Biochem 215 lab 1 PDF

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Dzeneta Pajazetovic Monday 6pm February 26, 2018 Pavan Patel

Lab 1: The Regulation of pH with Buffers

Objectives: Buffers help solutions resist pH changes when acids or bases are added. The purpose of this experiment is to learn how to prepare buffers and to understand the use of buffers. There were two ways to prepare buffers using both water and Tris and phosphate buffers. Buffers contain extreme biological importance because they help resist pH change which is necessary for all biochemical processes because they only function normally when the pH remains close to 7. In this lab, both the pH meter and its electrode was calibrated. The pH of distilled water was then adjusted to 7.00. Next, 100ml of an acetate buffer (.2M) was prepared using 0.2 M acetic acid and 0.2M sodium acetate. After this is accomplished, a 100ml phosphate buffer is prepared using 100mg of sodium phosphate and 100ml of distilled water. Lastly a “Tris” buffer was titrated using HCl and NaOH to change the pH and compare the results to the titration of the distilled water alone. Procedures: The same procedure was used that was in the lab manual (pages 55-61). No changes were made to the procedure.

Results: I. Adjustment of pH meter to 7.0 pH of acid: 3.98 pH of base: 10.01 II. Initial distilled water pH: 7.06 -no adjustment needed III. Using 76mL of 0.2M acetic acid and 24mL of sodium acetate pH of solution: 4.06 IV Preparation of a phosphate buffer B: Used 105mgs of Na2PO4 Used 100ml of distilled H2O pH reading- 8.78 (8 drops of HCl added) C: Used 103mgs of Na2PO4 Used 100ml of distilled water pH reading: 4.81 (15 drops of NaOH) To raise to 1mL- 30 drops 30 more drops for 2mL D: 18 drops of HCl for pH 3.80 V. Titration of a tris buffer

B: Used 100ml of distilled water Used 104 mgs protonated- NH3+ MW- 157.6 g/mol pH- 2.50 C: Table 1

Tris-HCl NaOH added (drops) 1 5 10 11 12 13 14 15 16 17 18 19 20 21 24 29 34 39 41 43 44 45 46 47 49 51 55 60

Observed pH 2.52 2.56 3.05 3.33 5.63 6.17 6.52 6.94 7.11 7.25 7.36 7.48 7.54 7.61 7.86 8.29 8.78 10.51 10.77 10.92 10.97 11.04 11.08 11.13 11.20 11.26 11.35 11.46

Distilled water NaOH added (drops)

Observed pH

5 9 12 13 14 15 16 17 18 19 23 25 26 28 30 35

2.60 2.71 3.00 3.09 3.25 3.99 9.90 10.39 10.61 10.76 11.03 11.13 11.19 11.27 11.34 11.40

pH vs drops NaOH added 14 12 10

pH

8

Tris-HCl distilled water

6 4 2 0

0

10

20

30

40

50

60

70

Drops NaOH added

*added 5 drops to each Tris-NaOH added value so that Tris is on the right side of water*

Table 2: pH 3.0 3.5

Drops NaOH added to Tris 13 15

Drops NaOH added to distilled water 12 14

Delta drops (the difference) 1 1

4.0 4.5 5.0 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0 10.5 11.0

16 16.5 18 18 19 20 25 31 37 41 43 43 44 50

15 15 16 16 16 16 16 16 16 16 16 16 17 23

1 1.5 2 2 3 4 9 15 21 25 27 27 27 27

pH vs delta drops 12 10

pH

8 6 4 2 0

0

5

10

15

delta drops

Questions: 1)CO2+ H2O H2CO3  H+ + HCO32)

3)

0. 000017378M

20

25

30

4) 5) Sodium phosphate is monobasic because it only binds one H+ 6) The NaH2PO4 has a higher concentration of H+ so it will make the water more acidic when dissolved rather than Na2HPO4. 7) Stabilizing a buffered pH solution of 7 us to pH 4 is easier than trying to stabilize the pH at 7 because it was already set at a neutral pH. 8) In the first half of the data it takes a while before NaOH starts to increase the pH because of the initial OH that was added to the solution had a pH of 3.0 that was already neutralized by the H+ that was contained in the solution. 9) It has an exponential rise because pH is logarithmic. Logarithmic means that a change in one means a change in OH- or H+ by a factor of 10. 10) 4:5 11) It has one dissociable proton and this could be seen by the amount of plateaus in the graph. 12) The pK value is around 8.0-8.2. 13)

14) They play the best role between pH values of 6.5-8.3.

Conclusions and Discussions: As seen in graph one, the titration of Tris-HCl is increasing and the buffer is neutralizing the solution of H+ by factors of 10 up until pH 3.2. Then between 3.2 and 5.8, the buffering effect occurs. Buffer solutions are better when stabilizing pH because organic substances can produce H+ or OH- and the buffer can absorb these changes. It was also determined that changing the pH of a buffered solution is much more difficult than manipulating the pH of an unbuffered solution. The pKa of tris is around 8.0-8.2. One problem that was encountered during this experiment was the buffering of the distilled water solution. There was a dramatic jump in pH when the drops of NaOH were being added to the solution. The pH jumped from around 4 to 10 between the 9th and 10th drop. The cause for this increase is unknown but most likely due to error. The drops might have been added to quickly for the solution to adjust slowly to it. There also could have been contamination while performing

this experiment. Because of this problem, 5 drops of NaOH were added to the distilled water set of numbers because if the numbers stayed the way they were, the delta drops would have been negative and incapable of being graphed. Besides this challenge, I learned about buffers like tris, phosphate buffers, and how hard it would be to carry out experiments if there were no buffers. I also learned how to calculate buffers and how to fix your results if they are unexpected. Things like dropwise, size, and not adding enough are factors that can cause inaccurate results. There are many different ways to calculate pH, pKa, and molecular weight as well. The graphs also showed how pH is a logarithmic scale and the titration of water taught us this. Lastly this lab showed how important buffers are in biological sciences. In order for most things in our body to function they need to be at a normal pH of 7 and buffers help contain this pH without being affected by strong acids or bases. In biochemistry it is seen how pH sensitive reactions are and why we need buffers. When adjusting distilled water to a pH of 7.0, it was very simple because the distilled water that was used in the experiment already was at pH 7.06. So there was no adjusting that had to be done. When preparing the phosphate buffer, the pH of the solution had to be adjusted. The buffer was originally at a pH of 7.00 but then had to be stabilized to a pH of 4.0. To get it to a pH of 7, 16 drops of NaOH was added over a span of 4 minutes and the resulting pH was 7.05. Next 18 drops of HCl was added to this solution to get it to a pH of 4.0. This was pretty difficult to stabilize and the resulting pH was 3.80. When titrating Tris, it was pretty simple and the results were very accurate. A problem arose when distilled water was titrated because the pH jumped from 4.0 to about 10.0 suddenly in between drops. This could have been error due to contamination or the appropriate time was not accounted for in between adding drops which caused inaccurate readings....