Journal of Chromatography A, 964 (2002) 55–66
www.elsevier.com / locate / chroma
Retention of ionizable compounds in high-performance liquid
chromatography
14. Acid–base pK values in acetonitrile–water mobile phases
´ *
Sonia Espinosa, Elisabeth Bosch, Martı´ Roses
´
´ , Universitat de Barcelona, Diagonal 647, E-08028 Barcelona, Spain
Departament de Quımica
Analıtica
Received 22 March 2002; received in revised form 2 May 2002; accepted 3 May 2002
Abstract
Linear relationships between ss pKa values in acetonitrile–water mixtures and ww pKa values in pure water have been
established for five families of compounds: aliphatic carboxylic acids, aromatic carboxylic acids, phenols, amines, and
pyridines. The parameters (slope and intercept) of the linear correlations have been related with acetonitrile–water
composition. The proposed equations allow accurate estimation of the pKa values of any member of the studied families at
any acetonitrile–water composition up to 60% of acetonitrile in volume (100% for pyridines). Conversely, the same
equations can be used to estimate aqueous pKa values from chromatographic pKa values obtained from any acetonitrile–
water mobile phase between the composition range studied. Estimation of pKa values have been tested with chromatographic
literature data. 2002 Elsevier Science B.V. All rights reserved.
Keywords: Mobile phase composition; Retention behaviour; Acidity; pKa ; Dissociation constants; Carboxylic acids; Amines;
Phenols; Pyridines
1. Introduction
In previous works, the importance of proper pH
measurement in liquid chromatography mobile
phases has been highlighted [1–8]. Measurement of
the pH in the mobile phase after mixing aqueous
buffer and organic modifier has been recommended
[3–8]. This procedure provides fits of analyte retention to mobile phase pH much better than those
obtained when the pH is measured in the aqueous
buffer before mixing it with the organic modifier. In
*Corresponding author. Tel.: 134-93-402-1796; fax: 134-93402-1233.
´
E-mail address: marti@apolo.qui.ub.es (M. Roses).
addition, the pKa value obtained from the fits to pH
values measured after mixing is the thermodynamic
pKa value of the analyte in the mixed solvent used as
mobile phase. This is a significant advantage over
pKa parameters obtained from pH measurements
before mixing, which are only fitting parameters
without any physical meaning [9].
With proper measurement of mobile phase pH,
known thermodynamic pKa values can be used to
predict ionization of the analytes in the mobile phase
and thus, retention in the chromatographic system.
These pKa values refer always to the solvent system
used as mobile phase, not to the pKa values in water.
This is an important restriction to practical applicability of the method because the number of
0021-9673 / 02 / $ – see front matter 2002 Elsevier Science B.V. All rights reserved.
PII: S0021-9673( 02 )00558-7
56
S. Espinosa et al. / J. Chromatogr. A 964 (2002) 55–66
known pKa values in water–organic mobile phases is
very limited. However, in previous studies [10,11]
we established linear relationships between pKa
values in methanol–water mixtures and pKa values
in water for several families of compounds. These
relationships provide a good estimation of the pKa
value of the compound in any methanol–water
mixture from the pKa value of the compound in
water, which can be used to predict the ionization of
the compound in a chromatographic system that uses
pH buffered methanol–water mobile phases.
In this paper, we extend these relationships to
acetonitrile–water mobile phases. Methanol–water
and acetonitrile–water solvent mixtures are by far
the most used mobile phases in liquid chromatography. The relationships between pKa values established for these two solvent systems can be extremely useful for prediction of retention and optimization
of chromatographic separations of acid–base compounds. Additionally, they can be used for determination of the aqueous pKa value from the fitting pKa
value obtained by measurement of retention at
several mobile phase pH values. This method has
been recently used to estimate the aqueous pKa
values of water insoluble arilpropionic acids with
antiinflammatory properties from the chromatographic pKa values in methanol–water mobile phases [12].
2. Theory
The approach followed is based in the linear
relationship proposed by Chantooni and Kolthoff
[13] to measure the ‘‘resolution of acid strength’’ of
a family of compounds in a solvent (s) in reference
to water (w). In this approach, the pKa values of a
series of related compounds (commonly compounds
that share the same charge and functional acidic
group) in the solvent s ( ss pKa ) are plotted against the
pKa values of the same compounds in water ( ww pKa ).
A straight line (Eq. (1)) is usually obtained with a
slope value (a s ) that measures the ‘‘resolution of acid
strength’’ for the compounds in solvent s as regards
to water (slope unity), i.e. the ability of the solvent to
differentiate between the acidities of the compound’s
set
s
s
pKa 5 a s ww pKa 1 b s
(1)
The intercept of the correlation (b s ) is related to
the differences in basicities, dielectric constants, and
specific solvation interactions of the solute (e.g.
hydrogen bonding) between solvent s and water. The
first two differences depend only on the solvents
considered (s and w), but the specific solvation
differences depend also on the family of compounds
studied. The slope of the correlation (a s ) is related to
differences between specific solvation interactions,
which depend on the solvent and family of compounds studied. The larger the specific solvation of
the compound in solvent s, in reference to water, the
larger the slope value. Detailed explanations about
theoretical derivation of Eq. (1) have been given in
previous studies [10,11,14].
