In the Laboratory
An Easy and Fast Experiment for the Determination W
of the Equilibrium Constants of an Acid–Base Pair,
Free and Complexed with a Molecular Receptor
Elena Junquera and Emilio Aicart*
Departamento de Química Fisica I, Facultad de Ciencias Químicas, Universidad Complutense de Madrid, 28040-
Madrid, Spain; *aicart@eucmax.sim.ucm.es
Molecular complexation with artificial receptors is an
interesting tool widely used in the interpretation of biological
mechanisms based on molecular recognition processes (1, 2),
where competition between equilibria plays a relevant role.
A proper and accurate determination of the corresponding
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equilibrium constants is a necessary but not always easy task,
since several processes may be involved in the interaction,
and any of the properties analyzed is an overall result of this
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competition. This is very important because in such a case there
is more than one association equilibrium, and more than one
supramolecular entity is formed. The importance and rising
interest of supramolecular systems validates their introduction
to chemistry students; the picture presented to them, however,
is often quite obscure. This paper shows, through easy pH
experiments, how to determine the equilibrium constants of
two molecular receptor–substrate systems involved in a
competitive process. The experiment is appropriate for students
in the second semester of a junior-level physical chemistry class.
Equilibrium Constant Determination
When there is only one substrate species in solution that
can associate with the receptor, the determination of the bind-
ing constant is normally achieved by relating the percentage of
complex formed with any easily measurable physicochemical
property, such as conductivity, absorbance of light, fluorescence,
or 1H NMR chemical shifts (3). However, in many cases the
substrate is an ionizable weak acid; both the acid (HA) and its
conjugated base (A
) are present in solution, and both are able
to associate with the receptor (R), a situation that complicates
the usual procedure. Scheme I shows the set of equilibria that
occur on the formation of R–HA and R–A
complexes, assum-
ing a usual 1:1 stoichiometry.
Ka
HA H+ + A−
KR–HA KR–A−
+R
+R
Ka'
R–HA R–A− + H+
Scheme I
The equilibrium constants are related to the activities
of the species through these expressions:
Ka = (aH+ aA
)/aHA (1)
KR–HA = aR–HA /(aR aHA) (2)
KR–A
= a R–A
/(aR aA
) (3)
Ka′ = (a R–A
aH+)/aR–HA = Ka KR–A
/KR–HA (4)
JChemEd.chem.wisc.edu • Vol. 77 No. 9 September 2000 • Journal of Chemical Education
where only Ka, KR–HA, and KR–A
are independent, since Ka′
is related to them as eq 4 shows.
There are two possibilities for studying these association
equilibria. One is to buffer the pH of the medium so that
only one of the two substrate species, HA or A
, is in solution,
and determine KR–HA and KR–A
independently from the varia-
tion of a physicochemical property as a function of total [R]
at constant total [substrate] (4 ). It is important to ensure that
none of the buffer species is capable of being complexed by
R, because this could interfere in the final results. The other
possibility is to determine the Ka of the substrate and the
association constants KR–HA and KR–A
simultaneously, by
following the variation of the pH of an unbuffered aqueous
solution of the substrate as long as the receptor is added. Since
H+ is one of the species involved in Scheme I, this variation
is a faithful indication of the shifting of these equilibria as
long as the two complexes R–HA and R–A
are formed. This
method has clear advantages over the first one, since no buffer
solutions are needed and all the equilibrium constants of
Scheme I are obtained with only one titration, reducing the
number of experiments and the amount of substrate and
receptor—which is of great importance when they are very
expensive or even not commercially available.
This paper describes an experimental setup and a math-
ematical model, easily affordable in the laboratory, for the
simultaneous determination of all the equilibrium constants
of Scheme I through easy pH measurements. Several equa-
tions must be considered for such a purpose: eqs 1–3, and
the charge and mass balances for the receptor and substrate,
given by the following expressions:
[H+] = [A
] + [R–A
] + [OH
] (5)



[HA]total = [HA] + [A ] + [R–HA] + [R–A ] (6)


[R]total = [R] + [R–HA] + [R–A ] (7)
The activities in eqs 1–3 are related to the concentrations
shown in eqs 5–7 through the activity coefficients, assumed
to be unity for the neutral species and determined with the
extended Debye–Hückel theory for the charged species.
Thus eqs 1–7 allow the simultaneous determination of Ka,
KR–HA, and KR–A
as coefficients of the nonlinear regression
of the experimental pH values (=
log aH+) as a function of
total receptor concentration. The program to carry out the
numerical analysis is a conventional nonlinear fitting proce-
dure based on Newton–Raphson and Marquardt algorithms.
Equipment
Figure 1 shows a block diagram of specific apparatus for a
potentiometric technique.1 A pH–ion meter together with a
combined glass electrode was used for the pH measurements.
1215
 In the Laboratory

