Electrospray ionization mass spectrometry of terrestrial humic substances and their size fractions

Electrospray ionization mass spectrometry (ESI-MS) was used to evaluate the average molecular mass of terrestrial humic substances, such as humic (HA) and fulvic (FA) acids from a soil, and humic acid from a lignite (NDL). Their ESI mass spectra, by direct infusion, gave average molecular masses comparable to those previously obtained for aquatic humic materials. The soil HA and FA were further separated in size-fractions by preparative high performance size exclusion chromatography (HPSEC) and analyzed with ESI-MS by both direct infusion and a further on-line analytical HPSEC. Unexpectedly, their average molecular mass was only slightly less than for the bulk sample and, despite different nominal molecular size, did not substantially vary among size-fractions. The values increased significantly (up to around 1200 Da) after on-line analytical HPSEC for the HA bulk sample, at both pH 8 and 4, and for the HA size-fractions when pH was reduced from 8 to 4. It was noticed that HA size-fractions at pH 8 were separated by on-line HPSEC in further peaks showing average masses which progressively increased with elution volume. Furthermore, when the HA and NDL bulk samples were sequentially ultracentrifuged at increasing rotational speed, their supernatants showed mass values which were larger than bulk samples and increased with rotational speed. These variations in mass values indicate that the electrospray ionization is dependent on the composition of the humic molecular mixtures and increases when their heterogeneity is progressively reduced. It is suggested that the dominance of hydrophobic compounds in humic supramolecular associations may inhibit the electrospray ionization of hydrophilic components. Our results show that ESI-MS is reasonably applicable to humic substances only after an extensive reduction of their chemical complexity.