In this section we survey additional retrospective tests with reference calculations and analyze the results. For most of the reference calculations, simulations which had the greatest error in binding affinity had poor sampling/mixing of the states within replicas. Moreover, many of the free energy estimates were not converged, or converged to a value which disagreed with experiment at timescales up to 30 ns per window. Convergence to a value which differs from experiment may indicate force field problems. These errors was particularly prevalent for TrimerTrip and Cyclodextrin derivatives while also in the presence of a guest with a formal charge (Figures and). Interestingly, free energy estimates seemed to converge better for the GDCC dataset depending on whether the guest was positively or negatively charged (Figure ). In addition, in simulations for exoOA with guests with a negatively charged carboxyl group had poor mixing of states within replicas, while with positively charged guests mixing of states was generally better. To check the contribution of the charged protocol in mixing of states between replicas and estimate error in reference calculations, additional calculations for exoOA-g1, exoOA-g3, and clip-g1 were done.