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Department of Pure and Applied Chemistry College of Arts and Sciences Visayas State University

Date Performed: June 8, 2017 Date Submitted: June 16, 2017

Score: ________ Prepared by: BS Chem-2

Exercise No. 1 pH and BUFFER SYSTEM

Objectives:

To calibrate the pH meter.

To choose and prepare appropriate buffer systems.

To titrate an amino acid.

Results:

Factors Affecting Buffer Capacity

  • 1. Effect of Concentration of Buffer Table 1.1. Phosphate Buffer (0.1M), pH of 7.2

Buffer Type

 

Concentration

Original pH

Actual pH

 

Buffer +

 

Change in

(M)

NaOH (2mL)

 

pH

Phosphate

0.005

7.2

5.58

 

6.07

 

0.49

Phosphate

0.05

7.2

5.77

 

7.74

 

1.97

Table 1.2. Acetate Buffer (0.1M), pH of 4.7

 

Buffer Type

 

Concentration

 

Original pH

Actual pH

 

Buffer +

 

Change in

(M)

NaOH (2mL)

 

pH

Acetate

0.005

4.7

3.09

 

3.57

 

0.48

Acetate

0.05

4.7

3.36

 

4.23

 

0.87

Table 2.1. Ratio of Dihydrogen phosphate and Monohydrogen phosphate

 

Buffer Type

Concentration

 

Expected

 

Ratio

Buffer + NaOH

 

Change

 

(M)

 

pH

(Base:Weak Acid, in moles)

(2mL)

in pH

Phosphate

 

8.2

  • 0.1 9.77:1

 

4.02

0.32

Phosphate

 

6.2

  • 0.1 0.0977:1

 

11.37

5.67

Table 2.2. Ratio of Acetic acid and Acetate

 

Buffer Type

Concentration

 

Expected

 

Ratio

Buffer + NaOH

 

Change

 

(M)

 

pH

(Base:Weak Acid, in moles)

(2mL)

in pH

Acetate

 

3.7

  • 0.1 0.0902:1

 

4.02

0.59

Acetate

 

5.7

  • 0.1 9.016:1

 

11.37

4.53

  • 2. Effect of the Ratio of the Conjugate Base to the Weak Acid

Titration of an Amino acid (Alanine)

Table 3. Amount of NaOH added into the Alanine solution

Volume of NaOH (mL)

pH of the Solution

0.00

1.49

0.50

1.58

1.00

1.61

1.50

1.65

2.00

1.69

2.50

1.74

3.50

1.80

4.50

1.88

5.50

1.97

6.50

2.05

8.50

2.16

10.50

2.33

12.50

2.62

14.50

2.82

16.50

3.15

18.50

4.91

20.50

8.71

22.50

9.14

24.50

9.46

26.50

9.69

28.50

9.91

30.50

10.03

32.50

10.23

34.50

10.46

36.50

10.69

38.50

11.01

40.50

11.41

42.50

11.78

44.50

12.00

Graph 1. Tritration of an Amino acid

Titration of Amino acid (Alanine)

50 5 0 15 10 25 35 20 45 30 14 40 pH of the Solution
50
5
0
15
10
25
35
20
45
30
14
40
pH of the Solution
0
2
4
6
8
10
12

Volume of NaOH

Discussion

First task that was performed was to identify the effect of concentration of buffer. Two different buffers namely phosphate and acetate buffers with pHs of 7.2 and 4.7, and each buffer (both with

initial concentrations of 0.1M) were diluted to prepare two solutions of each buffer of different concentrations, 0.005M and 0.05M. First case was the phosphate buffer. As it can be seen in the table, at 0.005M the pH was 5.58. After adding NaOH, the pH was 6.07, therefore, the pH has increased by

  • 0.49. At 0.05M, the pH recorded was 5.77, and after adding NaOH the pH was 7.74. So, the pH has

increased by 01.97. For acetate, at 0.005M, the pH was 3.09 and after adding NaOH the pH became

  • 3.57. At 0.05M the pH was 3.36 and became 4.23 after adding NaOH. So, the pHs at 0.005M and 0.05M

had increased by 0.48 and 0.87.

