Development of Methods For The Determination of PK Values: Analytical Chemistry Insights

Analytical Chemistry Insights
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Development of Methods for the Determination

of pKa Values
Jetse Reijenga, Arno van Hoof, Antonie van Loon and Bram Teunissen
Department of Chemical Engineering and Chemistry, Eindhoven University of Technology, Eindhoven, The Netherlands.
Corresponding author email: [email protected]
Abstract: The acid dissociation constant (pKa) is among the most frequently used physicochemical parameters, and its determination
is of interest to a wide range of research fields. We present a brief introduction on the conceptual development of pKa as a physical
parameter and its relationship to the concept of the pH of a solution. This is followed by a general summary of the historical develop-
ment and current state of the techniques of pKa determination and an attempt to develop insight into future developments. Fourteen
methods of determining the acid dissociation constant are placed in context and are critically evaluated to make a fair comparison and
to determine their applications in modern chemistry. Additionally, we have studied these techniques in light of present trends in science
and technology and attempt to determine how these trends might affect future developments in the field.
Keywords: review, history, dissociation constant, pKa, pH
Analytical Chemistry Insights 2013:8 53–71
doi: 10.4137/ACI.S12304
This article is available from http://www.la-press.com.
© the author(s), publisher and licensee Libertas Academica Ltd.
This is an open access article published under the Creative Commons CC-BY-NC 3.0 license.
Analytical Chemistry Insights 2013:8 53

Reijenga et al
Introduction 1
History
The centennial of the concept and of the quantitative
measurement of pH was celebrated not long ago.1–3 pH = pKa = 6.0
By now, accurate and precise determination of pH
Parameter X
seems to have few secrets left. The related concept
of the acid dissociation constant (pKa) as a substance 0.5
property is recognized as being among the most com-
monly used parameters in modern-day chemistry.
Both pH and pKa are essential for understanding the
behavior of chemical substances in everyday life.
This realization came gradually and under different
names. The first notion of acids comes from ancient
0
Greece, where people noticed that some substances 0 5 10
tasted sour. This is also where the word “acid” comes pH
from; it is derived from the Greek word “oxein”
Figure 1. A classic example of a sigmoidal curve created by plotting a
which in Latin is “acere,” meaning “to make sour.” It measured quantity versus pH. The inflection point corresponds to pKa.
was also noted that acids and bases could color cer-
tain substances.
The first description of an equilibrium constant only non-dissociated acid to the situation where only
came from Guldberg and Waage in 1864 in their “law dissociated acid is present. A more specific example is
of mass action.” With the help of van’t Hoff’s work represented if parameter X denotes the degree of disso-
on osmotic pressures, Ostwald formulated his “dilu- ciation α ranging between 0 and 1, the inflection point
tion law” for solutions. Measurements of osmotic will be at α = 0.5, where pH equals pKa. The degree of
pressures and conductivity of solutions gave insight dissociation α for acids is defined as:
into the degree of dissociation.4
It was in 1907 that Henderson published a paper α = [A−]/([HA] + [A−]) (2)
first describing the relation between the hydrogen
ion [H+] and the composition of a buffer.5 In 1909, Combining Equations 1 and 2 leads to (for anions):
Sörenson6 proposed the more convenient pH and pKa
terms as the negative logarithm of [H+] and the equi- Log (α/(1 - α)) = pH - pKa (3)
librium constant, K, respectively. Although Henderson
defined K in terms of a concentration ratio in 1908, it However, for concentrated solutions or pKa’s near
was not until 1916 that Hasselbalch7 proposed their the extremes of the pH scale, it has been shown that
now famous equation (1), which remains the most there are significant differences between the predicted
commonly used equation to calculate pKa values: the and actual concentration of the hydrogen ion.8
Henderson-Hasselbalch equation. It relates pH and Since the Henderson-Hasselbalch equation only
pKa to the equilibrium concentrations of dissociated gives accurate results for dilute acids in aqueous
acid [A−] and non-dissociated acid [HA] respectively: solutions, another formula for the quantification of
acid strength was developed by Hammett.9 Instead of
pH = pKa + log ([A−]/[HA]) (1) measuring the concentration of the species present in
solution, Hammett described the strength of an acid
In many experimental methods to determine pKa val- as the relation between the hydrogen ion activity (a)
ues, a certain parameter is measured as a function of pH. and the activity coefficients (f) of the various species
This results in a characteristic sigmoid curve (Fig. 1) in solution. However, difficulty associated with accu-
from which the pKa may be determined by locating the rate determination of the parameters in this model has
inflection point. Generally speaking, for acidic compo- kept it from being as widely used as the Henderson-
nents X ranges from a bulk property of a solution of Hasselbalch equation.
54 Analytical Chemistry Insights 2013:8

Development of pKa measurements
Influences on pKa Measured pKa values also depend on the ionic

In spite of being universally referred to as a constant, strength of the solution under investigation. The ionic
the dissociation constant pKa is not in fact truly con- strength is defined as the sum of concentrations (c) of
stant; it depends on temperature (T), ionic strength (I), all ionic species, corrected for their charge number (z):
and the solvent dielectric constant (ε).
The temperature dependence of the pKa values is I = ½ Σ z2 ⋅ c (6)
sometimes fitted to a van’t Hoff type equation:
At extreme values of the pH scale, the contribu-
d ln Ka/dT = ∆H/RT2 (4) tion of H+ or OH− should also be included. Activity
coefficients (γ) of different ionic species in solution
Here ∆H is the enthalpy change of dissociation strongly depend on ionic strength (Debye-Hückel
and R is the gas constant. The value of ∆H is often theory). Because pKa depends on the activity coef-
negative. In the case where ∆H is independent of tem- ficients, the ionic strength will also influence pKa,
perature, plotting pKa versus 1/T will result in a linear especially at higher charge numbers (z). An example
plot. This is commonly used to interpolate to other of this dependency is shown in Figure 3.
temperatures.10,11 Extrapolation of experimental data Because the acid-base equilibrium occurs in solu-
is, of course, not advised. We can understand this by tion, the solvent composition can also influence
realizing that ∆H can be re-written as: the pKa values (Fig. 4). Measuring pH of mixtures
of organic solvents and water is in itself far from
∆H = ∆G + T∆S (5) straightforward, and are beyond the scope of the pres-
ent contribution. When regarding an acid dissociation
Here ∆G is the Gibbs free energy change and ∆S reaction, three thermodynamic steps are considered:
is the entropy change. The linear relation mentioned (1) the dissolution of the acid from the solvent into
only holds if ∆H .. T∆S. Moreover in some cases the gas phase, (2) the dissociation of the acid into
∆S also depends on temperature, resulting in even the ions, and (3) the solution step of the ions into the
more non-linear dependence of pKa on 1/T. An exam- solvent. In the first and the last step, the solvent is
ple of the pKa temperature dependency is shown in involved. When considering the influence of the sol-
Figure 2. vent, the difference between the solvation energies of
8.0 4.8
7.0
pKa of acetic acid
4.7
pKa
6.0
Imidazole 4.6
Histidine
5.0 Citric acid
Glutamic acid
Oxalic acid
4.0 4.5
0 20 40 60 0 0.1 0.2 0.3 0.4
Temperature (°C)
− Ionic strength (mole/I)
Figure 2. Various pKa values as function of temperature. Each component has an Figure 3. The dependency of the pKa of acetic acid on the ionic strength,
own dependency. Values in water at infinite dilution, data from Everaerts et al.12 at 18 °C in water. Data from Cohn et al.13

Reijenga et al
6 or other interaction between the analyte and these

3-Me benzoic acid
other components.
3-CI benzoic acid
5.5
3-NO2 benzoic acid
Complications and overview
5 Depending on the sample and matrix under inves-
tigation, the choice of technique can be a difficult
pKa value
4.5 one, even for the case of monovalent ions, to which

this paper is limited to. For multivalent components,
4 matters are more complicated as the pKa differences
are smaller. This is because all methods, except per-
3.5 haps for nuclear magnetic resonance (NMR), require
curve fitting in addition to the normal calculation
3
procedure for the respective technique. Investigat-
0 20 40 60 80 ing both acidic and basic pKa of amphoteric com-
Methanol % pounds also requires curve fitting in a much broader
range. For such compounds, especially peptides and
Figure 4. The dependency of pKa of different Benzoic-acids on the sol-
vent composition, methanol in water at 25 °C and I = 0.02 M. Data from proteins, the isoelectric point is often relevant for
Sarmini and Kenndler.14 identification purposes but beyond the scope of this
paper.
the acid and the dissociated acid influences the final Figure 5 displays an overview of the first time
pKa value. Hence the pH range of one solvent may these techniques where used for this purpose.
differ from that of another solvent.
It must be stressed that when performing pKa Techniques
measurements, all parameters mentioned will have Potentiometry
to be kept constant in order to produce a meaningful The simplicity and low cost of potentiometric titra-
result. tion has made it one of the most commonly used
It is often overlooked that this is also the case with methods for pKa determination. In a potentiometric
pH measurements. Before use, pH meters should be titration, a known volume of reagent is added step-
calibrated under the same conditions of temperature, wise to a solution of analyte. The change in potential
ionic strength, and solvent. Reporting pKa values in (E) upon reaction is consequently measured with the
literature also require that exact conditions of tem- use of two electrodes, an indicator, and a reference
perature, ionic strength, and solvent be stated. If these electrode. These are often integrated in what is now
details are omitted, it cannot be assumed they were commonly called a combined pH electrode.
measured in water, at room temperature, and extrapo- Plotting the potential versus volume subsequently
lated to infinite dilution. gives rise to a sigmoid curve, where the inflection
Many of the techniques mentioned in the subse- point gives the potential at equilibrium. With the use
quent section measure solutions in which not only the of standards with known pH, this potential can be lin-
analyte is present, but also various other components early converted into a pH, equaling pKa. Sigel et al
for buffering. One has to be sure that there is no ionic have, however, correctly noted that there are multiple
Potentiometry Solubility Electrophoresis Polarometry Fluorometry Computational
Spectrometry Conductometry Partition HPLC Calororimetry
Voltammetry
NMR and kinetic
1900 1910 1920 1930 1940 1950 1960 1970 1980 1990 2000
Figure 5. Timeline of the first notion of the various techniques to determine pKa (dissociation constant, acid strength).

