Titration of Amino Acids and Peptides

Kim C. Abian Hassien Alromali George Christopher L. Cabatay Arizaldo E. Castro Weena Ross P. Dygico De La Salle University-Dasmarias Dasmarias, Cavite ABSTRACT Titration of commercial Aspartame and an unknown amino acid was demonstrated using 0.20 g of each sample and addition of small increments (0.5-1.00 ml) of 0.2 M NaOH to the acidified amino acid-water solution. The titration curves of both amino acids where plotted and their experimental pKa and pI values were determined using the graphs. Results show that the unknown amino acid yielded experimental pKa values of 1.86, 4.45, and 9.39 and a pI of 3.16. Aspartame yielded pKa values of 2.11, 3.00, and 8.25 and a pI of 2.56. Computed percentage errors for the unknown sample ranged from 4.38% to 11.00%. On the other hand, Aspartames percent error range is 10.18%-22.28%. From the data, it was identified that the unknown amino acid is Aspartic Acid. It can be inferred that pKa is a vital information for the characterization of an unknown amino acid. Moreover, discrepancies between experimental pKa and pI values can be attributed to some factors like pH reading and abrupt addition of the titrant to the acidified solutions for titration. INTRODUCTION Amino acids are biologically important building blocks of one major type of biomolecules, Proteins. They aggregate as Proteins via peptide bond formation during condensation where water molecules are eliminated (1). They are also levorotatory organic compounds whose only optically inactive member is Glycine. Basically there are 20 primary amino acids that serve as the backbone of most proteins. These 20 amino acids have three letter representations and one letter symbols summarized by the following table: Amino Acid Glycine Alanine Valine Leucine Isoleucine Proline Phenylalanine Tyrosine Tryptophan Serine Three Letter Symbol Gly Ala Val Leu Ile Pro Phe Tyr Trp Ser One Letter Symbol G A V L I P F Y W S Amino Acid Threonine Cysteine Methionine Aspartic Acid Glutamic Acid Asparagine Glutamine Lysine Arginine Histidine Three Letter Symbol Thr Cys Met Asp Glu Asn Gln Lys Arg His One Letter Symbol T C M D E N Q K R H

Table 1. 20 Basic Amino Acids

In addition to this, an amino acid is characterized by three major groups- an alpha amino group, an alpha carboxylic group, and the variable R group (3). The varying R group gives an amino acid its identity. On the other hand, the basic amino group and the acidic carboxylic acid allow internal acid-base reaction and make an amino acid an amphoteric substance. Meaning, it can act as an acid and as a base in aqueous solutions (2). Amino acids occur and crystallize from neutral dipolar ions or known as zwitterions (2).

Figure 1. Formal and Zwitterion Structures of Amino Acid Furthermore, amino acids can be classified in basis with the polarity of their R groupshydrophobic class, polar neutral class, positively charged class, and the negatively charged class (2). Other classification scheme can also be employed when categorizing these organic compounds like the type of functional group present in the variable R group. Some examples are hydroxyl (Serine), alkyl (Alanine), and sulfur containing amino acids (Cysteine). The identity of the amino acid is related to its acid-base properties. One characterization of amino acids is their respective pKa or the negative logarithm of their acid dissociation constants. The 20 basic amino acids have their respective pKa values which are helpful in identifying an unknown sample. Titremetry or Titration is a laboratory method most commonly used in studying and exemplifying the acid-base properties of these low weight protein building blocks. During titration of an amino acid, the most acidic form is achieved first before titrating it with a basic titrant until a certain pH has been reached (end point) (1). Identified pKa can also be used to determine the pH of the amino acid in Zwitterion form. The mean of these two pKa values where change is slowest is the isoelectric pH of the amino acid (1). MATERIALS AND METHODS 0.20 g of unknown amino acid and 0.20 g of Aspartame were weighed and transferred to separate beakers for titration. Afterwards, 25 ml of distilled water was added to both set-ups to facilitate dissolution of the reactants. Acidification of both solutions was accomplished via addition of small increments of 0.2 M HCl using syringe while stirring action was performed. 1 ml of the acid was added per instance and pH was measured using a digital pH meter until the solutions reached a pH of 1.5 (1). After the solutions are acidified, small increments of NaOH were slowly added until the pH reading is 11.5. Data gathered was plotted at MS Excel to come up with two titration curvesthat of commercial Aspartame and the unknown amino acid sample. pKa of both reactants was observed and the unknown amino acid was identified base on the pKa plotted. Afterwards, titremetric profiles of the both samples were drawn, and the percent error of the pKa and the pI were calculated (1).

RESULTS AND DISCUSSIONS Vol. NaOH (ml) 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 pH 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.69 1.69 1.86 1.86 1.94 2.04 2.15 2.29 2.45 Vol. NaOH (ml) 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 pH 2.88 3.12 3.33 3.55 3.76 4.06 4.45 6.80 8.76 9.15 9.39 9.72 9.99 10.29 10.68 11.12 11.45

Table 2. Titration of the Unknown Amino Acid Table 2 shows the increasing pH measurements when the acidified unknown amino acid was titrated using 0.2 M of Sodium hydroxide. It can be observed that at 11 ml of the basic titrant, the pH reading of 1.86 plotted an inflection in the curve. This is true with pH readings at 24 ml and 28 ml which are 4.45 and 9.39. These three readings were identified as the pKa values of the unknown sample. Comparing these data with the theoretical pKa values of the 20 basic amino acids, it was identified that the unknown sample is Aspartic acid. The following graph shows the samples titration curve:

