5.1.11: Making buffers for enzyme assay
- Page ID
- 242459
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\(\newcommand{\avec}{\mathbf a}\) \(\newcommand{\bvec}{\mathbf b}\) \(\newcommand{\cvec}{\mathbf c}\) \(\newcommand{\dvec}{\mathbf d}\) \(\newcommand{\dtil}{\widetilde{\mathbf d}}\) \(\newcommand{\evec}{\mathbf e}\) \(\newcommand{\fvec}{\mathbf f}\) \(\newcommand{\nvec}{\mathbf n}\) \(\newcommand{\pvec}{\mathbf p}\) \(\newcommand{\qvec}{\mathbf q}\) \(\newcommand{\svec}{\mathbf s}\) \(\newcommand{\tvec}{\mathbf t}\) \(\newcommand{\uvec}{\mathbf u}\) \(\newcommand{\vvec}{\mathbf v}\) \(\newcommand{\wvec}{\mathbf w}\) \(\newcommand{\xvec}{\mathbf x}\) \(\newcommand{\yvec}{\mathbf y}\) \(\newcommand{\zvec}{\mathbf z}\) \(\newcommand{\rvec}{\mathbf r}\) \(\newcommand{\mvec}{\mathbf m}\) \(\newcommand{\zerovec}{\mathbf 0}\) \(\newcommand{\onevec}{\mathbf 1}\) \(\newcommand{\real}{\mathbb R}\) \(\newcommand{\twovec}[2]{\left[\begin{array}{r}#1 \\ #2 \end{array}\right]}\) \(\newcommand{\ctwovec}[2]{\left[\begin{array}{c}#1 \\ #2 \end{array}\right]}\) \(\newcommand{\threevec}[3]{\left[\begin{array}{r}#1 \\ #2 \\ #3 \end{array}\right]}\) \(\newcommand{\cthreevec}[3]{\left[\begin{array}{c}#1 \\ #2 \\ #3 \end{array}\right]}\) \(\newcommand{\fourvec}[4]{\left[\begin{array}{r}#1 \\ #2 \\ #3 \\ #4 \end{array}\right]}\) \(\newcommand{\cfourvec}[4]{\left[\begin{array}{c}#1 \\ #2 \\ #3 \\ #4 \end{array}\right]}\) \(\newcommand{\fivevec}[5]{\left[\begin{array}{r}#1 \\ #2 \\ #3 \\ #4 \\ #5 \\ \end{array}\right]}\) \(\newcommand{\cfivevec}[5]{\left[\begin{array}{c}#1 \\ #2 \\ #3 \\ #4 \\ #5 \\ \end{array}\right]}\) \(\newcommand{\mattwo}[4]{\left[\begin{array}{rr}#1 \amp #2 \\ #3 \amp #4 \\ \end{array}\right]}\) \(\newcommand{\laspan}[1]{\text{Span}\{#1\}}\) \(\newcommand{\bcal}{\cal B}\) \(\newcommand{\ccal}{\cal C}\) \(\newcommand{\scal}{\cal S}\) \(\newcommand{\wcal}{\cal W}\) \(\newcommand{\ecal}{\cal E}\) \(\newcommand{\coords}[2]{\left\{#1\right\}_{#2}}\) \(\newcommand{\gray}[1]{\color{gray}{#1}}\) \(\newcommand{\lgray}[1]{\color{lightgray}{#1}}\) \(\newcommand{\rank}{\operatorname{rank}}\) \(\newcommand{\row}{\text{Row}}\) \(\newcommand{\col}{\text{Col}}\) \(\renewcommand{\row}{\text{Row}}\) \(\newcommand{\nul}{\text{Nul}}\) \(\newcommand{\var}{\text{Var}}\) \(\newcommand{\corr}{\text{corr}}\) \(\newcommand{\len}[1]{\left|#1\right|}\) \(\newcommand{\bbar}{\overline{\bvec}}\) \(\newcommand{\bhat}{\widehat{\bvec}}\) \(\newcommand{\bperp}{\bvec^\perp}\) \(\newcommand{\xhat}{\widehat{\xvec}}\) \(\newcommand{\vhat}{\widehat{\vvec}}\) \(\newcommand{\uhat}{\widehat{\uvec}}\) \(\newcommand{\what}{\widehat{\wvec}}\) \(\newcommand{\Sighat}{\widehat{\Sigma}}\) \(\newcommand{\lt}{<}\) \(\newcommand{\gt}{>}\) \(\newcommand{\amp}{&}\) \(\definecolor{fillinmathshade}{gray}{0.9}\)Lab #11 Preparation and properties of a buffer
CHEM 0103 Chemistry of the life sciences (follow-up will be lab #12, enzyme activity)
