Spectrophotometric Determination of the pKa of BTB

1. Spectrophotometric Determination of the pKa of BTB Lab 7

2. Outline • Purpose • Bromothymol Blue • Indicators • Reaction • Equilibrium Expression • pKa Determination • Procedure • Safety Concerns • Waste • Next Lab Reminder

3. Purpose • 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

4. Indicators Consider a generic indicator (HIn).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. Figure 1. Example absorbance spectra of an acid base indicator in (A) acidic solution, (B) basic solution , and (C) solution of intermediate pH.

5. BTB Solutions BTB indicator in pH acidic, neutral, and basic solutions (left to right).

6. Bromothymol Blue • 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.

7. Bromothymol Blue • BTB is also used for observing photosynthetic activities or respiratory indicators (turns yellow as CO2 is added). • A common demonstration of BTB's pH indicator properties involves exhaling through a tube into a neutral solution of BTB. As carbon dioxide is absorbed from breath into the solution, forming carbonic acid, the solution changes color from green to yellow.

8. Indicators • Bromothymol blue is a pH indicator with a pKa of 7.1. • We want to determine this experimentally.

9. Indicators • The pH at which an indicator changes color depends on its pKa. • An indicator is chosen such that the pKa of the indicator is that of the equivalence point pH. • The color change takes place over a pH range of the pKa ± 1.

10. Reaction HIn In- + H+ yellow blue low pH high pH

11. Equilibrium Expression Ka = pH = pKa + log pH = pKa when = 1

12. pKa Determination We graph: y = mx + b; log = m(pH) + b Where = 1, log = 0 pH = pKa = -

13. Procedure • Prepare 8 solutions using the indicated concentration of BTB and buffer solutions A through H. • Measure the absorbance of each solution. • Measure the pH of each solution. • Make up a spreadsheet and graph. • Determine the pKa of BTB.

14. Safety Concerns • Reagents: • Potassium Phosphate / Sodium Phosphate • Bromothymol blue • Ingestion: • Vomiting, lethargy, diarrhea, cardiac effects, CNS effects, respiratory effects, tingling in extremities, irritation of the GI tract, and burns • Skin Contact: • May act as a sensitizer. Inflammation, pain, burns, scarring, and redness • Inhalation: • Respiratory irritation, coughing, shortness of breath, bronchitis, pneumonitis, and pulmonary edema • Eye Contact: • Irritation, redness, pain, blindness, and corneal damage

15. Waste Dispose of waste in the appropriate waste receptacles. • Acid and basic solutions / waste need to be disposed in the acid/base waste container in the fume hood. • Solutions with a pH between 6 and 8 can be disposed down the drain.

16. Lab 8 Reminder • Lab 8 is next.