TitrationCurveof Unknownno. 1
14 12 10 8 6 4 2 0 1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 Titratio n Curve of Unkno wn

Figure 2. Titration Curve of Unknown Amino Acid

From Table 3, percentage errors of 11.00%, 15.28%, and 4.38% were calculated using the experimentally obtained pKa from the graph and the theoretical pKa values of Aspartic acid. Moreover, the difference between the theoretical and experimental isoelectric pH was also calculated and it was found out that a 6.04% error exists between them. Table 3 below summarizes the percent errors of pKa and pI values for Aspartic Acid: Experimental Theoretical % Error pKa1 1.86 2.09 11.00 pKa2 4.45 3.86 15.28 pKa3 9.39 9.82 4.38 pI 3.16 2.98 6.04 Identity of the Unknown Amino Acid: Aspartic Acid Table 3. Theoretical and Experimental pKa values of Amino Acid Sample 1 Vol. NaOH (ml) 1 2 3 4 5 6 7 8 Table 4. Titration of Aspartame Table 4 shows the increasing pH measurements when the acidified Aspartame solution was titrated using 0.2 M of Sodium hydroxide. The respective inflection points determined as the experimental pKa values are 2.11 at 8 ml, 3.00 at 9.5 ml, and 8.25 at 10ml. The following graph shows the samples titration curve: pH 1.51 1.53 1.56 1.61 1.68 1.76 1.90 2.11 Vol. NaOH (ml) 9 9.5 10 10.5 11 11.5 12 pH 2.71 3.00 8.25 10.68 11.18 11.38 11.52

Titration Curve of Aspartam e

14 12 10 8 6 4 2 0 1 2 3 4 5 6 7 8 9 9.5 10 10.5 11 11.5 12 Titration Curve of Aspartam e

Figure 3 . Titration Curve of Aspartame

From Table 5 below, percentage errors between the experimental and theoretical pKa values of Aspartame were computed and resulted to a percent error range of 15.30%-22.28%. In addition to this, the pI differences were also noted and a 10.18% error was determined. These discrepancies between the experimental and theoretical pKa values of the identified amino acid sample and Aspartame can be attributed to several factors. One possible source of error is reading the pH measurements every addition of small amounts of Sodium hydroxide. Failure to push the read button from the digital pH meter will definitely result to an incorrect reading. Another factor that can be considered is the abrupt addition of the basic titrant. Sudden addition of the titrant will result to drastic changes in pH and the exact inflection points will be altered. pKa1 pKa2 pKa3 pI Experimental 2.11 3.00 8.25 2.56 Theoretical 1.83 3.86 9.82 2.85 % Error 15.30 22.28 15.99 10.18

Table 5. Theoretical and Experimental pKa values of Aspartame From the plotted graphs, we can also infer that the buffering zones for both samples are between the two inflection points where pH change is slowest. For the identified Aspartic Acid sample, the buffering zone is between the pH levels of 1.86 and 4.45 while for Aspartame, the buffering region is between 2.11 and 3.00. Moreover, these buffering zones also contain their isoelectric pH. Aspartic acid has an experimental pI of 3.16 while Aspartame has 2.56. Based on these values, both can be used as a buffer on acidic pH.

Figure 4. Experimental Titremetric Analysis of Aspart

Aspartame is an artificial sweetener used as a replacement for sugar in several drinks and several low-calorie or sugar free foods being sold in the market. Its fundamental health benefits include: regulating carbohydrate intake particularly in diabetic patients to enable them to still eat sweets without in any way affecting their bodys blood sugar; aiding in weight control since aspartame has fewer calories as compared to their sugar-flavored counterparts; and providing healthy nutritional plans since aspartame can replace or reduce the calories and sugar in beverages and foods while simultaneously maintaining a rich and great taste (4). On the other hand, despite all the possible health benefits of aspartame, it obviously also has several side effects in which physical weakness is one of the common or major one. In relation to this, based from a research, aspartame has at least 92 kinds of side effects. Aspartame can indeed pose those numerous side effects because based primarily on its characteristics it can travel to all over the body and be able to deposit in any tissues and be dissolved into solution once it is consumed. Hence, it can cause acute and immediate reactions. Among the side effects of aspartame are: eye blindness or decrease in night vision, insomnia, mental depression and anxiety, feeling of irritability, increase in blood pressure level, short duration of breath, migraines and headaches, feeling dizzy and drowsy, loss in memory and confusion, skin allergies, pain in abdomen, diarrhea, peptic ulcer, hypothyroidism, epilepsy, lyme disease, and lupus (5). REFERENCES

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Legaspi, G.A.2011. Essentials of Biochemistry Laboratory. Philippines (Evaluation copy) Lehninger, A.L.1976. Biochemistry. 2nd Ed. NY: Worth Publishers, Inc. Proteins, Peptides, and Amino Acids.(n.d.). Retrieved from http://www2.chemistry.msu.edu/faculty/reusch/VirtTxtJml/proteins.htm Retrieved on July 5, 2011 Aspartame Side Effects. 2011. Retrieved from http://sideeffectsdb.com/aspartame-side-effects/ Retrieved on July 5, 2011

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(5) Benefits of Aspartame.(n.d.). Retrieved from

http://benefitof.net/benefits-of-aspartame/ Retrieved on July 5, 2011