Drug Pathways and Chemical Concepts

Prof. Sally Boudinot

11. Spectrophotometric Determination of pKa of Bromothymol Blue

Please remember these concepts:

  1. Many processes can be at equilibrium.   But with changes in condition - concentration, temperature -  the system will no longer be at equilibrium and will adjust to try to get there again.
  2. The equilibrium concentrations of H3O+ and OH- are vanishingly small in pure water. 
  3. A weak acid or a weak base drug, in water, will disassociate to some extent.  The pH of the drug solution  will depend upon the pKa.
  4. Buffers stabilize pH.  This stabilized acidity determines the form of drug disassociation in systems.  The Henderson-Hasselbach equation conveniently handles drug ionization questions for buffered systems like the body.
Lab Guide:

This is the sequence of steps in this laboratory determination:

  1. Prepare buffer phosphate solutions with varying pH.
  2. Use an indicator (bromothymol blue) as the weak acid whose pKa we will determine.
  3. read the pH of EACH sample.
  4. read samples at the wavelengths to determine amount of ionized.
  5. read samples at wavelength to determine amount of unionized.
  6. graph data to determine where the concentrations are equal.
Objective(s): Upon completion of this laboratory, the student should be able to determine the dissociation constant of a chemical moiety based on the degree of ionization of its acidic and basic components.

One of the most important of properties of a drug molecule is its acidity constant. For many drugs, this property can be related to physiological activity because solubility, rate of solution, extent of binding, and rate of absorption are affected by the extent of dissociation at various pH's. 

For an acidic drug, the following reaction expresses this disassociation:

HA  +H2O <==> H3O+ + A-

The determination of an acid dissociation constant requires the estimation of the equilibrium concentration of the acid and base forms of the drug. It can be seen mathematically by using the Henderson-Hasselbach equation:

If the concentration of the acid and base forms are equivalent, the pKa of the drug will be equal to the pH of the solution in which those forms are found.

pH = pKa + log (base/acid)

If the acid/base ratio is 1:1, then the log of that number will be zero, and 

pH = pKa

In the following procedure, the acid dissociation constant of an acidic dye will be determined by estimating the point at which the acid and base forms are equal.  The ionized and unionized forms of the dye have different absorption in the visible portion of the electromagnetic spectra and we will use the spectrophotometer to determine these absorptions.  We will then determine the pH of the solution at which the two forms are present at equal concentrations.

A basic knowledge of spectrophotometry is beneficial to the understanding of the principles of this laboratory exercise.

Procedure: Spectrophotometric Determination of pKa Utilizing Bromothymol Blue 

Bromothymol Blue (B) is a weak acid whose acid and base forms have different absorption spectra.

HB  +H2O <==> H3O+ + B-
yellow, 550nm                                   blue, 450nm

Both species of Bromothymol Blue absorb in the visible region but at different wavelengths.  We will determine the specific absorption maxima of each species under highly acidic conditions where only HB is present and highly basic conditions where only B- is present.  Then use that data to determine the pH at the point when both species are present at equal concentration.

Under ideal conditions, the absorbance of light by a substance is proportional to the molar concentration of that substance.  This is called Beer's Law

Using the stock solutions provided, prepare the following dilutions, each with a total of 25 ml, as described in the table. The solutions should be prepared in small beakers, or other vessel in which the pH can be conveniently measured. A portion of each sample should then be assayed spectrophotometrically, at both 450nm and 550 nm. Data should be recorded in the table

Plot absorbance vs. pH for each of the two wavelengths examined.

Connect the points with a smooth curve. The midpoint of the straight portion of each curve corresponds to the point at which the acid and base species are present in equal concentration.  The midpoint of each straight portion should intersect.   At  that point, the pH equals the pKa. Determine the acid dissociation constant from this graph. 


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Prof. Sally Boudinot
College of Pharmacy
University of Georgia
Athens, GA