We study the isotope effect by liquid chromatography (LC) with a variety of separation media under reversed and normal phase conditions using the protiated and/or deuterated compounds as the solutes. Results of reversed phase LC (RPLC) suggested that the protiated compounds were more hydrophobic than that of deuterated compounds due to the isotope effect based on the hydrogen bonding between hydrogen atoms of isotopologues and hydroxy groups in the mobile phase. The importance of the hydrogen bonding was also supported by the separation of isotopologues with a silica stationary phase on normal phase LC (NPLC), where the deuterated compounds showed stronger hydrogen bonding to hydroxy groups on silanol. Additionally, we investigated the difference of the strength between CH-π and CD-π interactions. Comparison of free energies of isotopologues by RPLC suggested that the CH-π interaction was slightly stronger than CD-π interaction. Finally, we demonstrated the separation of a few isotopologues on NPLC using a column coated with C70-fullerene, which is capable of strong π-based interactions, by effective CH/CD-π interactions.
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