Lab 2 - Bromothymol Blue Equilibrium PDF

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Chemistry 163

Equilibrium Constant of Bromothymol Blue Purpose: In this experiment, you will determine the equilibrium constant, Ka, for the acid-base indicator bromothymol blue from spectrophotometric data. Although you will not carry out the experiment, you will receive a set of data that has been collected for you. You will then analyze the data and prepare a report of results.

Learning Objectives: Upon successful completion of this laboratory experiment, you should be able to: -

Determine the most appropriate wavelength of maximum absorbance (λmax) to study a compound that changes color during a reaction. Use spectrophotometric data and Beer’s Law to calculate the concentration of a compound in solution. Calculate the equilibrium constant for an acid-base indicator. Confirm that the equilibrium constant does not depend on reactant and product concentrations.

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Background Indicators are substances whose solutions change color due to changes in pH. They are usually weak acids or bases, but their conjugate base and acid forms have different colors due to differences in their absorption spectra. Indicators typically have complicated structures, as you can see in the two forms of bromothymol blue at the bottom of this page. For simplicity, we will represent the formula for the acid form of the indicator by the formula HB-, the base form by the formula B2- and its ionization in aqueous solution by the equilibrium equation: HB−(aq) + H2O(l) ⇌ B2-(aq) + H3O+(aq) When bromothymol blue is in its acidic form it is yellow, and when it is in its basic form it is blue. Because the acidic and basic forms of the indicator are different colors, the equilibrium constant (Ka) for bromothymol blue can be determined using a spectrophotometer.

SO3

SO3

Br

+

O

Br

H 2O

OH

+

O

+

H3 O

O

Br

Br

BromothymolBlue (acidic form) yellow

BromothymolBlue (basic form) blue color

HB-

B2-

Note that the acidic form is a negatively charged anion, and that the base form becomes doubly Page 1

Last Modified 1/25/2020

negative after loss of a proton. Sometimes the acid form of an indicator is neutral, and other times it has a charge, as in this case. The equilibrium constant for this reaction (Ka) is then: ฀฀฀ ฀ =

[฀฀2− ][฀฀3 ฀฀+ ] [฀฀฀฀− ]

In this experiment we will actually calculate the pKa, which is just another way that chemists represent the equilibrium constant. pKa = -log(Ka). Note that stronger acids have lower pKa values.

Method To determine the Ka we must measure the equilibrium concentration of all three species. Here is how that will be done. The hydronium ion concentration will be maintained at a constant value by making up solutions of the indicator in a buffer. A buffer, as you will soon learn, is a solution that maintains a constant pH. So if the indicator is prepared using a buffer of pH 6, the hydronium ion concentration will be constant at 10-6 M even as the concentrations of the indicator and its conjugate base fluctuate. To prepare the solutions, a stock solution of the indicator will be used. As the indicator dissociates to form its conjugate base, the total concentration of the weak acid and the conjugate base remain constant. That means if we can measure the equilibrium concentration of one form of the dye – which we will do spectrophotometrically – we will be able to calculate the concentration of the other form of the dye by subtraction. Let’s look at an example to illustrate this. Imagine that a stock solution of the dye with a concentration of 55.0 µM is diluted x 15/50 with a pH 6 buffer. As noted above, this means that the hydronium concentration will remain constant at 10-6 M regardless of any additional hydronium ion that is gained or lost as a result of the equilibrium. HBI



55.0 ฀฀฀฀ ฀฀

15 50

= 16.5 ฀฀฀฀

H3O+

+ B2-

Total M of HB- + B2-

10-6 M

0

16.5 ฀฀฀฀ + 0 = ฀฀฀฀. ฀฀ ฀฀฀฀

C

−฀฀

−฀฀

+฀฀

E

16.5 − ฀฀

10-6 M *

16.5 + ฀฀

(16.5 − ฀฀) + (16.5 + ฀฀) = ฀฀฀฀. ฀฀ ฀฀฀฀

* Remember, hydronium ion concentration is constant in a buffered solution. Observe that- in this example - the sum of the acid and base forms of the dye will always be 16.5 µM, even as the amount of dissociation (฀฀ ) varies. As with all equilibrium constants, Ka is a constant that depends only on temperature. Thus, even though a solution may contain different amount of B2-, H+ and HB-, their ratio should always give the same constant value in accordance with the Law of Mass action. You will test this idea by determining a value of Ka for several solutions that are buffered at different pH values. You will confirm (or refute) the proposal that Ka remains the same as the pH of the solution changes.