acid strength-models in use, each giving a different Completely automated potentiometric pH meters
H+ concentration for a given pH.15 for a wide range of applications and with complicated
Increasing understanding of electrochemical pro- calibration software are widely available. Because of
cesses in the late 19th century gave rise to the first this and the simplicity and relatively low costs asso-
potentiometer. This consisted of a platinum working ciated with the potentiometric pH-meter, it will prob-
electrode and a reference electrode and used a H2/H+ ably remain in use in the foreseeable future. This of
equilibrium to determine its standard reduction poten- course only holds for those analytes that are available
tial, which is by definition 0 V. The first description of in sufficient quantity and purity.
the use of a setup to determine equilibrium constants
was made by Denham in 1908.16 Conductometry
It was not long until the cumbersome hydrogen The determination of acid dissociation constants by
electrode was replaced by the familiar glass conductometry relies on the assumption that strong
electrode. Completely automated and self-adjusting electrolytes are completely dissociated at all concen-
pH-measuring equipment by Keeler is known from trations, while weak electrolytes only attain complete
as early as 1928.17 Because of difficulty in obtaining dissociation at infinite dilution. A measurement of the
reliable results, the first working pH-meter was only conductivity of a sample yields a value that is the sum
constructed in the 1930’s.18 of the independent contributions of all ions present in
Although the glass electrode has become widely the solution:
used, it has been proven that at higher temperatures or
more extreme pH’s the results deviate from theoreti- Λ = Σ λi (7)
cal predictions. The “alkaline-error” is certainly the
Here, Λ represents the equivalent conductance and
most widely known. Because temperature influences
λi represents the specific conductivity contribution of
not only the measurement but also the pKa itself, it is
ionic species i. The equivalent conductance is depen-
of vital importance to conduct the titration at constant
dent on the number of ions in solution, and reaches its
temperature. A good review about the various errors
limiting value Λ0 at infinite dilution, where, by defini-
of the electrode itself is given by Gardiner19 while
tion, activity coefficients are unity.
Benett20 discusses the various ways to determine pKa
Λ is a measurable quantity and Λ0 can be obtained
from the measured potential.
from extrapolation in a plot of Λ versus the square root
Traditional potentiometric titrations have a lower
of the analytical concentration as shown in Figure 6. It
concentration limit of about 10−4 M, however various
can also be seen from Figure 6, however, that this linear
methods where developed to extend this range. Another
practical complication is the pKa-measurement of sub-
stances with a low water-solubility. One example is 30
extrapolation of measurements in solvent mixtures.21

These require, however, a relatively high co-solvent
concentration, and are inherently inaccurate and time 20
consuming. An alternative approach using surfactants
Soidum formate
(mS)
is proposed22 which avoids tedious extrapolating while

Hydrochloride acid
giving the same level of accuracy (± 0.2 pKa value).
V
Sodium chloride
A precise determination of the pH from the titra- 10
Formic acid
tion slope has also been difficult in the earlier period
of its use. However, over the years various software
programs have become available to minimize most
of the previous mentioned errors. Potentiometric 0
titration requires a relatively large amount of sample 0 0.2

√c (√M)
0.4 0.6
compared with separation methods such as high per-

Figure 6. Plots of Λ versus √c required for pKa determination of formic
formance liquid chromatography (HPLC) and capil- acid at room temperature in water. Data from Saxton and Darken24 and
lary electrophoresis (CE). Landolt-Börnstein et al.25

Reijenga et al
extrapolation does not hold for weak acids (or bases). to non-aqueous systems where such a measurement
This is because for these electrolytes, the assumption of pH would be impracticable. It also means that the
that all ions are independent of their counter ions does method, unlike many others which express their mea-
not hold as these species are not fully dissociated. sured quantity as a function of pH, is not constrained
Though the limiting conductance of a weak elec- by the precision of the pH electrode.
trolyte cannot be directly extrapolated, their value can The conductometric method offers a relatively fast
still be obtained quite readily. Even for weak electro- and reliable method of determining the pKa and is also
lytes, the Kolhrausch law23 of the independent migra- capable of attaining a high degree of precision with
tion of ions holds. This law can be stated as meaning pKa deviations as small as ± 0.01–0.03 units.27 The
that at infinite dilution, each ion makes a specific main downside of the method is that conductomet-
contribution to the conductivity regardless of the ions ric measurements are nonspecific, ie, different ions
associated with it. It is not possible to measure the cannot be measured separately. This requires working
limiting conductance of ions directly, but Λ0 for a salt with pure compounds.
may be expressed as a linear combination of its ionic Foundational work that enabled the develop-
components. ment of the conductometric method was undertaken
Expressing the limiting conductance of a salt in by Friedrich Kohlrausch. Among the key ideas he
terms of ionic contributions is useful as it allows the developed was the use of alternating current to pre-
calculation of the limiting conductance of a weak elec- vent electrolysis during conductivity measurements.
trolyte. In order to do this, the limiting conductance of His work examining a variety of electrolyte solutions
other relevant salts of a strong electrolyte is extrapo- led to the law of independent migration of ions that
lated. For a weak acid (formic acid) relevant plots are is ascribed to him. Building on this early work, and
shown in Figure 6. One would obtain the ionic contri- further contributions by Ostwald and Arrhenius, the
butions for the limiting conductance of the acid from method reached maturity during the late 1930s, and
the salt of its conjugate base and from hydrochloric the accuracy of the method approached that of mod-
acid, subtracting the value for sodium chloride to ern methods. The incorporation of Debye-Hückel
eliminate the ionic contributions of the sodium and activity coefficients enabled the calculation of true
chlorine counter ions in the reference compounds. thermodynamic pKa values, and the calculation of
This yields the sum shown in Equation 8: these coefficients for a range of ions was published
during this period.28 In contrast to the earlier period,
Λ0HA = Λ0NaA + Λ0HCl - Λ0NaCl (8) which focused mainly on obtaining thermodynamic
dissociation constants, experiments performed after
Once the limiting conductance is known, the degree
this period were additionally concerned with corre-
of dissociation (α) for a weak electrolyte is given by
lating this data with the structure of the organic acids
α = Λ/Λ0. An apparent dissociation constant K′ can
being studied.29
be obtained directly from the degree of dissociation
Work on the conductometric method came to a
using Ostwald’s dilution law by expressing it in terms
virtual standstill after the outbreak of World War II,
of a concentration quotient, as shown in Equation 9.26
and further research was practically abandoned after
In order to obtain the true thermodynamic acid disso-
the end of the war. Renewed activity would not come
ciation constant Ka, one has to correct for the activity
until the 1970s, when two new conductance equations
of the ions:
were published. These equations enabled the study
K′ = Λ2 ⋅ c/(Λ0 ⋅ (Λ0 - Λ)) (9) of asymmetrical electrolytes, and even mixtures of
electrolytes.30–33
Equation 9 yields the dissociation constant for Since then, it has been remarked that the spectacu-
a given analytical concentration (c) and a series of lar interest for the method that existed during its early
measurements would yield the dissociation constant period has waned with time, leaving the subject a some-
as a function of ionic strength. It is worth noting that what unfashionable topic of research. With the devel-
this determination does not require knowledge of opments made over its history, however, the method
the pH of the solution, making it easily applicable has achieved a high degree of precision.26,34,35

