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: Ingestion: Skin Contact: Inhalation:
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.