Spectrophotometric titration curves, for

Spectrophotometric titration curves for the titration of an analyte, A, with a titrant, T, to form a product, P, in the presence of a visual indicator. Titration curves are shown for cases where (a) only A absorbs (b) only T absorbs (c) only P absorbs (d) A and T absorb (e) P and T absorb and (f) only the visual indicator absorbs. 

Spectrophotometric titration curve for the complexation titration of a mixture. 

The titration of a mixture ofp-nitrophenol (pfQ = 7.0) and m-nitrophenol pK = 8.3) can be followed spectrophotometrically. Neither acid absorbs at a wavelength of 545 nm, but their respective conjugate bases do absorb at this wavelength. The m-nitrophenolate ion has a greater absorbance than an equimolar solution of the p-nitrophenolate ion. Sketch the spectrophotometric titration curve for a 50.00-mL mixture consisting of 0.0500 M p-nitrophenol and 0.0500 M m-nitrophenol with 0.100 M NaOH, and compare the curve with the expected potentiometric titration curves. 

Sketch the spectrophotometric titration curve for the titration of a mixture of 5.00 X 10 M Bi + and 5.00 X 10 M Cu + with 0.0100 M EDTA. Assume that only the Cu +-EDTA complex absorbs at the selected wavelength. 

Fig. 15. Spectrophotometric titration curves for RNase, 1.9 mg/ml, in 0.08 M KC1. The temperature for each titration is given in the figure. Reproduced from Hermans and Scheraga (377).

Redox potentials have been determined for each of the steps of reduction of the trypsin-solubilized reductase (403) step 1, one electron consumed, Eo = —109 mV step 2, two electrons consumed. Eg = —276 mV and step 3, one electron consumed. Eg = —371 mV at pH 7.0, 26°. As expected, the redox potential of step 3 is more negative than the potential of the NADPH-NADP+ couple and was determined from the dithio-nite titration. The overall potentiometric—spectrophotometric titration curves could be very closely fitted with a computer-generated curve based on the assumptions of four one-electron reduction steps and octinction coefficients of 4.9 and 4.5 mM cm for the semiquinones, FliH and rijH the Eg values assumed for steps 2 and 3 were —270 and —290 mV. The precise fit was very sensitive to all of the assumptions (40 ) ... 

With several other proteins, such as bovine serum albiunin (Tanford and Roberts, 1952), lysozyme (Tanford and Wagner, 1954), and/3-lacto-globulin (Tanford and Swanson, 1957), pK shifts of the phenolic OH groups of tyrosine residues are observed, but these are of a qualitatively different nature. Thus, the tyrosines of any one of these proteins cannot be readily differentiated into a normal and an abnormal variety, since the spectrophotometric titration data for these proteins are reversible and fall on single smooth curves, in contrast to the situation with RNase. On the other hand, the tyrosine residues of ovalbumin show comparable behavior to the three abnormal tyrosine groups of RNase (Crammer and Neuberger, 1943). About 2 of the total of 9 tyrosine residues appear to titrate normally, but the remainder are not titrated up to pH 12. At pH 13, these anomalous tyrosines become titratable, and this is accompanied by the irreversible denaturation of the ovalbumin molecule. 

Figure 13 shows the data for the three phenolic groups of ribonuclease which ionize reversibly (Tanford etal., 1955a), based on spectrophotometric titration curves such as Fig. 11. A straight-line plot is obtained, in agreement with Eq. (14). The values of w are 0.112, 0.093, and 0.061, respectively, at ionic strengths 0.01, 0.03, and 0.15. (The salt used to produce the ionic strength was KCl, and there is evidence that neither K" nor CF is bound to an appreciable extent. The use of Zn as abscissa is therefore presumably acceptable.) Comparison with the calculated values of Table III shows that the experimental values are lower than predicted by about 20%. Such a deviation must be considered almost within the error of calculation. [If the radius of the hydrodynamically equivalent sphere (19 A) had been used as the basis of calculation, the calculated values of w would have become 0.119, 0.096, and 0.066, respectively.]...

Titration curves for bovine a-chymotrypsinogen have been determined under a variety of conditions by Wilcox (1961). The results are summarized in Table IX. The spectrophotometric titration of the phenolic groups is shown in Fig. 4. 