We shall use here the same notation recommended
by the IUPAC [15] for pK and pH definition we have
used in previous works. A lower-case left-hand
superscript in pH or pK term indicates the solvent (w
or s) in which measurements are being made; a
lower-case left-hand subscript indicates the solvent
in which the ionic activity coefficient g is referred to
unity at infinite dilution (w or s). Notice that the
possibility of choosing two different standard state
solvents for the ionic activity coefficients leads to
two different scales for pH measurement in nonaqueous or mixed solvents [16]. The ss pH scale refers
to pH measured in solvent s with pH standardization
in the same solvent s, and the sw pH scale refers to pH
measurement in solvent s with pH standardization
with aqueous reference buffers. ww pH scale refers to
pH measurement in water.
Linear relationships for resolution of acid strength
have been well established for the pK values of
families of compounds in different solvents in reference to the pK values in water [10,11,14,17–23]. In
two previous studies, we applied them to the available ss pKa data in methanol–water mobile phases
[10,11]. The ss pKa values of 121 acid–base compounds belonging to six different chemical families
in several methanol–water compositions were fitted
to Eq. (1) and the a s and b s parameters of the
equation were obtained for each compound family
and solvent composition. The a s and b s sets of
values obtained for each family were related to
solvent composition through polynomials. For mobile phase compositions measured in volume fraction
of methanol (vMeOH ), the equations take the forms:
S. Espinosa et al. / J. Chromatogr. A 964 (2002) 55–66
2
1 1 a 1 vMeOH 1 a 2 v MeOH
a s 5 ]]]]]]]
2
1 1 a 3 vMeOH 1 a 4 v MeOH
(2)
2
b 1 vMeOH 1 b 2 v MeOH
b s 5 ]]]]]]]
2
1 1 b 3 vMeOH 1 b 4 v MeOH
(3)
where a 1 , a 2 , a 3 , a 4 , b 1 , b 2 , b 3 , and b 4 were fitting
parameters constant for all acids of the same family
at all methanol–water mixtures.
This approach shall be applied here to the available data for acetonitrile–water mixtures in order to
establish equations for the different families of
compounds, which should allow an accurate estimation of the compound pKa value at a given acetonitrile–water mobile phase from the pKa value of the
compound in water.
3. Results and discussion
3.1. pKa values in pure acetonitrile
The resolution of acid strength for the families of
compounds studied was first investigated in pure
acetonitrile. The available literature ss pKa values in
pure acetonitrile [24–26] were collected (Table 1)
and plotted against its ww pKa values in water [27,28].
Fig. 1 depicts the plots obtained. It is evident that the
compounds can be divided in five families that
follow five different straight lines, with only four
outliers of the 101 pairs of pKa data points plotted.
These five families are aromatic carboxylic acids,
aliphatic carboxylic acids, phenols, pyridines, and
amines. The parameters of the straight lines obtained
are given in Table 2.
All families present a resolution of acid strength in
acetonitrile larger than in water (a s .1). This behaviour is usual in solvents with a poor hydrogen
bond donor acidity, such as acetonitrile. In a good
hydrogen bond donor solvent, e.g. water, the negative charge of the anion (carboxylic acids and phenol
families) or the lone electron pair of the neutral base
(amines and pyridines) are stabilized in the oxygen
or nitrogen atom of the acid–base group by hydrogen
bonding. However, in a poor hydrogen bond donor
solvent, the negative charge or lone electron pair can
be easily delocalized along the structure of the
57
molecule. In this instance the effect of the substituents in the molecule is more important and
produces a larger variation of the acid–base strength
(pKa values) [10,11,13,14].
The families with a larger resolution of acid
strength are aliphatic carboxylic acids and phenols,
whereas aromatic carboxylic acids and the protonated bases, amines and pyridines present a lower
resolution of acid strength. In contrast with pure
methanol [14] and other alcohols [17,19], ortho
effect (variation of the resolution of acid strength
caused by substituents in ortho position) was not
observed in acetonitrile.
3.2. Acetonitrile–water mobile phases
The same compound families investigated in pure
acetonitrile were studied in acetonitrile–water mixtures. The available ss pKa data in acetonitrile–water
mixtures were compiled for round percentages of
acetonitrile in volume and they are given in Table 3.
Most data were taken from a previous compilation
[29]. The equations given in that study for each
compound were used to calculate the ss pKa value of
each compound at each round percentage of acetonitrile. The early compilation for neutral acids was
complemented with the ss pKa data of some amines
[31] and substituted pyridines [32]. The ss pKa data for
these substituted pyridines were determined in the
molality scale at a constant ionic strength of 0.01
mol kg 21 . These ss pKa data were converted to
thermodynamic ss pKa values (zero ionic strength) in
the molarity scale used through all this work by
¨
using the densities and Debye–Huckel
parameters
for acetonitrile–water mixtures given elsewhere [4].
Finally, the linear equations proposed in a previous
study [6] and the cubic equations given by Sarmini
and Kendler [30] to relate the sw pKa values of some
compounds to acetonitrile composition were used to
calculate the sw pKa of these compounds at the round
acetonitrile percentages. ss pKa values of the compounds were calculated from the sw pKa values and the
d parameter of the corresponding acetonitrile–water
mixture (determined in a previous work [4]) by
means of the following relationship:
s
s
pKa 5 sw pKa 2 d
(4)
The a s and b s parameters of the correlations
S. Espinosa et al. / J. Chromatogr. A 964 (2002) 55–66
58
Table 1
Acid–base pKa values of compounds in pure water and pure acetonitrile
Acid
H2O
pKa a
MeCN
pKa
Ref.