Voltammetry therefore a reference with known characteristics is

In voltammetry, a changing potential is applied over a usually added. The shift of the peaks of the reference
sample solution and the resulting current is measured. upon addition of the acid is then used to determine
When the potential reaches the reduction poten- the pKa value.42,43 Care has to be taken since the ref-
tial of the analyte this will give rise to an increase erence will influence the ionic strength of the solu-
in current, followed by a decrease due to depletion tion. Additionally, its concentration should be in
of the molecule. In the case of cyclic voltammetry, the same order as that of the analyte to give optimal
for example, this will lead to results much like those sensitivity.
shown in Figure 7. More recently, advanced techniques have been
A typical voltammetry setup usually consists of developed such as those measuring the surface pKa
3 electrodes: reference, working, and auxiliary elec- of self-assembled monolayers.43 Voltammetry has
trode. The working and auxiliary electrodes act as not been used extensively for the measurement of
the anode and cathode, respectively, while the refer- pKa values. The reason for this is the need for an
ence electrode acts as a fixed point to measure the electro-active molecule which is soluble in a con-
applied voltage. Modern-day voltammetric meth- ductive solvent. Also, it is often necessary to recali-
ods can be extremely sensitive; with specific tech- brate the apparatus when different samples are used.
niques it is possible to accurately measure very low Besides these “operational” drawbacks, polarography
concentrations.36,37 also requires the use of mercury electrodes.
The earliest use of voltammetry was the use of On the other hand, voltammetry has some
a dripping mercury electrode in 1922 by Jaroslav advantages. It is of particular use in measurements
Heyrovsky to develop the first polarograph.38 As early of pKa values in less polar solvents, something which
as 1941, voltammetric methods were deployed to is often difficult to do accurately with the use of
determine local pH at the electrode surface.39 However techniques such as potentiometric titration.41 When
the use of voltammetry to specifically determine bulk optimized, it is usually not very time-consuming and
pKa values came into use in the 1960s.40 relatively cheap in comparison to other techniques such
When the pKa of a substance is being determined as NMR, separation methods, and spectrometry.
voltammetrically, one could in principle measure the Another advantage over other techniques is that it
electrochemical response of the molecule itself.41 is able to conduct a quantitative measurement of dif-
However, accurate knowledge of the electrochemi- ferent oxidation states.44
cal behavior of the sample substance is required and
Calorimetry
All calorimetric methods work by the same principle:
a physical or chemical process takes place in a sam-
ple and the amount of heat evolved is measured. For
the measurement of pKa values, a technique called
Isothermal Titration Calorimetry (ITC) has been used.
Here, a regular acid-base titration is carried out inside
the calorimeter while the energy needed to keep the
Current
temperature constant is measured. It is also one of the

oldest analytical techniques. The first recorded model
was made by Lavoisier and Laplace in 1783.45
In recent years, the ITC-method has been used to
measure the dissociation constants of peptides and the
influence of binding on the specific ionizable groups.
This method also calculates the pKa indirectly from a
Potential measured enthalpy change ∆H.46
Figure 7. Example of a cyclic voltammetric curve. The upper line repre-
A related technique was developed which
sents an increase in potential, the lower line the following decrease. measures the pKa directly: Isothermal Titration

Reijenga et al
icrocalorimetry (ITM). Here the reagent for the

M existing literature. Based on his work, further knowl-
ionizable groups is added in equivalent amounts edge of the pH-chemical shift relation was described
and at once. The resulting heat released upon reac- and published.49
tion is measured in buffer solutions with different pH When the pH dependent acid-base equilibrium is
values (Fig. 8). By plotting the minima or maxima fast on the NMR time scale, a mean chemical shift of
versus pH, a sigmoid curve is obtained from which the HA and A− groups can be measured. Because the
the pKa can be determined from the inflection point.47 equilibrium is pH dependent, the chemical shift will
Uncertainties are in the range of 0.05–0.15 pKa units also change with the pH level. For this situation, the
depending on the calorimetric technique.47 pKa can be written as Equation 10:
Nuclear magnetic resonance pKa = pH + log [(δA - δobs)/(δA - δHA)] (10)

Before NMR was used to determine acid dissociation
constants, the technique was already applied to deter- The pKa value can be determined by plotting the
mine the site of deprotonation of an acid, or the site of δ-component shown above versus the pH. This yields
protonation of a base. In these cases, the temperature a familiar sigmoid curve where the pKa is located at
was decreased to such extent, that the acid-base equi- the inflection point.
librium was slow on the NMR time-scale, so that two Instead of performing a continuous titration, a
separately peaks would be observed for HA and A−. known amount of a strong acid or base is added to
In 1957, Grunwald et al48 used NMR to deter- different samples (constant volume titration), from
mine the pKa of mono-, di- and trimethyl-amine, thus which the pH is consequently calculated. This how-
determining the chemical shift of the triplet from ever neglects the influences of the target molecule on
the protons in the CH3 group(s) as a function of pH. the pH and the risk of a systematic error is added.50
A linear correlation was found between the chemical The main advantage of the NMR technique is that
shift and the acid-base ratio. Experiments could be it is possible to measure mixtures, even when impuri-
carried out in water by using a reference measurement. ties are present, because mole fractions are observed
A sigmoid curve is obtained from which the pKa was instead of the total acid concentration, in contrast to
calculated.48 potentiometric titrations. When observing the chemi-
Pioneering work was performed by Lee et al,49 cal shift of one characteristic group, no other groups
who determined the pKa values of a wide range of are involved, so even the pKa values of diprotic
functional groups with good concurrence towards acids with pKa values close together can be observed
separately, provided that the chemical shifts do not
5 overlap. These individual constants are referred to
Time [min] as “microscopic pKa values” and their determina-
0 tion is one of the most promising applications of this
technique. Also, performing NMR with 2H, 13C, 31P or
0 5 10 15
other nuclei with an electromagnetic moment can be
−5
used to measure the pKa.
Cal/Time
With this characterization method, even more

−10 complex molecules, such as enzymes can be fully
characterized. So, the pKa of individual acid sites on
−15 complex molecules can be determined, making this
a promising technique for further development. This
−20 was discovered by Rabenstein and Sayer in 1976 who
determined microscopic dissociation constants for
polyprotic acids by means of curve fitting.51
−25
The main errors for the NMR method are found
Figure 8. Curves created after a typical direct measurement of pKa
with the use of calorimetry. The amplitude of the minimum at different
to be caused by imprecision of the chemical shift and
pH values is proportional to the degree of dissociation. the pH level, which is calculated and not measured.

The pKa can be measured precisely up to ±0.05 log sociation for acids is expressed in terms of mobility,
units.52 one obtains the relation shown in Equation 13:
For all NMR measurements, a reference compound
is required to acquire a ‘lock’ for the apparatus; this meff = (1 - α) ⋅ m0 + α ⋅ md (13)
processes is called shimming. Because an internal
reference can interfere with the acid-base equilib- Here, meff is the effective mobility, md is the
rium, an external reference is recommended, either mobility of the fully dissociated species, and m0 the
with an extra measurement or a second co-axial sam- mobility of the non-dissociated species (which equals
ple tube. zero). For bases, a similar equation is derived. A sig-
Control of temperature in NMR is often not a prob- moid curve is obtained by plotting meff vs. pH, with
lem, but when performing NMR the fact that energy the inflection point of α = 0.5 at pH = pKa.
dissipation can occur must be taken to account; Model equations can be derived for weak acids
locally, at the vibrating nucleus of interest the tem- and bases with any number of ionizable centers by
perature can rise. rewriting Equation 13. These models for species
Glaser et al presented the first fully-automated with up to three ionizable centers were summarized
NMR apparatus for measuring the pKa.52 This method in a 2004 review article by Poole.52 The pKa may be
also has good concurrence with available data in determined by numerically fitting the resulting rela-
literature. With this automation and stronger mag- tions to a plot of mobility versus pH through nonlin-
nets, the NMR technique is a promising technique ear regression, as shown in Figure 9 for the case of
to measure multiple enzymes at specific (de-)proto- 2-aminopyridine.54
nation sites within a reasonable time frame. For less The shape of these curves depends only upon md
complex acids and bases with only one protonation and the position depends only upon pKa, therefore
site, however, this technique is rather expensive. this regression directly yields the pKa value(s) of
interest. Remarkably, few data points are needed to
Electrophoresis provide an accurate fit for these models, and a preci-
In electrophoresis, charged species are separated sion of ± 0.03–0.08 can be achieved depending on
under the influence of an electric field, migrating with the analyte, placing the method roughly on par with
a velocity proportional to their size-to-charge ratio. potentiometry in terms of precision.55
The ratio of the linear velocity vi and field strength E The method has a number of key advantages
is defined as the electrophoretic mobility mi: in comparison to more traditional alternatives.
mi = vi/E (11) 6
The relation with previously discussed conducto-

5
metric methods and electrophoretic ones is impor-
tant: in the latter we measure individual mobilities mi
meff*10-4 [cm2/Vs]
4
(Equation 11) whereas in the former we measure the
sum of mobilities of all ions together (Equation 7).
3
They are interrelated on the basis of individual ions
using the Faraday constant F:
2
mi = λi/F (12) Regression results

1
Raw data
The use of electrophoresis for the determination of
the pKa value depends on the differing mobilities of the 0
protonated and deprotonated forms of the analyte. As 3 4 5 6 7 8 9 10
the two forms exist in fast equilibrium, a net mobility pH
is measured that can be related to the degree of dis- Figure 9. Curve fitted to data for electrophoretic mobility of
sociation (α) of the analyte. When the degree of dis- 2-aminopyridine as a function of pH, data from Poole et al.54

