Flow of liquid monomolecular films is important in a variety of applications, such as lubrication, lithography, coatings, and microfluidics. In these systems, flow behavior is strongly affected by the interaction between the flowing macromolecules and the underlying substrate. The latter changes molecular conformation, causeing alignment of molecules, and it also mediates the flow rate through molecular friction. Here, we study the effect of interfacial interaction on the flow rate of individual macromolecules that results in the fractionation of flowing macromolecules with respect to their size. This fractionation contradicts the current model of spreading of a 2D fluid, which can have great implications on numerous technologies. The experiments were done by placing a drop of polymer melt onto a mica surface and using AFM to measure the composition of the monolayer as it spreads away from the drop. AFM allows for realtime, non-destructive measurement of flow properties of this monolayer on a molecular scale. Two possible mechanisms of fractionation are considered: fractionation that originates at the drop precursor film interface, and fractionation that occurs at the precursor film substrate interface as the drop spreads. It was found that the fractionation which occurs during the flow of molecules along the surface dominates. This fractionation yields interesting consequences to the fields mentioned above. In microfluidics in particular, it gives a better understanding of how molecules will spread through channels, knowledge that becomes increasingly important as the channel sizes are reduced.