3.00

2.95

2.90

pH
2.85

2.80

Figure 1. Block diagram of the potentiometric technique. (1)

Metrohm 713 pH-Ion meter; (2) and (3) combined glass electrode;
(4) measurement cell; (5) cover; (6) polypropylene stirrer; (7) 2.75
0.000 0.002 0.004 0.006 0.008
Metrohm 665 Dosimat digital buret; (8) thermostatted bath.
[β-CD] / M
Figure 2. pH values of aqueous solutions of salicylic acid at a
The aqueous unbuffered solution of the substrate is placed constant concentration of 4.04 mM as a function of β-CD con-
in a measuring cell, with a cover to prevent evaporation of the centration, at 25 ºC.
solution. A digital buret is filled with the receptor solution,
which will be added to the cell in successive additions. A
polypropylene stirrer, activated by an external motor that
rotates at constant velocity, guarantees thorough mixing. The
measuring cell and buret cylinder are thermostatted with 3.05 T = 15 °C
recirculating water from a thermostatted bath. The combined T = 20 °C
glass electrode was calibrated with three buffer solutions of T = 25 °C
pH 4, 7, and 9. 3.00
T = 30 °C

Experimental Procedure T = 40 °C

In the experiment shown as an example, the receptor is 2.95

the β-cyclodextrin (β-CD) and the substrate is the salicylic

acid. β-CD is a torus-shaped oligosaccharide built up with 7
pH

α(1→4) linked α- D -glucopyranose units. Its hydrophobic 2.90

cavity allows for the encapsulation of an apolar substrate of

suitable medium size, with the formation of an inclusion
complex characterized by the stoichiometry and the association 2.85

constant. This capability has made β-CD one of the most

important and widely used drug carriers (5). Salicylic acid is a
well-known hydroxybenzoic acid with various pharmaceutical 2.80

applications. As a weak acid, it is dissociated in aqueous

solution and its aromatic ring is of appropriate size to fit
inside the β-CD cavity. Thus, salicylic acid and β-CD are 2.75
0.000 0.002 0.004 0.006 0.008
examples of an acid–base pair and a molecular receptor, re-
spectively, and have been chosen to show the suggested model. [β -CD] / M
pH measurements were performed on aqueous unbuffered Figure 3. pH values of aqueous solutions of salicylic acid at a
solutions2,3 of salicylic acid at a constant concentration of around constant concentration of ca. 4 mM as a function of β-CD con-
4 mM, as a function of β-CD concentration, at 25 °C (Fig. 2). centration at different temperatures.
From these pH values, the dissociation constant of the acid
(Ka) and the association constants of the complexes formed
by the cyclodextrin and the ionized (KR–A
) and un-ionized Table 1. Values of Equilibrium Constants in Scheme I
(KR–HA) forms of the substrate were determined.4 The values, for the ␤-Cyclodextrin–Salicylic Acid–Water System
reported in the third line of Table 1, lead to some conclusions [Substrate]/
T/ ºC 103 Ka KR–HA /M
1 KR–A¯ /M
1
that can be discussed by the teacher and the students: (i) the mM
value of Ka is in excellent agreement with literature (8 ), 4.05 15 1.2 ± 0.1 1160.± 120 120.± 20
corroborating the efficacy of the model; (ii) since the statistics 4.00 20 1.2 ± 0.1 940.± 90 110.± 20
of the fits are fairly good, 1:1 stoichiometries are confirmed 4.04 25 1.3 ± 0.1 780.± 80 90.± 20
for the inclusion complexes R–HA and R–A
, as initially assumed
3.96 30 1.2 ± 0.1 630.± 60 70.± 15
in the model; (iii) the acid species of the substrate, salicylic
acid, binds the β-CD much more favorably than its ionic 3.92 40 1.4 ± 0.1 450.± 40 45.± 10

1216 Journal of Chemical Education • Vol. 77 No. 9 September 2000 • JChemEd.chem.wisc.edu

 In the Laboratory

students. It is usually attributed to the contribution of a clear

R–HA hydrophobic effect, van der Waals contacts between the host
R–A− and the guest, and the solvent reorganization, as noncovalent
60 intermolecular forces responsible for the overall stability of
the complexes (2, 5). Notice in Table 2 that it is the entropic
term that makes the CD–salicylic acid complex more stable
than the CD–salicylate, because the entropy change is more
R lnK / (J mol-1 K-1)

unfavorable for the anion while the enthalpy change is equally

45
favorable for both the anion and acid.

