Novaspec Spectroscopy

The cuvette should remain in the waterbath for at least three minutes for the solution's temperature to equilibrate.

Use
Nova
Spec

Determination of ΔG°, ΔH°, and ΔS°
for the acridine / acridinium ion equilibrium.

In this experiment you will determine the equilibrium constant for the acid dissociation of acridinium ion at 10°C, Room Temperature (25°C), and 40°C. You will use these results to calculate ΔG° for the reaction at 25°C, and to calculate the average values of ΔH° and ΔS° for the reaction between 10°C and 40°C.

The equilibrium constant for the acid dissociation is defined as

K_a = [acridine][H₃O⁺] / [acridinium ion]

We can determine [H₃O⁺] by measuring the solution's pH. The values for the [acridine] and [acridinium ion] will be determined via spectrophotometry.

We have created five aqueous acridine solutions of differing pH. When we speak of an acridine solution, we implicitly understand that the original acridine is now in equilibrium with acridinium ion. Each solution was made by pipetting 5.00 mL of 0.002 M aqueous acridine solution into a 50.00 mL volumetric flask. The flasks were then filled to the mark with solutions of differing pH.

First we need to determine room temperature. A thermometer has been carefully rinsed with deionized water and dryed with a paper towel and has been inserted into the acidic acridine solution. Record the temperature of this solution to 0.1 degrees. We will assume that the remaining solutions are at the same temperature. When you are done, press the 'done' icon.

Now that we know the temperature of the acridine solutions we will measure the pH of those three that are buffered at different pHs. We will assume that the pH of the strong acid solution is ~2 and that the pH of the strong base solution is ~12.

We will measure the pH of the remaining three acridine solutions using a pH meter.

The pH meter uses an electrode to sense the concentration of H₃O⁺ in aqueous solutions. In order to give accurate results, the pH meter must be calibrated using a solution with a known pH. For this experiment we will calibrate the pH meter using a standard buffer solution prepared to have a pH of 7.00. Following the calibration, we will use the pH meter to measure the pH of our three buffered acridine solutions.

To begin the calibration process, press the 'CAL' button. This will place the meter into calibration mode. Once the display settles, use the 'Up' and 'Down' buttons to set the target pH of 7.00. Press 'Yes'.

Next, select the reference solution and then lower the probe. Wait about 10 seconds and then press 'Yes' to complete the calibration.

Once you are completely done, press the 'Done' icon in the lower right hand corner. Note that you can redo the calibration at any time.

READY

CAL

YES

Lower pH Probe

Raise pH Probe

Select pH 7.00 Buffer

Select Buffer '1'

Select Buffer '2'

Select Buffer '3'

Rinse the pH probe using deionized water initially and each time you switch solutions, then dry with a clean tissue.

7.00

CALIBRATING

SLP

Now that the pH meter is calibrated we are ready to measure the pH of the three buffer solutions. You saw during the calibration that it is important to clean the pH probe before use and between samples. Failing to do so could contaminate your solutions and give false readings.

Select each buffer solution in turn and record the measured pH of each. When you are done, press the 'Done' icon.

When you are done using the pH meter it is important to clean and dry the probe. Also you must replace the cap that covers the probe tip. If you fail to do this, the probe will dry out and become useless.

Before continuing, please make sure that you have measured and recorded the pH of each of the three buffer solutions. Once you pass this point by pressing the 'next' button, you will be unable to make any further pH measurements.

The next step of the experiment involves determining the acridine and acridinium ion concentrations in each of the three buffer solutions. This will be done using a NovaSpec spectrophotometer.

You will measure the absorptivity of the five solutions using light with a wavelength of 403 nm. The Beer-Lambert law relates the observed absorbance to the concentration of the dissolved species.

A = ε C ℓ

Consult your lab manual for a detailed explanation of how the concentrations are determined from the absorbance measurements.

A spectrophotometer works by shining light of a particular wavelength through a sample holder called a cuvette which holds our solution. The instrument shows how much of the light is absorbed. In our case, part of this absorbtion is due to the acridine and acridinium ion. But some light is also absorbed by the water in which the acridine was dissolved, and also by the material used to make the cuvette.

Our first task will be to determine how much light is absorbed by the cuvette and water.

Cuvette 'A' Buffer 1

Cuvette 'B' Deionized Water

Fill
Cuvette
'A'

Fill
Cuvette
'B'

Use
Nova
Spec

Warm
Cuvette
'A'

Cool
Cuvette
'A'

Warm
Cuvette
'B'

Cool
Cuvette
'B'

Select a solution with which to fill the cuvette

Deionized
Water

Acidic
Solution

Buffer
Solution
'1'

Buffer
Solution
'2'

Buffer
Solution
'3'

Basic
Solution

Work Sheets - Do Not Hand In

Absorbance Readings

Partner's blank correction:

0.023

	Cold		Room Temperature		Hot
Measured Temperature:	10.0C		10.0C		10.0C
A_{buffer 2}	0.023	0.023	0.023	0.023	0.023	0.023
average	0.023		0.023		0.023
corrected	0.023		0.023		0.023
A_{buffer 3}	0.023	0.023	0.023	0.023	0.023	0.023
average	0.023		0.023		0.023
corrected	0.023		0.023		0.023
A_base	0.023	0.023	0.023	0.023	0.023	0.023
average	0.023		0.023		0.023
corrected	0.023		0.023		0.023

You have been given two cuvettes. Each cuvette has two optically clear sides through which the light shines. You should only handle the cuvette by the rough, non-clear sides. Fill each of the cuvettes with deionized water. Use the two 'Fill Cuvette' buttons to do this.

