Anal. Chem.
2002, 74, 3809-3818
Retention of Ionizable Compounds on HPLC. 12.
The Properties of Liquid Chromatography Buffers
in Acetonitrile-Water Mobile Phases That
Influence HPLC Retention
Sonia Espinosa, Elisabeth Bosch, and Martı́ Rosés*
Departament de Quı́mica Analı́tica, Universitat de Barcelona, Diagonal 647, E-08028 Barcelona, Spain
The addition of acetonitrile to aqueous buffers to prepare
RP HPLC mobile phases changes the buffer properties
(pH and buffer capacity). This variation is studied for ace-
tate, phosphate, phthalate, citrate, and ammonia buffers
in acetonitrile-water mixtures up to 60% in acetonitrile
(v/v). Equations are proposed to relate pH and buffer
capacity change of these buffers to the initial aqueous pH
value and to the volume fraction of acetonitrile added. It
is demonstrated that the pH change of the buffer depends
not only on the initial aqueous pH of the buffer and on
the percentage of acetonitrile added but also on the
particular buffer used. The proposed equations allow an
accurate prediction of this ionization for the studied
buffers. Since the retention of acid/base compounds
shows a strong dependence of their degree of ionization,
the equations are used to predict the change in this
ionization with addition of acetonitrile when the RP HPLC
mobile phase is prepared. This prediction allows estima-
tion of the retention of an acid/base compound in a
particular acetonitrile-water buffered mobile phase.
Buffers are widely used for pH control of chemical processes.1
In reversed-phase high-performance liquid chromatography (RP
HPLC) separations of compounds with acid/base properties,
appropriate buffered solutions are needed for calibration of the
electrode system used to measure pH and for control of the
mobile-phase pH, since the latter influences analyte ionization and
thus retention.
pH standardization of electrode systems in water and in the
most used RP HPLC mobile phases has been substantially
achieved. The IUPAC has endorsed rules for pH standardization
in water and in water-organic solvent mixtures of moderate to
high permittivities and proposed some buffered solutions for pH
standardization in these solvents.2-4 Some additional buffers in
* Corresponding author. Fax: 34 93 402 12 33. E-mail: marti@apolo.
qui.ub.es.
(1) Bates, R. G. Determination of pH: Theory and Practice, 2nd ed.; Wiley: New
York, 1964.
(2) IUPAC Compendium of Analytical Nomenclature. Definitive Rules 1997, 3rd
ed.; Blackwell: Oxford, U.K., 1998.
(3) Mussini, T.; Covington, A. K.; Longhi, P.; Rondinini, S. Pure Appl. Chem.
1985, 57, 865-876.
(4) Mussini, P. R.; Mussini, T.; Rondinini, S. Pure Appl. Chem. 1997, 69, 1007-
1014.
10.1021/ac020012y CCC: $22.00 © 2002 American Chemical Society
Published on Web 07/03/2002
acetonitrile-water have been studied by Barbosa et al.5-7 On the
basis of IUPAC rules and recommendations, we have studied the
different pH scales that are employed in pH measurement of RP
HPLC mobile phases. Three different pH scales have been used.
The most common is the aqueous pH scale (wwpH), which is
obtained when the electrode system is calibrated with aqueous
buffers and the pH measured in the RP HPLC aqueous buffer
before mixing it with the organic modifier. If the electrode system
is calibrated with aqueous buffers, but the pH is measured in the
mobile phase obtained after mixing the aqueous buffers with the
organic modifier, the pH scale in the mobile-phase solvent (s)
relative to water (w) as standard-state solvent is obtained (swpH).
Finally, if the electrode system is calibrated with buffers prepared
in the same mixed solvent used as mobile phase (s) and the pH
is measured in the mobile phase (s), the pH scale in the mobile
phase referred to the same mobile phase as standard-state solvent
is obtained (sspH). Whenever possible, we recommend sspH and
s
wpH scales because they give much better general relationships
between pH and retention.8-10 The swpH scale is specially recom-
mended because of its simplicity of measurement, since it does
not require pH standards for each different mobile-phase composi-
tion.
However, the main acid/base properties of the buffers used
to control the pH of RP HPLC mobile phases has not been
systematically studied. In a pioneer work, Bates discussed the
properties of acid/base aqueous buffers and its applicability to
chemical processes.1 Bates remarked that in choosing a suitable
buffer system, one should not only consider the pH required but
should also take into account the nature of the reaction to be
regulated. A high buffer capacity is essential for the control of an
acid/base reaction, and a low dilution value is desirable if dilution
of the medium is likely to occur. The dilution value is defined as
the increase of pH suffered by a solution when it is diluted with
an equal volume of pure solvent. Salt effects and temperature
changes may be important in some instances. The chemical nature
(5) Barbosa, J.; Sanz-Nebot, V. Anal. Chim. Acta 1993, 283, 320-325.
(6) Barbosa, J.; Butı́, S.; Sanz-Nebot, V. Talanta 1994, 41, 825-831.
(7) Barbosa, J.; Sanz-Nebot, V. Mikrochim. Acta 1994, 116, 131-141.
(8) Canals, I.; Portal, J. A.; Bosch, E.; Rosés, M. Anal. Chem. 2000, 72, 1802-
1809.
(9) Espinosa, S.; Bosch, E.; Rosés, M. Anal. Chem. 2000, 72, 5193-5200.
(10) Canals, I.; Oumada, F. Z.; Rosés, M.; Bosch, E. J. Chromatogr., A 2001,
911, 191-202.
Analytical Chemistry, Vol. 74, No. 15, August 1, 2002 3809
of the buffer materials is a key factor, because the buffer cannot Table 1. Properties of Relevant Interest for pH
form insoluble compounds or undesired side reactions. All these Measurement in Acetonitrile-Water Mixtures at 25 °Ca
factors, cited by Bates, should be considered in the selection of s
% MeCN A a0B spKap δ
RP HPLC buffers. In addition, the compatibility of the buffer with
the detection method used must be also considered; e.g., the 0 0.528 1.52 14.00 0.00
buffer must not absorb in the UV region in analyte UV detection 10 0.566 1.55 14.24 -0.01
20 0.604 1.59 14.47 -0.03
or form volatile compounds for mass spectrometry detection. 30 0.655 1.63 14.74 -0.04
In this work, we shall study the acid/base properties of some 40 0.712 1.68 15.08 -0.14
of the buffers most used to control the pH of acetonitrile-water 50 0.791 1.74 15.48 -0.22
60 0.877 1.80 15.90 -0.46
RP HPLC mobile phases. RP HPLC buffers are commonly
prepared in water and later mixed with the organic modifier a A and a B, Debye-Hückel equation parameters; spK , autopro-
0 s ap
(acetonitrile in this instance). When an aqueous buffer is diluted tolysis constant of the solvent mixture (pK values); δ, interconversion
s s
parameter between spH and wpH scales (eq 1).
with acetonitrile, its acid/base properties (namely, pH and buffer
capacity) are altered because of both the dilution and the change
of solvent. The properties change depends on the particular type
and composition of the buffer prepared. An understanding of the drogenphosphate/disodium hydrogenphosphate (wwpH 7.02). The
changes produced by addition of acetonitrile to the most typical pH readings provide directly the swpH value of the solution.
RP HPLC aqueous buffers seems essential in order to foresee
the suitability of the buffer for a practical RP HPLC separation.
RESULTS AND DISCUSSION
Relationships between the pH of a Buffer Measured in
EXPERIMENTAL SECTION
Apparatus. Potentiometric measurements were taken with a the Different pH Scales. As pointed out in the introduction, the
Ross combination electrode Orion 8102 (glass electrode and a three procedures used in RP HPLC to measure the pH of buffers
reference electrode with a 3.0 mol‚L-1 KCl solution in water as lead to three different pH quantities. If one calibrates the electrode
salt bridge) in a Crison micropH 2002 potentiometer with a system with aqueous buffers and measures the pH of the RP
precision of (0.1 mV. All the solutions were thermostated HPLC aqueous buffer before mixing it with the organic modifier,
w
externally at 25 ( 0.1 °C. The retention data were measured on wpH is obtained. However, the pH of the solution changes after

