Isolation of the stable fraction (the core) of the humic acid Fabrizio Adani a, * , Giuliana Ricca b , Fulvia Tambone a , Pierluigi Genevini a a Dipartimento di Fisiologia delle Piante Coltivate e Chimica Agraria, Universita` degli Studi di Milano, Via Celoria 2, 20133 Milano, Italy b Dipartimento di Chimica Organica e Industriale, Centro di Studio delle Sostanze Naturali del CNR, Universita` degli Studi di Milano, Via Venezian 21, 20133 Milano, Italy Received 19 January 2006; received in revised form 7 April 2006; accepted 9 April 2006 Available online 2 June 2006 Abstract Humic acid consists of a recalcitrant (unhydrolysed fraction) (the core) and labile (hydrolysable fraction) fraction. Core-humic acid (core-HA) isolation was performed by treating source material with apolar and polar solvents (organic solvents + acid hydrolysis) before alkaline extraction. Leonardite, soil Ah horizont and dry blood were chosen for this study because of their different origin and degree of humification. Chemical analysis (elemental analysis, total acidity, E 4 :E 6 ), spectroscopic analysis (DRIFT and 1 H NMR), and complete mass balance were used to investigate the effect of purifying humic acids. The results obtained showed that purification produced a slight modification of Leonardite humic acids as was expected for these highly humified organic matrices. On the other hand, about 500 g kg 1 of soil humic acids were lost by purification. The fractions lost mainly consisted of carbohydrates. Dry blood showed the presence of humic acids that contrasted with its origin, thus indicating the limitations of the common analytical methods used for HA extraction. Nevertheless, in practice, purification caused the complete dis- appearance (914 g kg 1 of HA was lost) of these HAs. The results obtained in this work suggest that the HA fraction isolated (named core-HA) effectively represents the HA structure pro- posed by the existing literature, since the purification proposed was able to eliminate the adsorbed organic molecules (interference mate- rials) coating the HA structure. Ó 2006 Elsevier Ltd. All rights reserved. Keywords: Core-humic acids; DRIFT; Humic acids; 1 H NMR; Purification 1. Introduction Humic substances (HS) are composed of chemically complex, non-biochemical organic components, which are largely hydrophilic, amorphous, dark colored, and resis- tant to chemical and biological degradation (Schnitzer, 1991). While previously thought of as being high molecular weight polymers, currently HS are thought to be com- pounds of relatively low molecular weight that form supra- molecular associations (Sutton and Sposito, 2005). This new view of the HA does not conflict with the previous def- inition of the chemical structures of HA (Schulten, 1995), but suggests that these structures do not represent repeat- ing units of high molecular weight polymers, but low molecular weight structures physically associated to form aggregates. The contemporary HA chemical structure model sug- gests the presence of a biologically relatively resistant, and highly substituted core of an aromatic nature consist- ing of heterocyclic structures (Schulten, 1995). Addition- ally, carbohydrates, fatty acids and amino acid are also present in HA (Rozenbaha et al., 2002). These molecules are not constituents of HA but are interference materials (IMs) co-extracted by the alkaline treatment (Genevini et al., 2002). Previously, multiple attempts have been made to elimi- nate the interference materials (IMs) from the humic acid 0045-6535/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.chemosphere.2006.04.032 * Corresponding author. Tel.: +39 0250 316546; fax: +39 0250 316521. E-mail address: fabrizio.adani@unimi.it (F. Adani). www.elsevier.com/locate/chemosphere Chemosphere 65 (2006) 1300–1307