In the Laboratory

The Determination of the pKa of Multiprotic, Weak Acids W

by Analyzing Potentiometric Acid–Base Titration Data
with Difference Plots
Arno Kraft
Department of Chemistry, Heriot-Watt University, Edinburgh EH14 4AS, United Kingdom; a.kraft@hw.ac.uk

Acid–base titrations are used routinely in medicinal • No simplifying approximations are used.
chemistry to determine pKa values as well as the lipophilicity • Bjerrum plots are very easy to use for the approximate
parameter, log P, of a compound. With the knowledge of the determination of pKa values.
pKa, distribution plots can be calculated that show the ex-
Difference plots or formation curves were introduced by
tent to which a potential drug candidate is ionized at physi-
Niels Bjerrum (14) as a graphical method for the calculation
ological pH (i.e., 7.40 in the case of blood). The logarithm
of stability constants of metal–ligand complexes. They work
of the partition coefficient P of a drug between two immis-
equally well when a proton rather than a metal cation binds
cible solvents, such as water and octanol, serves as an indica-
reversibly to a ligand base, L. For this, an average number
tor of how easily a drug is able to cross cell membrane barriers.
n–H of dissociable protons bound to a weak acid LHn is plot-
Properties such as lipophilicity, solubility, and permeability
ted as a function of pH (10, 15–17). The normalized vari-
through membranes are pH dependent and must be opti-
able n–H has values in the range 0 ≤ n–H ≤ n, where n is the
mized during drug development. The rapid and reliable de-
number of dissociable protons. At low pH, the weak acid is
termination of pKa has therefore considerable importance beyond
fully protonated, and n–H equals n. At high pH, the acid be-
being a simple laboratory experiment for undergraduates.
comes fully ionized, and n–H approaches zero.
Difference Plots Analysis of Experimental Data
A number of articles in this Journal have dealt with the The equations for a Bjerrum plot are easily derived for
calculation of potentiometric titration curves (1–4) and the a monoprotic acid LH (n = 1) that dissociates into its conju-
usefulness of Gran plots (5–7) for identifying the end point gate base L
and a proton. The equilibrium
of a titration and for determining the pKa of a weak acid or Ka
base. Whereas the calculation of theoretical titration curves LH L  + H
occasionally converges to a false minimum (usually as a re-
has the dissociation constant Ka
sult of ill-chosen start values), Gran plots are restricted in
their application to monoprotic acids or bases. Especially [H +] [ L− ]
when polyprotic acids are analyzed, difference plots provide Ka = = 10− pK a (1)
[LH]
an alternative for the analysis of potentiometric acid–base ti-
tration data. This is also the procedure implemented in the and the pKa is, as usual, defined as the negative logarithm of
more sophisticated, automated instruments employed nowa- Ka. The fraction of protons still bound to the acid, n–H, can
days by the pharmaceutical industry (8). However, to the best be written as
of our knowledge, only one report so far in this Journal has
referred to difference plots in connection with the acid–base nH =
moles of bound H +
=
[LH]
total moles of weak acid [ L− ] + [LH] (2)
titration of monoprotic acids (9).
This paper discusses several examples that illustrate the
advantages offered by difference plots (also called difference Electrical neutrality demands that the concentration of the
curves, formation curves, or Bjerrum plots): cations must equal the concentration of the anions at all times
during a titration, and hence
• The pKa’s of multiprotic weak acids are readily esti-
mated even if the individual ionization constants are [H +] + [Na+] = [OH− ] + [Cl − ] + [ L− ] (3)
close to each other (10).
• A difference plot can often reveal values of pKa < 4 or
Note that the Na+ ions originate from the titrant (NaOH),
> 10 although the titration curve may not show dis-
while the Cl
ions may be introduced by the deliberate addi-
tinct inflection points and buffer regions (10, 11).
tion of HCl (a strong acid that is sometimes used to ensure
complete protonation of the weak acid LH) prior to titra-
• The determination of the pKa(s) and log P of a com- tion. The autoionization of water
pound can be combined; this requires a second titra-
tion to be carried out in the presence of octanol or Kw
H2O H + OH
another partitioning solvent (12).
• Small errors in weighed quantities of ligand and strong gives rise to the ion product Kw
acid are readily detected and often rectified.
K w = [H +] [OH− ] = 10− pK w (4)
• Difference plots can provide starting values for further
refinement (13). that equals 10
13.787 at 25 C and 0.1 M ionic strength (pKw

