Available at www.sciencedirect.com journal homepage: www.elsevier.com/locate/watres Differences in the molecular composition of fulvic acid size fractions detected by size-exclusion chromatography–on line Fourier transform ion cyclotron resonance (FTICR–) mass spectrometry Thorsten Reemtsma a,Ã , Anja These a , Andreas Springer b , Michael Linscheid b a Department of Water Quality Control, Technical University of Berlin, Sekr KF 4, Strasse des 17 Juni 135, 10623 Berlin, Germany b Department of Chemistry, Humboldt-Universita ¨t zu Berlin, Brook-Taylor-Strasse 2, 12489 Berlin, Germany article info Article history: Received 7 April 2007 Received in revised form 21 June 2007 Accepted 24 June 2007 Keywords: Natural organic matter Aquatic fulvic acids Ultrahigh-resolution mass spectrometry Size-exclusion chromatography FTICR–MS abstract Size-exclusion chromatography was coupled to electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (SEC–FTICR–MS) to separate a fulvic acid isolate into three size fractions and to determine the elemental composition of fulvic acids in these fractions. Molecular formulas of about 3000 ions in the mass range of 200–700 Da were derived, many of which occur in all three fractions and follow the same system of elemental composition. Product ion spectra generated by SEC coupled to quadrupol-time- of-flight-MS (Q-TOF-MS) confirmed that the ions of all three fractions are basically polycarboxylates with hardly any other functional moiety. However, SEC–FTICR–MS revealed that the ions generated from the high molecular weight (HMW) fraction are enriched in carboxyl groups and are more aromatic as compared with the low molecular weight (LMW) fraction. These findings support the idea that the HMW fulvic acids are formed from LMW fulvic acids. The shift in the relative frequency of ions from the LMW to the HMW fraction is in line with different interaction mechanisms: HMW fulvic acids may be aggregates held together by electrostatic interaction of the carboxylate groups via hydrogen bonds or with polyvalent cations or by hydrophobic interaction of their carbon backbone, or consist of LMW fulvic acids covalently bound to each other or to (aliphatic) alcohols. & 2007 Elsevier Ltd. All rights reserved. 1. Introduction With a portion of up to 30–50% of total dissolved organic matter (DOM) in natural waters, fulvic acids can be con- sidered the largest class of mobile organic carbon on the earth (Abbt-Braun and Frimmel, 2002). Understanding fulvic acid properties and dynamics in aqueous solution would be substantial to understand DOM transport and dynamics. To date fulvic acids are only operationally defined by their sorption and ion-exchange properties towards certain poly- meric sorbents that are used for their enrichment from aquatic samples (Leenheer, 1981; Aiken et al., 1992). Owing to a lack of knowledge at the molecular level, it is still a matter of debate as to whether humic substances do really exist as a chemically distinct class of compounds or whether they have to be considered a mixture of diverse classes of compounds ARTICLE IN PRESS 0043-1354/$ - see front matter & 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.watres.2007.06.063 Ã Corresponding author. E-mail address: thorsten.reemtsma@tu-berlin.de (T. Reemtsma). WATER RESEARCH ] ( ]]]] ) ]]] – ]]] Please cite this article as: Reemtsma, T., et al., Differences in the molecular composition of fulvic acid size fractions detected by size-exclusion chromatography–on line Fourier transform ion.... Water Res. (2007), doi:10.1016/j.watres.2007.06.063