In©this©work©is©described©the©investigation©of©bivalent©versus©monovalent©enantioselective molecular© recognition© in© the© context© of© enantioselective© separations.© Electrospray© ionizationmass© spectrometry© (ESI-MS)© and© tandem© mass© spectrometry© (MS/MS)© are© used© for© evaluating enantioselective©systems©through©the©measurement©of©(1)©relative©solution-phase©binding constants© via© titration© and© (2)© relative© gas-phase© binding© via© collision© threshold© dissociation.© In HPLC,© a© cinchonane-type© chiral© stationary© phase© (CSP)© based© on© tert.-butylcarbamoylquinine provides© vastly© increased© retention© and© enantioselectivity© for© separation© of© bivalent© versus monovalent© alkoxy-benzoyl-N-blocked© leucine© enantiomers.© The© bivalent© enantiomers© are© able to© span© and© simultaneously© interact© with© multiple© interaction© sites© on© the© CSP© surface,© leading to© enhanced© separation.© ESI-MS© titration© measurements© also© show© an© increased© avidity© for binding©between©bivalent©selector©and©bivalent©selectand,©compared©with©the©monovalent system.© However,© enhanced© enantioselectivities© measured© in© HPLC© for© the© bivalent© system cannot© be© reproduced© by© MS© due© to© inherent© mechanistic© differences.© Assumed© discrepancies in© relative© response© factors© also© give© rise© to© systematic© errors© which© are© discussed.© The© results of© MS/MS© gas-phase© experiments© show© that© enantioselectivity© is© essentially© lost© in© the© absence of© solvation,© but© that© dissociation© thresholds© can© provide© a© measure© of© relative© dissociation energy©in©the©bivalent©interaction©system©compared©to©the©monovalent©counterpart.©Such measurements© may© prove© useful© and© efficient© in© better© understanding© multivalent© interactions,© in© line© with© current© theoretical© considerations© of© effective© concentrations© and© ion© trap effects.© This© is© the© first© application© of© mass© spectrometric© methods© for© assessing© increased avidity© of© binding© in© multivalent© enantioselective© molecular© recognition