Reijenga et al
Firstly, only a very small amount of the sample is s pecifically for the determination of mobilities and
required for a measurement, on the order of microlitres pKa values. The potential sensitivity of the method had
in terms of volume, and with detection limits in the already been demonstrated61 and further refinements
10−6 M range. This allows for the processing of poorly of the technique would be made over the next few
soluble species without much difficulty. Since elec- years. A general methodology for the determination of
trophoresis is a separation technique, impure samples pKa values by CE was proposed in 1992, and further
can be readily processed and as the molecules are refined by the addition of terms concerning the rate of
measured directly, exact knowledge of sample con- electroosmotic flow and a method of handling poten-
centrations is not required. In addition, commercially tial discontinuities between buffer electrolytes.62,63
available equipment is capable of automatic opera- An important step was made by Ishihama64 when he
tion without requiring modification, allowing large studied multivalent compounds and derived relation-
numbers of measurements to be conducted at speed. ships between pKa and zone mobility that were more
This makes the method quite suitable for screening easily applicable than the earlier equation proposed
applications.56 A potential problem of the method is by Kiso.58
the requirement of preparing buffer solutions for each Developments over the next decade would include
pH to be measured, since the buffer compounds must further examinations of the pKa equations, comparing
be carefully chosen to avoid undesirable interactions different regression techniques, and better experimen-
between buffer and analyte. tal methods.65 Improvements included using charged
The relationship between the electrophoretic polymer coatings of the capillary wall to influence
mobility of an acid or base and the pH of the back- the rate, influencing the electroosmotic flow to enable
ground electrolyte was already considered when measurements at a lower pH,66 and the use of pressure
Consden and Martin56 published their paper on iono- applied over the capillary to shorten measurement
phoresis in 1946, which discussed the separation of times for screening purposes.67 Recent work includes
two analytes based on a difference in their pKa val- efforts undertaken by Fuguet et al68–70 to improve the
ues. This relation was explicitly applied to the estima- internal standard method first proposed by Gluck and
tion of acid dissociation constants by Waldron in the Cleveland.65
1950s57 and by Kiso et al in the 1960s58 using a paper
strip as a supporting medium. However, despite the High performance liquid chromatography
known advantage of requiring only minute amounts The first observation that the time of elution can
of sample, both methods were found to be impractical be changed by adjusting the pH level was made by
in application. Improvements over the next decades Singhal.71 By changing the pH, the performance of
would eventually lead to the introduction of the fused the HPLC was optimized, in this case ion-exchange
silica capillaries. chromatography.
By the end of the 1980s, isotachophoresis was With the advance of reversed-phase HPLC in the
considered a viable method for the determination late 1970s, advanced models were made relating the
of dissociation constants. While the method showed capacity factor (k) to the degree of dissociation (α).
reduced precision in comparison to potentiometric or An excellent overview of the principle is presented
conductometric methods, this was offset by its faster by Horváth,72 who presented a generally applicable
measurement times and lower sample requirements.59 equation for acidic components:
However, determining pKa values by isotachopho-
resis was not without disadvantages which are dis- k = (1 - α) ⋅ k0 + α ⋅ k-1 (14)
cussed in detail by Beckers et al.60 It was remarked
that the calculations required are quite laborious as in which k0 and k-1 are the capacity factors on the
the zones formed during the separation all have dif- non-ionic and the ionic species respectively, where
ferent parameters (pH, ionic strength, and even tem- in reversed-phase HPLC k0 .. k1. Plotting k vs. pH
perature). To avoid the complexities and limitations gives a sigmoid curve with the inflection point of
of isotachophoresis, it was proposed that the simpler α = 0.5 at pH = pKa. The similarity of Equations 13
method of capillary zone electrophoresis be used and 14 are immediately obvious.

A solid theory was formulated by Foley73,74 where There are two main techniques used to determine
the pH dependence of the capacity factor follows the log P. One is the “shake-flask” technique78 where an
dissociation curve. Again, some recommendations octanol/water mixture together with the sample is
and optimizations were formulated to improve selec- shaken in a separation funnel, after which the con-
tivity, retention, and efficiency. centration in one or both of the phases is measured.
As long as the analyte has a chromophore for Another is the so called Filter probe79 which was
detection, this method works fine, even with sam- later improved to the “filter chamber technique.”80
ples that are not 100% pure, as HPLC is a separation These are automated separating funnels connected to
method. On the other hand, the full range of α from 0 a pump which directs the layer of choice to an analyt-
to unity cannot always be utilized, because k0 is often ical device (usually a spectrophotometer) and back.
unacceptably large. Of course, the addition of organic In some specific cases, the technique used to deter-
modifiers decreases all k values but hugely compli- mine pKa by P is largely unaffected by the choice
cates matters if we desire aqueous pKa values. of solvent.77 Although it might seem that log P is a
relatively simple quantity to determine, it has been
Partition and distribution proven that there are still many sources of errors,78
The first investigation into the division of a substance one of which being the case of mutual miscibility of
between two immiscible solvents was carried out by the two phases.
Berthelot and Jungleisch in 1872. They came to the The use of partition coefficients to determine pKa
important conclusion that the ratio of the concentra- is not used extensively. Partition coefficients are
tion was a constant, e.g., it does not depend on the however still very much in use in drug development
relative amounts of solvents used. Following up on as they give information about the uptake of a certain
this research, Nernst concluded in 1891 that the par- drug in various parts of the body.
tition coefficient (P) was only a constant if a single A particularly illuminating example of this fol-
substance was considered.75 lows. In some surgeries, the body temperature and
Later insight further revealed that for ionizable blood pH values deviate from normal values. This
components, the partition coefficient depended on naturally affects the log P value and drug efficacy.
the pH of the aqueous phase. In the limiting case Thurlkill et al81 reported an additional aspect for the
where the ionization is completely suppressed by pH case of Fentanyl, a local anesthetic. It was found that
(for bases, for example, at high pH) a distribution its pKa significantly depends on temperature, which
coefficient (D) can be defined mathematically (for might lead to further complications in drug adminis-
bases): tration, in other words a temperature and blood gas
dependent bioavailability.
D = [B]o/[B]w (15)
P = [B]o/([B]w + [BH+]w) (16) Solubility

In 1945, Krebs82 described the relationship between
Here B is the uncharged and BH+ the charged spe- pH, pKa, and the solubility of sparingly soluble weak
cies, subscript o refers to the organic and subscript acids and bases, where a derivation of the Henderson-
w, the water phase. Several derivations for pKa from Hasselbalch equation was used to describe the
P and D are found in literature,76,77 but all of them behavior. A drawback of this method at that time was
result in roughly the same expression (for bases) as the solubility of the non-ionized analyte had to be
they combine Equations 1, 15, and 16: known, which was not often the case.82
This theory was further expanded by Zimmerman
pKa = log ((P - D)/D) + pH (17) et al83 who made mathematical derivations by which
it was possible so determine the solubility of the neu-
These are usually determined by adding a known tral analyte and the pKa. By these means, the solubil-
amount of sample to an organic solvent/water mix- ity of the neutral compound was no longer required
ture, followed by the measurement of the concentra- and the use of solubility data to determine the pKa
tion in one of the phases. was much more applicable.83

Reijenga et al
A derivation of the Henderson-Hasselbalch equa- Therefore, solubility measurements for pKa determi-
tion allows us to determine the pKa from solubil- nations can be used for sparingly soluble compounds
ity data, the graphical representation is shown in which have a chromophore near the ionization
Figure 10. center.
log S = log S0 + log (10−pK−pH + 1) (18) UV/Vis spectrometry

Well before 1900, it was already known that a change
Here S0 is equal to the intrinsic solubility. When
in acidity could lead to color changes of natural sub-
pH .. pKa or pH ,, pKa assumptions can be
stances. Spectrometry with visible light made it pos-
made and linear log S/pH functions are obtained. By
sible to measure pKa values of acid/base indicators
extrapolating these two functions and calculating the
and this in turn was extended to the use of UV light to
intercept, the pKa can be calculated:
measure pKa’s of other components.
log S = (log S0 - pKa) + pH (19) A requirement for this UV/pH measurement is the
presence of a chromophore close to the ionization site
In the later years, pKa values for zwitterionic com- in the molecule. If this is fulfilled, then the spectra of
pounds were determined85 and a solid method for the dissociated and the non-dissociated form can be
the bi-functional bases and acids was formulated.86 expected to differ. In principle any wavelength can be
Avdeef84 found a special way of determining the pKa. used for the determination of pK, except at the isos-
Under certain conditions the pH does not change bestic point at which wavelength of both forms have
under addition of more titrant. This pH is known as the same molar absorptivity. The best choice however
the Gibbs pKa. is a wavelength at which the molar absorbtivities are
Nowadays, the solubility data are used to deter- as different as possible.
mine pKa values for a wide range of drugs, where The method was further improved by measur-
the pKa value is of great interest.87 The power of this ing the absorption of two different wavelengths at a
technique is that poorly soluble drug can be analyzed variable pH. The ratio in absorption at those two wave-
at very low concentrations, in the order of µM, and lengths is plotted against the pH. In this way, a sig-
with a precision up to ±0.5 in log units.84 A draw- moid curve is obtained and the pKa can be determined
back is that for most solubility determinations, a time from the inflection point as normal. One of the wave-
frame of 1–24 hours is required where a measure- lengths has to be assigned to the chromophore and the
ment with spectroscopy only takes a few minutes. other wavelength should be invariant under change
of pH (if this is possible). By using a 2nd wavelength
as reference, change in total concentration will not
affect the final result and activity/concentration issues
Acid pK 4.8 and assumptions are bypassed.
Base pK 3.2 This method was introduced by Holmes and
Snyder in 192588 when the “decomposition” constant
of a dye was measured. This was then further elabo-
log S
rated by Flexser et al89 in 1935 by determining differ-

ent ionization constants. In the 1960s, Wigler et al90,91
were the first to determine pKa values of di-protic
compounds.
Up to this point, the pKa values calculated required
prior knowledge of experimental data, such as the
0.5
absorption coefficients of the neutral and ionized
0 2 4 6 8 compound. By measuring over a whole wavelength
pH range, Allen et al92 were able to determine the pKa
Figure 10. A plot of the pH dependence of the solubility of an acid with
values without this prior knowledge. The measure-
pKa 4.8 and a base with pKa 3.2. ments could also be done much faster. This method

