6. Determination of the acid dissociation constant of phenytoin
Method principle
Dissociation of weak acids including phenytoin, i.e. 5,5-diphenylimidazolidin-2,4-dione, an
antiepilepsy drug, can be expressed by Henderson-Hasselbach equation, which can be written in the
form
pKa
= pH + log ([HA]/[A]),
[1]
where [HA] is the equilibrium concentration of non-dissociated form of the acid and [A]
is the
equilibrium concentration of its dissociated form.
Spectrophotometric determination of dissociation constant requires significant changes of
absorption spectrum in dependence on pH. The optimal wavelength which is needed to be chosen is
that, which gives the maximum difference of absorbances measured in strongly acid and strongly
alkaline solutions respectively, i.e. between absorbances of fully dissociated and fully nondissociated
forms of a compound. Thus we will measure the absorbance of the compound in its
fully dissociated form (in this task in solution of NaOH of concentration 0.01 mol.l-1
) and
consequently in the form in which dissociation is completely suppressed (here in solution of HCl of
concentration 0.01 mol.l-1
). For absorbance of a substance, the Lambert-Beer's law is valid
A = ε.c.l , [2]
where A is absorbance, ε is molar absorptivity, and l is length of the layer through which the light
goes (i.e. cuvette width). Absorbance of phenytoin in fully dissociated form, i.e. in solution of a
strong base, can be expressed
AA=
εA.
[A].
l = εA.
cN
. l , [3]
where cN is total concentration of phenytoin, and, because all the substance is under these conditions
dissociated, [A]
= cN
. Absorbance of phenytoin in fully non-dissociated form can be expressed
NH
NH
O
O
+ B
-
N
-
NH
O
O
+ BH
M.W. = 252.27
similarly
AHA
= εHA
. [HA] . l = εHA
. cN
. l , [4]
because all the substance is in non-dissociated form which means [HA] = cN
.
Further we will determine absorbances of the set of phenytoin solutions with pH graded so
that the substance will be almost completely in form HA in the most acid one and completely in
form Ain
the most alkaline one. The range of pH values could be chosen so that the numeric value
of pH which corresponds with assumed or estimated value of pKa, which is to be determined, was
set in the middle of this range. Such estimation of pKa value can be based on analogy, or can be
performed by calculation by means of a suitable software. The particular absorbance of every of the
set of nitrofurantoin solutions can be expressed by the equation
A = εA.
[A]
. l + εHA
. [HA] . l [5]
The resolution of equations [2] - [5] gives for the ratio of equilibrium concentration in
investigated solutions [HA] / [A-
]
[HA] / [A]
= (AA
- A) / (A - AHA
) [6]
The substitution the formula [6] for [HA]/[A]
in equation [1] results in the equation
pKa
= pH + log {(AA
- A) / (A - AHA
)} [7]
which must be enumerated for every buffered solution. The final value of pKa is acquired as mean
of all partial values.
Procedure
1.Preparation of solutions
The set of stock buffer solutions of concentration 0.2 mol.l-1
and pH 7.8, 8.0, 8.2, 8.4, 8.6, 8.8 and
9.0 is diluted with distilled water into the set of basic buffer solutions of concentration 0.01 mol.l-1
.
The stock solutions of hydrochloric acid and sodium hydroxide respectively are diluted into the
basic solutions of the concentration 0.01 mol.l-1
too. 100.00 ml of the stock solution of phenytoin of
concentration 0.005 mol.l-1
in ethanol is prepared in a suitable volumetric flask. 0.5 ml of the
phenytoin stock solution is then pipetted into every of the set of nine 50 ml volumetric flasks. The
flasks are then filled up to the mark with the appropriate basic solution of hydrochloric acid, buffer
or sodium hydroxide respectively. The set of comparison solutions (blanks) is prepared similarly
but 0.5 ml of pure ethanol is pipetted into every volumetric flask instead of the phenytoin stock
solution.
2. Measurement on a Hewlett-Packard 8453 UV-VIS spectrophotometer
Switching-on and setting-up of both spectrophotometer and its controlling computer are
performed by a lecturer or a laboratory assistant. We measure absorption spectra in the ultraviolet
region in range from 220 to 320 nm. We determine absorbances at the wavelength of 236 nm.
Procedure of the proper measurement
Only one cuvette (cell) is used for whole measurement. In order to minimize errors caused
by changes of solutions in it, it is recommended measure the particular samples together with their
blanks in order of increasing or decreasing pH, so that the measurement starts with blank and
sample containing hydrochloric acid, continues with those of pH 7.8, 8.0, 8.2, 8.4, 8.6 and 9.0 and
finally blank and sample with sodium hydroxide is measured. Or contrariwise, the measurement can
start with solutions with hydroxide, continue with buffers of pH from 9.0 to 7.8 and finish with
solutions with hydrochloric acid.
1. The cuvette is at least twice poured with a comparison solution (blank), then it is filled with
this solution and placed into the cuvette holder of the spectrophotometer. The cuvette is
fixed by the lever at the side of the holder. The spectrum of a blank is then measured by
click to the button Blank. The blank spectrum appears in a window entitled Lasts blank
spectrum. A sample spectrum is measured similarly, with a cuvette at least twice washed
and then filled with a sample solution, by clicking Sample button. The window Sample
information appears and it is needed to write HCl or NaOH or pH of the particular buffer
into the field Sample information in the window Sample information and confirm OK. Thus
absorbances of all 8 solutions are measured. All the acquired spectra together with a table
of absorbances are printed by clicking the button Print results.
3. Calculation of pK
a
The substitution of particular determined values of absorbance in the equation [7] gives the partial
values of pKa. The resulting pKa value is then calculated as the arithmetic mean of these values.
Tasks:
1. Determine pKa of disociaciation of N-H of imidazolidinedione ring of phenytoin.
2. What will be the percentage of dissociated form of the compound in blood plasma at pH =
7.4 ?
3. What will be the percentage of dissociated form of the compound in small intestine at pH =
8.0 ?
4. What will be the percentage of dissociated form in stomach at pH = 1.0 ?