To compare calculated absolute hydration free energies to experimental data, we also ran AHFE calculations on a subset of the Mobley Lab FreeSolv database molecules. Unlike molecules from the Protein-Ligand Benchmarks set, these molecules have generally not been previously demonstrated to have partial charge variations that impact computed properties like binding free energies meaningfully; rather, these molecules are simply present in this widely used test set and are tested here to determine whether they are subject to conformational variation. We chose to examine them here in part to allow comparison to experimentally measured properties, and to see to what extent similar partial charge variation issues appear in this set. Thus, for each of the 642 molecules in the database, we generated 50 conformers using rdkit v2024.03.2 conformer generator starting from a random seed. Each conformer was charged with AmberTools AM1-BCC charges using open -toolkit v0.14.3 and AmberTools v23.6 resulting in 50 sets of partial charges for each molecule. We calculated the partial charge variation using the maximal q bond di erence across all partial charge sets for each molecule and ordered the molecules based from highest to lowest maximal q bond di erence. We manually chose 12 molecules of the top 25 molecules with the highest maximal q bond di erence by avoiding molecules that had high similarity to those already chosen.