Base on the results from tables 1.1 and 1.2, the pHs of the buffers increase with increasing concentrations. Upon addition of a strong base namely sodium hydroxide, NaOH, big changes on the pHs occurred indicating that the pHs of the buffer solutions are already outside the range of their buffering capacities, due to the acids present in the buffer systems are almost used up to react with the added base causing the pHs of the buffers to rise.

As shown in table 2.1, 9.77 moles dihydrogen phosphate is to 1 mole monohydrogen ratio, in enough water, is required to make a 250 mL buffer solution with a pH of 8.2 and ratio of 0.0977 mole dihydrogen phosphate is to 1 mole monohydrogen phosphate ratio, in enough water, is required to make a buffer solution with a pH of 6.2, both were prepared from a stock phosphate buffer solution with an initial concentration of 0.1M. After adding 2 mL of NaOH into the two different buffers, the pHs were 4.2 (at pH 8.2) and 11.37 (at pH 6.2), therefore, the two pHs of the buffers changed by 0.32 and

acid is to 9.016 moles acetate ratios, in enough water, were required to make two 250 mL acetate buffer solutions with pHs of 3.7 and 5.7 respectively. Each acetate buffer was prepared from a stock acetate buffer solution with an initial concentration of 0.1M.

After adding 2 mL 0.1M NaOH, the pHs of the buffer solutions had increased, except for the one phosphate buffer (pH of 8.2) where its pH decreased after adding 2 mL 0.1M NaOH. Their pHs changed by 0.32 and 5.67 for phosphate buffers with pHs of 8.2 and 6.2, and 0.59 and 4.53 for acetate buffers with pHs of 3.7 and 5.7. As it can be seen in the values of pHs, it can be said that there were some errors committed upon preparing the buffer solutions with corresponding pHs. One error is maybe the volumes of the stock buffer solutions used to prepare the said buffers were not precise in order to achieve the required pHs and another one is improper use of the pH meter, or the glass electrode was not washed properly after dipping it from one solution to the other.

Table 3 shows the volume of NaOH added into the amino acid buffer solution, specifically alanine, resulting to a change in pH of the buffer little by little after increment addition of the strong base. By plotting the values for volumes of NaOH and pHs, the student has obtained a curve shown in graph 1. From the graph, the first pKa is 2.25 and the second pKa is 8.71 and these pka values are close to the theoretical pKas of alanine which are 2.33 (pKa1) and 9.1 (pKa2). By looking at table 3, the pH is increasing as the amino acid was being titrated with NaOH and it is evident in the titration curve in the graph. The 2.33 is the pKa of carboxylic group, which is lower than the second pKa (9.1). This means that carboxylic group is the first one to give up a proton the most readily. While the amine group is the last one to give up a proton because amine group is relatively stable with a positive charge and generally prefer to have more hydrogens attached.

Conclusion

To calibrate a pH meter, it is appropriate to use buffer solutions with pHs of 4, 7, and 10, which are the ideal pHs of buffer solutions. Rinse first the electrode with distilled water and dry it with a piece of tissue paper before placing it in a buffer solution with pH 7, 4, or 10, and rinse again with distilled and dry the electrodes with tissue paper before and after placing it different acids/bases to avoid contaminating the buffer solution and errors when obtaining the pHs of the solutions.

To have an effective buffer solution, one must make

it

out of

a

weak acid/base and its

conjugate acid/base or its salt with a pH close/equal to the pKa in order to achieve a maximum

buffering capacity.

Upon titrating an amino acid, there will be two or more pKas that can be obtain. Depending on what amino acid is used in titration because amino acid has many functional groups that can be deprotonated.

Reference(s) and Link(s):

Ferrier, Denise. Lippincott’s Illustrated Reviews Biochemistry. Sixth edition.

Answers to Questions.

1. In protein precipitation, 2 L of 5 mM buffer solution with pH 5.2 is needed in the isolation of albumin. Which among the following buffer solutions is best fitted for the said purpose? Justify your answer.