w
w
s
s
Aliphatic carboxylic acids
Trifluoroacetic acid
Trichloroacetic acid
Oxalic acid (pK1 )
Dichloroacetic acid
Dichloroacetic acid
Cyanoacetic acid
Malonic acid (pK1 )
Chloroacetic acid
Fumaric acid (pK1 )
Tartaric acid (pK1 )
Glycolic acid (pK1 )
Succinic acid (pK1 )
Glutaric acid (pK1 )
Adipic acid (pK1 )
Nonanedioic acid (pK1 )
Acetic acid
Butyric acid
0.23
0.63
1.27
1.33
1.33
2.46
2.85
2.87
3.02
3.04
3.83
4.16
4.34
4.43
4.53
4.76
4.82
12.7
10.6
14.5
13.2
15.8 b
18.0
15.3
18.8
18.6
14.9
19.3
17.6
19.2
20.3
20.9
22.3
22.7
[24]
[24]
[24]
[24]
[25]
[24]
[24]
[25]
[24]
[24]
[24]
[24]
[24]
[24]
[24]
[25]
[24]
Aromatic carboxylic acids
2,6-Dinitrobenzoic acid
2,6-Dihydroxybenzoic acid
2,4-Dinitrobenzoic acid
2-Nitrobenzoic acid
2,4-Dichlorobenzoic acid
3,5-Dinitrobenzoic acid
3,5-Dinitrobenzoic acid
2-Chlorobenzoic acid
o-Phthalic acid (pK1 )
2-Hydroxybenzoic acid
4-Nitrobenzoic acid
2,4,6-Trimethylbenzoic acid
3-Nitrobenzoic acid
3,4-Dichlorobenzoic acid
m-Phthalic acid (pK1 )
3-Bromobenzoic acid
3-Bromobenzoic acid
4-Bromobenzoic acid
Benzoic acid
Benzoic acid
3,4-Dimethylbenzoic acid
4-Hydroxybenzoic acid
1.14
1.22
1.42
2.22
2.72
2.82
2.82
2.94
2.95
2.98
3.43
3.44
3.49
3.60
3.62
3.82
3.82
3.97
4.21
4.21
4.41
4.58
15.8
12.6 b
16.1
18.2
18.4
17.2
16.9
19.0
14.3 b
16.9 b
18.7
20.5
19.2
19.0
19.3
19.5
20.3
20.3
20.1
20.7
21.2
20.8
[24]
[24]
[24]
[24]
[24]
[24]
[25]
[24]
[24]
[24]
[25]
[24]
[24]
[25]
[24]
[24]
[25]
[24]
[24]
[25]
[25]
[24]
Phenols
2,4,6-Trinitrophenol
4-Chloro-2,6-dinitrophenol
2,6-Dinitrophenol
2,4-Dinitrophenol
3,4-Dinitrophenol
4-Nitrophenol
0.65
2.97
3.71
4.02
5.42
7.16
11.0
15.0
16.0
16.0
17.9
20.7
[25]
[24]
[24]
[24]
[24]
[24]
Acid
Amines
2-Nitroaniline
3-Nitroaniline
Aniline
p-Toluidine
Benzylamine
Trimethylamine
1,5-Pentanediamine (pK1 )
1,3-Propanediamine (pK1 )
Isobutylamine
Butylamine
Methylamine
Propylamine
Ethylamine
Dimethylamine
Triethylamine
Triethylamine
1,4-Butanediamine (pK1 )
Tributylamine
Diethylamine
Dibutylamine
Pyridines
2-Chloropyridine
2-Bromopyridine
2-Hydroxypyridine
3-Cyanopyridine
4-Cyanopyridine
2-Acetylopyridine
3-Bromopyridine
3-Chloropyridine
3-Acetylopyridine
3-Hydroxypyridine
Pyridine
Pyridine
3-Methylpyridine
4-Ethylpyridine
2-Methylpyridine
4-Methylpyridine
3-Aminopyridine
3,5-Dimethylpyridine
3,4-Dimethylpyridine
2,3-Dimethylpyridine
2-Aminopyridine
2,6-Dimethylpyridine
2,4-Dimethylpyridine
4-Aminopyridine
H2O
pKa a
MeCN
pKa
Ref.
w
w
s
s
2.17
3.49
4.61
5.08
9.33
9.81
10.25
10.30
10.43
10.61
10.62
10.69
10.70
10.73
10.78
10.78
10.80
10.90
10.98
11.30
4.9
7.6
10.6
11.3
16.8
17.6
19.1
19.7
17.9
18.3
18.4
18.2
18.4
18.7
18.7
18.5
20.1
18.1
18.8
18.3
[24]
[24]
[25]
[24]
[24]
[24]
[24]
[24]
[24]
[25]
[24]
[24]
[25]
[24]
[24]
[25]
[24]
[25]
[25]
[25]
0.49
0.71
1.25
1.38
1.90
2.76
2.84
2.84
3.55
4.75
5.17
5.17
5.58
5.87
5.91
5.93
6.03
6.15
6.47
6.57
6.66
6.68
6.70
9.06
6.8
7.0
8.3
8.0
8.5
9.6
9.5
10.0
10.8
12.6
12.6
12.3
13.7
13.6
13.9
14.5
14.4
13.9
14.7
14.8
14.7
14.4
15.0
18.4
[26]
[26]
[26]
[26]
[26]
[26]
[26]
[26]
[26]
[26]
[26]
[25]
[26]
[25]
[26]
[26]
[26]
[26]
[26]
[26]
[26]
[26]
[26]
[26]
S. Espinosa et al. / J. Chromatogr. A 964 (2002) 55–66
59
Table 1. Continued
Acid
2-Nitrophenol
4-Cyanophenol
3-Nitrophenol
2-Bromophenol
3,4-Dichlorophenol
3-Chlorophenol
4-Bromophenol
4-Chlorophenol
Phenol
3-Methylphenol
4-Methylphenol
2-Methylphenol
a
b
H2O
w
a
w pK a
MeCN
pKa
Ref.
s
s
7.24
7.95
8.39
8.42
8.63
9.08
9.37
9.42
9.98
10.00
10.25
10.28
22.0
22.7
23.8
23.9
24.0
25.0
25.5
25.4
26.6
26.5
27.5
27.5
[24]
[24]
[24]
[24]
[24]
[24]
[24]
[24]
[24]
[24]
[24]
[24]
Acid
H2O
pKa a
w
w
MeCN
pKa
Ref.
s
s
From Refs. [27,28].