Fig. 149. Spectrophotometric titration curves of the three phenolic groups in lysozyme at 25°C. The points are average values of the data obtained at three wavelengths 290, 295, and 300 m/ - Dashed curve the ionization of the phenolic groups after the protein was heated in 9 M urea at 60° for 24 hours. At pH 13.0, a value of 1.0 was assumed for the degree of ionization a. This curve is similar to that reported by Tanford and Wagner (1954) for the ionization of the phenolic groups in KCl solution, and to curves obtained in urea without heating (not shown). Solid curve GU at 25°C., the points on this curve having been determined after the protein had been in solution for about 2 hours. This sample was not heated (Donovan el al., 1960).

For the different values of pAHX and pA H+ see the summary Table 4.5 later of pKa data in various solvents of low e in comparison with pAa(H20). The mutual agreement of pffHX values obtained by spectrophotometry, DVP, potentiometry and titration was reasonably good the typical form of the curves for titration of the dinitrophenols with TMG can be explained by homoconjugation and more especially by its influence on the potentiometric measurements, calculated on the basis of simple dissociation hence the major discrepancies in the spectrophotometric and potentiometric pK values. In order to... 

Fig. 4. H-dependence of the visible absorption spectrum of bovine Co(II) carbonic anhydrase. The broken curve represents the basic spectral form, from ref. (58) (near infrared) and at pH 11.6 (visible). The solid curves were obtained in imidazole-sulfate buffers, ionic strength 0.1, pH 7.80, 7.00, 6.55, 6.10, respectively. The spectrum of lowest intensity was obtained by extrapolation and represents the acidic spectral form. Insert Spectrophotometric titration at 640 nm. The curve was calculated for the titration of a single group with pKa = 6.6...

A colorimetric method based on the inhibitory effect of EDTA on the Mn(II) catalyzed oxidation of malachite green by periodate was reported [32]. An alternative method based on using Fe(III) instead of Mn(II) was proposed [33]. The reduction of the absorbance of Bi(III) bromo-pyrogallol red tenside ternary complex upon the addition of EDTA has been exploited for its determination. Calibration curves obtained at 650 nm were linear over the range of 0.2-6 pg/mL of EDTA [34]. EDTA in ophthalmic solutions could be assayed by spectrophotometric titration using Mg(II) as the titrant and Arsenazo I as the indicator. The working range was 0.05-2 pg/mL [35]. 

A spectrophotometric titration of the phenolic groups of myosin and its subunits has been reported by Stracher (1960). The data resemble those shown for ribonuclease in Fig. 11. About two-thirds of the tyrosine residues are titrated normally, and about one-third appear inaccessible in native myosin. An interesting feature is that 6 M urea has no effect at all on the titration curve. 

A useful test for spectrophotometric titrations is to compare the apparent tyrosyl ionization from absorptivity versus pH measurements at several wavelengths. A good illustration is found in Tanford and Wagner s (1954) study on lysozyme. Their measurements at 2880, 2900, and 2950 A resulted in nonidentical titration curves. From these results, they concluded that .. . the observed changes in light absorption are not a true... 

The apparent pKa for chlorprothixene has been determined spectrophotometrically to be 8.4 (10). The apparent pKa has also been determined from the titration curve in an isopropanol water (1 1) mixture and found to be... 

F lo. 124. Titration curves of the amino group of tryptophan at the temperatures indicated. The open symbols represent spectrophotometric data [a = AD293/(AZ)jg3)miix] and the closed symbols direct titration data [a = [OHw)/(OHM)maxl- The curves are theoretical ones for the pK values indicated by the vertical strokes and are drawn to fit the spectral data. The pK values are plotted against /T in the inset. The reference pH values for the spectrophotometric titrations are approximately 6 (Hermans el al., 1960). 

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). 

In systems such as the 2- and 6-hydroxypteridine series, rapid potentiometric or spectrophotometric pA determinations on neutral solutions usually give values near to the acidic pA of the hydrated series. (Exceptions include 2-hydroxy-4,6,7-trimethyl-, 6-hydroxy-7-methyl-, and 4,6-dihydroxy-pteridine, where the neutral solution contains comparable amounts of hydrated and anhydrous species. In such cases, rapid potentiometric titrations show two well-defined and separated curves, one for the hydrated, the other for the anhydrous, species.) Similarly, from solutions of the anion, an approximate pA value for the anhydrous species is obtained. For convenience, the anhydrous molecule is referred to as HX, its anion as X , the hydrated neutral molecule as HY, and its anion as Y, and the two equilibrium constants are defined as follows ... 

The verification of Lamber Beer s law is shown in Figure 4. Organic solutions of cerium were prepared by extracting cerium (IV) from titrated aqueous solutions and standardized by beta counting of Ce tracer. Standardization curves were plotted from three values for further spectrophotometric determinations of cerium (IV). 

---