Determination of an unknown amino acid from a titration curve

Abstract Experiment 11 used a titration curve to determine the identity of an unknown amino acid. The initial pH of the solution was 1.96, and the pKa's found experimentally were 2.0, 4.0, and 9.85. The accepted pKa values were found to be 2.10, 4.07, and 9.47. The molecular weight was calculated to be 176.3 while the accepted value was found to be 183.5. The identity of the unknown amino acid was established to be glutamic acid, hydrochloride. Introduction Amino acids are simple monomers which are strung together to form polymers (also called proteins). These monomers are characterized by the general structure shown in figure 1. Fig. 1 Although the general structure of all amino acids follows figure 1, the presence of a zwitterion is made possible due to the basic properties of the NH2 group and the acidic properties of the COOH group. The amine group (NH2) is Lewis base because it has a lone electron pair which makes it susceptible to a coordinate covalent bond with a hydrogen ion. Also, the carboxylic group is a Lewis acidic because it is able to donate a hydrogen ion (Kotz et al., 1996). Other forms of amino acids also exist. Amino acids may exists as acidic or basic salts. For example, if the glycine reacted with HCl, the resulting amino acid would be glycine hydrochloride (see fig. 2). Glycine hydrochloride is an example of an acidic salt form of the amino acid. Likewise, if NaOH were added, the resulting amino acid would be sodium glycinate (see fig. 3), an example of a basic salt form. Fig. 2 Fig. 3 Due to the nature of amino acids, a titration curve can be employed to identify an unknown amino acid. A titration curve is the plot of the pH versus the volume of titrant used. In the case of amino acids, the titrant will be both an acid and a base. The acid is a useful tool because it is able to add a proton to the amine group (see fig. 1). Likewise the base allows for removal of the proton from the carboxyl group by the addition of hydroxide. The addition of the strong acid or base does not necessarily yield a drastic jump in pH. The acid or base added is unable to contribute to the pH of the solution because the protons and hydroxide ions donated in solution are busy adding protons to the amine group and removing protons from the carboxyl group, respectively. However, near the equivalence point the pH of the solution may increase or decrease drastically with the addition of only a fraction of a mL of titrant. This is due to the fact that at the equivalence point the number of moles of titrant equals the number of moles of acid or base originally present (dependent on if the amino acid is in an acidic or basic salt form). Another point of interest on a titration curve is the half-equivalence point. The half-equivalence point corresponds to the point in which the concentration of weak acid is equal to the concentration of its conjugate base. The region near the half-equivalence point also establishes a buffer region (Jicha, et al., 1991). (see figure 4). Fig. 4 The half-equivalence point easily allows for the finding of the pKa values of an amino acid. A set pKa values can be extremely helpful in identifying an amino acid. Through a manipulation of the Henderson-Hasselbalch equation, the pH at the half-equivalence point equals the pKa. This is reasoned because at the half-equivalence point the concentration of the conjugate base and the acid are equal. Therefore the pH equals the pKa at the half-equivalence point (see figure 5.) Fig. 5 [base] pKa= pH - log ------- [acid] [base] log -------- = log 1 = 0 [acid] therefore, pH = pKa However, many substances characteristically have more than one pKa value. For each value, the molecule is able to give up a proton or accept a proton. For example H3PO4 has three pKa values. This is due to the fact that it is able to donate three protons while in solution. However, it is much more difficult to remove the second proton than the first. This is due to the fact that it is more difficult to remove a proton from a anion. Furthermore, the more negative the anion, the more difficult to remove the proton. The trapezoidal method can be employed to find the equivalence points as shown if figure 6. The volume of titrant between two equivalence points is helpful in the determination of the molecular weight of the amino acid. Fig. 6 The purpose of experiment 11 is to determine the identity of an unknown amino acid by analyzing a titration curve. The experiment should lend the idea that the following may be directly or indirectly deduced from the curve-- the equivalence and half equivalence points, pKa values, the molecular weight and the identity of the unknown amino acid. Experimental The pH meter was calibrated and 1.631 grams (.0089 moles) of the unknown amino acid was weighed and placed in a 250-mL volumetric flask. About 100 mL of distilled water was added to dissolve the solid. The flask was gently swirled and inverted to insure a complete dissolution of the solid. The solution was diluted with distilled water to the volume mark on the flask. Then, one buret was filled with 0.100 M HCl