Multiangle light scattering (MALS) is a well-established technique used to determine the size of macromolecules and particles. In this study, different extrapolation procedures used in MALS were investigated with regard to accuracy and robustness in the obtained molar mass and rms radius. Three different mathematical transformations of the light scattering function referred to as the Debye, Zimm, and Berry methods for constructing the Debye plot were investigated for two idealized polymer shapes, homogeneous spheres and random coils, with radii from 25 to 250 nm. The effect of the angular interval used for the extrapolation was investigated, as was the robustness of the different transformations toward errors in the measured light scattering intensity at low angles. For an rms radius less than 50 nm, the relative error in molar mass due to extrapolation was less than 1% independent of the method used. For larger radii, the error increased and the extrapolation procedure became more critical. For random coil polymers, the Berry method was superior in terms of accuracy and robustness. For spheres, the Debye method was superior. The Zimm method was inferior to the others. The different extrapolation methods were evaluated and compared on experimental data from a size exclusion chromatography-MALS analysis of an ultrahigh molar mass poly(ethylene oxide) (PEO). The PEO data qualitatively verified the calculations and stressed the importance of optimizing the extrapolation procedure after careful evaluation of the experimental data. A discussion of how to detect erroneous data in an experimental Debye plot is given.

Enzymatic hydrolysis of carboxymethyl cellulose (CMC) has been studied with purified endoglucanases Hi Cel5A (EG II), Hi Cel7B (EG I), and Hi Cel45A (EG V) from Humicola insolens, and Tr Cel7B (EG I), Tr Cel12A (EG III), and Tr Cel45Acore (EG V) from Trichoderma reesei. The CMC, with a degree of substitution (DS) of 0.7, was hydrolyzed with a single enzyme until no further hydrolysis was observed. The hydrolysates were analyzed for production of substituted and non-substituted oligosaccharides with size exclusion chromatography (SEC) and with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF-MS). Production of reducing ends and of nonsubstituted oligosaccharides was determined as well. The two most effective endoglucanases for CMC hydrolysis were Hi Cel5A and Tr Cel7B. These enzymes degraded CMC to lower molar mass fragments compared with the other endoglucanases. The products had the highest DS determined by MALDI-TOF-MS. Thus, Hi Cel5A and Tr Cel7B were less inhibited by the substituents than the other endoglucanases. The endoglucanase with clearly the lowest activity on CMC was Tr Cel45Acore. It produced less than half of the amount of reducing ends compared to Tr Cel7B; furthermore, the products had significantly lower DS. By MALDI-TOF-MS, oligosaccharides with different degree of polymerization (DP) and with different number of substituents could be separated and identified. The average oligosaccharide DS as function of DP could be measured for each enzyme after hydrolysis. The combination of techniques for analysis of product formation gave information on average length of unsubstituted blocks of CMC.

No abstract

Flow field-flow fractionation (FlFFF) was used to characterize the aggregation of a charged, amphiphilic copolymer, MMA 550, prepared from poly(styrene-co-methyl methacrylate-co-maleic anhydride) and poly(ethylene oxide) monomethyl ether. MMA 550 has a charged hydrophobic backbone and hydrophilic grafts which give the polymer a complex behavior in aqueous solution. FlFFF turned out to be a powerful tool for separation and size determination of different polymer aggregate populations in various media. The hydrodynamic diameter of the polymer was obtained from FlFFF measurements in water and pH-adjusted solutions. In pure water, the majority of the polymer molecules had a hydrodynamic diameter of 3-4 nm, which was also found at higher pH values (pH 9). When the pH was lowered, the hydrodynamic diameter increased rapidly. This suggested the formation of large micelles, probably polymolecular, as an effect of the reduced charge repulsion.