Spectrophotometric Determination of pK

A pH-sensitive dye induces two different absorption spectra for the protonated (AH+) and deprotonated (A) forms. This means that at low pHs, we observe an absorption spectrum of the protonated form only, while at a high pHs only the absorption spectrum of the deprotonated form is recorded. However, this is not always true, since many dyes can absorb in both forms, protonated and deprotonated. The absorption spectra maxima of the two forms are different. The protonated form displays an absorption spectrum with a peak that is shifted toward short wavelengths in comparison with the absorption spectrum of the deprotonated form of the dye. 

Therefore, one method to determine the pK of a dye is to plot the peak position of the absorption spectrum as a function of pH. A sigmoid curve is obtained the pK is equal to the pH at the inflection point. 

a third method exists. This involves calculating the concentrations of both the protonated and deprotonated forms at different pHs. The pK will correspond to the pH where the concentrations of both forms are equal. The derivation of the mathematical equation used to determine the pK can be obtained as follows. The variation of the optical density of the dye with the pH is sigmoidal. At low pHs in the presence of the protonated form only, the optical density (OD(ah)) is 

At high pHs, we observe the optical density of the deprotonated form only (OD(a)), which is 

When the dye is completely protonated or deprotonated, each of the concentrations C(ah) and C(A) will be equal to the total concentration, C, of the dye. At an intermediate pH where both protonated and deprotonated forms are present, the optical density is 

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Saurina, J., Hernandez-Cassou, S., Tauler, R., Izquierdo-Ridorsa, A. A., Spectrophotometric determination of pK. values based on a pH gradient flow-injection system, Anal. Chim. 

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

Hwang H, Dasgupta PK. 1985. Spectrophotometric determination of trace aqueous sulfate using barium-beryllon II. Mikrochim Acta 1 313-324. 

Equation (2) stresses the acid-base nature of the cation-pseudobase equilibrium and the pK + value, which is analogous to the pKa value for a Br nsted acid, denotes the pH at which the heterocyclic cation and pseudobase are present at equal concentrations. Experimental techniques for the spectrophotometric or potentiometric determination of pK values for pseudobase formation are identical to the methods for determination of pKa values for Br nsted acids.72... 

Spectrophotometric titrations of proteins Side chain of many amino acids contain ionizable groups which are available for titration. Determination of pK values for proton dissociation from these ionizable groups often provides a good indication about the position of these amino acids in the protein. This can best be understood by taking into account the example provided below.. ... 

It may seem odd that the technique of spectrophotometric titration, so extensively applied to the tyrosyl residues of proteins, has not been used in the study of protein sulfhydryl groups. The only study of this sort known to the author is the determination of the ionization constant of —SH grouj of thiolated gelatin (introduced by reaction with A -acetylhomocysteine thiolactone). By measuring the change in absorptivity as a function of pH at 2380 A, Benesch and Benesch (1958) found the apparent acidity of the thiol groups of the modified gelatin (pK = 9.8) to be nearly the same as that of an appropriate model compound, A -acetylhomocysteine (pK = 10.0). 

Where the compounds are poorly soluble or where the quantity of compotmd available is small, pK values are calculated from ultraviolet (UV) spectrophoto-metric measurements. The method simply relies on the change in UV spectra at different pH values. An adaptation of the filter probe method (described next) is used. Sample concentrations down to 4 mg/400 mL may be determined (approx. 0.000025 M). Sirius Analytical Instruments supplies the D-PAS probe, which enables spectrophotometric determinations using the GLpfCa instrument (Table 6). 

The first reported electrochemical study on dihydrodiazepines was a potentiometric determination carried out on the 5,7-dimethyl derivative. A value of pK between 13 and 14 was obtained in this way.21 The technique has since been applied to 6-nitro-5,7-diphenyldihydrodiazepine, giving a pKa of 6.7.36 For other dihydrodiazepines with very high pK values, spectrophotometric methods have proved more convenient and often more accurate.24... 

The most common methods used for pharmaceutical pK values are based on pH measurements, Eqs. (3-8). Thus, they cannot be interpreted with greater accuracy than 0.02 pKa unit [see the definition in Section 2.1, Eq. (1)]. This level of precision and accmacy should always be the aim in determining pK values for inclusion in the drug sciences literature. Potentiometric titrations [Eqs. (3-4)] are often performed with this level of accmacy, primarily for compounds with either a single ionization step or for multiple ionizations with >4 log units between the pKa values. The careful use of precise pH meters (e.g., the series of Beckman Research models, or the corresponding Radiometer, Orion, or Metrohm instruments) for the determination of pH data means that reproducibility for replicate measurements may be rather better than 0.02. In the author s experience, these instruments may be calibrated with a reproducibility of 0.002 pH imit, which can be maintained (with proper temperature control and exclusion of CO2) for at least 8 h. This does not imply accuracy of 0.002 pH unit, which is not possible according to the current definition of pH. Spectrophotometric [Eq. (5)] and solubility-pH dependence [Eqs. (7-8)] methods are potentially capable of similar accuracy, but often do not give results better than 0.05 pKa unit, due to the inevitable inclusion of additional sources of error from the absorbance or concentration measurements. 