showed good concurrence with previous single wave- Polarimetry

length methods93 and was later highly automated by Determination of an acid dissociation constant by
Saurina et al.94 polarimetry involves the measurement of the optical
rotation of plane polarized light by the sample solu-
Fluorometry tion as a function of pH. This method depends on the
It can be argued that fluorometry is a specific form difference in optical rotation between the ionized and
of spectrometry as any fluorescence is the result of non-ionized forms of an analyte.
light absorption, while the reverse is not the case. Only very few examples of polarimetry were
The use of fluorescence spectroscopy to determine found. A determination of the pKa values of tartaric
pKa values depends on the difference in the fluores- acid was performed by Katzin and Gulyas97 using this
cence spectrum between a free acid or base and its method. They expressed the optical rotation of the
conjugated form. While fluorometry can potentially sample as the sum of the optical rotation of the ionic
be more sensitive and selective than conventional fractions derived from the analyte.
spectrometry, it has the disadvantage of being only When the fractions are expressed as a function of
applicable to fluorescent analytes. Additionally, it is the acid dissociation constants and the solvated pro-
known that the pH dependence of fluorometry often ton concentration, the pKa values may be determined
does not agree with those obtained from spectrom- by curve fitting of a plot of the optical rotation against
etry or other methods. The reason for this is that the the pH, where small differences in pKa inhibit accu-
former depends on excited state proton exchange rate determination.
as well as the ground-state equilibrium. This causes While polarimetry was shown to be a reason-
problems because the pKa for the ground state and ably sensitive method to determine the pKa values,
pKa* for the excited state can differ quite strongly, the method has a number of significant drawbacks.
and the position of the inflection point in a fluo- Firstly, the method can only be applied to optically
rometric titration will depend on both values as active analytes, and samples must be relatively pure
well as the kinetics of the excited state proton enantiomers as the presence of the opposite enantiom-
transfer.95 Another problem may occur when buf- ers will cause a reduction in the sensitivity of the
fer components lead to pH-dependent fluorescence measurement with a corresponding drop in precision.
quenching. Secondly, the determinations are carried out at high
In spite of these challenges, a successful determi- concentrations meaning that relatively large amounts
nation of the ground-state pKa values of sparingly of sample are required.
soluble N-heterocyclic bases was accomplished by
Rosenberg et al.96 They made use of dilute solutions Kinetic method
of a strong acid or base to perform a pH titration; The kinetic method of determining pKa values
strong buffers were not used in order to prevent depends on measuring the rate of reaction of a con-
reactions of the buffer ions with the highly reactive trol reaction that is influenced by the pH of its reac-
excited state forms of the aromatic analyte. By care- tion medium. While determinations based on reaction
fully selecting the excitation wavelength, they were kinetics are rarely encountered in literature, Bunnett
able to obtain a titration curve in which the inflec- and Nudelman98 demonstrated a method by which
tion point corresponded to the ground-state pKa. The the principle might be applied systematically. Their
more precise of the two methods suggested involved method involved a reference reaction, the reaction of
choosing an excitation wavelength such that only the thiophenoxide ion with 2,4-dinitrofluorobenzene to
conjugate acid can absorb, meaning that no excited- form a conjugated double ring system, that could be
state proton transfer can occur to the free base, in easily monitored using absorption spectrometry.
order for the fraction of conjugate acid to be mea- The rate of this reaction is determined by the dis-
sured directly. The bottom line is that the linear sociation of thiophenol to yield the free ion, and this
relationship between fluorescence intensity and the dissociation is repressed by an increase in the solvated
degree of dissociation is not as common as in absorp- proton concentration. When the dissociation constant
tion spectrometry. of thiophenol is known, performing the reaction in a

Reijenga et al
buffering solution of the analyte allows for the calcu- It compares the results obtained by their own method
lation of the pKa. The assumption is made that the sol- with those published by Schüürman et al100 a year
vated proton concentration, and thus the position of earlier. It is remarked that the methodology used in
the thiophenol/thiophenoxide equilibrium, is entirely describing the molecules in the system and the com-
controlled by the unknown buffer, and that the reac- putational methods used to determine the properties
tion rate thus depends only on the buffer composition can drastically influence the outcome of the calcula-
and the pKa of the buffering acid. tion, illustrating the difficulty in deriving ab initio
The method as described seemingly has several systems. Another major downside of these calcula-
advantages. Firstly, no exact knowledge of the pH is tions compared to other computational techniques is
required since the solvated proton concentration is their enormous cost, making their application in any
directly linked to the analytical composition of the context where large numbers of molecules must be
buffering solution and it in turn is directly linked to investigated impractical.
the thiophenoxide concentration. A second advantage Semi-empirical quantum-mechanical (QM) meth-
is that the pKa can, in principle, be determined in a ods are based on the same formalisms as the ab initio
single measurement. In spite of this, the method still calculations, but make use of further approximations
remains complicated and not generally applicable. and obtain many parameters from experimental data.
A good treatment of these methods was given by
Computational method Tehan.101 The method works by taking information
Finally, we consider the method of computational from a large database of known molecules and using
chemistry. This method is unusual when compared to this information to obtain approximate QM parameters
those previously described in that it is a mathematical for the calculation. While these methods are much faster
method rather than an experimental one. Computation than ab initio calculations, the results of the calcula-
requires no sample and is in principle unrestrained in tion are only reliable if the molecule being computed
terms of physical conditions, but the accuracy of the is similar enough to those stored in the database. More
values obtained depends entirely upon the model used recently, there has been much work on the development
to perform the calculations. of quantum structure-property relationship (QSAR)
Since the science of computational chemistry based methods.102 These may be considered a further
began to take shape in the early 1970s with the devel- development of the semi-empirical methods described
opment of efficient software for the calculation of above in the sense that empirical and ab initio data are
molecular orbitals and the development of the first correlated with the contribution of certain fragments
molecular mechanics methods, many different meth- within a molecule. This dataset is then used for further
ods have been developed to computationally estimate calculations. The most recent methods make use of
molecular properties. Several of these methods have Molecular Tree Structured Fingerprints to describe the
been evaluated for the determination of acid dissocia- chemical neighborhood of an ionizable center.103
tion constants. In terms of the accuracy of their predictions, com-
The most precise results available are generally putational methods suffer where multiple competing
obtained from ab initio calculations, meaning that models exist, since the choice of parameters and start-
these calculations tend to converge to an exact math- ing assumptions can dramatically affect the outcome
ematical solution. However, since the equations used of the calculations. In addition, there are often sig-
to describe the system are entirely derived from theo- nificant deviations from literature values as shown in
retical principles, any flaw in the model will still pro- Figure 11.
duce a result that may deviate from its physical value. Despite these problems, there is still considerable
For instance, considerable difficulties exist when interest in computational pKa determination methods
attempting to calculate the pKa value in a solvated in such fields as drug discovery, where the method
or aqueous phase, since the problem of how to accu- might be used to reduce uncertainty of the physical/
rately take solvent-analyte interactions into account chemical properties of (modifications of) mole-
remains largely unsolved. An article by da Silva et al99 cules without even having to synthesize all of them.
published in 1999 compares several methodologies. However, for the time being the method is purely of

7 Comparison and Discussion

Comparing the incomparable is usually difficult and
6 can only be done in a qualitative way as it depends
on many factors, among which those specific for each
5 analyte and experimental facilities available. This is
even more so as the introduction and application of the
Calculated pKa
4 different methods described in this paper cover many

decades. As such, they also provide a fascinating his-
3 torical cross-section of chemical experimentation. We
are, however, able to make a tabulated comparison of
2 the methods discussed, see Table 1.
The amount and concentration of analyte available
1 are the most obvious factor for method selection. If
only minute amounts in organic micro-synthesis are
0 available for characterization, most of the methods
0 1 2 3 4 5 6 7 cannot be used. Other sample restrictions include,
Experimental pKa for example, overlapping pKa’s, very low solubility,
lack of a chromophore, etc. If the available sample
Figure 11. Plot of calculated versus experimentally observed pKa for a
set of 143 aliphatic carboxylic acids, data from Schüürmann et al.101 has impurities, only separation methods such as
HPLC and particularly electrophoresis can be used
successfully. Measuring in the extremes of the pH
interest as an estimator, since the reliability of the scale is sometimes impossible. Financial factors must
results leaves much to be desired. It may be expected also be taken into consideration. It is not fiscally
that the method will find more use in the future as the responsible to invest in NMR equipment for the pur-
computational power available increases and better pose of determining only a few pKa values annually.
models are derived, but it shall be some time before In regards to precision, for a very rough estimate
computational methods are able to compete with (± 1 pK unit), the only thing needed is a software
actual measurement in terms of accuracy. There are package,104,108 however experienced chemists are
several software packages available (ACD/labs was often able to estimate this from the structural formula.
one of the first) and five of these are compared in a Additionally, if the pure analyte is available as an acid
recent review by Manchester et al.104 and as a salt of a strong base, a 1:1 solution of both
Table 1. Overview of the strong (++) and weak (--) points of the methods discussed. Precision based largely on Xie et al,47
Manchester et al,105 Barbosa et al,106 and Beltrain et al.107 T, I and ε denotes effects of Temperature, Ionic Strength and
organic modifier, respectively.
Amount/conc Restrictions pKa range Costs/time Precision T, I and e

Potentiometry - + -- ++ + +
Conductometry - ++ - + + -
Voltammetry - + + + + +
Calorimetry - ++ ++ + + +
NMR - - ++ -- ++ -
Electrophoresis ++ + + + ++ ++
HPLC ++ + - + + +
Solubility - - + -- + -
Spectrometry + + + + ++ ++
Fluorimetry + - + + -- +
Polarimetry + -- + + + +
Kinetic -- -- - - + +
Computational ++ - ++ - -- -