This lab consists of making a buffer by adding either HCl or NaOH to a solution of ammonium acetate. The ammonium ion (a weak acid with pKa near 9) together with its conjugate base, ammonia, can act as a buffer according to this equilibrium
\[\ce{NH4+ <=> NH3 + H+}\tag{1}\]
Likewise, the acetate ion a weak base together with its conjugate acid, acetic acid, pKa near 5, can act as a buffer according to this equilibrium:
\[\ce{CH3COO- + H+ <=> CH3COOH}\tag{2}\]
Task 1:
Make a buffer: Combine ammonium acetate (weak acid/base, c = 0.1 mol/L) with HCl (strong acid, c = 0.1 mol/L) or NaOH (strong base, c = 0.1 mol/L) at the following volumes:
| Group | Label | Volumes to be mixed |
| H | 5:5 recipe | 10 mL ammonium acetate, 5 mL HCl, add water to fill to 40 mL |
| N | 9:1 recipe | 10 mL ammonium acetate, 1 mL HCl, add water to fill to 40 mL |
| C | 1:9 recipe | 10 mL ammonium acetate, 9 mL HCl, add water to fill to 40 mL |
| O | 5:5 recipe | 10 mL ammonium acetate, 5 mL NaOH, add water to fill to 40 mL |
| P | 1:9 recipe | 10 mL ammonium acetate, 1 mL NaOH add water to fill to 40 mL |
| S | 9:1 recipe | 10 mL ammonium acetate, 9 mL NaOH, add water to fill to 40 mL |
Think about what tools you will use to measure the volumes accurately. Once you made the buffer, estimate the pH with pH paper, and accurately measure it using the pH electrode. Keep this buffer in a 50 mL tube, labeled with your group name, and the pH measured with the pH electrode. You will use this solution for tasks 2, 3, and 4, and next week for the enzyme assay lab.
Task 2:
Dilution test: Take 2 mL of buffer from task 1, add 18 mL of water, and measure pH with the pH electrode. This is best done in a second 50 mL tube. After you measured the pH, you can discard the solution in the sink, and rinse the
Task 3:
Adding acid test: Take 10 mL of buffer from task 1, add 100 uL of HCl (c=0.1 mol/L) to buffer, and measure the pH with the pH electrode. How much did it change?
Task 4:
Adding base test: Take 10 mL of buffer from task 1, add 100 uL of NaOH (c=0.1 mol/L) to buffer, and measure the pH with the pH electrode. How much did it change?
Task 5:
Analyze your results, answering the following question:
- What are the weak acid and conjugate base in your solution that form the buffer, i.e. is your buffer based on reaction 1 or reaction 2? The answer will be different depending whether your recipe asked you to add HCl or NaOH.
- Does your buffer contain more weak acid or more weak base of the buffering substance. What is the ratio of your weak acid and conjugate base in the buffer you made? How is that reflected in the name of the recipe shown in the table above?
- How did the pH change when you diluted your buffer? Did the pH move toward more neutral, or away from it? What pH would you expect for an extreme dilution (one drop of your buffer into a swimming pool of pure water at pH 7)?
- Comparing pH changes in task 3 and task 4, did your buffer protect better against adding acid or adding base?
- Looking at everyone's data (this is for the reflection part), which group had the best buffer?