Excluded from the correlations.
between ss pKa values in acetonitrile–water and ww pKa
values in water (Eq. (1)) are given in Table 2 for the
families and solvent compositions studied. The variation of these parameters for the families studied
with the composition of acetonitrile–water mixtures,
compared with the variation of the same parameters
in methanol–water mixtures, is depicted in Figs. 2
and 3. As compared with methanol–water, acetonitrile–water mixtures exhibit larger a s values (except
for aromatic carboxylic acids), that demonstrate a
larger resolution of acid strength. This is already
Fig. 1. Plot of the ss pKa values in pure acetonitrile vs. the ww pKa
values in pure water. Symbols: (앳) aliphatic carboxylic acids,
(n) aromatic carboxylic acids, (s) phenols, (h) amines, (3)
pyridines.
expected because the resolution of acid strength in
pure acetonitrile is much larger than in pure methanol.
The a s and b s values have been fitted to solvent
composition in terms of the volume fraction of
acetonitrile according to Eqs. (2) and (3). The
parameters obtained for the fits are given in Tables 4
and 5.
The parameters of Tables 4 and 5 allow calculation of the slope and intercept of the correlation
between ss pKa values in acetonitrile–water mixtures
and ww pKa values in water for the families of compounds studied. The ss pKa value of any member of
these families, including compounds not studied in
the original set, in any acetonitrile–water composition can be precisely estimated from the pKa value of
the compound in water. Conversely, the pKa value of
the compound in water can be calculated from the
s
s pKa value of the compound at any acetonitrile–
water composition. The studied ss pKa data cover the
range 0–60% of acetonitrile in volume (plus pure
acetonitrile), except for the pyridines studied by
Pawlak [32] which comprise all the acetonitrile–
water composition range. Thus, accurate estimation
of pKa values is restricted to these acetonitrile–water
composition ranges.
3.3. Estimation of pKa values and degrees of
protonation of pyridines in acetonitrile–water
mixtures
Estimation of pKa values and degrees of ionization
S. Espinosa et al. / J. Chromatogr. A 964 (2002) 55–66
60
Table 2
Parameters of linear Eq. (1) for each family of compounds studied and acetonitrile–water composition
Acetonitrile (v / v)
0%
10%
20%
30%
40%
50%
60%
100%
Aliphatic carboxylic acids
as
1.000
bs
0.000
SD
–
r
–
1.076
20.089
0.18
0.987
1.095
0.091
0.21
0.984
1.118
0.289
0.24
0.979
1.146
0.507
0.29
0.972
1.183
0.744
0.33
0.965
1.235
0.997
0.39
0.956
2.135
10.784
1.62
0.893
Aromatic carboxylic acids
as
1.000
bs
0.000
SD
–
r
–
0.953
0.374
0.13
0.981
0.933
0.717
0.19
0.960
0.930
1.008
0.24
0.939
0.998
1.130
0.28
0.931
1.046
1.313
0.30
0.926
1.193
1.273
0.33
0.929
1.511
14.095
0.60
0.933
Phenols
as
bs
SD
r
1.000
0.000
–
–
–
–
1.163
20.804
0.15
0.998
1.186
20.672
0.13
0.998
1.215
20.508
0.17
0.998
1.213
20.120
0.16
0.998
1.216
0.378
0.21
0.996
1.706
9.455
0.42
0.996
1.000
0.000
1.011
20.144
0.14
0.999
1.029
20.418
0.12
0.999
1.044
20.661
0.16
0.999
1.050
20.760
0.19
0.998
1.073
21.011
0.23
0.997
1.080
21.007
0.28
0.996
1.479
2.842
0.81
0.983
42.20%
1.008
20.570
0.15
0.997
66.07%
1.000
20.848
0.14
0.997
81.41%
0.999
20.779
0.10
0.998
92.11%
0.970
20.166
0.16
0.996
94.30%
1.042
20.071
0.14
0.997
–
–
Amines
as
bs
SD
r
–
–
Pyridines
as
bs
SD
r
0%
1.000
0.000
–
–
in acetonitrile–water mixtures with the method proposed is illustrated with the set of 16 pyridines
studied by McCalley [33,34] by liquid chromatography. The pyridines were studied in isoelutropic
mixtures of methanol (55%), acetonitrile (40%) and
tetrahydrofuran (25%) in combination with a phosphate buffer at aqueous pH 7.0. Table 6 presents the
aqueous pKa values of the pyridines as reported by
McCalley [33,34]. We have also calculated the
degree of ionization of these pyridines (aHB ) at the
aqueous pH 7.0 of the phosphate buffer through the
equation:
HB 1
1
a 5 ]]]
1 5 ]]]]
B 1 HB
1 1 10 pH2pK a
(5)
According to the aqueous pKa and pH of the
buffer, pyridines should be partially protonated,
especially the most basic dimethylpyridines. However, McCalley found that pyridines behaved chro-
100%
1.314
6.136
0.29
0.996
matographically as completely unprotonated in 55%
methanol, 40% acetonitrile and 25% tetrahydrofuran
mobile phases [34]. Based on potentiometric and
spectrophotometric measurements he deduced that
the pKa value of pyridines decreased and the pH
value of the buffer increased with the addition of the
organic modifier to the aqueous buffer, resulting in a
much lower degree of protonation of the pyridines.
