A Novel Method for the Determination of Carbonyl Groups in Cellulosics by Fluorescence Labeling. 2. Validation and Applications J ¨ urgen R ¨ ohrling, † Antje Potthast,* ,† Thomas Rosenau, † Thomas Lange, † Andrea Borgards, ‡ Herbert Sixta, ‡ and Paul Kosma* ,† Christian-Doppler-Laboratory, University of Agricultural Sciences Vienna, Muthgasse 18, A-1190 Vienna, Austria, and Lenzing AG, R & D, A-4860 Lenzing, Austria Received March 18, 2002; Revised Manuscript Received May 27, 2002 Fluorescence labeling with the marker carbazole-9-carboxylic acid [2-(2-aminooxyethoxy)ethoxy]amide was shown to be a promising approach toward the accurate determination of carbonyls in cellulosic materials. Combined with gel permeation chromatography in DMAc/LiCl with fluorescence/multiple-angle laser light scattering/refractive index detection, the method yields carbonyl profiles relative to the molecular weight of the cellulosic material. The derivatization procedure can be carried out either homogeneously in DMAc/ LiCl or advantageously as heterogeneous derivatization in aqueous buffer. The heterogeneous carbonyl group determination, offering shorter reaction times and increased simplicity as compared to the homogeneous approach, was comprehensively validated. The carbonyl content in numerous dissolving pulps of different provenience has been determined, including pulps with carbonyl contents additionally increased by oxidative treatment. The method was also applied to follow bleaching sequences and oxidative treatments of pulps. Introduction Responding to a lack of reliable and accurate methods for the determination of carbonyl groups, such as keto, 1,2- diketo, and aldehyde structures, in pulps and other cellulosics, a novel approach was developed, based on fluorescence labeling of carbonyls with the marker carbazole-9-carboxylic acid [2-(2-aminooxyethoxy)ethoxy]amide (“CCOA”). It was considered imperative that the procedure can be incorporated into gel permeation chromatography (GPC) systems, so that the labeling procedure would provide carbonyl profiles of the respective cellulosics relative to their molecular weight, when combined with multiangle laser light scattering (MALLS), refractive index (RI), and fluorescence detection. Furthermore, it was demonstrated that the labeling does not lead to any cellulose degradation under the prevailing conditions. 1 Two versions of the labeling method were elaborated, a homogeneous procedure in DMAc/LiCl (2.5%, w/v) and a heterogeneous derivatization in aqueous buffer solution. Both variants have been thoroughly optimized with regard to reaction conditions (temperature, reagent ratios, catalysts, stability of reagent, and labeled products), completeness of conversion, and reproducibility. The homogeneous procedure requires prolonged reaction times, which can, however, be reduced by recording a reaction kinetics and subsequently extrapolating toward complete conversion. In addition, for each sample the laborious and time-consuming cycle of precipitation, washing, and redissolution of the pulp is required to remove excess marker. The heterogeneous labeling is advantageous for various reasons: it is completed at much shorter derivatization times, recording kinetics is unnecessary, the precipitation-redissolution procedure be- comes obsolete, and the results are as consistent as those obtained according to the homogeneous working procedure. The heterogeneous method (and only this one) thus clearly has the potential to become a routine method in pulp and cellulose chemistry. To place the labeling approach at the cellulose chemists’ disposal as a general analytical method, several tasks, must be performed beforehand, which will be delineated in this second part of our studies: validation of the heterogeneous labeling procedure; selection of reference pulps; testing of the general applicability of the method by determining the carbonyl content of multiple pulps; application of the method in examples to demonstrate its use in cellulose and pulping chemistry. Materials and Methods Chemicals, general analytics, GPC, and high-performance liquid chromatography (HPLC) systems components were used as described previously. 1 The GPC system, a modifica- tion of the setup described by Schelosky et al., 2 consisted of fluorescence, MALLS, and refractive index (RI) detectors, with automatic injection and four serial columns. Molecular weight distribution (MWD) and related polymer-relevant parameters were calculated by software programs, based on a refractive index increment of 0.140 mL/g for cellulose in DMAc/LiCl (0.9%, w/v). The following general GPC parameters were used: eluant, DMAc/LiCl (0.9%, w/v); flow, 1.00 mL/min; columns, four, PL gel mixedA ALS, 20 † University of Agricultural Sciences Vienna. ‡ Lenzing AG. 969 Biomacromolecules 2002, 3, 969-975 10.1021/bm020030p CCC: $22.00 © 2002 American Chemical Society Published on Web 07/17/2002