Dissociation constants, spectrophotometric

SPECTROPHOTOMETRIC DETERMINATION OF THE pAT VALUE OF AN INDICATOR (THE ACID DISSOCIATION CONSTANT OF METHYL RED)... 

Discussion. The dissociation of an acid-base indicator is well suited to spectrophotometric study the procedure involved will be illustrated by the determination of the acid dissociation constant of methyl red (MR). The acidic (HMR) and basic (MR-) forms of methyl red are shown below. 

Spectrophotometric determination of the pK value of an indicator (the acid dissociation constant of methyl red) 718... 

Tam, K. Y., Takacs-Novak, K. Multiwavelength spectrophotometric determination of add dissociation constants. Part 11. First derivative versus target fador analysis. Pharm. Res. 1999, 16, 374-381. 

In this way Kolthoff and Bruckenstein59 determined spectrophotometrically at 25° C for acid-base equilibria in glacial acetic acid the following ionization and dissociation constants of the bases ... 

Mitchell, R. C. Salter, C. J. Tam, K. Y., Multiwavelength spectrophotometric determination of acid dissociation constants. Part III. Resolution of multi-protic ionization systems, J. Pharm. Biomed. Anal. 20, 289-295 (1999). 

In a series of papers (Cohen and Lach, 1963 Lach and Cohen, 1963 Lach and Chin, 1964a,b Pauli and Lach, 1965 Lach and Pauli, 1966), Lach and co-workers used a similar technique to evaluate the effect of the cycloamyloses on the solubilities of a variety of pharmaceuticals. Plots of the solubility of the pharmaceutical against the concentration of added cycloamylose were usually linear with slopes ranging from 0 to 2.25. In theory, these slopes can be related to the dissociation constants for the cycloamylose-substrate complexes if the stoichiometries of the complexes can be determined (Thoma and Stewart, 1965). This technique, however, is inferior to the spectrophotometric method to be discussed presently. 

By correlating the observed spectral changes with the concentrations of added cycloamylose, dissociation constants of the cycloamylose-substrate adducts may be calculated (Rossotti and Rossotti, 1961). Values of the dissociation constants determined in this manner for a variety of complexes are presented in Table II. In most cases, stoichiometries of the complexes have been shown to be 1 1 from the presence of distinct isosbestic points in the spectrophotometric titrations. In a few cases, additional spectral perturbations are observed as the cycloamylose concentration is increased, indicating more complex modes of association. Methyl orange, for example,... 

The proton dissociation constants, of two series of 3,7-bis(arylazo)-2,6-diphenyl-1 //-irnidazo[l,2-7]pyrazoles, in the ground state and the excited state were determined by the spectrophotometric method and utilizing the Forster energy cycle, respectively. These constants were correlated by the Hammett equation and the results of such correlations with spectral data indicated that both series of compounds exist in solution almost exclusively in the l//-bis-(arylazo) tautomeric form A <2002T2875> (Scheme 3). 

Drug dissociation constants are experimentally determined by manual or automated potentiometric titration or by spectrophotometric methods.40 Current methods allow determination of pXa values with drug concentrations as low as 10 to 100 pM. For highly insoluble compounds (concentration <1 to 10 pM), the Yesuda-Shedlovsky method41 is commonly used where organic cosolvents (i.e., methanol) are employed to improve solubility. The method takes three or more titrations at different cosolvent concentrations, and the result is then extrapolated to pure aqueous system. The dissociation constant can also be determined with less accuracy from the pH-solubility profile using the following modification of Henderson-Hasselbach equation ... 

Cessna, A.J. and Grover, R. Spectrophotometric determination of dissociation constants of selected acidic herbicides, J. Agric. Food Chem., 26(l) 289-292, 1978. 

Nitraminomethyltriazole described in Ref 2 was obtained as col rosettes or ndls, mp 212-13° with decompn. Williams(Ref 5) gives mp 206-7°. Was prepd by Henry from l-acetamido-3-nitroguanidine as described in Ref 2,p 5344- Spectrophotometric studies of dissociation constant are discussed in Ref 4 and IR absorption spectra in Ref 3a. 

Spectrophotometric studies of dissociation constants of tetrazoles, triazoles and nitro-guanidines)... 

Figure 6.6 Spectrophotometric titration of the binding of inositol hexaphosphate (IHP) to methemoglobin (Methb). The complex has an increased absorbance at 512 and 649 nm, and no increases at 640,618,588, and 599 ran. The concentration of methemoglobin (20 fjM) is about 14 times higher than the dissociation constant of 1.4 f.iM for the complex. The intersection of the slope of the increase in absorbance with the maximum value gives the stoichiometry (1, in this case). Note that this simple procedure cannot be used if the protein is not initially present at such a high concentration relative to the dissociation constant, since the assumption is that all the added ligand is bound to the protein for the early additions.

In contrast, spectrophotometric measurements via indicator dyes sense concentrations and yield nonthermodynamic dissociation constants (concentrations rather than activities). 

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