From Eqs. (1)–(3) and the parameters of Tables 4
and 5 and the similar parameters given in the
literature [11] for methanol–water one can calculate
the ss pKa values of the pyridines in 40% acetonitrile
and 55% methanol mobile phases. The equations
give a s 50.959 and b s 5 21.013 for 55% methanol
and a s 50.996 and b s 5 20.583 for 40% acetonitrile,
which led to the ss pKa values given in Table 6. pKa
values in 40% acetonitrile and 55% methanol are
about 0.6 and 1.3 pK units lower than in water,
respectively. The variation of the pH of the aqueous
buffer by addition of methanol or acetonitrile can be
S. Espinosa et al. / J. Chromatogr. A 964 (2002) 55–66
61
Table 3
s
s pK a values of the compounds studied in acetonitrile–water mixtures
Acetonitrile (v / v)
0%
10%
20%
30%
40%
50%
60%
Ref.
Aliphatic carboxylic acids
2,3-Dibromopropionic acid
Chloroacetic acid
2,3-Dichloropropionic acid
2-Chloropropionic acid
Tartaric acid (pK1 )
Citric acid (pK1 )
3-Bromopropionic acid
Cinnamic acid
Acetic acid
Valeric acid
Isobutyric acid
Propionic acid
2.17
2.87
2.89
2.90
3.03
3.13
4.02
4.44
4.76
4.80
4.85
4.88
2.46
3.01
2.52
3.14
3.21
3.31
4.26
4.74
4.95
5.13
5.23
5.11
2.76
3.22
2.77
3.40
3.38
3.49
4.52
4.94
5.17
5.46
5.62
5.35
3.10
3.44
3.06
3.69
3.59
3.68
4.82
5.19
5.42
5.82
6.03
5.63
3.48
3.67
3.40
4.03
3.83
3.90
5.16
5.52
5.73
6.20
6.48
5.96
3.90
3.89
3.79
4.43
4.11
4.16
5.57
5.97
6.10
6.63
6.96
6.36
4.39
4.13
4.25
4.90
4.46
4.45
6.04
6.59
6.57
7.09
7.48
6.85
[29]
[29]
[29]
[29]
[29]
[29]
[29]
[29]
[29]
[29]
[29]
[29]
Aromatic carboxylic acids
2-Nitrobenzoic acid
o-Phthalic acid (pK1 )
4-Nitrobenzoic acid
3-Nitrobenzoic acid
1-Naphthoic acid
3-Chlorobenzoic acid
3-Hydroxybenzoic acid
3-Methylbenzoic acid
Benzoic acid
2.19
2.89
3.45
3.49
3.69
3.79
4.00
4.21
4.21
2.58
3.08
3.50
3.54
4.07
3.88
4.23
4.41
4.49
2.95
3.26
3.82
3.75
4.44
4.06
4.50
4.68
4.77
3.29
3.45
4.10
4.01
4.79
4.29
4.79
4.98
5.05
3.60
3.68
4.45
4.39
5.24
4.64
5.19
5.39
5.44
3.89
3.93
4.85
4.77
5.67
4.99
5.57
5.78
5.75
4.16
4.23
5.39
5.31
6.26
5.49
6.10
6.34
6.25
[29]
[29]
[29]
[29]
[6]
[30]
[30]
[30]
[6]
Phenols
Resorcinol
Phenol
2,4-Dichlorophenol
2,4-Dinitrophenol
b-Naphthol
2-Nitrophenol
3-Bromophenol
4-Chlorophenol
3-Methylphenol
3-Aminophenol (phenol)
9.81
9.98
7.85
4.07
9.57
7.24
8.87
9.42
10.00
9.99
–
–
–
–
–
–
–
–
–
–
10.51
10.80
8.18
4.07
10.27
7.40
9.63
10.11
11.06
10.87
10.81
11.13
8.56
4.25
10.72
7.71
10.02
10.47
11.35
11.20
11.13
11.69
9.02
4.51
11.32
8.06
10.46
10.90
11.73
11.57
11.48
11.89
9.50
4.81
11.59
8.57
10.80
11.21
12.11
12.14
11.92
12.38
10.14
5.25
12.08
9.20
11.25
11.66
12.65
12.81
[6]
[6]
[6]
[6]
[6]
[6]
[6]
[6]
[6]
[6]
Amines
2,6-Dimethylaniline
4-Chloroaniline
Aniline
p-Toluidine
N-Ethylaniline
N,N-Dimethylbenzylamine
Ammonia
Ethanolamine
Triethylamine
3.95
4.00
4.61
5.08
5.12
8.91
9.29
9.48
10.66
3.79
3.68
4.56
5.07
5.28
8.73
9.27
9.47
10.63
3.60
3.58
4.38
4.86
4.98
8.54
9.21
9.45
10.54
3.43
3.40
4.20
4.73
4.77
8.35
9.17
9.41
10.41
3.36
3.25
4.10
4.72
4.71
8.29
9.19
9.38
10.30
3.21
3.27
3.99
4.53
4.41
8.11
9.21
9.44
10.33
3.24
3.39
4.03
4.54
4.33
8.14
9.34
9.60
10.33
[6]
[6]
[6]
[6]
[6]
[6]
[31]
[31]
[31]
42.20%
8.61
5.91
5.47
5.38
5.07
4.78
4.39
2.87
66.07%
8.30
5.56
5.13
5.03
4.72
4.39
4.10
2.62
81.41%
8.31
5.74
5.17
5.10
4.81
4.44
4.13
2.68
92.11%
8.63
6.21
5.59
5.55
5.20
4.86
4.77
3.10
94.30%
9.39
6.78
6.10
6.08
5.72
5.32
5.17
3.49
[32]
[32]
[32]
[32]
[32]
[32]
[32]
[32]
Pyridines
4-Aminopyridine
2-Aminopyridine
4-Methylpyridine
2-Methylpyridine
3-Methylpyridine
Pyridine
3-Hydroxypyridine
3-Acetylopyridine
0%
9.06
6.66
5.93
5.91
5.58
5.17
4.75
3.55
62
S. Espinosa et al. / J. Chromatogr. A 964 (2002) 55–66
Fig. 2. Variation of the slope of the linear correlations between ss pKa values in acetonitrile–water or methanol–water and the ww pKa in pure
water with solvent composition for the studied series of acids. Acetonitrile–water, symbols as in Fig. 1. Methanol–water: (♦) aliphatic
carboxylic acids, (m) aromatic carboxylic acids without ortho substituents, (.) ortho-substituted aromatic carboxylic acids, (d) phenols,
(j) amines, (1) pyridines.
