Titration curve spectrophotometric

As the four microscopic constants cannot be determined by a titration curve, spectrophotometric analysis (UV absorption of R-S ) was necessary. The pX (8.65) of cysteine betaine (ionization of a thiol in the presence of a positive nitrogen) and the pK (8.75) of S-methyl cysteine (ionization of an amino group in the presence of neutral sulfur) closely mimic the and /c2 dissociation pathways and suggest that these values should be close to each other... 

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. 

Determination of iron(III) in the presence of aluminium. Iron(III) (concentration ca 50 mg per 100 mL) can be determined in the presence of up to twice the amount of aluminium by photometric titration with EDTA in the presence of 5-sulphosalicylic acid (2 per cent aqueous solution) as indicator at pH 1.0 at a wavelength of 510 nm. The pH of a strongly acidic solution may be adjusted to the desired value with a concentrated solution of sodium acetate about 8-10 drops of the indicator solution are required. The spectrophotometric titration curve is of the form shown in Fig. 17.23. 

Each of the curves in Figure 5-3 exhibits two or three pH regions in which the slope of the logarithmic plot is approximately —1, with intermediate regions where the slope is small or zero. Lewis and Hanson (1967) showed that in the case of (E,)-4-nitrobenzenediazoate the portion of the curve with slope —1 at relatively high pH was consistent with the acidity constant K3 of the (E )-diazohydroxide determined either by titration or spectrophotometrically, the relevant results being (by... 

The same transition may also be examined as a function of pH at constant temperature (277). Some typical curves are shown in Fig. 15. These spectrophotometric titration curves are functions also of ionic strength as seen in Fig. 16. The transition temperature is not significantly... 

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).

EDTA was determined in the urine of patients treated with Na2CaEDTA upon formation of its Fe(II) complex with 2,4,6-tripyridyl-s-triazine at pH 4.5 [30], Concentrations ranging from 0.1-15.7 pM of EDTA in urine could be accurately determined. EDTA can be standardized by spectrophotometric titration with electrolytically pure Cu(II) at pH 5 without an indicator [31]. The break of the titration curve obtained at 700 nm is used as the end-point of the titration. 

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 ) ... 

Fiq. 6. Spectrophotometric titrations at 296 nv of RNase in 0.2 M KCl in HjO (dashed curve) and in KCl in ethylene glycol (solid curve and data). The upper mv and pH scales refer to the HsO titration and the lower scale to the ethylene glycol. Forward and back titration data in ethylene glycol show that the titration curve is reversible, unlike that in HiO. (Sage and Singer, 1958, 1962.)... 

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. 

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... 

Figure 6.6.2 (a) Spectrophotometric titration curve of the tetracationic porphyrins 1-4) with the tetraanionic porphyrinate TPPS" (5) in water/methanol 1 1. Pure water can also be used as a solvent, but the absorption bands then are slightly broader, the isosbetic points less sharp, (b) The corresponding Job plot, (c) Fluorometric titration of the isomer 1-4) mixture with CuTPPS" in water.The assumed molecular structure of the dimer is also indicated. (From Endisch et al., 1996.)... 

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... 

Figure 11. Molecular hysteresis in polynucleotide—triplex organizations. Cyclic spectrophotometric acid—base titrations. Absorbance (A) as a function of pH c = 1.67 10 M triplex, (o

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). 

Since the three extra carboxyl groups appear to be interacting with lysyl rather than with phenolic groups, the abnormally high pK a of the phenolic groups remain to be explained. While the nature of the interactions which make the phenolic groups of lysozyme abnormal is as yet unknown, it is possible to disrupt these interactions with GU at 25 C. Analysis of the spectrophotometric titration curve of Fig. 149 indicates that the... 

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).

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