stock solution and another buret was filled with 0.100 M NaOH. A pipet was used to add 25.00 mL of the unknown amino acid solution to a 100-mL beaker. The solution's initial pH was established to be 1.96 by the pH meter. The electrode was left in 100-mL beaker with the unknown amino acid solution. In the accurate titration curve, the acid was added in 0.5 mL increments until the pH of the solution was 1.83. As the titrant was added the pH of the solution was recorded on a data sheet. Also, a graph of pH versus the mL of titrant added was plotted. After the addition of the acid, a new 25 mL aliquot of unknown solution was added to a clean 100-mL beaker. The base was then used to titrate the solution. It was added in 0.20 to 1.0 mL increments depending on the nature of the curve. (The nature of the curve was somewhat expected because previously an experimental titration curve was established. This curve used increments of up to 2.0 mL.) The base was added until the pH reached 12.03. Results Table 1 shows the pH endpoints for both the titration with the acid as well as with the base. It also shows the initial pH. Table 1 also shows the experimentally determined and accepted molecular weight and pKa values for the glutamic acid, hydrochloride. Tables 2 and 3 show the amounts of base and acid added to the unknown solution (respectively) and the pH which corresponds to that amount. Figures 7 and 8 represent the exploratory titration and the accurate titration curves (respectively). Figure 9 represents the structure of the unknown amino acid, glutamic acid, hydrochloride. Table 1 pH of endpoints pKa values (experimental) pKa values (accepted) initial pH Molecular weight identity of unknown 1.83 2.0 2.10 1.96 176.3 (expt.) Glutamic acid, hydro-chloride 12.03 4.0 4.07 183.5 (accepted) 9.85 9.47 Table 2 Accurate Titration for NaOH Fig.9 total mL of 0.10 M NaOH pH of solution 0.00 1.96 1.0 2.05 3.0 2.26 5.0 2.5 7.0 2.84 9.0 3.28 10.0 3.53 11.0 3.77 13.0 4.14 14.0 4.39 15.0 4.56 15.5 4.66 16.0 4.78 16.5 4.93 17.0 5.13 17.5 5.63 17.7 5.99 17.8 6.52 18.0 7.93 18.2 8.18 18.4 8.50 18.5 8.56 19.0 8.83 21.0 9.44 22.0 9.62 23.0 9.82 23.5 9.93 24.0 9.98 24.5 10.12 25.0 10.21 25.5 10.37 26.0 10.52 26.5 10.69 27.0 10.86 27.5 11.06 28.0 11.22 28.5 11.37 29.0 11.41 29.5 11.53 30.0 11.58 31.0 11.71 33.0 11.85 36.0 12.03 Table 3 Accurate Titration for HCl total mL of 0.10 M HCl pH of solution 0.00 1.96 0.5 1.93 1.0 1.91 1.5 1.87 2.0 1.85 2.5 1.83 Discussion The initial pH of the unknown solution was 1.96. This information was helpful in determining the identity of the unknown amino acid because only a three of the nine unknowns were acidic salts. (Acidic salt forms of amino acids are capable of having pH values of this degree.) However, more information was required before the determination could be conclusive. The unknown produced three equivalence points and therefore, three pKa values. Therefore, one of the three remaining amino acids one could be omitted from the uncertainty, because it contained only two pKa values. However, after examining the pKa values of the unknown, it was apparent that they were remarkably similar to those of glutamic acid, hydrochloride. The unknown's pKa values were 2.0, 4.0, and 9.85, while the glutamic acid's pKa values were 2.10, 4.07, 9.47. At this point, the identity of the amino acid was conclusive. However, as a precautionary measure, the molecular weight of the amino acid was calculated and found to be 176.3 amu. The calculated value corresponds well with the known value of 183.5 amu. There are a few errors that can be held accountable for the small deviation from the accepted values. First, the pH meters never reported a definite value; most times the meter would report a floating number. Therefore, one have no way of knowing which reported pH was more correct. Also, the method by which the equivalence points was extremely crude. It called for a series of rough of estimations. These estimations then led to the equivalence point. Then the use of the equivalence point was used to determine the half-equivalence point. This point was then used to find the pKa. The deviance from accepted values of the pKa values occur because of the compounded series of crude estimates which were required. Likewise, the deviance of the calculated molecular weight can be attributed to these crude vehicles, because the change in volume (between equivalence points) were used in calculation. Conclusion The identity of an unknown amino acid was determined by establishing a titration curve. The equivalence and half-equivalence point, the pKa values, and the molecular weight were directly or indirectly found through the titration curve. The equivalence points were found through a crude method known as the trapezoidal method. The establishment of the equivalence points gave rise to the half equivalence points and the D volume (used in calculating the molecular weight). The half-equivalence points were directly used to find the pKa values of the unknown. The molecular weight could also be calculated. This data led to the determination of the identity of the unknown amino acid--glutamic acid, hydrochloride.