Briana Halbert Activity 8: Spectrophotometric Determination of the pka of the Bromothymol Blue November 5, 2013 Purpose The purpose

of this activity is to study the characteristics and the factors that influence the acid properties of a weak acid. A series of buffered bromothymol blue (BTB) solutions with increasing pH is prepared. The ratio [In-] / [HIn] is determined with the aid of spectrophotometry by measuring the absorbance for each solution at a wavelength of 635 nm. The pKa of BTB is then determined by plotting log ([In] / [HIn]) versus pH. In- = basic form of the indicator HIn = acidic form of the indicator Theory Bromothymol blue is a weak acid and in aqueous solution the following reaction occurs: HIn + H2O In- + H3O+ In a very acidic solution bromothymol blue will be present as the protonated form HIn (Le Chtletiers Principle). In very basic solution bromothymol blue will be present as the unprotonated from In- (Le Chtletiers Principle). Acid-base indicators are compounds that are simply weak acids (or bases) that exhibit different colors depending on whether they are present in solution as their acidic form (HIn) or as their basic form (In ). As the pH of a solution containing the indicator changes, the equilibrium shown below will be driven either towards reactants (HIn), or products (In ) causing the solution color to change depending on the concentration of each form present. For example, in strongly acidic solution, most of the indicator will be present in the form HIn, causing the solution color to correspond to that of HIn. In strongly basic solutions, most of the indicator will be in the In form, resulting the color 2 dominating the solution. At intermediate pH values, the solution color will be a mix of color 1 and color 2, depending on the relative amounts of HIn and In present. HIn Color 1 = H + + Color 2 In (1)

Quantitatively, the relationship between the pH (really the hydronium ion

concentration) and the relative amounts of HIn and In in solution is described by the acid dissociation constant (Ka) for the indicator, as shown in Equation 2, where the square-bracketed terms represent the molar concentrations of each species in solution (M). (2) + In practice, we typically measure the pH of a solution, rather than the [H ], therefore, it is useful to perform a few mathematical manipulations to the Ka expression to get things in terms of experimentally-determined quantities. We begin by taking the negative logarithm of both sides of the equation, resulting in the following: (3)

That equation can be rewritten as (4) An examination of Equation 4 suggests that if we are able to monitor the relative concentrations of HIn and In , it should be possible to determine the Ka for the indicator (or any weak acid for that matter). Procedure A pipet was used to deliver 1 mL of the bromothymol blue solution in each 25-mL two, labeled volumetric flask. 5 mL of distilled water and 4 drops of concentrated HCl was added to one of the flasks. The resulting solution had a pH of 1. The second flask required the addition of 12 drops of 4M sodium hydroxide solutions and was filled to the mark. The resulting solution should have a pH of 13. The absorption spectra were obtained of the two solutions between 365 and 575 nm. The monochromater was adjusted of the instrument to 575 nm. The curvet was filled with distilled water. The instrument was adjusted to read 0% transmittance then the shutter was opened and the instrument was adjusted to read 100% transmittance. the sample was rinsed and the sample curvet was filled with the sample and the spectrum was recorded. On a piece of chart paper, the spectra was recorded of the pH 1 and the pH 13 bromothymol blue solution. The recorded spectra of one of the solution absorbs strongly but the pH 13 solution absorbs weakly, 10, 25 mL volumetric flask were labeled and a pipet contained 1 mL of the bromothymol blue solution was placed in each flask. The volumes of 0.10 M Na2HPO4 solution and 0.10 M KH2PO4 solution were added to each flask. Each solution was diluted to the mark. The pH meter was used to measure the eact pH of

pH 1 solution and the 10 solutions prepared as shown in the table. The pH of each solution recorded. The percent transmittance were measured and tabulated of each of the solutions at the two-selected wavelength. Data Figure 1. Raw Data of pH Solution

Solution pH1 pH13 1 2 3 4 5 6 7 8 9 10

pH 1.4 12.8 4.6 5.8 6.2 6.6 6.8 7.2 7.8 8.8 7.8 7.2

(A) 0.950 0.150 1.230 1.220 1.130 0.865 0.935 1.180 0.351 0.209 0.396 0.532 Absorbance (A) 0.554 0.692 0.865 0.975 0.95 0.77 0.51 0.306 0.189 0.11 0.069

(%) 11.0 70.8 6.0 6.0 7.4 13.8 11.6 6.6 44.6 62.0 40.2 29.2

(A) 0.069 1.460 0.037 0.097 0.198 0.289 0.590 1.210 1.350 1.660 1.400 1.170

(%) 85.2 3.4 92.0 80.0 63.4 51.4 25.8 6.2 4.4 2.2 4.0 6.8

Wavelength (nm) 365 385 405 425 445 465 485 505 525 545 565

Transmittance (%) 28 20.4 13.8 10.6 11 17 30.8 49.4 64.8 77.8 85.2

Figure 2. Raw Data of pH 1 to 1.4 Figure 3. Raw Data of pH 13 to 12.8

Wavelength (nm) 365 385 405 425 445 465 485 505 525 545 565

Absorbance (A) 0.444 0.544 0.464 0.288 0.150 0.171 0.297 0.484 0.710 1.050 1.460

Transmittance (%) 36.0 28.6 34.4 51.6 70.8 67.4 50.4 32.8 19.4 8.8 3.4

Figure 4. Absorbance vs pH

Absorbance vs pH
1.800 1.600 1.400 1.200 1.000 0.800 0.600 0.400 0.200 0.000 0 2 4 6 pH 8 10 12 14 Absorbance

Figure 5. pH vs

pH vs log([In-/HIn])
14 12 10 pH 8 6 4 2 -2 -1.5 -1 0 -0.5 0 log([In-/HIn]) y = 2.1302x + 7.4917 R = 0.6376 0.5 1 1.5

Calculation There were no necessary calculations for this Activity. HIn Yellow Low pH Equilibrium Expression In- + H+ blue high pH

=1 pKa Determination We graph: ( )

Discussion At a low pH the indicator is completely in the HIn form and the absorbance is at a maximum at wavelength 1 (Figure 1A). At a high pH the indicator is completely in the In- form and the absorbance is at a maximum at wavelength 2 (Figure 1B). At an intermediate pH, the solution contains appreciable concentrations of both HIn and In-, and exhibits an absorbance spectrum containing contributions from both forms. pH indicators are useful to estimate the pH of a solution and to visually detect the equivalence point of an acid / base titration. Bromothymol blue is mostly used in measuring the pH of substances that would have relatively low acidic or basic levels (near a neutral pH). It is often used in pools, fish tanks, or measuring the presence of carbonic acid in a liquid.

Figure 3. Example absorbance spectra of an acid base indicator in (A) acidic solution, (B) basic solution , and (C) solution of intermediate pH. Conclusion Bromothymol blue is a pH indicator with a pKa of 7.1. Assignment A1corr = 0.735 0.132 = 0.603 A2corr = 0.569 0.021= 0.548