Reijenga et al
will by definition have a pH equal to the pKa within analyte and buffer ions. These are checked with
the range 4 , pH , 10. analyte behavior in two different buffers of same pH
On the other hand, precisions claimed in literature but different buffering constituents. Ionic strength
generally refer only to specific analytes under inves- dependence is checked by adding sodium chloride
tigation, and as we all know, are sometimes brightly buffer made with the analyte under investigation.
colored. Three decimals is impossible and two deci- Temperature dependence is most easily checked
mals is considered very good. The last column in by measuring a buffer of the analyte at different
Table 1, in our view, is a most important one for rea- temperatures.
sons mentioned in section 1.2. In most cases, pKa are A laboratory where accurate pKa determinations
measured under conditions imposed by the method of are essential would be wise to invest in equipment
choice. These are not always the conditions relevant and experience of CE, complemented with spectrom-
for the eventual application of the analyte. As already etry, pH, and conductivity meters. All of the former
indicated in the Fentanyl example,81 physiologically should be well thermostated.
relevant pKa values of drugs inherent to their appli- Besides those, it would be advised to monitor future
cation are those in water at 37 °C and with an ionic developments in pKa estimation software, especially
strength of 0.9% NaCl (154 mM). Thermodynamic those programs that can be fed with experimental data
values at 25 °C and infinite dilution are irrelevant for more accurate prediction (rather than estimation)
in this area. In a recent 700 page compilation109 of of similar structures.
aqueous pKa values of drugs, only half are listed with
details of temperature and ionic strength. Author Contributions
The most versatile method able to accurately mea- Entire literature search and first draft versions of man-
sure under these conditions appears to be CE. The uscript chapters: AHM van Loon, AJF van Hoof, AJP
theoretical basis is straightforward and well under- Teunissen. Jointly developed the final structure, illus-
stood and the experiments can use a high degree of trations and revised chapters of the paper: AHM van
automation.110–112 In the world of drug characterization Loon, AJF van Hoof, AJP Teunissen, JC Reijenga.
and analysis, the method is well established.113 The Made critical revisions and approved final version:
pK range in which reliable values can be determined JC Reijenga. All authors reviewed and approved of
is expanding to very acidic regimes114,115 unavailable the final manuscript.
to other techniques.
Funding
Conclusion and Recommendations Author(s) disclose no funding sources.
Being able to actually measure under the conditions
relevant for the application is a great advantage, but Competing Interests
alas often impossible due to limitations of the tech- Author(s) disclose no potential conflicts of interest.
nique used. Extrapolation to the desired conditions is
often possible at the cost of accuracy and precision Disclosures and Ethics
inherent to all extrapolations. As a requirement of publication the authors have
Depending on the sample purity, a choice can be provided signed confirmation of their compliance
made between a spectrometric method and a separa- with ethical and legal obligations including but not
tion method. HLPC is time consuming in the case of limited to compliance with ICMJE authorship and
small dissociation constants (α) whereas CE is faster competing interests guidelines, that the article is
and cheaper. In the absence of a chromophore for neither under consideration for publication nor pub-
optical detection, CE has the additional advantage lished elsewhere, of their compliance with legal and
over chromatography in that it is possible to apply ethical guidelines concerning human and animal
indirect detection.116,117 research participants (if applicable), and that per-
Whichever method is used, matrix effects can mission has been obtained for reproduction of any
always occur, especially ionic interaction between copyrighted material. This article was subject to

blind, independent, expert peer review. The review- 25. Landolt-Börnstein, Zahlenwerte und Funktionen. 6. Auflage, II. Band, 7.
Teil, Elektrische Eigenshaften II. Springer Verlag, Berlin; 1960.
ers reported no competing interests. 26. Apelblat A. Dissociation constants and limiting conductance of organics
acids in water. J Mol Liq. 2002;95:99.
27. Gelb RI. Conductometric determination of pKa values. Oxalic and squaric
References acids. Anal Chem. 1971;43(8):1110–3.
1. Camoes MF. The quality of pH measurements 100 years after its definition. 28. Kielland J. Individual activity coefficients of ions in aqueous solutions.
Accred Qual Assur. 2009;14(10):521–3. J Am Chem Soc. 1937;59:1675–8.
2. Camoes MF. A century of pH measurement. Chem Internat. 2010;32:2. 29. Dippy JFJ. The dissociation constants of monocarboxylic acids; their mea-
3. Belyustin AA. The centenary of glass electrode: from Max Cremer to F.G.K. surement and their significance in theoretical organic chemistry. Chem Rev.
Bouche. Journal of Solid State Electrochemistry. 2011;15:47. 1939;25:151.
4. Brock WH, editor. The Fontana History of Chemistry. Fontana press; 1992. 30. Quint J, Viallard A. The electrophoretic effect for the case of electrolyte
5. Henderson LJ. Concerning the relationship between the strength of mixtures. J Solution Chem. 1978;7(7):525–31.
acids and their capacity to preserve neutrality. Am J Physiol. 1908; 31. Lee WH, Wheaton RJ. Conductance of symmetrical, unsymmetrical and
21:173. mixed electrolytes. Part 1.—Relaxation terms. J Chem Soc, Faraday Trans II.
6. Sörenson SPL. Enzymstudien. II: Mitteilung. Über die Messung und die 1978;74:743–66.
Bedeutung der Wasserstoffionenkoncentration bei enzymatischen Prozessen. 32. Lee WH, Wheaton RJ. Conductance of symmetrical, unsymmetrical and
Biochemische Zeitschrift. 1909;21:131–200. mixed electrolytes. Part 2.—Hydrodynamic terms and complete conduc-
7. Hasselbalch KA. Die Berechnung der Wasserstoffzahl des Blutes aus der tance equation. J Chem Soc, Faraday Trans II. 1978;74:1456–82.
freien und gebundenen Kohlensäure desselben, und die Sauerstoffbindung 33. Lee WH, Wheaton RJ. Conductance of symmetrical, unsymmetrical
des Blutes als Funktion der Wasserstoffzahl. Biochemische Zeitschrift. and mixed electrolytes. Part 3.—Examination of new model and analy-
1916;78:112–44. sis of data for symmetrical electrolytes. J Chem Soc, Faraday Trans II.
8. Po HN, Senozan NM. Henderson—Hasselbalch equation: its history and 1979;75:1128–45.
limitations. J Chem Educ. 2001;78:1499–503. 34. Apelblat A. Representation of electrical conductances for polyvalent elec-
9. Hammett LP. The theory of acidity. J Am Chem Soc. 1928;50:2666. trolytes by the quint-viallard conductivity equation. Part 1. Symmetrical 2:2
10. Monzyk B, Crumbliss AL. Acid dissociation constants (Ka) and their type electrolytes. Dilute aqueous solutions of alkaline earth metal sulfates
temperature dependencies (∆Ha, ∆Sa) for a series of carbon- and nitro- and transition metal sulfates. J Solution Chem. 2011;40(7):1209–33.
gensubstituted hydroxamic acids in aqueous solution. J Org Chem. 1980; 35. Kroflič A, Apelblat A, Bešter-Rogač M. Dissociation constants of para-
45:4670. bens and limiting conductances of their ions in water. J Phys Chem B.
11. Poth-Brink C, Crumbliss AL. Temperature-dependent acid dissociation 2012;116(4):1385–92.
constants (Ka, .DELTA.Ha, .DELTA.Sa) for a series of nitrogen-substituted 36. Kalvoda R, Fresenius J. Review of adsorptive stripping voltammetry-
hydroxamic acids in aqueous solution. J Org Chem. 1982;47(7):1171–6. assessment and prospects. Anal Chem. 1994;349:565.
12. Everaerts FM, Beckers JL, Verheggen TPEM. Isotachophoresis, Theory, 37. Zuman P. Current status of polarography and voltammetry in analytical
Instrumention and Applications. Elsevier Scientific Publishing Co., chemistry. Anal Letter. 2000;33(2):163–74.
Amsterdam; 1976. 38. Bard AJ. The rise of voltammetry: from polarography to the scanning elec-
13. Cohn EJ, Heyroth FF, Menkin MF. The dissociation constant of acetic acid trochemical microscope. J Chem Educ. 2007;84(4):644–50.
and the activity coefficients of the ions in certain acetate solutions. J Am 39. Kolthoff IM, Orleman EF. The Use of the dropping mercury electrode
Chem Soc. 1928;50:696–714. as an indicator electrode in poorly poised systems. J Am Chem Soc.
14. Sarmini K, Kenndler E. Capillary zone electrophoresis in mixed 1941;63(3):664–7.
aqueous-organic media: effect of organic solvent on actual ionic mobilities, 40. Breslow R, Balasubramanian K. The pKa of triphenylcyclopropene. Electro-
acidity constants and separation selectivity of substituted aromatic acids. chemical determination of an inaccessible equilibrium constant. J Am Chem
I. Methanol. J Chromatogr A. 1998;806:325–35. Soc. 1969;91:5182.
15. Sigel H, Zuberbuhler AD, Yamauchi. Comments on potentiometric pH titra- 41. Barette WC Jr, Johnson HW Jr, Sawyer DT. Voltammetric evaluation of the
tions and the relationship between pH-meter reading and hydrogen ion con- effective acidities (pKa’) for Brönsted acids in aprotic solvents. J Am Chem
centration. Anal Chim Acta. 1991;225:63–72. Soc. 1984;56:1890.
16. Denham HG. The electrometric determination of the hydrolysis of salts. 42. Kim H-s, Chung TD, Kim H. Voltammetric determination of the pKa of
J Chem Soc Trans. 1908;93:41–63. various acids in polar aprotic solvents using 1,4-benzoquinone. J Electro-
17. Stock JT. Early industrial pH measurement and control. Bull Hist Chem. analytical Chem. 2001;498(1–2):209–15.
1991;10:31–4. 43. Gupta N, Linschitz H. Hydrogen-bonding and protonation effects in electrochem-
18. Beckman AO, Fracker HE. United States patent 2085761. 1936. istry of quinones in aprotic solvents. J Am Chem Soc. 1997; 119(27):6384–91.
19. Gardiner WC, Sanders HL. Errors of the glass electrode. Indust Eng Chem. 44. Zhao J, Luo L, Yang X, Wang E, Dong S. Determination of surface pKa of SAM
1937;9:274–8. using an electrochemical titration method. Electroanalysis. 1999; 11(15):1108–13.
20. Benet LZ, Goyan JE. Potentiometric determination of dissociation constants. 45. Heinze J. Cyclic Voltammetry-“Electrochemical spectroscopy”. Ang Chem.
J Pharm Sci. 1967;56:665–80. 1984;23:831–47.
21. Avdeef A, Comer JEA, Thomson SJ. pH-Metric log P. 3. Glass electrode 46. Fenby DV. Heat: its measurement from Galileo to Lavoisier. Pure Appl
calibration in methanol-water, applied to pKa determination of water-insol- Chem. 1987;59(1):91–100.
uble substances. Anal Chem. 1993;65(1):42–9. 47. Xie D, Gulnik S, Collins L, Gustchina E, Suvorov L, Erickson JW. Dissection
22. Ravichandiran V, Devajaran V, Masilamani K. Determination of ioniza- of the pH dependence of inhibitor binding energetics for an aspartic protease:
tion constant (pKa) for poorly soluble drugs by using surfactants: a novel direct measurement of the protonation states of the catalytic aspartic acid
approach. Der Pharmacia Lettre. 2011;3(4):183–92. residues. Biochemistry. 1997;36(51):16166–72.
23. Kohlrausch F, Göttinger Nachrichten. Über das Leitungsvermögen der in 48. Tajc SG, Tolbert BS, Basavappa R, Miller BL. Direct determination
Wasser gelösten Elektrolyte in Zussamenhang mit der Wanderung ihrer of thiol pKa by isothermal titration microcalorimetry. J Am Chem Soc.
Bestandteile. 1876;13:213–4. 2004;126(34):10508–9.
24. Saxton B, Darken LS. The ionization constants of weak acids at 25° from 49. Grunwald E, Loewenstein A, Meiboom S. Rates and mechanisms of pro-
conductance measurements. A method of extrapolating the data. J Am Chem tolysis of methylammonium ion in aqueous solution studied by proton mag-
Soc. 1940;62(4):846–52. netic resonance. J Chem Phys. 1957;27:641.

