Lignins calibration standards

Recently, efforts have been made to produce calibration standards of higher molecular weight that are chemically similar to lignins, by step-wise syntheses (12), anion-initiated polymerization of quinonemethides (13), and preparative HPSEC of acetylated lignins (14). Knowledge of the molecular weights of these materials is either built into the method of preparation or determined by absolute methods such as sedimentation equilibrium measurements. 

The effects caused by the electrolyte nature of lignin sulfonates are eliminated by using a 0.5M sodium chloride solution as eluent. This eluent is made 0.1M with respect to Tris-HCl and buffered to pH 8 with hydrochloric acid in order to dissolve the proteins used as calibration standards (Fig. 5). 

Figure 6 shows that the proteins used for calibration elute in the same way as lignin sulfonates, which justifies the use of proteins as calibration standards. A comparison between Figures 4 and 6 shows that elution with an electrolyte solution fractionates lignin sulfonates in the range 3 000-80 000 dalton, but that elution with water fractionates those in the range 20 000-100 000 dalton. 

Because of the very high molar masses of enzymically polymerized lignin sulfonates, Sephacryl S-300 is used as the gel matrix. The fractionation range is 10 000 to 1 000 000 dalton. Even in this case proteins of known molar mass can be used as calibration standards (Figs. 7 and 8). 

Values for system peak parameters were found using a narrow distribution polystyrene standard (PS68K) before calculating MWD data for the lignin samples from universal calibration. To check software and instrument operation, several narrow MWD polystyrene and one broad MWD polymethylmethacrylate standards were treated as unknown samples and subjected to analysis with the universal calibration curve assembled from all polymer standards files. It was found that the MWD could be estimated for the recalculated polymer standards with errors between 5 and 10% of the original value indicated by the supplier of the standard (e.g., Mw for PS11K and Mw and M for PMMA17K-6). 

Table I illustrates the molecular weight averages found from universal calibration (narrow standards) for four aspen lignins and two quinonemethide-derived polymers using the Unical software. The poly-dispersities were the same within the experimental errors for all lignin samples. In contrast, the polydispersities found for the quinonemethide-derived polymers by universal calibration were near 1.1. The molecular weight averages found for the four acetylated lignins studied by universal calibration were substantially larger than those determined from previous work using conventional GPC (e.g., AESE and BM lignins from refs. 7 and 12 had approximately one-third those values found by HPSEC-DV in the present study).

Table I. MWD of Acetylated Aspen Lignins and Model Compounds from Universal Calibration with Narrow Standards 1...

As a result of the dependence of universal calibration on column elution behavior (i.e., anomalous behavior due to adsorption or exclusion), the contribution of the polymer core and shell components (33,34) to hydrodynamic behavior must be fully understood if competent analysis of block copolymers and branched heteropolymers is to be made. It is hoped that with the advent of appropriate MW, composition, and branched polymer standards, the limits of fit of universal calibration to biopolymers such as lignin can be judged. 

Apart from one compound (II), the lignin model compounds that had free phenolic groups eluted at close to the retention times predicted by the calibration curve from the polymer standards and not from the derive tized model compounds. This could simply be a result of the underivatized models having a similar variation in hydrodynamic volume with molecular weight as the polymer standards. However, it is to be expected that solvation of the underivatized model compounds should occur with THF as solvent (10), with hydrogen bonding of one THF molecule to each under-... 

Of the methods for determining lignin in solution based on a specific chemical reaction, that involving nitrosation, the so-called Pearl-Benson method, has found the widest application. In this procedure, reaction of the phenolic units in lignin with acidified sodium nitrite leads to the formation of a nitrosophenol which, upon addition of alkali, is tautomerized to an intensely colored quinone mono-oxime. The absorbance of the latter structure is measured at 430 nm and related to lignin concentration by calibration with a standard lignin. The procedure described below is essentially that developed by Barnes et al. (1963), who modified the original Pearl-Benson method (Pearl and Benson 1940) to improve its sensitivity. 

Fig. 8.1.5. Calibration of styragel columns with polystyrene standards ( ) and with lignin samples (o). The molecular weights of the latter were determined by vapor phase osmometry. (Mansson 1981)...

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