a 15 cm × 4.6 mm i.d. Xterra MS C18 5-µm (Waters) column with
a flow rate of 1 mL/min in an Isco (Lincoln, NE) model 2350 dual-
pump system with a 20-µL loop valve. A Shimadzu (Kyoto, Japan)
model SPD-10Avp UV-visible detector was used set at 254 nm.
All data were taken by triplicate at 25 °C with the potentiometric
cell and the column thermostated with water jackets.
Chemicals. Acetonitrile was RP HPLC gradient grade from
Merck and water purified by the Milli-Q plus system from
Millipore. The studied buffers were prepared from orthophos-
dilution of the aqueous buffer with the organic modifier. If the
electrode system is calibrated with aqueous buffers, but the pH
is measured after mixing the RP HPLC aqueous buffers with the
organic modifier, swpH is directly obtained. Finally, if the elec-
trode system is calibrated with buffers prepared in the same
solvent composition used as mobile phase, and the pH is measured
in this same mobile-phase composition (i.e., after mixing aqueous
buffer and organic modifier), the quantity obtained is sspH.
s s
wpH can be easily converted to spH by means of eq 1 which

phoric acid (Merck, 85%, for analysis), citric acid (Fluka Chemica,
for analysis), ammonia (Merck, 25%, for analysis), or sodium
dihydrogenphosphate monohydrate (Merck, for analysis) using
hydrochloric acid (Merck, 25%, for analysis) and sodium hydroxide
(Merck, for analysis) to adjust the pH to the desired value. The
chromatographied samples were 2-nitrophenol (Fluka, >99%),
3-bromophenol (Schuchardt, 90%), 2,4,6-trimethylpyridine (Merck,
96%) and N,N-dimethylbenzylamine (Merck-Schuchardt, for syn-
thesis). Care should be taken in the handling and disposal of these
samples. They are harmful by inhalation, in contact with skin, and
if swallowed. Wear suitable protective clothing. Neutralize 2,4,6-
trimethylpyridine and N,N-dimethylbenzylamine with acid before
its disposal. N,N-dimethylbenzylamine is harmful to aquatic
organisms and may cause long-term adverse effects. Avoid its
release to the environment.
Procedure. Aqueous buffers were prepared by addition until
the desired pH of concentrated solutions of sodium hydroxide or
hydrochloric acid to 0.01 mol‚L-1 aqueous solutions of phosphoric
acid, citric acid, sodium dihydrogenphosphate, or ammonia.
Acetonitrile-water buffers were prepared by addition of acetoni-
trile to the aqueous buffers. In all instances, the electrode system
was calibrated using the usual aqueous standard reference buffers
of potassium hydrogenphthalate (wwpH 4.00) and potassium dihy-
h j - log(swγH0) ) swpH - sspH
δ)E (1)
includes the difference of the liquid-junction potentials (or Ej,
expressed in pH units and assumed to be constant), together with
the primary medium effect - log(swγ0H). The addition of the two
terms leads to the useful quantity δ.1,8-10
The δ term is a constant value for each mobile-phase composi-
tion. Table 1 reports these values for some acetonitrile-water
mixtures, as well as other parameters of interest for pH estimation
in acetonitrile-water mixtures. The literature reports equations
to estimate these parameters for other acetonitrile-water com-
positions not given in the table.9,11,12
However, the difference between wwpH and sspH (or swpH)
depends not only on the mobile-phase composition but also on
the particular buffering solution employed.
Buffer solutions may be prepared from strong or weak acids
or bases.1 If an aqueous buffering solution is prepared from a
strong monoprotic acid of ca concentration, the pH is
(11) Rosés, M.; Ràfols, C.; Bosch, E. Anal. Chem. 1993, 65, 2294-2299.
(12) Bosch, E.; Fonrodona, G.; Ràfols, C.; Rosés, M. Anal. Chim. Acta 1997,
349, 367-376.

