WaterSubject

With a portion of up to 30–50% of total dissolved organic matter (DOM) in natural waters, fulvic acids can be considered the largest class of mobile organic carbon on the earth. 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 polymeric sorbents that are used for their enrichment from aquatic samples. 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 associated with each other by intermolecular forces such as hydrogen bonding, electrostatic attraction, van der Waals and ?–? interaction.

Ultra high-resolution mass spectrometry as provided by Fourier transform-ion cyclotron resonance–mass spectrometry (FTICR–MS) is a very powerful technique to investigate molecules in complex mixtures of natural origin such as fulvic acids, DOM and atmospheric organic matter. Recently, it was shown that a mass resolution and accuracy of 0.1 mDa should be sufficient to resolve all theoretically possible isobaric ions in a mass range up to 500 Da and to unambiguously detect their elemental composition.

In addition to molecular formula determination by FTICR–MS, information on the structure of DOM molecules has been obtained by quadrupol-time-of-flight- mass spectrometry (Q-TOF-MS). As far as fulvic acids are concerned, these investigations have provided evidence that the operationally defined fraction of DOM called fulvic acids is dominated by a chemically distinct class of compounds. This class is made up of polycarboxylates of various degrees of aromaticity and molecular mass, with a limited number of hydroxy groups and hardly any other functional moiety. These fulvic acid molecules form a highly regular pattern in terms of elemental composition and repeating units.

Now that individual molecular species in such complex mixtures of DOM become visible, there is a new chance to address biogeochemical questions such as sources and formation processes of DOM or the reactivity of DOM molecules. DOM is also of great importance for the water sector, as it may limit the quality of source waters used for drinking water production, may require considerable efforts to be partly removed and may also interact with nutrient and trace pollutant removal. Therefore, an improved knowledge on the nature of DOM at the molecular level as well as on the dynamics of these molecules by using FTICR–MS may contribute to our understanding of water quality problems and of water treatment processes.

Although FTICR–MS provides unsurpassed mass resolution, it cannot distinguish between molecular ions and fragment ions, if these have the same elemental composition and both enter the interface at the same time. In such a case, chromatographic separation by size-exclusion chromatography (SEC) is essential to assign the detected ions to (precursor) molecules of a certain size or mass. We have previously used SEC coupled to low-resolution triple-quad-MS or to high-resolution Q-TOF-MS. With that approach fulvic acid isolates were fractionated into three chromatographic signals, which were denoted as high molecular weight (HMW), moderate molecular weight (MMW) and low molecular weight (LMW) fraction. Using an organic carbon detector (SEC–DOC) parallel to SEC–MS and intercalibrating both systems it was shown that HMW compounds are detected less sensitively than LMW compounds by means of electrospray–MS: while the largest amount of organic matter was eluting in the HMW fraction its MS signal was comparatively weak. The total ion intensity of this HMW fraction could be increased by increasing the mass spetrometers cone voltage, i.e. the potential held constant to ionize the analytes and to accelerate them towards the mass spectrometers aperture. But then the m/z ratio of the ions underlying this chromatographic signal decreased. Decreasing m/z values for the ions generated at increasing cone voltage has been observed for infusion-ESI–MS analyses of technical polymers.

Various factors of the electrospray ionization process may be responsible for differences between those molecular arrangements present in the solution and those ions entering the mass spectrometer. The electrophoretic field invoked by the high-voltage electrical field present at the capillary tip forces charge separation. During the evaporation of spray droplets, an increase in electrolyte and analyte concentration per unit volume occurs, while the repeated formation of offspring droplets and the drastic decrease in droplet volume may finally isolate one molecule from the next. During solvent evaporation, the pH of the liquid phase and its dielectric constant may change drastically. It is likely that these phenomena influence the appearance of non-covalently bound arrangements of molecules, whether they are held together by electrostatic or hydrophobic interaction. Following the ionization of molecules and their transfer into the gas-phase, collision with residual gas molecules can induce fragmentation, i.e. the rupture of weak covalent bonds.

It should be noted, however, that ESI is a soft ionization technique that has been shown to be able to ionize many fragile molecules without a significant extent of fragmentation. Moreover, it may also be inevitable that the shape of a large and complex molecular arrangement changes during its transformation from a polyelectrolyte in solution to a singly charged ion in the gas phase.

We here report on the use of SEC–FTICR–MS to study the elemental composition of ions generated from different molecular size fractions of fulvic acid in solution. Differences and similarities in the elemental composition of individual ions of different size fractions may provide information on the interrelationship of fulvic acids of lower and higher molecular weight and may provide further indications on the mechanism that hold larger fulvic acids together. We also illustrate different approaches of exploiting the data provided by SEC–FTICR–MS analysis on large sets of natural organic matter.