This is the first of two consecutive labs in which the procedures will be significantly different from the lab manual. In each instance, however, you will still need to do the pre lab and read the general procedures (sections II and III especially) in the manual. You will NOT be doing the microscope procedures until next week.
Educational Objectives: to understand the concept of pH and the functioning of buffers. You should be able to calculate the pH from the hydrogen ion concentration and calculate the hydrogen ion concentration given the pH. You should understand the significance of pK and how to determine the pK from a titration of a buffer.
Introduction to pK: While this is not meant to be a substitute for a chemistry class, it is important that you have some idea of the significance of the pK value for a buffer system. Frp, lecture, you should have some understanding of the pK. In addition, it is important that you understand that the pK is the pH at which the buffer systems works best. For example, a commonly used buffer in biological experiments is HEPES. It had a pK of 8.49. This means that HEPES action as a buffer is best at pH 8.49. So if you need a solution with a pH of 8.5, for example, HEPES would be a good choice as your buffer. If, however, you need a solution with a pH of 5.0, HEPES is not a good buffer at that pH as this pH is not close to 8.49. Instead, you should select a buffer in which the pK is close to 5.0.
In this lab, you will titrate two different buffer solutions. Using the titration curves that you generate, you will estimate the pK for each buffer solution.
Experimental Objectives: to determine the pK of two buffers: acetic acid/acetate system and the TRIS buffer system.
Read and complete the pre-lab activities. Do the prelab quiz, of course. You will not be doing the microscope procedures this week – you will need to do them for Lab 4.
Read the procedures of the lab and prepare your lab note book with the Introduction and Methods and Materials, as you should every lab. The new procedures are outlined below.
Procedures:
Prepare a 0.3 M solution of sodium acetate. The formula weight for sodium acetate is 82.03 g/mole. For each titration, you will need 30 ml of acetate solution and you will be performing 3 titrations (that’s a total of 90 ml). Make enough of the 0.3 M acetate solution so that you will have sufficient volume. Before you come to lab, you will need to calculate how much sodium acetate you will need to weight out to prepare a sufficient volume of acetate solution for the three titrations. If you do not remember how to do this, reread lab 2.
Titrate 30 ml of the sodium acetate solution. Do this by adding 1 ml (what is the best way to deliver 1 ml?) of 1.0 M HCl solution and record the pH. Continue adding HCl (one ml at a time) until the pH is below 3.0.
Repeat step 2 with a fresh 30 ml of sodium acetate. Then repeat a third time.
Prepare a 0.3 M solution of TRIS and titrate 30 ml using the same procedures as with the sodium acetate solutions. The formula weight for TRIS is 121.14 g/mol.
The purpose of the lab is to estimate the pK of acetate and of TRIS. How to do the analysis is outlined in the next section. You performed three titrations for each buffer. This allows you to determine the pK three different times for each buffer. This will entail having three different graphs for each buffer. Your results should include each graph. In addition, you must indicate on the graph how you found the pK. It should be something like that shown in Figure 1, however, the lines used to find the pK may be drawn in with a ruler and pen. They must be clearly labeled. Also, the y axis must be sufficiently labeled to accurately read the pK. Note in Figure 1, the scale is set at 0.2 pH units. You scales must no farther apart than this. Note: to change the scales on a graph in Excel, right click on the y axis and select the ‘Scales" tab.
Your report should also include a table showing the values of the pK for each buffer and the average pK for each buffer.
You must also include a table indicating what volume of HCl was added to completely titrate the buffers. Since you performed three titrations for each buffer, you should have three volumes for each buffer. Include a table showing the amount of HCl needed for each buffer as ell asthe average volume of HCl for each buffer.
Feel free to include any other suitable statistics you feel should be included.
Be sure to indicate what the pK means with regard to these buffers and explain the results dealing with the volume of HCl.
Sample Data Analysis:
The following data was obtained when titrating the buffer X. Table 1 contains the actual data from the titration; Figure 1 is a plot of the same data.
|
Table 1: Titration 1 of Buffer X |
|
|
ml of 1.0 HCl added |
pH |
|
0 |
8.5 |
|
1 |
7.5 |
|
2 |
7.1 |
|
3 |
6.9 |
|
4 |
6.7 |
|
5 |
6.6 |
|
6 |
6.5 |
|
7 |
6.3 |
|
8 |
6.1 |
|
9 |
5.7 |
|
10 |
4.5 |
The purpose of the titration was to estimate the value of the pK for Buffer X. Look at the graph, note that at the beginning and at the end of the titration, the pH changes more rapidly upon addition of the acid than it does in the middle (this was discussed in your pre-lab). The titration is complete when the pH begins to change rapidly again (compare the change in pH between 9 - 10 ml of HCl compared to 8-9 ml). Theoretically, the pH at which the buffer is half titrated is the pK. To estimate the value of the pK from the graph, determine how many milliliters of acid were required to reach the endpoint and completely titrate Buffer X In this instance, it took 10 ml to reach the endpoint. Divide this volume by 2, in this instance, 10/2 = 5. Next, on the graph, find the point on the x axis that represents 5 ml (or one half of the volume of HCl) and draw a vertical line perpendicular to the x axis so that the line goes through the titration curve (this is depicted as an arrow in Figure 1. Mark the point where the line you drew intersects your titration curve, then draw a horizontal line (parallel to the x axis) from the intersection point to the y-axis (see the arrow in Figure 1). The value of the y axis at this point is the pK, in this instance, you should see that the pK is 6.6 (this is the pH where the horizontal line intersects the y axis).
Real Data:
The following figure was taken from a recent paper. It shows the change in ionization of a protein as a function of pH. You should be able to explain what is going on with the protein (remember that the only two ionizable groups are amino groups and carboxyl groups.
