Heiko Thielking

Correlation between immunological activity, molar mass, and molecular structure of different (1→3)-β-d-glucans

(1-->3)-beta-D-Glucans are said to be potential biomedical drugs against bacterial or viral infections and also show antitumour activity. These substances seem to enhance the activity of the immune system, but today there is no accepted mechanism, and not even any agreement on the parameters which influence the activity. Therefore, glucans with different structures and/or varying molar mass were characterized by multi-angle laser-light scattering coupled with size-exclusion chromatography in order to obtain the molar mass distribution and to gain an idea of the structure in solution. After ensuring that all samples were free of pyrogens, the Tumour-necrosis-factor-alpha release activity and the superoxide-anion released from human blood monocytes were measured in groups of samples with comparable polydispersity and degree of substitution. All glucans investigated, regardless of molar mass and solution structure, stimulate the investigated immunological measures more than a commercially available biomedical drug used for comparison. The greatest magnitude of molar mass was found to be about 550,000 g/mol for all the glucans investigated. Contrary to the cited literature, helical structures were not essential, and not even advantageous, for immunological activity.

Characterization of aqueous car☐ymethylcellulose solutions in terms of their molecular structure and its influence on rheological behaviour

A set of car☐ymethylcellulose (CMC) samples was varied in degree of substitution (DS) from 0.71 to 2.95, with the partial degree of substitution being raised uniformly at positions C2, C3 and C6, so that a homologous series of CMC was made available. A further set of samples varied in molar mass from 200 000 to 2 000 000 g mol−1 at a constant DS of ∼ 1. The chemical structural parameters were determined by n.m.r. spectroscopy, showing that quantification of the degree of substitution at position C6 may be distorted by impurities of glycolic acid. The mean molar masses, for establishing the [η]-M relationship, were determined by multiangle laser light scattering preceded by size exclusion chromatography. The influence of concentration on zero-shear viscosity was given by η0 ∝ c4.3, whereas the influence of the molar mass was determined by η0 ∝ M3.9. An increasing DS within a homologous series influences the viscosity in different solvents (H2O 0.01 M NaCl, 0.1 M NaCl) at low polyelectrolytic concentrations. Surprisingly, the viscosity for all the CMC samples can be predicted by the single equation: η0[Pas]=8.91×10−4+1.30×10−5cMW0.9+5.33×10−8c2MW1.8+4.60×10−15c4.34MW3.91 at T = 298 K in 0.01 M NaCl. Increasing the DS up to a value of ∼ 1 improves the solubility characteristics (increases the viscosity), whereas above DS ≈ 1 this effect is overlapped by degradation of the molar mass. It was also possible to quantitatively determine the viscoelasticity as a function of frequency and molecular parameters.

On-Line Coupling of Flow Field-Flow Fractionation and Multiangle Laser Light Scattering for the Characterization of Macromolecules in Aqueous Solution As Illustrated by Sulfonated Polystyrene Samples

Seven sulfonated polystyrene standards (18 000-3 000 000 g/mol), taken as model substances for macromolecular polyelectrolytes, were dissolved in aqueous 0.1 M sodium nitrate solution and characterized by multiangle laser light scattering coupled on-line to flow field-flow fractionation. The distributions of molar mass and root mean square radius and the diffusion coefficients were obtained for each sample using a constant field of force for separation. Relationships between molar mass and root mean square radius [〈R(G)(2)〉(z)(0.5) = (2.71 × 10(-)(2))M(w)(0.56)] or diffusion coefficient [D = (7.10 × 10(-)(8))M(w)(-)(0.68)] were calculated. To investigate the static analytical range of this novel hyphenated technique a mixture of all seven samples was fractionated applying a programmed field. The relationship obtained between root mean square radius and molar mass was used to calculate a Mark-Houwink equation [[η]calcd = (2.99 × 10(-)(2))M(w)(0.68)]. To verify this result, the intrinsic viscosities for all samples were measured at low shear rate and found to be in good agreement [[η]calcd = (2.77 × 10(-)(2))M(w)(0.67)].