3810 Analytical Chemistry, Vol. 74, No. 15, August 1, 2002

 w
) -log aH ) -log ca - log wwγH
wpH
where γH stands for the activity coefficient of the hydrogen ion,
which is usually estimated by means of the Debye-Hückel
equation,
log γH ) -AI1/2/(1 + a0BI1/2)
where I is the ionic strength of the solution. Table 1 reports A
and a0B values for acetonitrile-water mixtures.
Later, the solution is diluted with the organic modifier and the
concentration of the acid changes to φH2Oca (neglecting volume
contraction), and the activity coefficient is ssγH, where φH2O is the
volume fraction of water in the mobile phase. The pH in the scale
relative to the acetonitrile-water mixtures is then
s ∆pH ) (∆pKa - log φH2O)/2
spH ) -log aH ) -log ca - log φH2O - logssγH
and the difference ∆pH is
∆pH ) sspH - wwpH ) -log φH2O - ∆ logγH
where
∆log γH ) log ssγH - log wwγH
The variation on the activity coefficients is very small because
the increase in the Debye-Hückel A parameter with acetonitrile
content (Table 1) is balanced by the decrease in the acid
concentration (ionic strength). For example, an initial acid
concentration of 0.1 mol‚L-1 in water has log wwγH equal to -0.11,
whereas when it is diluted to 50% acetonitrile, the term log ssγH is
-0.12. Thus, ∆log γH is meaningless and it will not further be
considered in this paper. Notice that we use the same IUPAC
nomenclature for ionic activity coefficients as for the related pH
and pK terms. A left-hand superscript indicates the medium where
the quantity is measured or determined (w for water and s for an
acetonitrile-water mixture). A left-hand subscript indicates the
standard-state medium (the solvent where activity coefficients are
taken as equal to unity at infinite dilution).
If the aqueous buffer is prepared from a strong monoprotic
base (e.g., KOH), the pH will depend also on the autoprotolysis
constant of the medium (Kap), because the hydrogen ion activity
is directly calculated from this constant and from the activity of
the lyate ion of the solvent (aOH in water, aS in general), i.e., aH )
Kap/aS.13-15 The activity of the lyate ion is equivalent to the
concentration of the strong base ([OH-]) corrected by the activity
coefficient. Addition of the organic modifier to the aqueous buffer
changes the concentration and activity of the lyate ions in the
term log φH2O, but it also changes the autoprotolysis constant of
(13) Sücha, L.; Kotrly, S. Solution Equilibria in Analytical Chemistry; Van Nostrand
Reinhold: London, 1972.
(14) Skoog, D. A.; West, D. M.; Holler, F. J. Fundamentals of Analytical Chemistry,
7th ed.; Saunders: Forth Worth, TX, 1996.
(15) Laitinen, H. A.; Harris, W. E. Chemical Analysis. An Advanced Text and
Reference, 2nd ed.; McGraw-Hill: New York, 1975.
the medium on a ∆pKap term (see Table 1). The overall pH change
(2)
is
∆pH ) ∆pKap + log φH2O (7)
where ∆pKap is the difference between the negative logarithms
(3) of the autoprotolysis constants of the solvent used as mobile phase
and water. Table 1 reports pKap values for acetonitrile-water
mixtures, which show that the term is important for these
solutions because the pH of the buffer will change more than 1
unit for high acetonitrile percentages.
A common case in RP HPLC is buffers prepared from a weak
acid, e.g., acetic acid. The hydrogen activity is approximately13-15
aH ) (Kaca)1/2, where Ka is the acidity constant of the acid. It can
be derived that
(8)
(4)
where ∆pKa is the difference between the pKa values of the acid
in the mobile-phase solvent and in water:
(5)
∆pKa ) sspKa - wwpKa (9)
If the initial aqueous solution is a weak base (e.g., NH3), the
(6) ∆pH also includes the variation in the autoprotolysis constant:
∆pH ) ∆pKap - (∆pKb - log φH2O)/2 )
(∆pKap +∆pKa + log φH2O)/2 (10)
The most common RP HPLC buffers are prepared from an
acid at concentration ca and its conjugated base at concentration
cb (e.g., acetic/acetate, NH4+/NH3, dihydrogenphosphate/hydro-
genphosphate, etc.). The Henderson-Hasselbach equation can
usually be applied:13-15
pH ) pKa + log(cb/ca) (11)
and the pH variation is
∆pH ) ∆pKa (12)
The pH difference does not include the dilution term log φH2O
because it affects to ca and cb to the same degree.8
Often, a buffer is prepared with a solution of an acidic salt.
For instance, the pH of 0.05 mol‚kg-1 potassium hydrogenphtha-
late solution has been adopted as the primary reference value
standard for pH calibration,2-4 sodium hydrogencarbonate buffer
is used in many biological studies,1 and sodium dihydrogencitrate
and sodium hydrogencitrate solutions are used in RP HPLC. The
pH of these solutions is practically equal to the average of the
two pKa values involved, and thus the pH variation is
∆pH ) (∆pKa1 + ∆pKa2)/2 (13)
Analytical Chemistry, Vol. 74, No. 15, August 1, 2002 3811
Table 2. swpKa Values of Acids in Acetonitrile-Water Mixtures
s
wpKa in % of acetonitrile by volume
acid/base pair 0 10 20 30 40 50 60 mpK
acetic/acetate 4.74 4.93 5.14 5.40 5.62 5.93 6.16 2.30
phosphoric/dihydrogenphosphate 2.21 2.38 2.59 2.76 2.97 3.20 3.29 1.87
dihydrogenphosphate/hydrogenphosphate 7.23 7.39 7.57 7.78 7.94 8.16 8.27 1.78
phthalic/hydrogenphthalate 2.92 3.07 3.23 3.41 3.54 3.71 3.77 1.51
hydrogenphthalate/phthalate 5.39 5.73 6.08 6.49 6.85 7.27 7.60 3.68
citric/dihydrogencitrate 3.16 3.30 3.46 3.64 3.76 3.94 3.99 1.48
dihydrogencitrate/hydrogencitrate 4.79 4.94 5.11 5.31 5.46 5.69 5.82 1.73
hydrogencitrate/citrate 6.42 6.61 6.82 7.07 7.26 7.52 7.67 2.12
ammonium/ammonia 9.29 9.26 9.18 9.13 9.05 8.99 8.88 -0.60