Once you have filled the two cuvettes, the 'Use NovaSpec' button will appear. Press it to continue.

Use the + and - buttons on the NovaSpec to set the frequency of the light used to 403nm.

Open the sample holder lid and insert 'cuvette A' then close the lid. The absorbance reading is due to the cuvette and the water solvent. Press the 'set ref' button to treat this reading as zero (like taring a balance). Next, remove the sample and insert 'cuvette B'.

When you've completed these steps press the 'done' button.

If the reading is positive, then 'cuvette A' is our reference and the current reading is our blank correction. We would use 'cuvette B' for all further measurements.

If the reading is negative then 'cuvette B' is the reference so press 'set ref' again. Reinsert 'cuvette A' and record that reading as the blank correction. In this case we would use 'cuvette A' for further measurements.

Once you've determined the reference cell and recorded the blank correction, press the 'done' button.

Remove the cuvette that is currently in the NovaSpec and close the lid. Press the 'Leave NovaSpec' button to return to the lab bench.

At this point you are ready to start measuring the absorbances of the acridine solutions. We will guide you through the process for one sample and will then leave you to finish the rest. Remember to use the cuvette which is not the reference.

Fill the non-reference cuvette with the acidic acridine solution and press the 'Use NovaSpec' button.

There is some variability in positioning when inserting a cuvette. If you remove and insert a cuvette, the reading you obtain will vary slightly. To reduce this error you should insert the cuvette, take an absorbance reading, and then remove the cuvette two times. By averaging the different readings, you will obtain a more accurate value.

When you have recorded the two measurements, remove the cuvette, close the lid, and press the 'Leave NovaSpec' button.

In order to complete the experiment we need to determine the temperature dependence of the acridine / acridinium ion equilibrium. We will do this by measuring the absorbance of each of our solutions at both a higher temperature and a lower temperature. We will assume that the absorbance of the buffer solutions does not change significantly.

We'll begin by warming the solution in our non-reference cuvette.

Press the 'warm cuvette' button to continue.

We will warm the solution in the cuvette by swirling it in a bath of warm water. It takes about 3 minutes for the temperature to equilibrate. For this computer lab you can assume that this time has already elapsed.

Record the temperature of the water bath and then press the 'Use NovaSpec' button.

Insert the cuvette and measure the absorbance two times. It is important to work efficiently since the temperature of the solution in the cuvette will begin to drift immediately.

When you are done, remove your cuvette, close the lid and press the 'Leave NovaSpec' button.

Next we will cool the solution in the cuvette and measure its absorbance.

Press the 'Cool Cuvette' button for the non-reference cuvette.

Again, the cuvette is swirled in a bath of cold water for at least three minutes. Record the temperature of the waterbath and then press the 'Use NovaSpec' button.

Measure the absorbance of the cooled cuvette two times, just as we have done previously.

When you are done, remove the cuvette, close the lid, and press the 'Leave NovaSpec' button.

By this point you should have measured the absorbance of the acidic acridine solution at three different temperatures.

You should now repeat these steps with buffer solution 1.

The results for the remaining three solutions have already been recorded by a classmate. You can choose to use their results or record you own. When you choose 'Fill Cuvette' there is a yellow note on which you can click to see the classmate's results.

You should now proceed to repeat these steps with the buffer solution 1, and any other solutions for which you would like to record results.

Fill your cuvette with a solution.
Measure its absorbance at room temperature.
Warm the cuvette and measure the absorbance.
Cool the cuvette and measure the absorbance.

When you are completely finished, press the 'done' icon.

If you are completely done measuring the absorbance of the required acridine solutions at each of the three temperatures, press 'All Done'. If you'd like to continue working, press 'Continue Working'.

Some Questions to Consider

A figure in your lab manual shows the absorption spectrum of Acridine and Acridinium Ion. Can you explain why you operated the spectrophotometer at 403nm?

Consider the Acridine/Acridinium Ion equilibrium. Should the absorbance at 403nm increase or decrease with increasing pH?

Pay attention to the trend in K_a with temperature. Can you explain whether or not this trend is reasonable?

Record the absorbance data for the buffer solution 1 and any other solutions for which you'd like to record data. You can use your classmate's data for the final three solutions.

Fill your cuvette with a solution.
Measure its absorbance at room temperature.
Warm the cuvette and measure the absorbance.
Cool the cuvette and measure the absorbance.

When you are completely finished, press the 'done' icon.