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Fluorescence and amino acid characteristics of molecular size fractions of DOM in the waters of Lake Biwa

Dissolved organic matter (DOM) in the waters from Lake Biwa, Japan was fractionated using tangential flow ultrafiltration, and subsequently characterized by fluorescence properties and amino acids. While major dissolved organic carbon (DOC), UV absorbance (Abs), humic-like fluorescence (Flu) and total hydrolyzed amino acids (THAA) occurred in the less than 5 kDa molecular size fraction, they were not evenly distributed among various molecular size fractions. Flu/Abs ratios increased, and THAA/DOC ratios decreased with decreasing molecular size. Humic-like fluorescence occurred in all molecular size fractions, but protein-like fluorescence only occurred in the 0.1 μm-GF/F fraction. Subtle differences in amino acid compositions (both individuals and functional groups) were observed between various molecular size fractions, this may indicate the occurrence of DOM degradation from higher to lower molecular weight. The results reported here have significance for further understanding the sources and nature of DOM in aquatic environments.

Ultraviolet absorbance titration for determining stability constants of humic substances with Cu(II) and Hg(II)

We describe an ultraviolet (UV) absorbance titration method that can be used to determine complexing capacities (C(L)) and conditional stability constants (logK) of humic substances (HSs) with metal ions such as Cu(II) and Hg(II). Two fulvic acids (FA) and one humic acid (HA) were used for this study. UV absorbance of HSs gradually increased with the addition of Cu(II) or Hg(II) after blank correction, and these increases followed the theoretical 1:1 (ligand:metal ion) binding model. The results from the absorbance titration calculation for HSs with Cu(II) and Hg(II) compared well with those from fluorescence quenching titration. The titration of the model compound l-tyrosine with Cu(II) proved the validity of this method, and the K and C(L) were within 2.3% and 7.4% of the fluorescence quenching titration. The results suggest that the UV absorbance titration can be used to study the binding capacities of HSs and/or dissolved organic matter (DOM) with trace metals. The advantages and disadvantages of the absorbance titration method were also discussed.

Molecular size distribution characteristics of the metal–DOM complexes in stream waters by high-performance size-exclusion chromatography (HPSEC) and high-resolution inductively coupled plasma mass spectrometry (ICP-MS)

In this work, the complexation of the metals Fe, V, Ce, Th, U, Mo, Cu, Ni, Co, Cr, Zn, Pb and Cd with different molecular size (MS) fractions of dissolved organic matter (DOM) in natural waters was investigated. In order to assess the MS distribution of the metal–DOM complexes, DOM samples from stream waters were concentrated by reverse osmosis, and were then analyzed by high-performance size-exclusion chromatography (HPSEC) coupled with on-line UV-Vis absorbance and high-resolution inductively coupled plasma mass spectrometry (ICP-MS). The MS distribution of overall DOM and its metal-bound complexes was evaluated. The results indicate the following order of decreasing number-averaged molecular weight: Cu > Ni > (Co, Zn, Cr) > Pb > Cd for the DOM-bound complexes of transitional metals, which is consistent with Irving-Williams series, and (Fe, V, Ce) > Th > U > Mo for the DOM-bound complexes of the other metals. The results suggest that the metal distribution among the different MS fractions was closely related to metal binding strength; metals with high binding strength were distributed more in the larger MS fractions, and metals with low strength were distributed more in the smaller MS fractions. Possible mechanisms for these observations were discussed. This study is significant to the understanding of metal complexation with DOM in natural waters.

High-performance liquid chromatographic fractionation and characterization of fulvic acid

High-performance immobilized metal ion affinity chromatography (HP-IMAC) was used to fractionate humic substances (HS) based on their affinity for the immobilized copper(II) ion using acidic and glycine eluents. The work was carried out with two naturally occurring aqueous fulvic acids and commercially available Suwannee River fulvic acid. The IMAC-fractionated HS were then characterized by reversed-phase high-performance liquid chromatography (RP-HPLC) and size exclusion chromatography. The results showed that the affinity HS fraction eluted first using an acidic pH = 2 eluent exhibited a relatively high hydrophilic character, whereas the fraction eluted later using a glycine eluent exhibited both a higher hydrophobic character and larger molecular size. On the other hand, the HS fraction with no affinity for the immobilized copper had low molecular size. The affinity of the HS fraction for copper(II) increased with increasing molecular weight. Based on the composite results of three different HS, it is evident that strong relationships exist between affinity, molecular weight, and hydrophilic/hydrophobic properties during the HP-IMAC fractionation. The results presented here have significance for understanding the nature of chemical interactions at the molecular level between dissolved organic matter and trace metals. IMAC, coupled with other liquid chromatographic strategies, is a promising tool for chemical fractionation and characterization of HS.

Fractionation and characterization of fulvic acid by immobilized metal ion affinity chromatography

Abstract Immobilized metal ion affinity chromatography (IMAC) has been used to separate humic substances (HS) based on their affinity for metal ions. The work was carried out with a stream fulvic acid (FA) as a representative HS. The effect of pH and ionic strength on the retention of HS was investigated, and comparison of different metal ions (Cu 2+ , Ni 2+ , Co 2+ and Cd 2+ ) for IMAC of HS was examined. pH dependence of retained HS and retention volume, and salt-promoted complexation were found; these results are similar to those reported for some proteins using IMAC in previous studies. The HS binding capacity and retention volume in four metal ions studied for IMAC were in the order: Cu 2+ >Ni 2+ >Co 2+ >Cd 2+ , which agrees with the Irving–Williams series. The HS retained on the Cu 2+ -IMAC was eluted with eluent of decreasing pH value, and the fractions collected were characterized by UV–VIS and fluorescence spectra, and fluorescence quenching titration. The results show that HS fractions eluted with lower pH eluent have a higher conditional stability constant ( log K Cu – HS ′ ) for copper(II), red-shifted maximum excitation (Ex) and emission (Em) fluorescence wavelengths (shifted towards longer wavelengths), lower fluorescence (Flu)/absorbance (Abs) ratios, spectral slope and E 265 nm /E 365 nm , indicating that the stronger affinity HS fractions may have relatively higher molecular size and a higher proportion of compounds absorbing and fluorescing at longer wavelength. Based on the results obtained, IMAC appears to be a promising tool for chemical separation of HS. The results will help in the understanding of the nature of HS and their metal binding characterization, and in modelling natural environments.