Buffer solutions

pKa

Acetate buffer

4.73

Tri-(hydroxymethyl)-aminomethane

8.08

Phosphate buffer

7.20

Acetate buffer is the one best fitted buffer because its pKa is the one closest to the needed pH of 5 mM buffer solution. If pH is close or equal to pKa, a buffer solution can achieve its maximum buffering capacity. Thus, it can absorb hydronium or hydroxide ions without a significant change in pH.

2.

Appendix

Appendix Calibration of pH Meter Prepared 0.05M and 0.005M Phopsphate Buffers Titration of Amino Acid (Alanine)

Calibration of pH Meter

Appendix Calibration of pH Meter Prepared 0.05M and 0.005M Phopsphate Buffers Titration of Amino Acid (Alanine)

Prepared 0.05M and 0.005M Phopsphate Buffers

Appendix Calibration of pH Meter Prepared 0.05M and 0.005M Phopsphate Buffers Titration of Amino Acid (Alanine)

Titration of Amino Acid (Alanine)

 

NaOH

               
 

Vol.

pH

Change Vol.

Change pH

dVol/dpH

eq vol.

pKa Vol

 

pKa

 

0.00

1.49

 

0.50

0.090

5.56

6.50

9.50

 

2.25

 

0.50

1.58

 

0.50

0.030

16.67

12.50

20.50

 

8.71

 

1.00

1.61

 

0.50

0.040

12.50

28.50

  • 0 0

 
 

1.50

1.65

 

0.50

0.040

12.50

0

  • 0 0

 
 

2.00

1.69

 

0.50

0.050

10.00

0

  • 0 0

 
 

2.50

1.74

 

1.00

0.060

16.67

0

  • 0 0

 
 

3.50

1.80

 

1.00

0.080

12.50

0

  • 0 0

 
 

4.50

1.88

 

1.00

0.090

11.11

0

  • 0 0

 
 

5.50

1.97

 

1.00

0.080

12.50

0

  • 0 0

 
 

6.50

2.05

 

2.00

0.110

18.18

0

  • 0 0

 
 

8.50

2.16

 

2.00

0.170

11.76

0

  • 0 0

 
 

10.50

2.33

 

2.00

0.290

6.90

0

  • 0 0

 
 

12.50

2.62

 

2.00

0.200

10.00

0

  • 0 0

 
 

14.50

2.82

 

2.00

0.330

6.06

0

  • 0 0

 
 

16.50

3.15

 

2.00

1.760

1.14

0

  • 0 0

 
 

18.50

4.91

 

2.00

3.800

0.53

0

  • 0 0

 
 

20.50

8.71

 

2.00

0.430

4.65

0

  • 0 0

 
 

22.50

9.14

 

2.00

0.320

6.25

0

  • 0 0

 
 

24.50

9.46

 

2.00

0.230

8.70

0

  • 0 0

 
 

26.50

9.69

 

2.00

0.220

9.09

0

  • 0 0

 
 

28.50

9.91

 

2.00

0.120

16.67

0

  • 0 0

 
 

30.50

10.03

 

2.00

0.200

10.00

0

  • 0 0

 
 

32.50

10.23

 

2.00

0.230

8.70

0

  • 0 0

 
 

34.50

10.46

 

2.00

0.230

8.70

0

  • 0 0

 
 

36.50

10.69

 

2.00

0.320

6.25

0

  • 0 0

 
 

38.50

11.01

 

2.00

0.400

5.00

0

  • 0 0

 
 

40.50

11.41

 

2.00

0.370

5.41

0

  • 0 0

 
 

42.50

11.78

 

2.00

0.220

9.09

0

  • 0 0

 
 

44.50

12.00

 

0

0

0

0

  • 0 0

 
 

Titration of Amino acid (Alanine)

 
 

20.00

18.00

6.5,18.18 28.50,16.67 12.50,10.00
6.5,18.18
28.50,16.67
12.50,10.00
 

16.00

pH of the Solution

14.00

12.00

10.00

8.00

 
  • Vol.-pH

6.00

  • dvol/dpH

4.00

 
 

2.00

0.00

 

0.00

5.00

10.00

15.00

20.00

Volume of NaOH

25.00

30.00

35.00

40.00

45.00

50.00