Gel Permeation Chromatographic Study of the Molecular Weight Distribution of Tannins in the Wood, Bark and Leaves of Eucalyptus spp. E. Cadahfa / E. Conde / M. C. Garcfa-Vallejo* / B. Fernfindez de Sim6n Area de Industrias Forestates, CIFOR-INIA, Apdo. 8111, 28080 Madrid, Spain Key Words Gel permeation chromatography Tannins in eucalyptus bark Polyphenols Molecular weight distribution Summary The molecular weight distribution of tannins from the Wood, bark and leaves of Eucalyptus camatdutensis, E. globulus and E. rudis from two different Spanish provenances has been studied by high performance gel permeation chromatography, using the compounds' acetylated derivatives. The MW distribution profiles showed important variability depending on the type of vegetal tissue, the species and, in some cases, on the geographical provenance of the samples. Bark was the vegetal tissue that yielded tannins with the highest molecular weight, followed by wood and leaves. Tan- nins from wood and bark of E. camaldulensis were of higher molecular size than those from E. globulus and E. rudis; those in the leaves of E. globutus and E. camat- dulensis were similar in molecular size and larger than those in the leaves of E. rudis. Introduction Vegetal tannins are singular among secondary metabo- lites because of their phenolic nature and their wide variety of molecular sizes. Both characteristics in- fluence their ability to complex with other plant meta- bolites, such as proteins [1, 21 and carbohydrates [1, 3]. The ability of tannins to complex with proteins has im- portant significance, not only in relation to vegetal physiology, but also in animal nutrition and in the in- dustrial application of the compounds. Our previous work [4] has demonstrated the abundance of tannins (proanthocyanidins and ellagitannins) in the Wood, bark and leaves of some species of Eucalyptus, and the high qualitative and quantitative variability of these compounds, depending on the vegetal tissue, the species and the tree provenance. The aim of this work was to study the molecular weight (MW) distributions of Eucalyptus tannins by gel per- meation chromatography (GPC) and the investigation of their variation with the vegetal tissue, the species and the sample provenance. We have studied the species E. gIobuIus, E. camaldulensis and E. rudis, which have dif- ferent suitabilities as raw material for the fabrication of cellulose pulp and paper. E. globulus and E. carnal- dulensis are grown extensively in Spain; E. rudis is grown only in experimental arboreta. Among the different size exclusion chromatographic techniques, GPC is the simplest and, in general, the most widely used in the analysis of tannins. In normal phase GPC, performed with non-polar polymers and or- ganic solvents, the chromatographic separation depends exclusively on the molecular size of the polymers, with no influence from other phenomena such as adsorption, partition or ion exchange. It is, therefore, a fast, simple and appropriate method for the study of the MW distri- butions of polymeric molecules [5-7]. The presence of hydroxyl groups in the tannin molecu- les can promote interactions by adsorption on the gel or intermolecular linkage by hydrogen bonding, which would lead to incorrect estimation of the MW. These phenomena can be minimized by the use of very polar mobile phases, such as dimethylsulphoxide (DMSO), dimethylformamide (DMF) [8, 9], or tetrahydrofuran (THF) [10]. Another procedure is to protect the hydro- xyl groups as acetyl derivatives [6, 7, 11, 12], methyl derivatives [13, 14] or silyl derivatives [15]. The simultaneous use of an eluent such as THF and the acetyl derivatives provides better results for the GPC study of the MW distributions of tannins and other biopolymers, e.g. lignins [6, 7, I6, 17]. Experimental Standards THF solutions (1-2 mg mL -1) of polystyrene standards (Polymer Laboratories) with molecular weights in the Original Chromatographia Vot. 42, No. 1/2, January 1996 95 0009-5893/96/01 095-06 $ 03.00/0 9 1996 Friedr. Vieweg & Sohn Verlagsgesellschaft mbH