Studies on fluorescein—VIIThe fluorescence of fluorescein as a function of pH
1989; Elsevier BV; Volume: 36; Issue: 3 Linguagem: Inglês
10.1016/0039-9140(89)80213-9
ISSN1873-3573
AutoresHarvey Diehl, R. Markuszewski,
Tópico(s)Protein Interaction Studies and Fluorescence Analysis
ResumoThe relative fluorescence of fluorescein over the pH range 3–12 has been measured at 516 nm, with excitation at 489 nm. The relative fluorescence is essentially zero at pH 3, increases slowly between pH 4 and 5, rises rapidly between pH 6 and 7, reaches a maximum at pH 8, and remains constant at above pH 8. The curve of relative fluorescence as a function of pH lies somewhat above the corresponding curve describing the fraction of fluorescein present as the doubly charged anion, Fl2−, indicating much weaker fluorescence of the singly charged anion, HFl−, and very much weaker fluorescence by the neutral species, H2Fl. The fluorescence data have been used to calculate a value for the third dissociation constant. Because of the complexity of the system, one unknown dissociation constant and three (relative) fluorescence constants, a series of three variable regressions on the data was made. The final values were KHFl = 4.36 × 10−7 (μ = 0.10) for the third dissociation constant and KH2Fl = 0.8; κHFl = 5.7; κFl = 100.0 for the relative fluorescence constants.
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