I am trying to separate and detect zinc-terpyridine complex (Zn-terpy) from original terpyridine (Terpy) in HPLC. Terpy is a chromophoric chelater and forms a complex with metals like Zn as (Terpy2)Zn2+ in the presence of excess Terpy. Under the condition shown below, Terpy peak appears at 4 min at 254 nm, but no other peaks are observed. I am not familiar with chelate chemistry and also, this complex behavior in the HPLC system whether the complex dissociates or not. I would appreciate anybody here can help or suggest me. This would proceed my project significantly. Thanks.

Column: ODS-2 C18 4.6x150 (mm)
Mobile phase: CH3OH/pH6.9 TEA (70:30) 1.5 mL/min
Detection: 254 nm for Terpy and 333 nm for (Terpy2)Zn2+

Note: TEA=tetraethylamine

There isn't a lot of published data on this chelator but the Smith and Martell compilation on chelators does have a bit of information on this chelator. They do list a formation constant for the 1:1 complex with zinc as 6.0 but don't list any details on the 2:1 complex. However, judging from several other metals (iron, cobalt and nickel) which have tabulated data for the 2:1 complexes, it looks like the formation constant should be around 12 for the zinc 2:1 complex. Based on experience with other systems, I would say that this is too low to expect the complex to remain together during chromatography without adding some of the ligand to the eluent. Generally, for 1:1 complexes the formation constant needs to be at least fifteen in order to stay together in the absence of excess ligand. In the case of 2:1 complexes, one can get away with this only when the cumulative formation constant is greater than 20.

My guess is that the complex is quantitatively dissociating during the chromatographic process. You probably will need to add at least micromolar levels to the eluent in order to keep the complex together.

Thanks for a message above. Your guess may be true and I will try adding terpy in the mobile phase to prevent dissociation. Hopefully, I can post my success soon.