554 Journal of Chemical Education • Vol. 80 No. 5 May 2003 • JChemEd.chem.wisc.edu

 In the Laboratory

being the negative logarithm of Kw). With Curve Fitting

[H ] +
= 10 − pH (5) It follows from the definition of the dissociation con-
it can be used to denote the concentration of hydroxide stant Ka for a monobasic acid (eq 1) that the Bjerrum func-
tion given in eq 2 can be expressed alternatively as
Kw
[OH−] = = 10−pK w + pH (6)
[H +] [LH]  [H + ] 
[LH]  [L ]
−  K a 
more conveniently as a function of pH and pKw. Rearrang- nH = = =
ing eq 3 allows us to express [L–] in terms of the concentra- [L− ] + [LH] 
1 + [LH] − 

1
 +
+ [H ]
 (14)
tions of the other ionic species in solution  [L ]  K a 

[ L− ] = − [Cl − ] + [Na+] + [H +] − [OH−] (7)

and, with eqs 1 and 5, n–H hence becomes
By introducing the total ligand concentration Ltot = [L
] +
− pH
[LH], eq 2 can be rewritten as 10pK a
nH = pK a − pH (15)
1 + 10
− − −
nH =
[L ] + [LH] − [L ] =
Ltot − [L ]
(8) In this form n–H is a function of the pKa and pH, indepen-
[L− ] + [LH] Ltot
dent of any concentration other than that of protons.
With eq 7 we now obtain The general formalism becomes apparent if we consider
−
a diprotic acid next. The original Bjerrum method was for-
nH =
Ltot + [Cl ] − [Na+ ] − [H +] + [OH −] mulated with formation constants (the reciprocal of the cor-
Ltot
(9)
responding dissociation constants) that incidentally gave the
term “formation curve” its name, and the derivation simpli-
For a multiprotic acid (charges have been omitted for clarity)
fies if we, too, introduce the protonation or formation con-
moles of bound H + [LH] + 2[LH2 ] + … + n[LHn ] stants K1 and K2 for the stepwise formation of LH2 (again
nH = = (10)
total moles of weak accid [L] + [LH] + [LH2 ] + … + [LHn ] charges for anions have been omitted for clarity).

K1
the number of moles of protons bound to the weak acid H + L LH
amounts to n times Ltot.
−
K2
nH =
n Ltot + [Cl ] − [Na + ] − [H +] + [OH −] H + LH LH2
Ltot
(11)
The corresponding equilibrium constants are
The amounts of excess strong acid (the source of Cl
ions, A)
and of weak acid (L) are often obtained by weighing a solid [LH]
K1 = (16)
or by dispensing a known amount of a standardized solu- [H +][L]
tion. They are therefore more conveniently given in mmoles
rather than in the form of concentrations (that are going to
K2 =
[LH2 ]
change in the course of the titration anyway). Furthermore, (17)
we need to take into account that the initial volume V0 (in [H +][LH]
mL) is increased by the added v mL of titrant sodium hy- The fraction of protons bound, n–H, is
droxide (of concentration Cb). As the total volume at any
given point during the titration is V0 + v, n–H becomes
moles of bound H +
A Cb v + − nH =
− − [H ] + [OH ] total moles of weak aciid
V0 + v V0 + v
nH = n +
L (12) [LH]  [LH2 ] 
V0 + v [LH] + 2[LH2 ]  [L] + 2  [L] (18)
= =
[L] + [LH] + [LH2 ] [LH]  [LH2 ] 
By expressing the term,
[H+] + [OH–], as a function of pH 1+ 
 [L] +  [L]
and pKw (eqs 5 and 6) and by multiplying both numerator
and denominator with (V0 + v), we finally obtain
for a diprotic acid. Eqs 16 and 17 allow us to calculate the
nH = n +
(
A − Cb v − 10− pH − 10− pK w + pH (V0 + v ) ) (13)
ratios
L [LH] +
= K 1 [H ] (19)
The plot of n–H (as defined by eq 13) versus pH is called a
[L]
difference plot. The pKa of a monoprotic acid can be read and
from the difference plot at n–H = 0.5, while the pKa’s of a di-
protic acid are found at n–H = 1.5 and 0.5, and those of a [LH2 ] [LH2 ] [LH] + 2
triprotic acid at n–H = 2.5, 1.5, and 0.5 (see dashed lines in = = K 2 K 1 [H ] (20)
[L] [LH] [L]
Figures 2–4).

