2.3. Chemical analyses

The separated cork particles were ground in a Retsch SK knife mill, sieved, and the 40–60 mesh fractions were kept for analysis. Chemical summative analyses included determination of ash, soluble extractives in dichloromethane, ethanol and water, suberin, klason and acid soluble lignin, and the monomeric composition of polysaccharides, including determination of neutral sugars, uronic acids and acetates.

The ash content was determined after incineration at 525 °C [17]. The soluble extractible compounds were determined in a Soxhlet apparatus with a solvent sequence of dichloromethane, ethanol and water during 6 h, 16 h and 16 h (adapted from Ref. [18].

Suberin content was determined in the extractive-free material by use of methanolysis with sodium methoxide in absolute methanol [19]. The suberin extracts after pH adjustment to 6 were concentrated, suspended in water and extracted with dichloromethane in successive liquid-liquid extractions. The dichloromethane extracts were kept for determination of the long chain monomeric composition of suberin, and the water phase for quantification of glycerol. The suberin content of cork was determined as the solid mass loss after methanolysis and expressed in percent of the initial dry mass.

Klason and acid-soluble lignin, and carbohydrates contents were determined on the extracted and desuberinised materials by acid hydrolysis with 72 % sulphuric acid [20,21].

The polysaccharides content was determined by quantifying the total monosaccharide monomers released by the acid hydrolysis used for lignin determination using a Dionex ICS-3000 system in HPIC-PAD and an Aminotrap plus Carbopac PA10 column (250 × 4 mm) for monosaccharides and uronic acids, and by HIPCE-UV using a Waters 600 system with a Biorad Aminex 87H column (300 × 7.8 mm) for acetic acid. In the conditions used, mannose was eluted with xylose.