also calculated from pK variation. The pH of a
dihydrogen phosphate / hydrogen phosphate buffer
can be calculated in a good approximation by the
Henderson equation, which follows:
pH 5 pKa 1 log ([HPO 422 ] / [H 2 PO 42 ])
(6)
2
Since the [HPO 22
4 ] / [H 2 PO 4 ] ratio remains constant with the addition of organic modifier, the pH
variation is equal to the pKa variation of dihydrogen
phosphate acid. The variation of the pKa value of
dihydrogen phosphate in methanol–water and acetonitrile–water has been studied in previous studies
[2,29]. From the equations developed in these works,
we can calculate that the addition of methanol up to
55% in volume or acetonitrile up to 40% increases
the pKa value of dihydrogen phosphate in 1.22 pKa
units or 0.85 pKa units, respectively. Thus, the ss pH
values of the phosphate aqueous buffer of pH 7.0
should be 8.22 and 7.85 in 55% methanol and 40%
acetonitrile, respectively. The methanol calculation
agrees very well with the potentiometric measurements of McCalley [33] who determined an ss pKa
value of 8.25 for the same buffer in 55% methanol.
The acetonitrile buffer was not measured.
From the estimated ss pKa values of pyridines and
s
s pH values of the buffer, the degree of ionization of
the pyridines in the 55% methanol and 40% acetonitrile mobile phase has been calculated. The results
are given in Table 6 and they demonstrate that the
pyridines are practically unprotonated (less than 2%
protonated) in those mobile phases.
3.4. Estimation of aqueous pKa values from liquid
chromatography retention data
The proposed equations that relate pKa values in
acetonitrile–water mixtures with pKa values in water
S. Espinosa et al. / J. Chromatogr. A 964 (2002) 55–66
63
Fig. 3. Variation of the intercept of the linear correlations between ss pKa values in acetonitrile–water or methanol–water and the ww pKa in
pure water with solvent composition for the studied series of acids. Symbols as in Fig. 2.
Table 4
Parameters for prediction of the slope (a s ) of the linear correlations between ss pKa values in acetonitrile–water and the ww pKa values in pure
water (Eqs. (2) and (3))
Aliphatic carboxylic acids
Aromatic carboxylic acids
Phenols
Amines
Pyridines
a1
a2
a3
a4
SD
F
9.97
22.42
10.05
20.73
21.67
28.59
3.14
210.04
20.27
0.67
8.83
21.98
7.97
20.87
21.66
28.72
2.12
28.37
20.12
0.67
0.01
0.02
0.02
0.00
0.03
5464
362
386
3476
38
Table 5
Parameters for prediction of the intercept (b s ) of the linear correlations between ss pKa values in acetonitrile–water and the ww pKa values in
pure water (Eqs. (2) and (3))
Aliphatic carboxylic acids
Aromatic carboxylic acids
Phenols
Amines
Pyridines
b1
b2
b3
b4
SD
F
20.68
9.97
25.33
21.82
21.78
9.94
29.12
9.95
2.25
1.89
8.45
5.96
0.19
21.75
20.58
28.59
26.90
20.70
0.90
20.40
0.08
0.14
0.11
0.05
0.10
5152
2607
2406
1559
1293
S. Espinosa et al. / J. Chromatogr. A 964 (2002) 55–66
64
Table 6
pKa values of protonated pyridines in water and in isoelutropic methanol–water (55:45 v / v) and acetonitrile–water (40:60 v / v) mixtures and
degree of ionization (aHB ) of pyridines in the three solvents prepared from an aqueous phosphate buffer of ww pH57.00
Compound
H2O
pH57.00
w
w
w
w
Pyridine
2-Methylpyridine
3-Methylpyridine
4-Methylpyridine
2-Ethylpyridine
3-Ethylpyridine
4-Ethylpyridine
2,3-Dimethylpyridine
2,4-Dimethylpyridine
2,6-Dimethylpyridine
3,4-Dimethylpyridine
3,5-Dimethylpyridine
2-Propylpyridine
4-Isopropylpyridine
4-tert.-Butylpyridine
MeOH–H 2 O (55:45 v / v)
pH58.22
s
s
MeCN–H 2 O (40:60 v / v)
pH57.85
s
s
pKa
aHB
s
s
pKa
aHB
s
s
pKa
aHB
5.17
5.96
5.68
6.00
5.89
5.80
5.87
6.57
6.74
6.71
6.47
6.09
6.30
6.02
5.99
0.01
0.08
0.05
0.09
0.07
0.06
0.07
0.27
0.35
0.34
0.23
0.11
0.17
0.09
0.09
3.94
4.70
4.43
4.74
4.63
4.55
4.61
5.28
5.45
5.42
5.19
4.82
5.03
4.76
4.73
5.3E205
3.0E204
1.6E204
3.3E204
2.6E204
2.1E204
2.5E204
1.2E203
1.7E203
1.6E203
9.3E204
4.0E204
6.4E204
3.4E204
3.2E204
4.57
5.35
5.07
5.39
5.28
5.19
5.26
5.96
6.13
6.10
5.86
5.48
5.69
5.41
5.38
5.2E204
3.2E203
1.7E203
3.5E203
2.7E203
2.2E203
2.6E203
1.3E202
1.9E202
1.7E202
1.0E202
4.3E203
6.9E203
3.6E203
3.4E203
for families of compounds can be used to estimate
