In order to develop new porous materials for applications in gas separations such as natural gas upgrading, landfill gas processing and acetylene purification it is vital to gain understanding of host-substrate interactions at a molecular level. Herein we report a series of six isoreticular metal-organic frameworks (MOFs) for selective gas adsorption. These materials do not incorporate open metal sites and thus provide an excellent platform to investigate the effect of the incorporation of ligand functionality amide and alkyne groups on substrate binding. By reducing the length of the linker in our previously reported MFM-136, we report much improved CO/CH (50 : 50) and CO/N (15 : 85) selectivity values of 20.2 and 65.4, respectively (1 bar and 273 K), in the new amide-decorated MOF, MFM-126. The CO separation performance of MFM-126 has been confirmed by dynamic breakthrough experiments. inelastic neutron scattering and synchrotron FT-IR microspectroscopy were employed to elucidate dynamic interactions of adsorbed CO molecules within MFM-126. Upon changing the functionality to an alkyne group in MFM-127, the CO uptake decreases but the CH uptake increases by 68%, leading to excellent CH/CO and CH/CH selectivities of 3.7 and 21.2, respectively. Neutron powder diffraction enabled the direct observation of the preferred binding domains in MFM-126 and MFM-127, and, to the best of our knowledge, we report the first example of acetylene binding to an alkyne moiety in a porous material, with over 50% of the acetylene observed within MFM-127 displaying interactions (<4 Å) with the alkyne functionality of the framework.