Characterization of Acetyl Starch by Means of NMR Spectroscopy and SEC/MALLS in Comparison with Hydroxyethyl Starch

The properties of starch derivatives which may be used as plasma substitutes, are dependent upon the molecular structure. Seven acetyl starch (AS) samples were determined and compared with results from hydroxyethyl starch (HES) samples. The molar masses and their distributions were determined with the combination of size exclusion chromatography and light scattering. Slightly asymmetric distributions were determined with a polydispersity Mw/Mn = 2.4 and weight-average molar masses of Mw = 250,000–300,000 g/mol for six AS samples and Mw/Mn = 3.6 and a weight-average molar mass of 766,000 g/mol for one AS sample. The average degrees of substitution (DS) and the substitution pattern were determined by high resolution NMR spectroscopy. The AS samples investigated had a DS of 0.42 to 0.81, comparable to HES, but the regioselective substitution pattern revealed differences. While for HES the position C-2 is preferred and the position C-3 has nearly no substituent, for AS both positions, C-2 and C-3, are substituted likewise. Degradability by α-amylase was tested in the laboratory for AS as well as for HES having nearly the same degree of substitution and molar mass, but C-2/C-6 = 2 for AS and C-2/C-6 = 1.4 for HES. An exponential decrease in the molar mass was observed over time, down to a limiting molar mass Mw = 50,000 g/mol for AS and Mw = 30,000 g/mol for HES, the degradation of AS occurred more slowly.

Development and evaluation of methods for determining the pattern of functionalization in sodium carboxymethylcelluloses

SUMMARY: Sodium carboxymethylcellulose (NaCMC) with varying degrees of substitution (DS) was investigated with different analytical methods in order to characterize the functional group distribution. The following methods were tested and adapted: high-resolution 13 C NMR spectroscopy in the solid state ( 13 C CP/ MAS NMR) and 13 C NMR spectroscopy on solutions of NaCMC samples with a reduced molar mass. Partial degradation was accomplished by ultrasonic means and with the enzyme endoglucanase. Combining the two techniques resulted in the greatest reduction in molar mass and hence in the best spectral resolution. Analysis of the NaCMC fragments following ultrasonic and/or endoglucanase degradation also reveals another interesting experimental finding. It appears that ultrasonic degradation is favored at unsubstituted areas near the center of the chain. These methods were compared with the following already familiar techniques of analysis: titrimetric techniques; 13 C and 1 H NMR spectroscopy as well as HPLC on completely hydrolysed solutions (hydrolysis with perchloric acid, trifluoroacetic acid and sulfuric acid). All of the methods characterize the samples as a series with increasing DS, the values of which range from 0.9 to 2.4. Methods that permit analysis of the partial degree of substitution produced the distribution x 2 A x6 A x3. Therefore, they are in principle suited for determining the functionalization pattern of the NaCMC samples relative to one another. The most suitable method can therefore be selected according to the objectives and the apparatus available. However, the measured values do exhibit considerable spread, variances of approx. 20%, thus, place restrictions on using the values of DS or xi in absolute comparisons beyond these methods.

Determination of the Molar Mass and the Radius of Gyration, Together with their Distributions for Methylhydroxyethylcelluloses

Three molar mass series were produced by different methods of degradation (namely ultrasonic (seven samples), oxidation (seven samples) and autoclaving (eight samples)) from a methylhydroxyethylcellulose (MHEC) sample with an average degree of substitution (DS) of 1.3, a molar degree of substitution (MS) of 0.46, a radius of gyration of 67 nm and a weight-average molar mass, Mw, of 318,000 g/mol. The degraded samples were characterized in terms of their molar mass and particle size together with their respective distributions with a hyphenated apparatus consisting of size exclusion chromatography and multi-angle laser light scattering and concentration detector (SEC/MALLS/DRI) at 25 °C in 0.1 M NaNO3 solution (with 200 ppm NaN3 as antibactericide). The refractive index increment was determined as dn/dc = 0.135 cm3/g. It was possible to reduce the weight-average molar mass down to approximately 10% of the initial molar mass using all the methods. In a comparison of the three degradation methods it was shown that only ultrasonic degradation retains the monomodal distribution, whereas the other two degradation methods yield a bimodal molar mass distribution. Consequently, only ultrasonic degradation represents a suitable method for producing homologous molar mass series. An RG–M relationship of RG = 0.0511 × M0.56 was established for the sample used in this case, and from this it was possible to calculate an [η]–M relationship of [η] = 0.3587 × M0.68.

Hollow Beads of Sulfoethyl Cellulose (SEC) on the Basis of Polyelectrolyte Complexes

This article describes the preparation of hollow beads with good mechanical stability on the basis of polyelectrolyte-polyelectrolyte complexes of SEC as polyanion for the encapsulation of biocatalysts and other materials under moderate conditions.