the aqueous pKa values of members of the studied
families from the chromatographically determined
pKa values in particular acetonitrile–water mobile
phases. Tables 4 and 5 show that the standard
deviations of the fits of the experimental a s and b s
parameters for the different families to mobile phase
composition are 0.03 or less for a s and between 0.05
and 0.14 for b s . According to Eq. (1), these standard
deviations should produce errors about 0.2 pK units
or less in pKa estimation, which is the precision
expected for pKa measurement in non-aqueous and
mixed solvents.
The accuracy of the method for pKa estimation in
water from pKa values determined in chromatographic mobile phases has been tested with the chromatographic pKa data obtained in a previous work for
several compounds at different acetonitrile–water
mobile phases [4]. The compounds belong to four
different chemical families (aromatic carboxylic
acids, pyridines, phenols, and amines). The results
obtained are presented in Table 7. It can be observed
Table 7
Estimation of aqueous ww pKa values from chromatographic ss pKa values in different mobile phases by Eqs. (1)–(3)
Compound
Solvent
as
bs
s
s
Benzoic acid
20% MeCN
40% MeCN
60% MeCN
20% MeCN
40% MeCN
60% MeCN
40% MeCN
40% MeCN
50% MeOH
50% MeOH
50% MeOH
50% MeOH
50% MeOH
0.932
0.979
1.183
0.998
0.996
0.993
1.199
1.055
1.279
1.087
1.087
1.087
0.970
0.717
1.130
1.273
20.323
20.583
20.765
20.560
20.822
20.130
20.014
20.014
20.014
20.327
4.70
5.48
6.43
4.95
4.75
4.49
9.55
9.86
5.24
4.38
3.97
9.09
4.20
Pyridine
3-Nitrophenol
Triethylamine
Benzoic acid
2,4-Dinitrophenol
2,6-Dinitrophenol
3-Nitrophenol
Aniline
pKa
w
w
pKa (lit)
4.21
4.21
4.21
5.17
5.17
5.17
8.43
10.66
4.21
4.02
3.71
8.39
4.61
w
w
pKa (calc.)
4.27
4.44
4.36
5.28
5.35
5.29
8.43
10.12
4.20
4.04
3.66
8.37
4.67
D ww pKa
20.06
20.23
20.15
20.11
20.18
20.12
0.00
0.54
0.01
20.02
0.05
0.02
20.06
S. Espinosa et al. / J. Chromatogr. A 964 (2002) 55–66
that the predictions of aqueous pKa values for
benzoic acid, pyridine and 3-nitrophenol agree with
the literature aqueous pKa values in 0.2 pK units or
less. However, the pKa values for triethylamine
differ in 0.5 pK units. This discrepancy is attributed
to the value of the original chromatographic pKa data
in 40% acetonitrile (9.86) which differ considerably
from the potentiometric pKa data for the same
solvent composition (10.30, see Table 3).
Estimation of aqueous pKa values from acetonitrile–water mobile phases can be compared with
estimation from methanol–water mobile phases.
Table 7 reports also estimations of pKa values from
50% methanol mobile phase [3] for five compounds
(one aromatic carboxylic acid, three phenols, and one
amine). The agreement between aqueous literature
and estimated pKa values is better than for acetonitrile–water (less than 0.1 pK units of difference for
50% methanol). This is not surprising because the
pK data analyzed in methanol–water mixtures was
much more extensive and the values of the fitting
parameters are expected to be more robust.
65
accurate estimation of the compound ionization can
be very useful in optimization of conditions of
separation of complex mixtures of acid–base compounds (mobile phase pH and composition). The
simplicity of the equations proposed allows an easy
implementation in optimization algorithms and computer programs. On the other hand, the equations
proposed can be used for estimation of aqueous pKa
values from chromatographically determined pKa
values in a particular acetonitrile–water mobile
phase.
However, it must be emphasized that the relationships have been established between the aqueous
w
s
w pH scale and the solvent dependent s pH scale. This
means that the application of the estimation methods
to practical liquid chromatography problems requires
a proper measurement of the pH of the mobile phase.
This pH must be measured in the particular acetonitrile–water mixture used as mobile phase. The pHelectrode system can be calibrated with ss pH standards prepared in the same acetonitrile–water mixture used as mobile phase or with the usual aqueous
standards and converted to ss pH through d values [4].