The above examples show that the pH difference between the

true pH of the mobile phase and the pH of the aqueous buffer is
very dependent on the buffer solution and especially on the pKa
variation. Even if buffers of the same type are used (e.g., acid/
conjugated base), different acids and bases with different ∆pKa
values will be needed to cover an extensive pH range.
All ∆pH terms in the equations above are given in the sspH
scale. ∆pH variations in the swpH scale can be easily calculated
from them by adding the δ value for the corresponding acetoni-
trile-water mixture (eq 1).
Variation with Solvent Composition of the pKa of Acids
and Bases Used To Prepare Buffers. Solutions prepared from
strong acids and bases have larger buffer capacity than solutions
prepared from weak acids or bases at the same concentration.1
However, the pH ranges buffered by solutions of strong acids and
bases lay in the extremes of the pH scale (pH <3 for strong acids
Figure 1. Variation of the pKa values of buffer components with
and pH >11 for strong bases in the aqueous wwpH scale). These
the addition of acetonitrile in the absolute pH scale (swpKa): (])
pH ranges are seldom used in practical RP HPLC, and thus, acetic acid, (0) phosphoric acid, (9) dihydrogenphosphate, (2)
common RP HPLC buffers are prepared from weak acids and phthalic acid, (4) hydrogenphthalate, (×) citric acid, ([) dihydrogen-
bases that may have an acceptable buffer capacity at pH values citrate, (/) hydrogencitrate, and (O) ammonium.
close to the pKa of the acid/base pair employed.
We studied the variation of the pKa values of some acids and s
bases commonly used to prepare RP HPLC buffers (acetic, wpKa variations with volume fraction of acetonitrile are close to
phosphoric, and citric acids and ammonia) with the addition of linear, as we have been already observed in previous work.18-20
acetonitrile up to 60% in volume. We also included phthalic acid In this instance, the swpKa variation (∆swpKa) can be approxi-
because 0.05 m potassium hydrogenphthalate is the reference mately described by the equation
value standard adopted by the IUPAC for pH standardization of
∆swpKa ) mpKφMeCN (14)
potentiometric systems.2-4 The pKa values were taken from the
literature,16,17 except for the first pKa of phosphoric acid, which
was not available, and it has been determined here for the
where φMeCN is the volume fraction of acetonitrile in the mixture
acetonitrile-water mixtures studied. The procedure used to
and mpK the slope of the plots of Figure 1, which has been included
determine the pKa values of phosphoric acid in acetonitrile-water
in Table 2. mpK values are positive for neutral (acetic, phosphoric,
mixtures is reported elsewhere.18 The third pKa value of phos-
phthalic, citric) and anionic (dihydrogenphosphate, dihydrogen-
phoric acid in acetonitrile-water has not been studied because it
citrate, hydrogencitrate, hydrogenphthalate) acids and negative
lays in the most basic region of the pH scale, which is seldom
for cationic acids (ammonium). This has been already explained
used in RP HPLC separations. This complicates the potentiometric
in terms of the electrostatic interactions that contribute to the pKa
determination of pK, and probably because of this reason, it has
value.15,18,21 The swpH values of buffers prepared from these acid/
not been reported in the literature.
base pairs are expected to change in a similar way with the
The pKa values of the studied acids in the swpH scale are
addition of acetonitrile.
presented in Table 2, and the variation of the pKa values with the
Variation with Solvent Composition of the swpH of Buff-
volume fraction of acetonitrile added is presented in Figure 1. The
ers. To study the pH variation when acetonitrile is added to an
(16) Barbosa, J.; Beltrán, J. L.; Sanz-Nebot, V. Anal. Chim. Acta 1994, 288, 271- aqueous buffer of a fixed pH value in water, we calculated the pH
278.
(17) Rondinini, S.; Nese, A. Electrochim. Acta 1987, 32, 1499-1505. (19) Espinosa, S.; Bosch, E.; Rosés, M. J. Chromatogr., A 2002, 945, 83-96.
(18) Espinosa, S.; Bosch, E.; Rosés, M. Anal. Chim. Acta 2002, 454, 157-166. (20) Espinosa, S.; Bosch, E.; Rosés, M. J. Chromatogr., A 2002, 947, 47-58.

3812 Analytical Chemistry, Vol. 74, No. 15, August 1, 2002


Figure 2. Variation of swpH of aqueous buffers prepared from
phthalic acid with the addition of acetonitrile. wwpH: (+) 2.54, (])
3.00, (2) 3.50, (×) 4.00, (0) 4.49, (O) 4.99, (b) 5.47, (4) 5.97, (f)
6.46, (9) 7.17, and ([) 8.55. Dashed lines are for swpH variation of
solutions of strong acids and bases. Overall aqueous buffer concen-
trations are 0.01 mol‚L-1.
of several of the acid/conjugate base buffer solutions of Table 2.
The overall ca + cb concentration has been fixed to 0.01 mol‚L-1.
The swpH values for different ca/cb ratios were calculated
through a procedure similar to that described by De Levie22 for
acid/base titrations of arbitrary mixtures, except that it incorpo-
rates the effect of the ionic activity coefficients.9,23
Figure 2 presents the results obtained for phthalic/hydrogen-
phthalate. The plots are close to linear. For initial aqueous pH
(wwpH) values between about 4.5 and 7, the slopes of the plots are
maximum and almost independent of the initial aqueous pH (the
lines are parallel). In this pH range, the hydrogenphthalate/
phthalate acid/base pair buffers the solution, and according to
the approximate eq 12, the ∆swpH variation should be equal to
the ∆swpKa variation of hydrogenphthalate in Figure 1 and
Table 2. A similar trend is observed for initial pH values between
3 and 3.5. These solutions are buffered by the phthalic/hydro-
genphthalate pair, which has a lower mpK value than that of
hydrogenphthalate/phthalate (Table 2), and thus the swpH varia-
tion is smaller. Between these two pH ranges, the slope of the
plot moves between the two mpK values of phthalic/hydrogen-
phthalate and hydrogenphthalate/phthalate. In fact, the pH
variation of a solution of pure hydrogenphthalate should follow
eq 13, and thus, the slope of this variation should be the average
of the mpK values of phthalic and hydrogenphthalate in Table 2
(pH 4 in Figure 2).
Therefore, the ∆swpH values obtained for buffers prepared
from phthalic acid/hydrogenphthalate/phthalate can be fitted to
(21) Rived, F.; Canals, I.; Bosch, E.; Rosés, M. Anal. Chim. Acta 2001, 439,
315-333.
(22) De Levie, R. Anal. Chem. 1996, 68, 585-590.
(23) Bosch, E.; Espinosa, S.; Rosés, M. J. Chromatogr., A 1998, 824, 137-146.
an equation similar to the one obtained for the variation of swpKa
values (eq 14), i.e.
∆swpH ) mpHφMeCN (15)
with an mpH value that depends on the initial wwpH of the buffer.
The most acidic buffer solution is the solution of pure phthalic
acid. The pH variation for this buffer is given by eq 8 and the
slope should be close to half the mpK value for phthalic/
hydrogenphthalate in Table 2, corrected by the change in the log
φH2O value. Figure 2 shows that the variation for this solution (pH
2.5) is the smallest observed. If a more acidic buffer is prepared
by addition of a strong acid, the pH would be determined by this
strong acid and the pH variation by eq 5. This depends only on
the log φH2O variation and for the swpH scale on the δ variation.
These terms do not show a linear dependence on φMeCN, although
their combination produces a very small pH variation, between
0.11 and -0.06 pH units in the studied range of 0-60% acetonitrile.
This would indicate that the swpH value of buffers prepared from
strong acids practically does not change with the addition of
acetonitrile (bottom dashed line in Figure 2).
The most basic buffer prepared from phthalic is the 0.01
mol‚L-1 solution of phthalate (pH 8.5 in Figure 2). The pH
variation for this solution is given by eq 10, which points out that
the slope obtained is intermediate between those obtained for
hydrogenphthalate/phthalate buffers and buffers prepared with
a strong base.
According to eqs 7 and 1, the pH variation for a strong base
should be
∆swpH ) ∆swpKap + log φH2O (16)
There is a good linear correlation between this ∆swpKap + log
φH2O term and the volume fraction of acetonitrile, φMeCN, in the
solvent mixture, which is
∆swpH ) (1.81 ( 0.03)φMeCN r ) 0.997
SD ) 0.03 F ) 1046 (17)
Therefore, the mpH slope for a strong base is 1.81 (top dashed
line in Figure 2).
The mpH values obtained for the swpH variation of the studied
buffers are presented in Figure 3. The interpretation of the mpH
variation for the different studied buffers follows the same trends
explained for phthalate buffers. Constant mpH values, close to mpK
values of Table 2, are obtained for initial wwpH values close to
each of the corresponding wwpKa values of the acid/base pair. For
buffers prepared from neutral or anionic acid/base pairs, the mpH
values are positive, and for initial wwpH values lower than the first
w
wpKa of the acid, the mpH values are between the mpK that
corresponds to this first wwpKa and zero.
For buffers prepared by a mixture of a cationic acid and the
corresponding neutral base (ammonium/ammonia), mpH values
are negative and close to the mpK values for the acid/base pair in
the initial pH range close to the pKa value. For higher pH values,
the mpH value increases toward the one that would be obtained
for a buffer solution prepared from a strong base (mpH ) 1.81).
Analytical Chemistry, Vol. 74, No. 15, August 1, 2002 3813