The commonly used methods of pK, determination, such as potentiometric or spectrophotometric titrations, do not generally give information on the sites of protonation. Since it is well known that the chemicals shifts of all atoms, e.g. hydrogen, fluorine, carbon or phosphorus, are related to the amount of charge present at the atom [3], they can be used to determine both the pK, values and the locus of protonation. 

Fio. 125. Spectrophotometrically determined titration curves of the carboxyl group of tryptophan at the temperatures and wavelength indicated. The curve is a theoretical one for the pK indicated by the vertical stroke. The reference pH s are approximately 6 (Hermans et al., 1960). 

The state of ionization of the imidazole residue linked to hemoglobin iron has no hitherto observed influence on the light absorption of hemi-, hemo-, or oxyhemoglobin. It seems possible, however, that very careful spectrophotometric determinations might reveal slight differences corresponding to the pK values of the heme-linked imidazole groups. 

Using weak bases (B) as indicators, which are partly converted in acidic solvents to the conjugated acids BH+, the Ho value is measured. Two typical indicators are the o-nitroanilinium ion (pK in water —0.29) and the 2,4-dinitroanilinium ion (pK in water —4.53)12. By means of spectrophotometric methods it is possible to determine the ratio of [BH+]/[B] for each indicator, therefore Ho can be calculated for any solvent system, provided that the pK value of this indicator in water (pA". i) is known. If Hq is known for a particular solvent system, the pKa values can be calculated for any acid-base pair. 

Dissociation constants of ionizable components can be determined using various methods such as potentiometric titrations [85] CE, NMR, [86] and UV spectrophotometric methods [87]. Potentiometric methods have been used in aqueous and hydro-organic systems however, these methods usually require a large quantity of pure compound and solubility could be a problem. Potentiometric methods are not selective because if the ionizable impurities in an impure sample of the analyte have a pK similar to that of the analyte, this could interfere with determining the titration endpoint. If the titration endpoint is confounded, then these may lead to erroneous values for the target analyte pKa. 

Visible and UV spectral data have been reported for the parent unsubstituted heterocycle (62JCS493). The apparent pK values of several derivatives of this system were determined using spectrophotometric methods. The parent heterocycle has a pKm of 2.51. Values of 1.14, -0.25, -0.53 and -0.02 were obtained for the pK, of the 2-methyl, 2,3-dimethyl, 2,3,6-trimethyl and 2,3,6,7-tetramethyl derivatives of this ring system, respectively (63JCS4304). ESR data for the parent heterocycle have also been reported (71JA5850). 

For pyridoxine pK and pK2 were determined spectrophotometrically as 4.94 and 8.89. These values, together with that of R given above, were used to estimate the microscopic constants that are given in Eq. 6-74/ Notice that the microscopic constants of Eq. 6-74 are not all independent if any three of the five equilibrium constants are known the other two can be calculated readily. In describing and measuring such equilibria it is desirable to select one pathway of dissociation, e.g., H2P —> HP(A) —> P, and to relate the species HP(B) to it via the pH-independent constant R. 

The potential advantages of selective nitration of tyrosyl residues in native proteins are numerous. The reaction is performed under mild conditions, giving rise to a 3-nitrotyrosyl derivative (pK 7), which in the acid form absorbs intensely at 350 nm. Hence, the nitrotyrosine content may be readily determined spectrophotometrically, as well as by amino acid analysis ( 2.2.3). The absorption spectrum of 3-nitro-tyrosine is highly sensitive to solvent polarity and exhibits significant optical activity in the long wavelength absorption band. Consequently, nitrotyrosyl residues can be utilized as indicators of conformational change, or of interactions of proteins with other macromolecules or small molecules (e.g. Kirschner and Schachman 1973). Any perturbation in the pK of nitrotyrosyl residues is readily determined spectrophotometrically. 

Ivakin et al. (1979) determined the second and third ionization constants of arsenious acid in NaCl and KCl solutions. The pK values were determined by two methods, potentiometric and spectrophotometric. The solutions had... 

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