Expt.

3 Spec pKa

CHM163 W06

Spectrophotometric Determination of the Ionization Constant of a Weak Acid

BACKGROUND If a substance appears colored when dissolved in solution, it is a potential candidate for analysis by colorimetry (determination of its concentration by analysis of its inherent color). This effect of color intensity increasing directly with increasing concentration is evident in every day examples like the color added to paints by increasing the amount of dye or pigment, and changing the color of food by adding more or less food color to it. In all of these cases, the coloring observed is due to the absorption of certain wavelengths of white light. The absorption or reflection of certain wavelengths of light will account for the colors that are observed in paints and in colored solutions. Mathematically, it is found that the amount of light absorbed by a specific sample depends on three things: 1) the concentration of the solution; 2) the distance the light travels through the sample; and 3) the natural ability of the specific substance to absorb light. Thus an equation can be written relating these things: A= bc (1) where A = absorbance = molar absorbtivity -- how well the material absorbs light b = path length through which the light passes c = concentration of the solution In general, the value for b will remain the same (use the same sample cell holder), and the value for is constant for a specific chemical at a given wavelength of light. Because the general equation for a straight line is y = mx + b if A is graphed against c, the result will be a straight line with a slope of b and a yintercept of zero. A solution that contains two colored species can also be analyzed using Beers Law (A= bc). Mathematically, the concentrations of the two species can be determined if two simultaneous equations can be developed that relate the two. In this experiment, the two unknown concentrations are determined by taking readings on each solution twice, using two different wavelengths of light. The two species that are being studied in this experiment are the two colored forms of specific indicators that are weak acids. If there are two species, HIn and In-, in a solution with absorbances AHIn and AIn, respectively, the total absorbance is A = AHIn + AIn. If the sample pathlength is combined with , then = b and Beers Law for a two component mixture becomes A1 = A1HIn + A1In = 1Hin [Hin] + 1In [In-] at 1 (2)

Page 1 of 4

Expt.3 Spec pKa

CHM163 W06

The constant does not change with concentration, but will change at different wavelengths and/or with a different absorbing species. So, at a second wavelength 2, the following equation for Beers Law would be true: A2 = A2HIn + A2In = 2HIn [HIn] + 2In [In-] at 2 (3)

There are now two equations that can be used to determine the concentrations of the two different colored unknowns that are present in the solution. These equations can be rearranged to allow determination of each concentration. [HIn] = ((A12In) ( A21In)) / ((2In 1HIn) (1In 2HIn)) [In ] = ((A2 1HIn) ( A1 2HIn)) / ((2In 1HIn) (1In 2HIn)) (4) (5)

In order to actually analyze a solution such as this, that has two colored components, three separate but related experiments must be done. First, the best wavelengths for analysis need to be chosen. Next, the four constants need to be determined, and finally, the values for the absorbances (A1 and A2) need to be measured at these wavelengths. The best wavelengths for the experiment are selected by measuring the absorbance vs. wavelength for each of the pure substances. The actual wavelengths are then chosen such that they will maximize the absorbances for each species. The values for the four constants can be determined by doing Beers Law plots of absorbance vs. concentration (of pure samples) at both wavelengths and determining the slopes of the lines generated. The final set of measurements will be collected by measuring the absorbances of solutions of the weak acid at different pHs.

Determining an equilibrium constant The reaction that is to be studied is the dissociation of a weak acid. This weak acid is an indicator that has different colors in its acidic and basic forms (HIn and In). HIn (acidic form) H++In(basic form) (6)

Ka = [H+] [In-] / [HIn] [H+] = Ka [HIn] / [In-] pH = pKa - log {[HIn] / [In-]}

(7) (8) (9)