Acknowledgment

30
We thank the DGES (M.E.C. of Spain), project No.
PB95-0356, for supporting this work.

W
Supplemental Material

15 Supplemental material for this article, including notes

3.2 3.3 3.4 3.5
T -1 / (10-3 K-1)
Figure 4. van’t Hoff plots for R–HA and R–A
complexes.
Table 2. Enthalpy (⌬H ⴗ) and Entropy (⌬S ⴗ) Changes
upon Binding (from Eq 8)
Receptor–Substrate .∆H °/kJ mol
1 .∆S °/J mol
1 K
1
β-CD–salicylic acid
29 ± 1
42 ± 4
β-CD–salicylate anion
29 ± 8
63 ± 20
counterpart, salicylate. This can be analyzed in terms of the
molecular factors affecting the inclusion process in each case—
solvent reorganization, electrostatic interactions, etc., although
more energetic information is required for such discussion.
van’t Hoff Analysis
The study can be completed with an analysis of the changes
in the enthalpy (∆H °), and entropy (∆S °) of the association
processes, which can be estimated through the dependence
of the binding constants on temperature, following the well-
known van’t Hoff equation:
∆H °
R ln K =
+ ∆S ° (8)
T
which assumes that ∆Cp° ≈ 0 in the temperature range studied.
Figure 3 shows the pH values of the β-CD–salicylic acid–
water system as a function of [β-CD] at five temperatures.5
Table 1 reports the equilibrium constants at these temperatures
obtained as previously explained. Figure 4 shows the plots of
the R ln K vs 1/T for the two complexes formed. The ∆H °
and ∆S ° values, determined from the slopes and intercepts of
the linear fits of these data and reported on Table 2, reveal that
both salicylic acid and salicylate species bind β-CD with favor-
able enthalpic terms (∆H ° < 0) dominant over unfavorable
entropic terms (∆S ° < 0). Thus, the encapsulation processes
are exothermic and enthalpy driven (|∆H °| > T |∆S °|). This
thermodynamic pattern and its comparison with a typical
hydrophobically driven process (|∆H °| ≈ 0, |∆S °| < 0, and
T |∆S °| > |∆H °|) (9) can be discussed by the teacher and
for the instructor and the nonlinear regression method to
determine the equilibrium constant, is available in this issue
of JCE Online.
Notes
1. An estimated price for the experimental equipment would
be around $4000.
2. Solutions must be freshly prepared with distilled, deionized
water, preferably from a Millipore Super-Q System. It is advisable
to sonicate initial solutions for 15 minutes in an ultrasonic bath to
guarantee their homogeneity.
3. A procedure for the preparation of solutions, which guar-
antees the constancy of the substrate concentration during the ti-
tration, is as follows: (i) 50 mL of aqueous solution of salicylic acid
4 mM is prepared; (ii) 20 mL of this substrate solution is placed
into the cell; (iii) the other 30 mL is used to prepare the β-CD
solution, at a concentration of 15 mM; (iv) this CD–substrate–
water solution is placed into the buret.
4. The constants A and B and the ion size parameters for H+
and OH
on the extended Debye–Hückel equation are taken from
the literature (6, 7). These parameters for salicylate species, free
(A
) and complexed (R–A
), have been estimated as 6 and 16 Å,
respectively, in terms of their geometry and size.
5. The van’t Hoff analysis can be done by several pairs of stu-
dents, each pair working at one temperature. A minimum of four
temperatures is recommended to obtain reliable energetic parameters.
Literature Cited
1. Fersht, A. Structure and Mechanism; Freeman: New York, 1985.
2. Attwood, J. L.; Davies, J. E. D.; MacNicol, D. D.; Vögtle, F.
Comprehensive Supramolecular Chemistry; Elsevier: Oxford, 1996.
3. Connors, K. A. Binding Constants: The Measurement of
Molecular Complex Stability; Wiley: New York, 1987.
4. Junquera, E.; Aicart, E. J. Inclusion Phenom. 1997, 29, 119.
5. D’Souza, V. T.; Lipkowitz K. B. Chem. Rev. 1998, 98, 1741.
6. Kielland, J. J. Am. Chem. Soc. 1937, 59, 1675.
7. Robinson, R. A.; Stokes, R. H. Electrolyte Solutions;
Butterworths: London, 1965.
8. CRC Handbook of Chemistry and Physics, 67th ed.; Weast, R. C.,
Ed.; CRC Press: Boca Raton, FL, 1986; p D-162.
9. Tanford, C. The Hydrophobic Effect. Formation of Micelles and
Biological Membranes; Wiley: New York, 1980.

JChemEd.chem.wisc.edu • Vol. 77 No. 9 September 2000 • Journal of Chemical Education 1217