Figure 3. Variation of the slope of the swpH vs volume fraction of
acetonitrile plot (Figure 2) with the initial aqueous pH of the buffer
(wwpH): (O) phthalic acid, (]) acetic acid, (0) citric acid, (4) phos-
phoric acid, and (×) ammonium. Continuous lines fitted through eq
18.
The type of variation with pH (initial wwpH) obtained for the
mpH values indicates that it should fit well the following model:
n
∑a 10 s(ipH-bi)
a0 + i + an+110s((n+1)pH-bn+1)
i)1
mpH )
n
1+ ∑10
i)1
s(ipH-bi)
+ 10 s((n+1)pH-bn+1)
The first term of numerator and denominator (a0 and 1)
predominates at low-pH values (pH , b1) and it is the limiting
mpH value for buffer solutions prepared from strong acids; i.e., a0
must be practically equal to zero. The last term (the n + 1 term)
predominates at very basic pH values (pH . bn), and it stands
out for the buffer solutions prepared from strong bases, i.e., an+1
) 1.81 (eq 17). The intermediate terms stand out for the different
acid/conjugate base pairs that can form buffer solutions and there
are as many as pKa values for the compound studied (one for
acetic acid and ammonia, two for phthalic and phosphoric acids,
and three for citric acids). Each one of these terms prevails in
the pH zone close to the pKa that relates the concentrations of
the acid/base pair. The ai values should be close to the mpK values
reported in Table 2, and the bi values a combination of the pKa
values of the corresponding acid/base system. s is a fitting param-
eter that accounts for the velocity of transition between the
different pH zones buffered by the different acid/base pairs of
the system.
Equation 18 is analogous to the equation used to fit retention
time and chromatographic hydrophobicity index (CHI) to aqueous
pH during gradient elution.24,25 A similar equation with two terms
and s ) 1 was used to fit the swpH change of ammonium acetate
buffers in methanol-water mobile phases.24
The results obtained by application of the model to the mpH
values for the different acid/base systems studied are reported
3814 Analytical Chemistry, Vol. 74, No. 15, August 1, 2002
in Table 3. a0 and an+1 were fixed to the theoretical values of 0
and 1.81 and all the other parameters were adjusted by nonlinear
regression. Table 3 shows that ai values are close to the
corresponding mpK values of Table 2 and agree with the mpH values
of the plateaus of the plots of Figure 3. The bi values agree with
the pH value of the inflection points of the plots and they are
related to the wwpKa values of the acid/base pair. For instance, for
citric acid, b1 ) 2.62 ≈ 2.58 ) (pKa1 - log c)/2; b2 - b1 ) 3.92 ≈
3.98 ) (pKa1 + pKa2)/2; b3 - b2 ) 5.29 ≈ 5.60 ) (pKa2 + pKa3)/2;
b4 - b3 ) 8.87 ≈ (pKa3 + pKap + log c)/2, taking into account that
c ) 0.01 mol‚L-1 and pKap ) 14. Very good fits have been obtained
for all studied buffers.
Equation 18 offers an easy way to predict how the pH of an
aqueous buffer will change with the addition of acetonitrile. It
helps to choose the best aqueous buffer that will give a desired
s
wpH value in a particular acetonitrile-water mobile phase. The
exact swpH required could be later finely adjusted by addition of a
small amount of a strong acid or base. The other buffer property
that must be considered is the buffer capacity of the aqueous
buffer and how it changes with the addition of acetonitrile.
Variation of Buffer Capacity with Solvent Composition.
The buffer capacity of the studied buffer systems at the different
pH values has been calculated for several acetonitrile-water
compositions obtained by addition of acetonitrile to an initial
aqueous buffer at overall 0.01 mol‚L-1 concentration. Buffer
capacity is defined by the differential eq 19;1,13 i.e., it is the reverse
β ) dcb/dpH
(19)
(18)
of the pH variation observed for the buffer when a small
concentration of a strong base is added. It can be easily calculated
with the algorithm used to calculate the pH of the buffer by simple
calculation of the pH change observed for the buffer when the
concentration of conjugate base (cb) is increased by a small
amount (e.g., 0.1%). The results obtained are plotted in Figure 4.
The type of β versus pH plots observed for buffers in
acetonitrile-water are very similar to the ones commonly ob-
served in water, which is a solvent extensively studied,1,13 and
much simpler than those observed in low dielectric constant or
poor solvating solvents, such as pure acetonitrile.26-28
Taking activity coefficients equal to unity, the function that
describes the buffer capacity for an acid/base buffer with up to n
acid/base equilibria (n Ka constants) can be written as1,13
[ ]
n Kaici[H+] Kap
β ) 2.303 [H+] + ∑ + 2
+ (20)
i)1 (K
ai + [H ]) [H+]
where ci is the concentration of one of the acid/base pairs of the
buffer. It is an additive function of all acid/base pairs that compose
the buffer.
(24) Canals, I.; Valkó, K.; Bosch, E.; Hill, A. P.; Rosés, M. Anal. Chem. 2001,
73, 4937-4945.
(25) Valkó, K.; Espinosa, S.; Du, C. M.; Bosch, E.; Rosés, M.; Bevan, C.; Abraham,
M. H. J. Chromatogr., A 2001, 933, 73-81.
(26) Bosch, E.; Rosés, M. Talanta 1989, 36, 615-621.
(27) Barbosa, J.; Bosch, E.; Cortina, J. L.; Rosés, M. Anal. Chim. Acta 1992,
256, 211-220.
(28) Rosés, M. Anal. Chim. Acta 1994, 285, 391-399.
Table 3. Parameters of Eq 18 for the Variation of the swpH Values of Buffers with the Addition of Acetonitrile

