In this paper, humic acid (HA), known to play a large role in the binding and transport of pesticides in soil, was immobilized on a chromatographic support. Then, the association of some herbicides and rodenticides with the main soil component HA was examined using this novel chromatographic column. It appeared that HA has a lower affinity for neutral than for charged pesticides. Moreover, the influence of various parameters was investigated on the pesticide retention in order to providevaluable information about both the binding mechanism and the utilization conditions of the HA column. For all the pesticides studied, a change was clearly vizualized in the HA-pesticide association mechanism at a critical value of the Na+ concentration in the bulk solvent, x(c), equal to 0.6 M. Around this value, the HA structure balanced between a flexible linear conformation for x < x(c) and a random coil form for x > x(c). This work confirmed the conformation change on HA immobilized on silica. As well, only for the charged pesticides, it was clearly pointed out that below a Na+ concentration equal to 0.3 M, the pesticide binding to HA decreased when the salt concentration was enhanced due to an ion pair formation and a competition effect between the sodium cation and pesticide to bind to the HA molecule. Furthermore, it was established that the HA column was stable during an extended period of time, indicating that the HA column could soon become very attractive to determine the risk assessment of pesticides.