PH curve indicators

In practice, we obtain the end point by extrapolating the two linear regions of the pH curve (the extrapolants should be parallel). A third parallel line is drawn, positioned exactly midway between the two extrapolants. The volume at which this third line crosses the pH curve indicates the end point. Knowing the volume V(end point) > we can calculate the concentration of the acid via a calculation similar to Worked Example 6.11. 

The pH dependence of Ks/Km is similar for step 1 and step 2 reactions as shown in Fig. 26b, but this similarity in the pH curves indicate only that the same titratable groups on the free enzyme and/or free substrate are involved in the two steps. As discussed explicitly by Usher et al. (522) the roles of the two histidines could be reversed and this would make no difference since the ratio of HE EH where these are the two singly protonated species is independent of pH. Similar ks and Ka curves for the two steps would also fail to prove identical roles for the two histidines. Since a pentacovalent species—whether it is a transient activated complex or a more stable intermediate—is common to the various alternatives, pK shifts deduced from ka curves could be the same. Both substrates are monovalent anions with low pK values so that 1 /Km, whether interpreted as an equilibrium binding value or as a function of the kinetic parameters mirroring the total occupancy of all the stable intermediates, could also be the same for both steps. The values for the reverse of step 2 would behave differently since the pj of 3 -CMP, for example, is 5.9. It should also be noted that ks/Km curves should be and are ionic strength dependent (508) in the same way that the His 12 and His 119 pK values are as observed by NMR (280). 

Use a pH meter to monitor the pH and then plot a titration curve. The center of the vertical region of the pH curve indicates the equivalence point (for example, see Figs. 8.1 through 8.5). 

A plot of the pH of the analyte solution against the volume of titrant added during a titration is called a pH curve. The shape of the pH curve in Fig. 11.4 is typical of titrations in which a strong acid is added to a strong base. Initially, the pH falls slowly. Then, at the stoichiometric point, there is a sudden decrease in pH through 7. At this point, an indicator changes color or an automatic titrator responds electronically to the sudden change in pH. Titrations typically end at this point. However, if we were to continue the titration, we would find that the pH... 

Now consider the overall shape of the pH curve. The slow change in pH about halfway to the stoichiometric point indicates that the solution acts as a buffer in that region (see Fig. 11.3). At the halfwayr point of the titration, [HA] = [A ] and pH = pfCa. In fact, one way to prepare a buffer is to neutralize half the amount of weak acid present with strong base. The flatness of the curve near pH = pKa illustrates very clearly the ability of a buffer solution to stabilize the pH of the solution. Moreover, we can now see how to determine pKa plot the pH curve during a titration, identify the pH halfway to the stoichiometric point, and set pKa equal to that pH (Fig. 11.8). To obtain the pfCh of a weak base, we find pK3 in the same way but go on to use pKa -1- pfq, = pKw. The values recorded in Tables 10.1 and 10.2 were obtained in this way. 

Figure 11.14 shows the pH curve of a diprotic acid, such as oxalic acid, H2C204. There are two stoichiometric points (B and D) and two buffer regions (A and C). The major species present in solution at each point are indicated. Note that it takes twice as much base to reach the second stoichiometric point as it does to reach the first. 

C18-0142. The amine group of an amino acid readily accepts a proton, and the protonated form of an amino acid can be viewed as a diprotic acid. The p Zg values for serine (H2 NCHRCO2 H, i = CH2 OH) are p ra(H3 N"") =9.1 and p (002 H) - 2.2. (a) What is the chemical formula of the species that forms when serine dissolves in pure water (b) If this species is titrated with strong acid, what reaction occurs (c) 10.00 mL of 1.00 M HCl is added to 200. mL of 0.0500 M serine solution. This mixture is then titrated with 0.500 M NaOH. Draw the titration curve, indicating the pH at various stages of this titration. 

Although Fig. 3.2 properly conveys the shapes of solubility-pH curves in saturated solutions of uncharged species, according to the Henderson-Hasselbalch equation, the indefinite ascendancy of the dashed curves in the plots can be misleading. When pH changes elevate the solubihty, at some value of pH, the solubihty product of the salt wiU be reached, causing the shape of the solubihty-pH curve to level off, as indicated in Fig. 3.2(a) for pH >8.38. 

Fig. 4 The logk0bs versus pH profile for each kinetic term in Eq. (38) k = 107, k2 = k2 = k4 = 107, 5 = k6 — 1 Ka — 10 6. Each number next to the curve indicates the slope of that portion of the curve.

Figure 6.10 High-throughput solubility-pH determination of chlorpromazine. The horizontal line indicates the set upper limit of solubility, where the compound completely dissolves and solubility cannot be specified. The points below the horizontal line are measured in the presence of precipitation and indicate solubility. The solubility pH curve was collected in the presence of 0.5 vol% DMSO, and is affected by the cosolvent (see text). [Avdeef, A., Cun Topics Med. Chem., 1, 277-351 (2001). Reproduced with permission from Bentham Science Publishers, Ltd.]...