JChemEd.chem.wisc.edu • Vol. 80 No. 5 May 2003 • Journal of Chemical Education 555

 In the Laboratory

and, with eq 18, we thus obtain Whereas the pKa of the monoprotic heterocyclic acid 1
can be deduced equally well by a Gran plot or a calculation
+
nH =
K 1 [H] + 2 K 1 K 2[H +]2 of the theoretical titration curve, the analysis of multiprotic
+ + 2 (21) acids is usually less straightforward. Figure 3 shows an ex-
1 + K 1 [H ] + 2 K 1 K 2 [H ]
ample of an incomplete difference plot that falls short of start-
More generally, n–H is found to be the weighted sum of the ing at its asymptotic value of n–H = n. Nevertheless, the first
mole fractions (distribution or alpha functions; ref 17–20) pKa value of glycine hydrochloride (2) can still be retrieved
αj of the protonated species LH, LH2, ..., LHn that are present from the difference plot at n–H = 1.5. Even the three overlap-
in solution ping pKa’s of tris(2-aminoethyl)amine or tren (3), an organic
n base that is protonated by excess HCl at the three peripheral
nH = ∑ j αj (22) amino groups (but not on the central nitrogen), are readily
j =1
deduced from a Bjerrum plot (Figure 4).
The expression for αj is calculated from
j
Detection of Common Concentration Errors
β j [H + ] and Electrode Problems
αj = n
1+
+ i
∑ βi [H ] (23)
Bjerrum difference plots are capable of revealing a num-
i =1
ber of systematic errors in potentiometric titrations as they
where the cumulative protonation constants βj are defined as will cause the plot of n–H versus pH to depart from the ideal
shape and value. These include (i) strong acid–base concen-
j tration errors (in A or Cb), (ii) ligand concentration errors
βj = ∏ Ki = K1 K 2 …K j (24) (in L), and (iii) nonideal responses of the electrode in ex-
i =1
treme pH regions < 3 or > 11. The topic has already been
A simple computer program using (un)weighted nonlinear discussed in some detail by Avdeef and co-workers (10). It
least-squares regression methods will be able to fit the theo- is, nevertheless, instructive to look at the examples shown in
retical function to the experimental data. The best results are Figure 5. Inaccuracies in the amount of added strong acid
obtained if n–H covers a full range from n to 0. The whole (A) shift the difference plot as a whole in the vertical direc-
procedure is easily implemented in, for example, an Excel tion (along n–H) as illustrated by Figure 5a–b, and corrections
spreadsheet. Minimization of the sum of residuals squared can be made by adjusting A in a pH region where n–H is sup-
(S) between the experimental n–H,exp (eq 13) and calculated posed to equal n. For the same reason, it is often advisable to
n–H,calc (eqs 15, 21, 22) add strong acid so that the titration begins at a pH where
the weak acid is completely protonated. The experimental
S = ∑ (nH, exp − nH, calc )2 data shown in Figure 2 misses out on this opportunity for
checking, and a separate titration had to be carried out, this
is then carried out with the Solver tool, a nonlinear least- time starting at around pH 3, to verify that the pKa extracted
squares routine that comes with the Microsoft Excel software. from the titration was indeed sound.
Two recent monographs are highly recommended for further
background reading and a thorough description of how to
implement all these equations into a spreadsheet (17, 18). A O
N
drawback in Excel’s Solver is that it gives no estimation of
error, but this can be provided by an add-on macro, such as CF3 O ⴙ OH
N H3N
Billo’s SOLVSTAT, which was also used to calculate the stan-
dard deviations for the pKa’s determined in this paper (4, 17). H ⴚ O
Cl

