Biochem Postlab 2

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Dec 6, 2023

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Title: pH Buffers and Ionization of Amino Acids Name: Bianca Binns Date: 9/19/23 Lab Partners: Isabel, Sabaa, and Laila Introduction: The purpose of this experiment is to convey the changes when a pH buffer is added to a base or acid. A pH buffer is a solution that counters the large changes in pH that are caused by small aliquots of acid or base. This is because the buffer contains a mixture of a weak acid and a conjugate weak base to balance it out in order to be presented as a salt. The Henderson- Hasselbalch equations, pH = pKa + log[conjugate base] / [acid], illustrates the relationship between the pH and equilibrium between conjugate acid and base pairs. This experiment will include the technique of titration which will help determine the equilibrium constants and molecular weight of an unknown amino acid. Through titration, we will be able to create a curve on a graph based on the amount of titrant added. Materials: - Given buffer solutions o (4,7,10) - pH meter - 0.10 M HCl - 0.10 M NaOH - Distilled water - Magnetic stirring bar

- Graduated cylinder - (4) beakers Methods: - Standardize the pH meter to have the electrode be set at a range of 4 to 7. Set the electrode using the given buffer solutions. - Weigh out 250 mg of Unknown C and dissolve it in 50 mL of distilled water - Use a magnetic stirring bar to mix the solution. Stop the mixer when recording the pHs - Press “measure pH” after submerging the probe into the liquid - Add small drops of .10 M HCl until the pH of your buffer is at 2. - Record the total volume of HCl added - Titrate the solution with standardized .10 M NaOH repeating the previous steps - Record the total volume of NaOH added and the pH after each addition - When the pH reaches to 7, standardize the pH meter and the electrode to be set at the range 7 to 10 - Keep adding NaOH until the pH does not increase anymore

Results & Discussion: Section A Table 1: Preparation of Solution Expected pH Value Measured pH Value 100mM HEPES 4.8 4.01 100 mM HEPES 6.8 6.3 - Made two 100 mM acetate buffers one with a pH of 4.8 and another with a pH of 6.8 - pKa for acetic acid = 4.74 - MW for sodium acetate x H20 = 136.1 g/mol Section C Table 2: Effects of Added Acid or Base Solution Acid/ Base pH 20 mL of water HCI 2.41 20 mL of water NaOH 11.55 20 mL of buffer with a pH of 4.8 HCI 3.10 20 mL of buffer with a pH of 4.8 NaOH 4.00 20 mL of buffer with a pH of 6.8 HCI 4.01 20 mL of buffer with a pH of 6.8 NaOH 5.07 According to Table 2, there seems to be some fluctuations for when base is added to the buffers. When base is added, the pH barely increases and does not go above the pH of the made buffers. This does not mean that the buffer is ineffective, there could be other factors that affected the pH levels like the pH of the distilled water. When acid is added, the pH of the buffer becomes more acidic hence the pH level being less than its actual pH level. Moreover, the two buffers seemed to be effective because effectiveness is determined by the buffer being +/- 1 of the pKa value which in this case is 4.74 . As shown, all the pH levels that has the buffer are within the range of 3.74 and 5.74, therefore it is in fact effective.

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Section D Table 3: Effects of Dilution Solution Acid/ Base pH 2.0 mL of buffer with a pH of 4.8 18 mL of water 3.50 2.0 mL of buffer with a pH of 6.8 18 mL of water 4.41 2.0 mL of buffer with a pH of 4.8 0.1 M of HCI 2.22 2.0 mL of buffer with a pH of 6.8 0.1 M of HCI 2.38 2.0 mL of buffer with a pH of 4.8 0.1 M of NaOH 6.94 2.0 mL of buffer with a pH of 6.8 0.1 M of NaOH 6.67 To reiterate, there must be an error with some of the materials because the pH levels were very low. When the buffer was mixed with distilled water, the pH level is in the acidic range being less than its own pH level. When the buffer was mixed with NaOH, the pH level was closer to being basic that it was in Table 2. I think this is because there is less buffer this time so the basicity of the NaOH can be shown now. When the buffer was mixed with HCl, the pH level of this solution was very acidic. Overall, between both tables, the pH levels were illustrated better when it was diluted rather than a normal addition of an acid or base. However, the buffer seemed to be ineffective for two solutions, 2.0 mL of buffer of with a pH of 4.8, and 2.0 mL of 6.8, according to Table 3. This is because it went over the range of +/- 1 of the pKa value, 4.74 . It can be inferred that there may be a relationship in dilution and buffer capacity. The buffers were more consistently effective in Table 2, in comparison to Table 3.

Section E Figure 1: Titration Curve of an Unknown Amino Acid Equivalent Point #1: Points: (10, 5.12), (8,3.25) 10-8/ 5.12-3.25 1.07 Equivalent Point #2: Points: (23, 8.56), (20, 7.05) 23-20/ 8.56-7.05 1.97 Equivalent Point #3: Points: (34, 11.04), (31, 9.93) 34-31/ 11.04-9.93 2.70

Figure 2: Comparison of Amino Acids v Unknown C Molecular Weight (g/mol) pKa 1 pKa 2 pKa 3 Unknown Amino Acid 147.05 2.02 4.58 9.71 Histidine 155.2 1.82 6.04 9.17 Glutamic Acid 147.1 2.19 4.25 9.67 Lysine 146.2 2.18 8.95 10.79 Since pH = pKa, our pKa values are 2.02, 4.58, and 9.71. These values support that our unknown amino acid is glutamic acid. This is because the pKa values for glutamic acid according to figure 2 are 2.19, 4.25. and 9.67 Volume of NaOH between equivalence point 1 and equivalence point 2 = 17 mL # of moles of NaOH = (.10 M x 17 L) / (1L/ 1000 mL) # of moles of NaOH = 0.0017 # of moles of amino acid = # of moles of NaOH # of moles of amino acid = 0.0017 Mass of unknown acid C = 250 mg  0.250 g Mass of unknown acid C/ # of moles of amino acid =. Molecular weight of unknown amino acid (0.250 g)/(0.0017 mols) = 147.05 g/mol Conclusion: Overall, the purpose of this experiment was accomplished. I was able to create my set of buffers through the Henderson-Hasselbalch equation, determine the effectiveness of my buffers, and use titration to create a curve to figure out my unknown amino acid. Table 1 and 2 conveyed that my buffers were effective despite some random error. Figure 1 illustrated the pKa and equivalence points that were similar to the pKas of glutamic acid. I can infer that there is a strong relationship between pH and buffers. Buffers allows the stability and balance of the pH.

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