Titration ferric-catechol

Figure 13. Distribution curve, as a function of pH, of the various species formed in the ferric-catechol titration experiment.

Titrations were performed as above except that 2 ymoles of ferric chloride and 3 Pmoles of rhodotorulic acid were mixed and neutralized prior to introduction of 2 ymoles of catechol type siderophore dissolved in ethanol. The eq.tiivalents of standard alkali required to neutralize the solution were then noted. 

Stock 10 mM solutions of enterobactin and a jrobactin in ethanol were prepared by dissolving, respectively, 3.736 mg in 0.56 ml and 3. 21 mg in 0.5 + ml. The ferric chloride was dissolved in methanol to give a 20 mM solution. A titration vial was loaded with 0.5 ml ethanol, 0.2 ml (2 ymoles) catechol solution, 0.1 ml (2 ymoles) ferric chloride solution and 0.5 n+1 water. The pH was raised to ca. 7 by addition of 0.1 N NaOH, at which point exactly 0.2 ml (2 ymoles) of solution of competing catechol was added and, when the latter was enterobactin, the pH readjusted to -7. The neutral solutions were sealed and stored overnight at room ten jerature to assure equilibration, although the latter appeared to be achieved immediately. 

The formation constants of the ferric complexes of these synthetic catecholate ligands have been determined spectrophotometri-cally by competition with EDTA, as described above for entero-bactln. The first three (most acidic) ligand protonation constants have been determined by potentlometrlc titration of the free ligand. The second, more basic, set of protonation constants are too large to be determined readily potentlometrically. Thus the proton-dependent stability constant is expressed as... 

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