Chemical base flow separation is a widely applied technique in which contributions of groundwater and surface runoff to streamflow are estimated based on the chemical composition of stream water and the two end-members. This method relies on the assumption that the groundwater end-member can be accurately defined and remains constant. We simulate solute transport within the aquifer during and after single and multiple river flow events, to show that (1) water adjacent to the river will have a concentration intermediate between that of the river and that of regional groundwater and (2) the concentration of groundwater discharge will approach that of regional groundwater after a flow event but may take many months or years before it reaches it. In applying chemical base flow separation, if the concentration in the river prior to a flow event is used to represent the pre-event or groundwater end-member, then the groundwater contribution to streamflow will be overestimated. Alternatively, if the concentration of regional groundwater a sufficient distance from the river is used, then the pre-event contribution to streamflow will be underestimated. Changes in concentration of groundwater discharge following changes in river stage predicted by a simple model of stream-aquifer flows show remarkable similarity to changes in river chemistry measured over a 9 month period in the Cockburn River, southeast Australia. If the regional groundwater value was used as the groundwater end-member, chemical base flow separation techniques would attribute 8% of streamflow to groundwater, as opposed to 25% if the maximum stream flow value was used.