The pulling coordinate dependence of hexadecane dimer dissociation in water was studied using AFM-based single molecule force spectroscopy. Hexadecanes were covalently bound to both the AFM cantilever and to the glass substrates through hydrophilic poly-(ethylene glycol) tethers. The polymer tether was attached either to the end of hexadecane or in the middle of the molecule. Experimentally studied configurations of hexadecanes tethered to the AFM probe and to the glass substrate include a symmetric end-attached configuration (EE), an asymmetric end-attached vs middle-attached configuration (ME), and a symmetric middle-attached configuration (MM). Kinetic parameters of the distance to the transition state barrier (barrier width) and activation energy of dissociation were extracted from the statistical analysis of double tether rupture events. The rupture force analysis employs a recently introduced two-bond model that corrects for errors induced by potential multiple simultaneous rupture events and accounts for the tether stiffening effects. Effects of the shape of intermolecular potential were considered by using the Bell−Evans and Hummer−Szabo force spectroscopy models. The activation energies to dissociation were similar for all configurations while the barrier width was significantly shorter for the MM and ME configurations than for EE configurations. Primitive models that include touching or merging spherical or cylindrical shapes were considered. These models were inconsistent with the extracted kinetic parameters. It is suggested that the observed anisotropy may be a result of conformational transition of hexadecane from extended to collapsed state during dimerization. A flexible four-bead model of hexadecane was introduced to account for conformational flexibility. Using the length and solvent accessible surface area of hexadecane, the four-bead model gave molecular dissociation parameters consistent with the experimental data. This suggests that conformational flexibility is an important factor in hydrophobic interactions between alkane chains
