Probing Local pH in Hydrophobic Domains of Humic Acids: An EPR Based Method

Despite the focused efforts by many labs during the last 30 years, vital aspects of the structure and fundamental physicochemical aspects of Humic Acids (HA) are largely unknown. It is currently accepted that humic acids contain both hydrophilic as well as hydrophobic domains which are the locus of sequestration of xenobiotic molecules. However vital information on the detailed nature, the accessibility of the hydrophobic domains is lacking. On the other hand, HA behave as polyelectrolytes which bear variable charge modulated by pH. It is considered that this pH-dependent charge is modulated by the local pKa of the chargeable structural groups i.e. carboxylates and phenolics. Traditionally only the water accessible hydrophilic fraction of the chargeable groups can be probed by H-binding or metal binding studies. However the function of chargeable groups in hydrophobic domains, local pKa, and their geometrical location are largely unknown. In this context we have developed a novel methodology by using the Spin-Probe method with Electron Paramagnetic Resonance (EPR) spectroscopy. A series of specific spin-probes with varying hydrophobicity as well as pH sensitivity were used and studied with EPR spectroscopy. The experiments were carried out in well characterized HA samples of varying molecular weight providing information of the local environment of the spin-probes, their mobility, and local pKa values.