A critical evaluation of an asymmetrical flow field-flow fractionation system for colloidal size characterization of natural organic matter

Abstract

Colloidal retention characteristics, recovery and size distribution of model macromolecules and natural dissolved organic matter (DOM) were systematically examined using an asymmetrical flow field-flow fractionation (AFlFFF) system under various membrane size cutoffs and carrier solutions. Polystyrene sulfonate (PSS) standards with known molecular weights (MW) were used to determine their permeation and recovery rates by membranes with different nominal MW cutoffs (NMWCO) within the AFlFFF system. Based on a ≥90% recovery rate for PSS standards by the AFlFFF system, the actual NMWCOs were determined to be 1.9kDa for the 0.3kDa membrane, 2.7kDa for the 1kDa membrane, and 33kDa for the 10kDa membrane, respectively. After membrane calibration, natural DOM samples were analyzed with the AFlFFF system to determine their colloidal size distribution and the influence from membrane NMWCOs and carrier solutions. Size partitioning of DOM samples showed a predominant colloidal size fraction in the <5nm or <10kDa size range, consistent with the size characteristics of humic substances as the main terrestrial DOM component. Recovery of DOM by the AFlFFF system, as determined by UV-absorbance at 254nm, decreased significantly with increasing membrane NMWCO, from 45% by the 0.3kDa membrane to 2–3% by the 10kDa membrane. Since natural DOM is mostly composed of lower MW substances (<10kDa) and the actual membrane cutoffs are normally larger than their manufacturer ratings, a 0.3kDa membrane (with an actual NMWCO of 1.9kDa) is highly recommended for colloidal size characterization of natural DOM. Among the three carrier solutions, borate buffer seemed to provide the highest recovery and optimal separation of DOM. Rigorous calibration with macromolecular standards and optimization of system conditions are a prerequisite for quantifying colloidal size distribution using the flow field-flow fractionation technique. In addition, the coupling of AFlFFF with fluorescence EEMs could provide new insights into DOM heterogeneity in different colloidal size fractions.