Examples 1 2
Experimental and calculated difference plots for a mono-,
di-, and triprotic acid (see Figure 1 for structures) are shown
ⴙ
in Figures 2–4. The corresponding Excel spreadsheets can be NH3
found in the Supplemental Material.W
The typical semilogarithmic, sigmoidal curve that is ⴙ
ⴙ NH3
common for many 1:1 binding events, here of course between ⴙ N H3N
the conjugate base L
of a weak acid and a proton, is dis- H3N ⴚ
played in Figure 2.1,2 It is instructive to note that similar for- 2 Cl
ⴚ
mation or binding curves are found throughout biochemistry, 3Cl NH3
ⴙ
pharmacology, and supramolecular chemistry, wherever the
extent of 1:1 receptor–ligand binding is plotted against the 3 4
logarithm of the ligand concentration (21). The Bjerrum-de-
termined and titration curve-determined (using de Levie’s for- Figure 1. Structures of the mono-, di-, and triprotic acids: 3-[3-
malism; ref 2) pKa, A, and L are identical within the error (trifluoromethyl)phenyl]-1,2,4-oxadiazol-5-one 1, glycine 2, tris(2-
limits. aminoethyl)amine 3, and ethylenediamine dihydrochloride 4.

556 Journal of Chemical Education • Vol. 80 No. 5 May 2003 • JChemEd.chem.wisc.edu

 In the Laboratory

Figure 2. Titration curve (left) and difference plot (right) for 3-[3-(trifluoromethyl)phenyl]-1,2,4-oxadiazol-5-one (22) 1 (L = 0.130 mmol) in
30 mL of water–methanol (2:1) with an ionic strength of 0.1 M KCl at 25 C; determined by titration with 0.5 mL of 0.5067 M NaOH in
0.01 mL steps. The addition of methanol as a cosolvent was necessary since 1 is almost insoluble in water in the protonated form. The
solid line is the fitted difference plot, and nonlinear least-squares regression using eq 15 gives a pKa of 4.60 ± 0.01 for the heterocyclic
acid.

Figure 3. Difference plot for glycine (L = 0.190 mmol) in 40 mL of
0.1 M aqueous KCl at 25 C, acidified with HCl (0.423 mmol) to
pH ≈ 2.2 prior to titration with 1.4 mL of 0.4905 M NaOH. Note
that, because protonation and dissociation constants are recipro-
cal to each other, log K1 = pKa,2 and log K2 = pKa,1 as the usual
convention is that Ka,1 ≥ Ka,2. The two pKa’s of 2.31 ± 0.01 and
9.63 ± 0.01 (obtained by fitting the experimental data to eq 21)
are in good agreement with reported literature values (2.35, 9.78;
ref 18).
Figure 4. Bjerrum difference plot for tris(2-aminoethyl)amine (L =
0.139 mmol) in 30 mL of 0.1 M aqueous KCl at 25 C. The solu-
tion was acidified with standardized HCl (0.532 mmol) to pH ≈
2.7 and then titrated with 1.4 mL of 0.4905 M NaOH. The three
pKa values of 8.33 ± 0.02 (log K3 = pKa,1), 9.48 ± 0.01 (log K2 =
pKa,2), and 10.19 ± 0.02 (log K1 = pKa,3) were calculated by non-
linear least-squares fitting to eq 22 (8.43, 9.47, 10.17; ref 23).