Reijenga et al
50. Lee DG, Cameron R. The basicity of aliphatic ethers. Can J Chem. 73. Horváth C, Melander W, Molnár I. Liquid chromatography of ionogenic sub-
1972;50(3):445–8. stances with nonpolar stationary phases (Solvophobic Theory of Reversed
51. Popov K, Rönkkömäki H, Lajunen LHJ. Guidelines for NMR measurements Phase Chromatography, Part II). Anal Chem. 1977;49:142–53.
for determination of high and low pKa values (IUPAC Technical Report). 74. Foley JP, May WE. Optimization of secondary chemical equilibria in liquid
Pure Appl Chem. 2006;78(3):663–75. chromatography: theory and verification. Anal Chem. 1987;59:102–9.
52. Rabenstein DL, Sayer TL. Determination of microscopic acid dissocia- 75. Foley JP, May WE. Optimization of secondary chemical equilibria in liquid
tion constants by nuclear magnetic resonance spectroscopy. Anal Chem. chromatography: variables influencing the self-selectivity, retention, and
1976;48:1141–6. efficiency. Anal Chem. 1987;59:110–6.
53. Glaser J, Henriksson U, Klason T. A 205Tl NMR titration study of the com- 76. Leo A, Hansch C, Elkins D. Partition coefficients and their uses. Chem Rev.
plex formation between Tl(I) and Cl- in aqueous solution. Act Chem Scand. 1971;71(6):525–616.
1986;A40:344. 77. Golumbic C, Orchin M, Weller S. Relation between partition coefficient and
54. Poole SK, Patel S, Dehring K, Workman H, Poole CF. Determination of ionization constant. J Am Chem Soc. 1949;71:2624–7.
Acid Dissociation Constants by Capillary Electrophoresis. J Chromatogr A. 78. Hasegawa J, Fujita T, Hayashi Y, Iwamoto K, Watanabe J. pKa determination
2004;1037(1–2):445–54. of verapamil by liquid-liquid partition. J Pharm Sci. 1984;73(4):442–5.
55. Gluck SJ, Cleveland JA Jr. Investigation of experimental approaches to the 79. Dearden JC, Bresnen GM. The measurement of partition coefficients. Quant
determination of pKa values by capillary electrophoresis. J Chromatogr A. Struct-Act Relat. 1988;7:133–44.
1994;680(1):49–56. 80. Tomlinson E. Filter-probe extractor: A tool for the rapid determination of
56. Babič S, Horvat AJM, Pavlović DM, Kaštelan-Macan M. Determination oil-water partition coefficients. J Pharm Sci. 1982;71(5):602–4.
of pKa values of active pharmaceutical ingredients. Trends Anal Chem. 81. Hersey A, Hill AP, Hyde RM, Livingstone DJ. Principles of method selec-
2007;26(11):1043–61. tion in partition studies. Quant Struct-Act Relat 8. 1989;288–96.
57. Consden R, Martin AJP. Ionophoresis in silica jelly: A method for the sepa- 82. Thurlkill RL, Cross DA, Scholtz JM, Pace CN, The pKa of Fentanyl
ration of animo-acids and peptides. Biochem J. 1946;40(1):33–41. Varies with Temperature: Implications for Acid-base Management Dur-
58. Waldron-Edward D. The micro determination of acid and base dissociation ing Extremes of Body Temperature. J Cardiothoracic Vas Anesthesia.
constants by paper electrophoresis. J Chromatogr. 1965;20:556. 2005;19(6):759–62.
59. Kiso Y, Kobayashi M, Kitaoka Y, Kawamoto K, Takeda J. A theoreti- 83. Krebs HA, Speakman JC. Dissociation constant, solubility, and the pH
cal study on the zone mobility-pH curve in paper electrophoresis of low value of the solvent. J Chem Soc. 1945;159:593–95.
molecular weight compounds with a dissociable proton and its application 84. Zimmerman I. Determination of pKa values from solubility data. Int J
to phosphorus compounds. J Chromatogr. 1968;36:215. Pharm. 1982;13:57.
60. Pospíchal J, Gebauer P, Boček P. Measurement of mobilities and dissociation 85. Avdeef A. Solubility of sparingly-soluble drugs. Adv Drug Del Rev. 2007;
constants by capillary isotachophoresis. Chem Rev. 1989;89(2):419–30. 59:568–90.
61. Beckers JL, Everaerts FM, Ackermans MT. Determination of absolute 86. Streng WH, Tan HGH. General treatment of pH solubility profiles pf weak
mobilities, pK values and separation numbers by capillary zone electro- acids and bases. II. Evaluation of thermodynamic parameters from the tem-
phoresis : Effective mobility as a parameter for screening. J Chromatogr. perature dependence of solubility profiles applied to a zwitterionic com-
1991;537:407–28. pound. Int J Pharm. 1985;25:135–45.
62. Terabe S, Yashima Y. Separation of oxygen isotopic benzoic acids by capil- 87. Zimmermann I. Dtermination of overlapping pKa values from solubility
lary zone electrophoresis based on isotope effects on the dissociation of data. Int J Pharm. 1986;31:69–74.
carboxyl group. Anal Chem. 1988;60:1673–7. 88. Bergström CAS, Luthman K, Artursson P. Accuracy of calculated
63. Cai J, Smith JT, El Rassi Z. Determination of the ionization constants of weak pH-dependent aqueous drug solubility. Eur J Pharm Sci. 2004;22(5):
electrolytes by capillary zone electrophoresis. J High Resol Chromatogr. 387–98.
1992;15(1):30. 89. Holmes WC, Snyder EJ. Spectrophotometric determination of hydrogenion con-
64. Cleveland JA, Benko MH, Gluck SJ, Walbroehl YM. Automated pKa centrations and of the apparent dissociation constants of indicators iv. 1-naph-
determination at low solute concentrations by capillary. J Chromatogr A. thol-2-sodium sulfonate indophenol1. J Am Chem Soc. 1925;47:2232–6.
1993;653:301–8. 90. Flexser LA, Hammet LP, Dingwall A. The Determination of Ioniza-
65. Ishihama Y, Oda Y, Asakawa N. Microscale determination of dissociation by Ultraviolet Spectrophotometry: Its Validity and its Application to
tion constants of multivalent pharmaceuticals by capillary electrophoresis. the Measurement of the Strength of Very Weak Bases1. J Am Chem Soc.
J Pharm Sci. 1994;83(10):1500–7. 1935;57:2103–15.
66. Gluck SJ, Cleveland JA Jr. Capillary zone electrophoresis for the determina- 91. Wigler PW. The kinetics of snake venom phosphodiesterase, with a
tion of dissociation constants. J Chromatogr A. 1994;680:43–8. new type of substrate, 3-pyridyl thymidine 5′-phosphate. J Biol Chem.
67. Gluck SJ, Steele KP, Benko MH. Determination of acidity constants of 1963;238:1767–71.
monoprotic and diprotic acids by capillary electrophoresis. J Chromatogr A. 92. Wigler PW, Wilson LE. Spectrophotometric determination of the acid disso-
1996;745:117–25. ciation constants of 3-hydroxypyridine. Anal Biochem. 1966;15(3):421–5.
68. Ishihama Y, Nakamura M, Miwa T, Kajima T, Asakawa N. A rapid 93. Allen RI, Box KJ, Comer JEA, Peake C, Tam KY. Multiwavelength spectro-
method for pKa determination of drugs using pressure-assisted capillary photometric determination of acid dissociation constants of ionisable drugs.
electrophoresis. J Pharm Sci. 2002;91:933–42. J Pharm Bio Anal. 1998;17:699–712.
69. Fuguet E, Ràfols C, Bosch E, Roses M. Fast high-throughput method for 94. Takacs-Novak K, Tam KY. Multiwavelength spectrophotometric determi-
the determination of acidity constants by capillary electrophoresis. J Chro- nation of acid dissociation constants: a validation study. Anal Chim Act.
matogr A. 2009;1216(17):3646–51. 2001;434:157–67.
70. Cabot JM, Fuguet E, Ràfols C, Roses M. Fast high-throughput method 95. Saurina J, Hernández-Cassou S, Tauler R, Izquierdo-Ridorsa A.
for the determination of acidity constants by capillary electrophoresis. II. Spectrophotometric determination of pK(a) values based on a pH gradient
Acidic internal standards. J Chromatogr A. 2010;1217(52):8340–5. flow-injection system. Anal Chim Acta. 2000;408(1–2):135–43.
71. Fuguet E, Ràfols C, Roses M. A fast high throughput method for the deter- 96. Schulman SG, Capomacchia AC. Variations of fluorescence quantum yields
mination of acidity constants by capillary electrophoresis. 3. Basic internal with pH or Hammett acidity. Near equilibrium vs. nonequilibrium excited
standards. J Chromatogr A. 2011;1218(25):3928–34. state proton exchange. J Phys Chem. 1975;79(14):1337–43.
72. Singhal RP, Cohn WE. Cation-exclusion chromatography on anion 97. Rosenberg LS, Simons J, Schulman SG. Determination of pKa values
exchangers: application to nucleic acid components and comparison with of N-heterocyclic bases by fluorescence spectrophotometry. Talanta.
anion-exchange chromatography. Biochem. 1973;12(8):1532–7. 1979;26(9):867–71.