Re(V) under these conditions are illustrated in Fig. 17. The curve represents the least-squares fit (.Ka values in Table II were used without compensation for possible temperature variation on values) of Eq. (22) to the data points. In Fig. 17, it is further illustrated (dashed line) that Eq. (24) holds for the data when Kal > [H+] > Ka2 and Eq. (25) (insert (a)) when Ka2 > [H+]. Note that the insert (a) shows a drawn line through the data points at higher pH values, indicating a negligible k0. Finally, at low pH values ([H+] > Kal) the exchange rate becomes independent of proton concentration according to Eq. (23). The... 

Fig. 10. (A) Far-ultraviolet CD spectra of fragment 206-316 of thermolysin at different temperatures in 20 raM Tris-HCl buffer, pH 7.2. Numbers near the curves indicate temperature in degrees Celsius. The peptide concentration was 0.2 mg/ml. (B) Temperature dependence of [0]/[0]o at 220 nm of fragment 206-316, where [0]o is the mean residue ellipiticity at 22°C. Reprinted with permission from Peptides Proceedings of the Fifth American Peptide Symposium, John Wiley Sons, Inc., 1977. Copyright by John Wiley Sons, Inc.

O O Sketch the pH curve for the titration of a weak acid with a strong base. Show the equivalence point on your sketch. Suggest an indicator that might be used, and explain your selection. 

Figure 6.8 shows three Bis of Hb [based on Table 6.2 from Imai (1982) for the same buffer conditions at 25 °C] at three different pH values of 9.1, 7.4, and 6.5. The overall appearance of these curves indicates that increasing the pH strengthens the cooperativity of the system. On the other hand, judging from the utility point of view, we see that the curve with the lowest pH has the highest utility value (which is about 0.2 this is quite small compared with values of the same system with added BPG and IHP, see below). 

Fig. 10.12 The effect of increasing amounts of goethite (surface area 51 m g ) on the electrophoretic mobility of kaolinite at various pH. The figures on the curves indicate the amount of goethite added (mg g ) (Venema and Glasauer, unpubl.).

The enzymatic activity versus pH is thus generally a bell-shaped curve as in Fig. 6. An autocatalytic effect may appear when the reaction products have an acid-base effect (often the case). A simple example is the glucose oxidase reaction, shown in Fig. 7. Notably, the rate versus product (H+) curve indicates an autocatalytic effect on the alkaline branch, that is, for pH>pH (see Fig. 6). Systems presenting analogous properties have been studied by R. Caplan et al.27 and we also learn more about them from D. Thomas in this volume. 

Fig. 5.6 Alkaline buffering of the corneal tissue. There is a considerable deviation of pH development from dilution curve, indicating buffering characteristics of the cornea...

Stearic acid films (Figures 6 and 7) show similar behavior except that the point at which the A V-A curve becomes horizontal and unaffected by compression is at higher pH—i.e., 12.3. The tt-A curves indicate con-... 

Fig. 22. Mixed mode retention. Logarithmic retention factor for oligoriboadenylic acids vs. phosphate concentration in the mobile phase, pH 6.3. The figures at the curves indicate the number of adenylyl phosphate residues in the samples. (From Ref. 74) with permission)...

FIGURE 11.13 Phenolphthalein can be used to detect the stoichiometric point of a weak acid-strong base titration, but methyl orange would give a very inaccurate indication of the stoichiometric point. The pH curves are superimposed on approximations to the color of the indicators in the neighborhoods of their end points. 

Cohen and Wilson ISO) measured the activity of the enzyme as a function of pZn over a broad range of zinc ion concentration using zinc ion buffers. The curve indicated that the activity depends upon two ionizations with constants lO7-66 and 10-10-22 in 1 M NaCl at pH 8.0, 25°C. The activity of the enzyme after the first ionization of zinc ion was 12% of the original. Using equilibrium dialysis with 65Zn(II) and 1,10-phenanthroline, Csopak 181) found that the first two zinc ions bound to the apoenzyme had a dissociation constant of 101178 in 0.1 M tris at pH 8.5 and 25°C. At the time Cohen and Wilson did their work, the enzyme was believed to contain only two zinc ions 36, 50). In the light of later developments described above, it would appear that the two ionizations might refer to pairs of zinc ions. 

It should be noted that the kinetics for the human B isoenzyme are more complicated in that the pH dependence indicates that additional groups influence the rate. Studies with the isoenzyme carboxymethylated at His-200 prepared from 13C-labelled bromoacetate show that the pH dependence of the 13C NMR signal can be fitted to a curve with two pKa values of 6.0 and 9.2, but not to a curve with a single pKa. The second group could be the imidazole side-chain of His-200. Paramagnetically shifted 13C NMR resonances in the modified Co11 human carbonic anhydrase-B have been located by a novel method 498 This should allow the confirmation of an earlier postulate that the carboxymethyl carboxylate is a ligand for zinc in the modified enzyme. 

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