Figure 5c shows how an error in the amount of weak
acid L, which is frequently the result of weighing inaccu-
racies at small scales (< 20 mg), gives rise to an offset at
high pH when n–H is supposed to approach zero. An acid-
ity error and a ligand error can be spotted independently,
since they affect opposite ends of the n–H scale. It is also
possible to treat both A and L as iteration parameters at
the same time when Excel’s Solver is used to optimize the
pKa’s. Finally, problems with the electrode, its calibration,
or an ill-chosen pKw result in a distorted Bjerrum plot that
no longer converges to a straight horizontal line at extreme
pH (Figure 5d). There is no way but to tackle the cause
of this problem.
The Bjerrum method allows minor errors in A, Cb, and
L to be detected (and even to be corrected), which makes it
highly suitable to produce a good set of estimates for pKa
values that are often quite close to the optimized results. The
carbonate error should however be reduced as much as pos-
sible, although small amounts of carbonate in the NaOH still
give satisfactory results as long as high-precision measure-
ments of pKa are not intended. Despite the simplicity of data
analysis, the value of the pKa’s as determined by difference
plots is surprisingly accurate.
Acknowledgments
We acknowledge the Royal Society for a grant to pur-
chase an automated titrator.

JChemEd.chem.wisc.edu • Vol. 80 No. 5 May 2003 • Journal of Chemical Education 557

 In the Laboratory

a b

c d

Figure 5. Bjerrum difference plot of ethylenediamine dihydrochloride 4 (L = 0.166 mmol) in 0.1 M aqueous KCl at 25 C, acidified with
HCl (0.053 mmol) to pH ≈ 3, and titrated with 1.0 mL of standardized 0.5067 M NaOH in 0.01 mL steps. (a) Fitted curve with error-free
data gave pKa values of 7.15 ± 0.01 and 10.04 ± 0.02 (literature 7.11, 9.96; ref 11). (b) A deviation (here by ±30 %) in the concentra-
tion of the strong acid leads to an offset in n–H. (c) If the concentration of the ethylenediamine dihydrochloride is incorrect (here by ±10 %),
the difference plot is compressed or stretched in the vertical (n–H) direction. (d) An electrode problem at high pH was deliberately created
by the omission to calibrate beyond pH 9; the experimental n–H then declines to reach the expected asymptotic value of zero.

W
Supplemental Material pHcorrected = pH + log f (25)
Experimental details and a description how Microsoft Granted that the ionic strength remains more or less constant, the
Excel Solver has been used to analyze titration data are avail- corrected pH will differ from the measured pH simply by a con-
able in this issue of JCE Online. stant (for a 0.1 M KCl solution: log f =
0.11, ref 17, 18)

Notes Literature Cited

1. For convenience most titrations were carried out with an 1.
automated titrator (Metrohm 702 SM Titrino), but the method 2.
worked equally well with a graduated 25-mL glass buret when stan- 3.
dardized NaOH was added manually in 0.3 mL steps (see Supple- 4.
mental MaterialW). Such a setup is frequently encountered in 5.
undergraduate laboratory courses. A total of 35 data points were
sufficient to determine the pKa of acetic acid as 4.72 ± 0.01 (litera- 6.
ture 4.76; ref 18). 7.
2. All titrations were carried out with 0.1 M KCl as back- 8.
ground electrolyte. A low ligand (ca. 0.0045 M) and a high titrant
NaOH concentration (0.5 M) ensured that volume changes dur- 9.
ing the titration were less than 5%. This way, the ionic strength is
determined by the “swamping” background electrolyte and does not 10.
change much during the titration. Ionic strength corrections are
taken into account by introducing a correction term, log f, for which 11.
values are obtained from Debye-Hückel theory (24). 12.
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McGraw-Hill: New York, 1961; Chapter 5.
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17. Billo, E. J. Excel for Chemists: A Comprehensive Guide, 2nd ed.;
Wiley-VCH: New York, 2001; Chapter 22.
18. de Levie, R. How to Use Excel in Analytical Chemistry and in
General Scientific Data Analysis; Royal Society of Chemistry:
Cambridge, 2001; Chapter 4.
JChemEd.chem.wisc.edu • Vol. 80 No. 5 May 2003 • Journal of Chemical Education
In the Laboratory
19. Waser, J. J. Chem. Educ. 1967, 44, 274–276.
20. Moya-Hernández, R.; Rueda-Jackson, J. C.; Ramírez, M. T.;
Vázquez, G. A.; Havel, J.; Rojas-Hernández, A. J. Chem. Educ.
2002, 79, 389–392.
21. Klotz, I. M. Ligand–Receptor Energetics: A Guide for the Per-
plexed; Wiley: New York, 1997; Chapter 5.
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