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Because bromothymol blue is highly colored, its concentration can be measured using spectrophotometry. Beer’s Law states that Absorbance is proportional to concentration when the wavelength, λmax, is held constant for all measurements. What λmax should we use for our experiments? Since one form of the indicator is blue and one form of the indicator is yellow, we expect there to be at least two different wavelengths of maximum absorbance (λmax), one for each form of the dye. There should be a maximum wavelength for the acidic (yellow) form and a maximum wavelength for the basic (blue) form. We could theoretically use either wavelength and choose to measure the equilibrium concentration of either form of the indicator. However, even though there are two potential λmax values we can use, we need to be concerned with interference which could bias our results. We need to be sure that if we use the λmax for yellow, that the blue form of the indicator does not have any absorbance at this wavelength. Likewise, if we use the λmax for blue, there should be no absorbance of the yellow form of the indicator that this wavelength. In other words, we need to find a wavelength of light that is only absorbed by one of the compounds, and isn’t absorbed by the other compound. This will be accomplished by taking full absorbance spectra for each form of the dye. From this it will be possible to determine which is the best wavelength to use. This idea will become clearer once you see the actual data.

Procedure The following are the steps that the lab technician will be using to collect your data. Familiarize yourself with the procedure so that you can properly interpret the results. A. Determine optimum wavelength The lab technician will 1. Calibrate the spectrometer using DI water as the blank. 2. Determine λmax for the acid (yellow) form of the dye by treating the dye with acid before recording the absorbance spectrum. 3. Determine λmax for the base (blue) form of the dye by treating the dye with base before recording the absorbance spectrum. 4. Both absorbance spectra will be plotted together on one graph and you will receive a copy. The lab technician will choose a λmax where one form of the dye absorbs strongly, and the other has little or no absorbance. The remaining data will be collected at this wavelength.

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B. Preparing Bromothymol Blue Standards and Collect Absorbance Data for the Standard Curve The lab technician will 1. Dilute a stock solution of bromothymol blue to prepare a set of standards. These standards will be diluted with either acid or base, depending on whether the technician wants to measure the absorbance of the yellow or the blue form of the dye. 2. Set up the spectrophotometer to collect absorbance data at the appropriate lambda max. 3. Calibrate the instrument with the blank (water). 4. Collect absorbance data for the standard solutions at the appropriate wavelength. C. Collect Equilibrium Concentration Data The lab technician will: 1. Prepare six test solutions by mixing 15.00 mL of stock bromothymol blue with buffers of different pH to make 50.00 mL of total solution. The pH of the test solutions will be: 6.25, 6.50, 6.75, 7.00, 7.25 and 7.50 2. The absorbance of each test solution will be measured and recorded.

Analysis You and your partner will receive a complete set of data that reflect the procedures followed above. Note that you will not receive the data until after you have completed the pre-lab quiz. To analyze the data you must use the data provided to: 1. Determine the concentration of each standard solution. 2. Prepare a graph of Absorbance vs concentration for the standard solutions. 3. Using the Absorbance data for the test solutions, find the concentration of the indicator in each test solution. 4. From the known total concentration of the indicator, find the concentration of the conjugate form. 5. Find the hydronium ion concentration in each test solution using the buffer concentration. 6. Find a value of Ka for each test solution. There will be a video posted that goes over how to do these calculations. You will need to enter your data and results into a Google Form for checking. Once your results are verified as correct, you will prepare and submit a post lab report. Page 4...