98. Katzin LI, Gulyas E. Dissociation constants of tartaric acid with the aid of 108. Babić S, Horvat AJM, Mutavdžić Pavlović D, Kaštelan-Macan M.
polarimetry. J Phys Chem. 1960;64:1739–41. Determination of pKa values of active pharmaceutical ingredients. Trends
99. Bunnett JF, Nudelman NS. An Independent Kinetic Method for Determin- Anal Chem. 2007;26:10431061.
ing Acid Dissociation Constants in Methanol. J Org Chem. 1969;34:2043. 109. ACD/pKa: Predict accurate acid/base dissociation constants from
100. da Silva CO, da Silva EC, Nascimento MAC. Ab Initio calculations of structure—the industry standard. Available at: http://www.acdlabs.com/
absolute pKa values in aqueous solution I. Carboxylic Acids. J Phys products/percepta/predictors/pka/. Accessibility verified Nov 2012.
Chem A. 1999;103(50):11194–9. 110. Brittain HG, editor. Profiles of Drug Substances, Excipients and Related
101. Schüürmann G, Cossi M, Barone V, Tomasi J. Predication of the pKa of Methodology. Vol 33. Academic Press; 2007.
carboxylic acids using the ab initio continuum-solvation model PCM- 111. Li SFY. Capillary Electrophoresis: Principles, Practice, and Applications.
UAHF. J Phys Chem A. 1998; 102:6706. Amsterdam: Elsevier; 1992.
102. Tehan BG, Lloyd EJ, Wong MG, et al. Estimation of pKa using 112. Altria KD, editor. Capillary Electrophoresis Guidebook: Principles,
semiempirical molecular orbital methods. Part 1: Application to phenols Operation, and Applications. Totowa NJ, USA : Humana Press; 1996.
and carboxylic acids. QSAR. 2002;21:457–72. 113. Foret F, Boček P. Capillary Electrophoresis: Instrumentation and
103. Chaudry UA, Popelier PLA. Estimation of pK(a) using quantum topologi- Methodology. VCH Weinheim; 1996.
cal molecular similarity descriptors: Application to carboxylic acids, ani- 114. Ahuja S, Jimida MI. Capillary Electrophoresis Methods for Pharmaceuti-
lines and phenols. J Org Chem. 2004;69(2):233–41. cal Analysis. Amsterdam: Elsevier; 2008.
104. Xing L, Glen RC, Clark RD. Predicting pK(a) by molecular tree struc- 115. Včeláková K, Zusková I, Kenndler E, Gaš B. Determination of cat-
tured fingerprints and PLS. J Chem Inf Comput Sci. 2003;43(3): ionic mobilities and pKa values of 22 amino acids by capillary zone
870–9. electrophoresis. Electrophoresis. 2004;25(2):309–17.
105. Manchester J, Walkup G, Rivin O, You Z. Evaluation of pKa estima- 116. Zusková I, Novotná A, Včeláková K, Gaš B. Determination of limiting
tion methods on 211 druglike compounds. J Chem Inf Model. 26 2010; mobilities and dissociation constants of 21 amino acids by capillary zone
50(4):565–71. electrophoresis at very low pH. J Chromatogr B. 2006;841:129.
106. Barbosa J, Barron D, Jimenez-Lozano E, Sanz-Nebot V. Comparison 117. Bruin GJM, van Asten AC, Xu X, Poppe H. Theoretical and experimental
between capillary electrophoresis, liquid chromatography, potentiometric aspects of indirect detection in capillary electrophoresis. J Chromatogr.
and spectrophotometric techniques for evaluation of pKa values of zwit- 1992;608:97.
terionic drugs in acetonitrile—water mixtures. Anal Chim Acta. 2001;437: 118. Doble P, Macka M, Haddad PR. Use of dyes as indirect detection probes for
309–21. the high-sensitivity determination of anions by capillary electrophoresis.
107. Beltran JL, Sanli N, Fonrodona G, Barron D, Ozkan G, Barbosa J. J Chromatogr A. 1998;804(1–2):327–36.
Spectrophotometric, potentiometric and chromatographic pKa values of
polyphenolic acids in water and acetonitrile-water media. Anal Chim Acta.
2003;484(2):253–64.

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Analytical Chemistry Insights

Development of Methods for the Determination

Keywords: review, history, dissociation constant, pKa, pH

Analytical Chemistry Insights 2013:8 53–71

This article is available from http://www.la-press.com.

© the author(s), publisher and licensee Libertas Academica Ltd.

Analytical Chemistry Insights 2013:8 53

54 Analytical Chemistry Insights 2013:8

Influences on pKa Measured pKa values also depend on the ionic

Analytical Chemistry Insights 2013:8 55

6 or other interaction between the analyte and these

4.5 one, even for the case of monovalent ions, to which

Potentiometry Solubility Electrophoresis Polarometry Fluorometry Computational

Spectrometry Conductometry Partition HPLC Calororimetry

56 Analytical Chemistry Insights 2013:8

extrapolation of measurements in solvent mixtures.21

is proposed22 which avoids tedious extrapolating while

titration requires a relatively large amount of sample 0 0.2

compared with separation methods such as high per-

Analytical Chemistry Insights 2013:8 57

58 Analytical Chemistry Insights 2013:8

Voltammetry therefore a reference with known characteristics is

temperature constant is measured. It is also one of the

Analytical Chemistry Insights 2013:8 59

­ icrocalorimetry (ITM). Here the reagent for the

Nuclear magnetic resonance pKa = pH + log [(δA - δobs)/(δA - δHA)] (10)

With this characterization method, even more

60 Analytical Chemistry Insights 2013:8

The relation with previously discussed conducto-

mi = λi/F (12) Regression results

Analytical Chemistry Insights 2013:8 61

62 Analytical Chemistry Insights 2013:8

P = [B]o/([B]w + [BH+]w) (16) Solubility

Analytical Chemistry Insights 2013:8 63

log S = log S0 + log (10−pK−pH + 1) (18) UV/Vis spectrometry

rated by Flexser et al89 in 1935 by determining differ-

64 Analytical Chemistry Insights 2013:8

showed good concurrence with previous single wave- Polarimetry

Analytical Chemistry Insights 2013:8 65

66 Analytical Chemistry Insights 2013:8

7 Comparison and Discussion

4 different methods described in this paper cover many

Amount/conc Restrictions pKa range Costs/time Precision T, I and e

Analytical Chemistry Insights 2013:8 67

68 Analytical Chemistry Insights 2013:8

Analytical Chemistry Insights 2013:8 69

70 Analytical Chemistry Insights 2013:8

Analytical Chemistry Insights 2013:8 71

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icrocalorimetry (ITM). Here the reagent for the