified which was rich in neutral sugars. Structural parameters of this component were determined by comparison of the experimental scattering behavior with master curves. β-Elimination of the fully esterified pectin was carried out to reveal the chemical nature of the particulate component.

Methodical aspects of characterization of alginate and pectate by light scattering and viscometry coupled with GPC

Abstract The reliability of light scattering depends on the complete removal of extraneous matter from the polymer solution. The use of membrane filters to clarify aqueous solutions is common practice. This paper studies the effect of the pore size of the membrane filter on the light scattering of several polyuronides. Alginate and pectates were fractionated by GPC on Sepharose gels, and the eluent was taken after filtration for light scattering and viscosity measurements. Light scattering data were represented as GUINIER plots to provide information about the scattering functions and scattering level. While for alginate satisfactory separation of molecularly dispersed polymer from extraneous material was achievable, heterogeneity of pectates did not allow an unequivocal differentiation of extraneous matter from ‘proper’ pectate. Dextran sulphate was included as a model substance which was free of extraneous material.

Physicochemical and chemical characterisation of chitosan in dilute aqueous solution

Static and dynamic light scattering as well as analytical ultracentrifugation and viscosity measurements were used to investigate the chain conformation of chitosans in salt-containing solutions (pH 4.5; ionic strength about 0.12 M). The samples of various degrees of acetylation were chemically homogeneous. The molecular-weight dependence of the radius of gyration has given clear evidence for a relatively flexible wormlike chain with a persistence length of about 6 nm irrespective of the degree of acetylation, where excluded-volume effects and the polydispersity of the samples were taken into account. In contrast, the interpretation of the hydrodynamic data via a “whole-body approach” according to the Wales-van Holde ratio suggested a strongly elongated chain conformation. The failure of the latter to properly reflect the chain conformation was ascribed to the high extent of draining. A nearly free-draining case can also account for the high scaling exponent of the relationship between intrinsic viscosity and molecular mass.

Static light scattering technique applied to pectin in dilute solution. Part III: The tendency for association

The tendency for association was studied by light scattering applied to a series of a high-methoxyl citrus pectin solution in phosphate buffer. To prevent artefacts, the clarified stock solution and buffer were mixed within the measuring cell. The measured data are presented as Zimm and master plots. A reversible increase of the apparent molecular weight with rising polymer concentration and slightly negative second virial coefficients were found in the case of the uncentrifuged, but filtered, solutions, indicating a slight tendency for association. The associates formed remain relatively small. This is independent of the pore size of the filters used for clarification. Prepurification by ultracentrifugation gave solutions which showed two-component behaviour and gave no hint of association.

Static light scattering technique applied to pectin in dilute solution. 2. The effects of clarification

Abstract The effects of purification/clarification by ultracentrifugation and/or membrane filtration were studied by light scattering, GPC plus off-line capillary viscometry and GLC sugar analysis using a high-methoxyl citrus pectin as model substance. The emphasis of light scattering measurements was put on analysing the angular dependence of the scattered light. For that reason the interpretation of the measured data involved the use of master curves in addition to Zimm or Guinier plots. The benefits and limitations of this method of data treatment for complex systems are discussed both in theory and practice. Due to the natural heterogeneity of pectins, purification or classification modified the original sample by removing the high molecular weight and dense tail, leaving a physically and chemically heterogeneous polysaccharide.

Static light scattering technique applied to pectin in dilute solution. Part I: Reliability of experiments and model calculations

Abstract During static light scattering studies of a commercial high-methoxyl citrus pectin in phosphate buffer, problems arising from sample clarification have been identified. Therefore, the effect of varying the pore size of the membrane filters between 5·0 and 0·2 μm was studied. Also, the effect of repeated filtration was investigated. Data are presented as Zimm and Guinier plots. The lower the pore size of the filter, the lower the average molecular weight, but the higher the negative second virial coefficients found. Results suggest that the negative virial coefficients are due to residual particulate matter. Model calculations suggest that a bimodal system consisting of a molecularly dispersed major component and smaller amounts of a high molecular weight particulate component is an appropriate description of the pectin sample. Dextran sulphate was used to demonstrate the behaviour of a ‘homogeneous’ non-aggregating polysaccharide.