acid s a0 b1 a1 b2 a2 b3 a3 b4 a4 N SD r F
ammonium 2.88 0.00 5.77 -0.61 16.41 1.81 23 0.01 0.998 2824
acetic 3.10 0.00 3.36 2.29 11.69 1.81 19 0.02 0.995 1097
phosphoric 1.62 0.00 2.21 1.42 6.85 1.75 16.78 1.81 21 0.02 0.997 939
phthalic 2.83 0.00 2.53 1.35 6.62 3.64 15.16 1.81 18 0.04 0.999 3151
citric 2.11 0.00 2.62 1.41 6.54 1.69 11.83 2.02 20.70 1.81 24 0.01 1.000 6921

Figure 4. Variation of the buffer capacity of the studied buffers with swpH and addition of acetonitrile. Acetonitrile percentages are indicated in
the figure.

The first and last terms of eq 20 describe the buffer capacity
of strong acids and bases, and the central terms, the buffer
capacity of the different weak acid/conjugate base pairs. There
are as many central terms as acid/base buffer pairs, i.e., acidity
constants. Each one gives a bell-shaped plot of β versus pH. When
these acidity constants are different enough, the buffer capacity
of each acid/base pair is independent of the ones of the other
phthalate solution is recommended by the IUPAC as a primary
standard for pH calibration in water and in acetonitrile-water
mixtures (as well in other solvents).2-4 The buffer capacity is given
by the minimum in the valley between the two peaks for phthalic
acid in Figure 4, and this minimum depends on the distance
between the two pKa values. From eq 20, it can be calculated that
then

acid/base pairs, and the maximum buffer capacity does not
depend on pKa values. The maximum1,13 is reached when [H+] )
Ka (or pH ) pKa) for which
βmax ) 0.576c (21)
This is the case for acetic, ammonium, and phosphoric acids.
The pKa1 value of the last one is very low and the contribution of
[H+] to eq 20 must be taken into account too. For these systems,
the addition of acetonitrile decreases c by dilution and the buffer
capacity decreases in the same ratio. That is to say, the buffer
capacity of a buffer in 50% acetonitrile is half the buffer capacity
in water.
But this is not the same when the different pKa values of the
acid/base system are close, such as in phthalic and citric acids.
The buffer capacity at a given pH depends on two or more acid/
base systems and on the pKa values of these systems in addition
to buffer concentration. For instance, a 0.05 mol‚kg-1 potassium
[ ]
β ) 4.606c
xKa1Ka2 (22)
(xKa1 + xKa2)2
Figure 1 shows that as acetonitrile is added the difference
between the two pKa values increases and therefore the minimum
of the valley in Figure 4 decreases in addition to the decrease by
dilution. The buffer capacity of this buffer in pure water have a
reasonable value, but Figure 4 shows that in 60% acetonitrile the
solution is almost not buffered. Despite IUPAC recommendations,
we do not recommend this buffer for calibration of electrode
systems in high acetonitrile percentages. A buffer prepared by
an equimolar mixture of hydrogenphthalate and phthalate would
be more advisable.
The same effect is observed for solutions of dihydrogencitrate
or hydrogencitrate although to a minor degree because Figure 1
shows that the variation of the difference between the pKa values
Analytical Chemistry, Vol. 74, No. 15, August 1, 2002 3815
Table 4. Comparison of Experimentally Measured swpH Values of Buffers with
s
wpH Values Calculated from Buffer Composition or Estimated by the Equations Developed in This Work

s a
wpH
% MeCN
0 2.29 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00
10 exp 2.36 3.07 4.11 5.18 6.20 7.21 8.14 8.97 9.96
calc 2.36 3.11 4.14 5.16 6.18 7.15 8.15 8.97 9.98
est 2.37 3.12 4.16 5.18 6.20 7.18 8.18 8.94 9.94
20 exp 2.44 3.20 4.28 5.38 6.42 7.44 8.36 8.89 9.88
calc 2.46 3.24 4.30 5.33 6.38 7.33 8.33 8.89 9.91
est 2.45 3.24 4.32 5.35 6.40 7.35 8.35 8.88 9.89
30 exp 2.55 3.36 4.47 5.58 6.65 7.62 8.51 8.78 9.78
calc 2.55 3.39 4.49 5.54 6.61 7.54 8.53 8.84 9.87
est 2.53 3.37 4.48 5.53 6.60 7.53 8.53 8.82 9.83
40 exp 2.65 3.50 4.65 5.76 6.81 7.77 8.63 8.71 9.69
calc 2.61 3.49 4.62 5.69 6.79 7.69 8.69 8.73 9.79
est 2.62 3.49 4.63 5.70 6.80 7.70 8.70 8.76 9.77
50 exp 2.74 3.62 4.80 5.91 6.94 7.90 8.78 8.57 9.59
calc 2.70 3.65 4.83 5.92 7.04 7.91 8.91 8.70 9.73
est 2.70 3.61 4.79 5.88 7.00 7.88 8.88 8.70 9.72
60 exp 2.83 3.75 4.94 6.05 7.04 8.03 8.86 8.44 9.45
calc 2.66 3.68 4.93 6.05 7.19 8.02 9.01 8.59 9.62
est 2.78 3.73 4.95 6.05 7.20 8.05 9.05 8.64 9.66

a exp, experimentally measured s pH; calc, s pH calculated from the buffer composition and the pK values of buffer components at the given
w w a
percentage of acetonitrile; est, swpH estimated from eqs 15 and 18 and the parameters of Table 3.