Page 2 of 4

Expt.3 Spec pKa

CHM163 W06

We can consider the last equation to be linear ( y = mx + b), so a graph of pH vs log {[HIn]/[In-]} will produce a straight line with an intercept equal to pKa where the y-axis is the pH and the x-axis is the log {[HIn]/ [In-]} The ratio of {[HIn] / [In-]} can be determined spectrophotometrically by combining equations 4 and 5 to get: {[HIn] / [In-]} = ((A12In ) ( A2 1In)) / ((A2 1HIn) (A1 2HIn )) (10) The plots of absorbance vs. wavelength have been made for you and are available from your instructor. These plots were made by taking 10 ml of the indicator solution and adding acid (HCl) or base (NaOH) to it to insure that only one of the two colored species would be present. With the help of your instructor, select the two best wavelengths for this study. These two wavelengths will be used for the rest of the experiment. The determination of the extinction coefficients ( ) is done by preparing a set of four solutions of varying concentrations of the acidic form and a set of four solutions of varying concentrations of the basic form of the indicator. Absorbance readings are then taken on these ( A vs. c ) and plotted. The slopes of these lines will be the values. The third part of this experiment involves preparing a set of solutions of varying pH that span the color change of the indicator in question. Readings are taken at the same wavelengths that were used for the earlier portion of the experiment. A plot of pH vs. log {[HIn] / [In-]} will produce a straight line with an intercept of pKa. PROCEDURE Work with a partner for this experiment. Your instructor will demonstrate the use of the Spectronic 20. If you still do nt understand how to use the instrument, ask an assistant to help you. Spectra of the Acid and its Conjugate Base The spectrum of the acid and also of its conjugate base are available from the course instructor. Discuss with the instructor the choice of the two wavelengths of light that are most suitable for your particular unknown. Determination of the Extinction Coefficients Using a graduated pipet, pipet 2.00, 4.00, 6.00, and 8.00 ml of the original indicator solution into individual 100 ml volumetric flasks and add 40 ml of 0.25 M HCl to each one. (NOTE: your instructor may advise you to use 1, 2, 3, and 4mL, instead) Dilute to 100 ml with deionized water. Prepare a second set of solutions (use the same volumes as the first set) using the original indicator solution and adding 40 ml of 0.25 M NaOH. Dilute these solutions to 100 ml with deionized water. Measure the absorbance of each of the eight solutions at the two wavelengths that were selected. Make a plot of absorbance vs. concentration for each wavelength for both HIn and In. Calculate the values of from the graphs ( is the slope of the line).

Page 3 of 4

Expt.3 Spec pKa

CHM163 W06

Determination of Ka To determine your value of Ka, you need to find the pH range in which your indicator undergoes its color change. The easiest way to do this, is to add approximately 0.5 ml of your original indicator solution to each of eight test tubes, and then add approximately 3 ml of a buffer solution to each test tube (starting with pH=2 and ending with pH=9). In consultation with your instructor, select the five or six pHs that span the color change. Pipet 5.0 ml of the original indicator solution into a volumetric flask, add 40 ml of one of the buffer solutions, and 40 ml of 0.25 M NaNO3. Dilute to volume. Repeat this procedure for all of the buffer solutions that were selected to span the color change. Measure the pH of the buffered indicator solutions that you have prepared. Measure the absorbance on each of these solutions at both of the wavelengths that were used in the determination of the extinction coefficients. Determine the value for pKa by plotting measured pH vs. log [HIn]/[In-] (all indicator solutions are 2.50 x 10 -4 M). Use literature sources to determine the pKa values for the following indicators and compare your value to these to determine which indicator you have: methyl orange, methyl red, bromocresol green, or bromothymol blue. Report The report should contain graphs of absorbance vs. concentration, and pH vs. log [HIn]/[In]. Remember that graphs should always have a title, numbered and labeled axes, and the axis scales should be selected so that the entire page is used. QUIZ QUESTIONS: 1. Calculate the individual concentrations of A and of B in the following mixture. compound A B Unknown mixture A at 365 nm 0.158 0.884 0.727 A at 470 nm 0.647 0.099 0.604 Concentration 1.00 x 10-4 2.00 x 10-4 ????????

2. What is the ratio of [HIn]/[In-] for your individual unknown in 0.10 M HCl? What is the ratio of [HIn]/[In-] for your individual unknown in 0.10 M NaOH? How is this related to the method that you used to determine your values of ? 3. For the last graph, what is the expected slope? Explain your answer. 4. List two possible sources of deviation from Beers Law (in other wo rds, what could have affected your analysis in such a way that the assumption that concentration is directly related to absorbance did not hold true?).

Page 4 of 4