4. Conclusions
It has been demonstrated that linear relationships
between ss pKa values in acetonitrile–water mixtures
and ww pKa values in water (Eq. (1)) hold for at least
five different families of compounds: aliphatic carboxylic acids, aromatic carboxylic acids, phenols,
amines and pyridines. The slopes and intercepts of
these correlations can be related to acetonitrile–water
composition by means of Eqs. (2) and (3). Combination of Eqs. (1)–(3) leads to simple relationships
between the aqueous ww pKa value of any member of
the studied families (even of those members not
included in the original compound sets) and the ss pKa
value of this member at any acetonitrile–water
composition up to 60% of acetonitrile in volume.
The established relationships have two main chromatographic applications. On one hand, they can be
used to estimate the ss pKa value of a compound in a
particular acetonitrile–water mobile phase from its
aqueous pKa value. From the estimated ss pKa value
and the pH of the mobile phase, the degree of
ionization of the acid–base compound in this particular mobile phase can be easily estimated. An
Acknowledgements
We are grateful for financial support from the
´ General de Investigacion
´ of the Spanish
Direccion
Government (project BQU2001-2882) and from the
Catalan Government (grant 2001SGR 00055).
References
´ Anal. Chem. 68
[1] E. Bosch, P. Bou, H. Allemann, M. Roses,
(1996) 3651.
´ I. Canals, H. Allemann, K. Siigur, E. Bosch, Anal.
[2] M. Roses,
Chem. 68 (1996) 4094.
´ Anal. Chem. 72
[3] I. Canals, J.A. Portal, E. Bosch, M. Roses,
(2000) 1802.
´ Anal. Chem. 72 (2000)
[4] S. Espinosa, E. Bosch, M. Roses,
5193.
´ E. Bosch, J. Chromatogr.
[5] I. Canals, F.Z. Oumada, M. Roses,
A 911 (2001) 191.
´ J. Chromatogr. A 947
[6] S. Espinosa, E. Bosch, M. Roses,
(2002) 47.
´ E. Bosch, Chromatographia 55
[7] I. Canals, J. Portal, M. Roses,
(2002) 565.
66
S. Espinosa et al. / J. Chromatogr. A 964 (2002) 55–66
´ J. Chromatogr. A 945
[8] S. Espinosa, E. Bosch, M. Roses,
(2002) 83.
[9] R.M. Lopes Marques, P.J. Schoenmakers, J. Chromatogr. 592
(1992) 157.
´ F. Rived, E. Bosch, J. Chromatogr. A 867 (2000)
[10] M. Roses,
45.
´ Anal. Chim. Acta
[11] F. Rived, I. Canals, E. Bosch, M. Roses,
439 (2001) 315.
`
´ E. Bosch, J. Pharm. Sci.
[12] F.Z. Oumada, C. Rafols,
M. Roses,
91 (2002) 991.
[13] M.K. Chantooni, I.M. Kolthoff, Anal. Chem. 51 (1979) 133.
´ E. Bosch, Anal. Chim. Acta 374 (1998)
[14] F. Rived, M. Roses,
309.
[15] IUPAC Compendium of Analytical Nomenclature, 3rd ed.,
Definitive Rules 1997, Blackwell, Oxford, 1998.
[16] R.G. Bates, in: 2nd ed, Determination of pH. Theory and
Practice, Wiley, New York, 1973.
´ Talanta 36 (1989) 627.
[17] E. Bosch, M. Roses,
[18] J. Barbosa, V. Sanz-Nebot, Talanta 36 (1989) 837.
`
´ Talanta 36 (1989) 1227.
[19] E. Bosch, C. Rafols,
M. Roses,
`
´ E. Bosch, Anal. Chim. Acta 338 (1997)
[20] C. Rafols,
M. Roses,
127.
[21] A. Fini, P. De Maria, A. Guarnerini, L. Varoli, J. Pharm. Sci.
76 (1987) 48.
´
[22] A.G. Gonzalez,
M.A. Herrador, Anal. Chim. Acta 356
(1997) 253.
´ S. Butı,
´ J. Barbosa, Anal. Chim. Acta 403 (2000)
[23] D. Barron,
349.
[24] K. Izutsu, IUPAC: Acid–base Dissociation Constants in
Dipolar Aprotic Solvents, Blackwell, Oxford, 1990.
[25] K. Sarmini, E. Kenndler, J. Biochem. Biophys. Methods 38
(1999) 123.
[26] D. Augustin-Nowacka, L. Chmurzynski, Anal. Chim. Acta
381 (1999) 215.
¨ W. Vogel, K. Andrussow, Dissociation Constants
[27] G. Kortum,
of Organic Acids in Aqueous Solution, Butterworths, London, 1961.
[28] D.D. Perrin, Dissociation Constants of Organic Bases in
Aqueous Solution, Butterworths, London, 1965, Supplement,
1972.
´ J. Chromatogr. A 824
[29] E. Bosch, S. Espinosa, M. Roses,
(1998) 137.
[30] K. Sarmini, E. Kenndler, J. Chromatogr. A 833 (1999) 245.
´ Anal. Chim. Acta 454
[31] S. Espinosa, E. Bosch, M. Roses,
(2002) 157.
[32] Z. Pawlak, J. Chem. Thermodyn. 19 (1987) 443.
[33] D.V. McCalley, J. Chromatogr. A 664 (1994) 139.
[34] D.V. McCalley, J. Chromatogr. A 708 (1995) 185.