of citric acid with addition of acetonitrile is much smaller than
that for phthalic acid. This buffer system provides good continuous
buffer capacity for aqueous wwpH values below 7, and we recom-
mend it for RP HPLC applications.
Notice that the addition of acetonitrile produces a shift in the
plots of Figure 4 toward higher swpH values for the neutral acids
and toward lower swpH values for the cationic acids (neutral
bases) because of the swpKa variation of the different compound
buffers (see Figure 1).
For the common RP HPLC buffers, the major factor that
decreases buffer capacity is the decrease of buffer concentration
when the organic modifier is added. This should be taken into
account in practical work, especially when working with a gradient
elution that may come to high organic modifier concentrations
or when one works isocratically with high organic solvent
contents.
Experimental Evaluation of the Model. The developed
model has been evaluated by preparation of several buffers at
round pH values and experimental measurement of the swpH of
these buffers. At each pH value, the 0.01 mol‚L-1 aqueous buffer
with the highest buffer capacity between all studied systems
(phthalic buffers excluded) was selected. The most acidic buffer
studied was a 0.01 mol‚L-1 solution of phosphoric acid with a
w w
wpH value of 2.29. Buffers at wpH values of 4.00, 5.00, and 6.00
were prepared from citric acid. Sodium dihydrogenphosphate
buffers cover wwpH 7.00 and 8.00. The most basic buffers were
prepared from ammonia (wwpH 9.00 and 10.00). Acetonitrile-
water buffers were prepared from these buffers by addition of
acetonitrile to obtain 10, 20, 30, 40, 50, and 60% of acetonitrile
(v/v) mixtures. The swpH of all these buffered solutions were
measured and the measured pH values compared with those
calculated from the buffer composition and with those estimated
by application of eqs 15 and 18. The results obtained are presented
in Table 4.
The agreement between the pH calculated from the buffer
composition and that experimentally measured is very good for
the phosphoric and citric buffers. As expected, the differences
between both pH values increase with the percentage of aceto-
nitrile added. Buffers prepared from ammonia give experimental
pH values somewhat lower than those calculated from the buffer
composition and the pKa value of ammonium. The average of the
differences between experimental and calculated pH for the
acetonitrile-water buffers is -0.01 ( 0.07 (mean ( SD), which
shows that the accuracy and precision achieved are very good.
The comparison between the measured pH values and those
estimated from eqs 15 and 18 and the parameters of Table 3
follows the same trends indicated in the previous paragraph for
the calculated pH values. In fact, the average of the differences
between experimental and estimated pH is -0.02 ( 0.07, which
shows that the precision and accuracy of this method are as good
as those of the pH calculation from buffer composition. The
advantage of the method is that the knowledge of the swpKa value
of the buffer components and the exact composition of this buffer
are not required. The parameters of Table 3 allow an easy
estimation of the pH variation of buffers prepared from the acids
and bases listed in the Table with an accuracy of (0.1 pH unit
for most compositions and with a maximum of (0.2 pH unit for
high acetonitrile percentages. This is indeed the maximum
precision that can be achieved for pH measurements in nonaque-
ous and mixed solvents.
Estimation of the Degree of Ionization and Prediction of
RP HPLC Retention. Retention in reversed-phase liquid chro-
matography is strongly dependent on the degree of ionization of
the analyte.8,10,29-37 In general, ionized compounds are poorly
retained in hydrophobic stationary phases, whereas nonionized
(29) Lopes Marques, R. M.; Schoenmakers, P. J. Chromatogr. 1992, 592, 157-
182.
(30) Schoenmakers, P. J.; Tijssen, R. J. Chromatogr., A 1993, 656, 577-590.
(31) Lewis, J. A.; Lommen, D. C.; Raddatz, W. D.; Dolan, J. W.; Snyder, L. R.;
Molnár, I. J. Chromatogr. 1992, 592, 183-195.
(32) Lewis, J. A.; Dolan, J. W.; Snyder, L. R.; Molnar, I. J. Chromatogr. 1992,
592, 197-208.
(33) Horváth, C.; Melander, W.; Molnár, I. Anal. Chem. 1977, 49, 142-154.

3816 Analytical Chemistry, Vol. 74, No. 15, August 1, 2002

Figure 5. Variation of the ionization of acid/base compounds with the addition of acetonitrile to aqueous buffers of wwpH 8.0. (A) H2PO4-/
HPO42- buffer; (B) NH4+/NH3 buffer. Compounds: (]) 3,5-dichlorophenol, (0) 2,4-dichlorophenol, (4) 2-nitrophenol, (×) 3-bromophenol, (9)
2,4,6-trimethylpyridine, and (b) N,N-dimethylbenzylamine.
compounds may be strongly retained. The ionization of an acid/
base compound in a particular RP HPLC buffer depends on the
pH of the buffer and on the pKa of the compound. For a compound
that has a unique acid/base equilibria ruled by the acidity constant
Ka, the mole fraction (R) of each one of the two species that coexist
in the mixture (HAz and Az-1) is
[HAz] 1 that addition of acetonitrile decreases the ionization of all
RHA ) ) (23)
z
[HA ] + [A z-1
] 1 + 10pH-pKa
[Az-1] 1 decreases because the pKa of the phenols increases to a larger
RA ) ) (24)
z
[HA ] + [A z-1
] 1 + 10pKa-pH
For a neutral acid (z ) 0), RA is its degree of ionization (R),
whereas for a neutral base (z ) +1), R ) RHA.
Since both the pH of the buffer and the pKa of the analyte
change with the addition of acetonitrile, the degree of ionization
changes as well.37 If the pH and pKa changes are close to linear,
they can be described by eqs 14 and 15 and the difference between
pH and pKa that determines the degree of ionization can be easily
estimated from
s
wpH - swpKa ) wwpH - wwpKa + (mpH - mpK)φMeCN (25)
which shows that the variation of the degree of ionization of the
analyte depends on the difference between the m values of buffer
pH and analyte pK. Equation 18 and the parameters of Table 3
allow estimation of the mpH of the buffers studied here, and in
previous works,19,20 we have reported the mpK values for several
compounds.
An illustrative example is given in Figure 5. An RP HPLC buffer
of wwpH 8.00 with acceptable buffer capacity can be prepared from
a hydrogenphosphate/phosphate buffer or from an ammonium/
(34) Bosch, E.; Bou, P.; Allemann, H.; Rosés, M. Anal. Chem. 1996, 68, 3651-
3657.
(35) Rosés, M.; Canals, I.; Allemann, H.; Siigur, K.; Bosch, E. Anal. Chem. 1996,
68, 4094-4100.
(36) Rosés, M.; Bolliet, D.; Poole, C. F. J. Chromatogr., A 1998, 829, 29-40.
(37) Sýkora, D.; Tesařová, E.; Popl, M. J. Chromatogr., A 1997, 758, 37-51.
ammonia buffer. The mpH values for these two buffers are 1.75
and -0.61, respectively. We consider the ionization of the following
compounds: 2-nitrophenol, 2,4-dichlorophenol, 3,5-dichlorophenol,
3-bromophenol, 2,4,6-trimethylpyridine, and N,N-dimethylbenzyl-
amine that have a wwpKa value close to 8 (6.64, 7.37, 8.14, 9.09,
7.49, and 8.93, respectively16,17). The corresponding mpK values
are 3.42, 3.82, 2.85, 2.97, -2.30, and -2.08.16,17 Figure 5 shows
compounds in both buffers, but the decrease is different. In the
phosphate buffer, the ionization of neutral acids (phenols)
degree than the pH of the buffer (mpK > mpH > 0). However, the
ionization of neutral bases decreases much more because the pH
of the buffer increases (mpH > 0) and the pKa of the base decreases
(mpK < 0).
This fact has been already reported in the literature for RP
HPLC separations. Sýkora et al.37 studied the effect of mobile-
phase pH with methanol-phosphate buffers in the retention of
19 neutral bases in several columns. They observed apparent shifts
of the retention versus pH plots (wwpH) toward pH values more
acidic than the wwpKa value of the base (∼2.5 pH units of
difference for 60% methanol). They demonstrated that the shifts
were a combination of the two individual shifts caused by the
change of dissociation of the phosphate buffer (which produces
a mobile-phase pH change) and by the change of the pKa of the
compound with addition of methanol to the aqueous buffer
solution.
McCalley38-40 studied the protonation of bases in methanol-
water, acetonitrile-water, and tetrahydrofuran-water and con-
cluded that half-protonation is obtained at aqueous pH values of
the phosphate buffer much lower than the aqueous wwpKa value of
the base. The same effect was observed by Neue et al.41 for a
phosphate buffer of wwpH 7.00 in 65% (v/v) methanol. At mobile-
phase pH 7.0 and for pKa values of bases (amitriptyline, doxepin,
propanolol) close to 9, the bases should be completely protonated.
(38) McCalley, D. V. LC-GC Eur. 1999, 638-650.
(39) McCalley, D. V. J. Chromatogr., A 1994, 664, 139-147.
(40) McCalley, D. V. J. Chromatogr., A 1995, 708, 185-194.
(41) Neue, U. D.; Serowik, E.; Iraneta, P.; Alden, B. A.; Walter, T. H. J.
Chromatogr., A 1999, 849, 87-100.
Analytical Chemistry, Vol. 74, No. 15, August 1, 2002 3817
Figure 6. Elution of a mixture of ionizable compounds with a 60% acetonitrile mobile phase prepared from aqueous buffers of wwpH 8.0. (A)
H2PO4-/HPO42- buffer; (B) NH4+/NH3 buffer. Compounds: 2-nitrophenol (1), 2,4,6-trimethylpyridine (2), 3-bromophenol (3), and N,N-
dimethylbenzylamine (4). Chromatograms for the individual compounds in each mobile phase are also given.
In this instance, a small variation of the pH of the mobile phase
should not influence ionization of the bases and therefore the
retention relative to the neutral compound acenaphthene should
not change. However, Neue noticed that the “apparent” pKa values
of the bases (caused by combination of the phosphate buffer pH
increase and base pKa decrease with the addition of organic
modifier) were found to be around 6.5-7, i.e., around the wwpH
value of the buffer. Therefore, in fact, the bases were more or
less half-protonated (R ≈ 0.5) and small variation of pH caused
appreciable variation of ionization and retention.
In the ammonia buffer, the decrease of ionization is the
contrary. The ionization of bases decreases slightly because the
pK of the bases decreases more than the pH of the buffer with
addition of acetonitrile (mpK < mpH < 0). The ionization of acids
decreases much faster because the pKa of the acid increases when
the pH of the buffer decreases (mpK > 0 > mpH). For instance, in
60% acetonitrile, 2-nitrophenol is highly ionized and N,N-dimeth-
ylbenzylamine almost not ionized in the phosphate buffer, but
2-nitrophenol is poorly ionized and N,N-dimethylbenzylamine quite
ionized in the ammonia buffer.
The different variation of the degree of ionization of ionizable
compounds with addition of acetonitrile to aqueous buffers of the
same pH value, but prepared from different buffer components,
may lead to significant differences in RP HPLC retention. This
has been tested for a mixture of 2-nitrophenol, 3-bromophenol,
2,4,6-trimethylpyridine, and N,N-dimethylbenzylamine eluted with
a mobile phase with a 60% acetonitrile prepared from the aqueous
phosphate or ammonia buffers at wwpH 8.00. The chromatograms
obtained are presented in Figure 6. Figure 5 shows that 3-bro-
mophenol and 2,4,6-trimethylpyridine are almost ionized in both
buffers and thus their retention time must be the same with both
mobile phases. N,N-Dimethylbenzylamine is almost not ionized
in the phosphate buffer and in fact it shows the largest retention
with this mobile phase (Figure 6A), but it is quite ionized in the
ammonia buffer and thus it shows the lowest retention (Figure
6B). For 2-nitrophenol, the reverse behavior is observed. It is
3818 Analytical Chemistry, Vol. 74, No. 15, August 1, 2002
highly ionized in the phosphate buffer where it shows the lowest
retention (Figure 6A), but it is poorly ionized in the ammonia
buffer where it shows the largest retention (Figure 6B).
CONCLUSIONS
This study points out that the pH and buffer capacity of RP
HPLC aqueous buffers change with the addition of organic modi-
fier (acetonitrile). The buffer capacity of the buffer decreases most-
ly by dilution of the buffer concentration, although the variation
of the pKa values of polyprotic acids used as buffer components
may decrease buffer capacity too. The variation of the pH value
of the buffer depends on the particular buffer components and
composition. This pH change may influence the RP HPLC sepa-
ration of acid/base compounds. A model has been proposed to
allow an accurate prediction of this pH change for buffers in aceto-
nitrile-water mixtures (phosphate, acetate, citrate, and ammonia
buffers). The model can be used to choose what aqueous buffer
will be the best to prepare an acetonitrile-water buffer with a
required pH value. It has also been used to predict the change in
the degree of ionization of acid/base compounds caused by the
addition of acetonitrile to a particular aqueous buffer in order to
prepare an RP HPLC mobile phase. The relative retention of the
different compounds can be assayed from this degree of ionization.
ACKNOWLEDGMENT
We are thankful for joint financial support from the MCYT
of the Spanish Government and FEDER of EU (projects
BQU2001-2882 and BQU2001-3226) and from the Catalan Govern-
ment (Grant 2001SGR00055). S.E. was supported by a grant from
the Catalan Government (1998FI 00639). We are also grateful to
the anonimous reviewers of this paper for helpful comments.
Received for review January 8, 2002. Accepted April 29,
2002.
AC020012Y