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Disclaimer: This article is for guidance and educational purposes only. The
author can accept no responsibility for loss or damage however caused. The
author recommends that manufacturers advice be consulted exclusively
when using any laboratory products.
PREFACE TO 2006 REVISION: This page was written in 1999 and can be
seen as summarising my practical knowledge of the field at that time. Things
have moved on particularly in the area of high throughput measurements. For
the latest in high throughput pKa and LogP measurements I suggest you
contact Sirius Analytical Instruments and for high throughput permeability
contact Pion Inc. I will continue to add things to this site on the use of
physical chemistry measurements in QSAR modelling. Please see section 1.7.
to 1.9.
Table of Contents:
Introduction
Contents
LogP/pKa measurement
techniques
Aqueous Titration
using Sirius
instruments
o Yesuda-Shedlovsky
experiment
o Ion Pair Log P's
o pKa by Manual
Titration
o pKa by U.V.
Spectroscopy
o pKa by Solubility
Method
o Filter Probe
Measurements
o Log D and Log P
by Filter Probe
Method
o Log P by Shake
Flask
o Log P by HPLC
References
Appendix 1 - Calculating
Log D and % ionised
Appendix 2 - Worked
example calculations
o
Introduction
The pKa or 'Dissociation Constant' is a measure of the strength of an acid or a base.
The pKa allows you to determine the charge on a molecule at any given pH.
The Partition Coefficient is a measure of how well a substance partitions between a lipid
(oil) and water.
pKa and Log P measurements are useful parameters for use in understanding the
behaviour of drug molecules. Different ionic species of a molecule differ in physical
chemical and biological properties and so it is important to be able to predict which ionic
form of the molecule is present at the site of action. The Partition Coefficient is also a
very useful parameter which may be used in combination with the pKa to predict the
distribution of a drug compound in a biological system. Factors such as absorption,
excretion and penetration of the CNS may be related to the Log P value of a drug and in
certain cases predictions made.
The measurement of pKa and Log P values are not straightforward. Experiments must be
very carefully performed under standard conditions to ensure the results are valid and
require interpretation of data which takes time and experience. In addition no one method
is available for all compounds due to problems of insolubility, lack of removable protons
and extreme values.
This guide gives the theoretical basis of the pKa and LogP parameters as well as
describing the techniques that can be used to measure them indicating which methods are
appropriate for problem samples. I have also briefly indicated the use of these
measurements in rational drug design.
For more information please see the References section.
The concentration of H+ and OH- ions, which are equal, are 1x 10-7 ions per litre The
equilibrium constant (or ion product ) for the dissociation of water, Kw, is
This equation sets the pH scale to 0-14, which gives a convenient way to express 14
orders of magnitude of [H+]. Any solution with pH>7 contains excess hydroxyl ions and
is alkaline; those with pH<7 are acidic, containing excess hydrogen ions
pH scale
1.2 Activity
A complication arises in electrochemistry due to the non-ideal nature of ions in solution.
The activity of an ion at infinite dilution is equal to its concentration but as the
concentration increases ionic attraction and incomplete hydration results in a drop in
effective concentration. This means the law of Mass Action is only valid when activities
are used in place of concentrations
Activity is defined as the "apparent concentration" of an ionic species, due to the
attraction which ions can exert on one another and the incomplete hydration of ions in
solutions that are too concentrated. The lower the concentration the less the interaction
becomes. At infinite dilution activity coefficients approach unity
The activity of a species X is equal to the product of its concentration and its activity
coefficient,
The pH from an electrode relates to {H+} not [H+] though below Ionic strength of 0.01
these terms are very close between pH 2 and pH 10
At pH 7 where {H+}={OH-} the voltage from the electrode is zero, this is called the
Isopotential Point. In theory this point is temperature independent. IUPAC-NBS
operational pH scale is defined as the pH relative to a standard buffer measured using
hydrogen electrode. In practice a pH electrode is calibrated with a standard pH 7.00
buffer to determine the isoelectric point and a standard buffer at either pH 4 or 9 to
determine the slope. Conventional pH meters will read accurately over a range 2.5 - 11.
Beyond this their accuracy is dubious.
In recent years Sirius Analytical Instruments have produced a series of dedicated
pKa/LogP instruments. In the PCA 101 pKa instrument the calibration is carried out in a
more sophisticated way adding empirical correction factors at the extreme ends of the pH
spectrum where the electrode behaviour is non-ideal. In this way measurements at pH 1
or 13 are possible. This is based on the work of Alex Avdeef (1)
The pKa or ionisation constant is defined as the negative logarithm of the equilibrium
coefficient of the neutral and charged forms of a compound. This allows the proportion of
neutral and charged species at any pH to be calculated, as well as the basic or acidic
properties of the compound to be defined.
"Thermodynamic Ionisation Constants" require the use of activities, being an "Infinite
Dilution" definition. The measurement of activities is highly impractical, so in practice a
high ionic strength swamping background electrolyte is used to give a "Constant Ionic
Medium" pH definition. This is closely related to the thermodynamic definition. Such
pKa values are independent of concentration and are of the type usually quoted in the
literature.
pKa = -log10(Ka)
for bases
pKa = -log10(Ka)
At a given temp these are Thermodynamic Ionisation constants, which are independent
of concentration. KTa. Since log 1 = 0 the pKa corresponds to the pH at which the
concentration of ionised and neutral forms are equal.
Ionisation constants that measured by Spectroscopy are "Concentration Ionisation
Constants" These constants are measured ignoring activity effects and are dependent on
concentration. It is therefore important that the concentration of the compound measured
is quoted. Comparison of different compounds is only valid if their concentrations are
identical.
These result from spectroscopic measurements where concentrations are used due to the
beer lambert law.
The "Thermodynamic" Ionisation Coefficient is related to the "Concentration" Ionisation
Coefficient by:
where f=activity coefficient
pKa values are temperature dependent in a non-linear and unpredictable way. Samples
measured by potentiometry are held at a constant temperature using a water jacket and
thermostated water bath. Spectroscopic values are measured at ambient temperature. No
pKa value should ever be quoted without the temperature. There is the additional
question of whether pKa values should be measured at biological temperature as well as
the standard 25 degrees. The former would have more meaning to biologists and the latter
to chemists. Standard practice is to measure pKas at 25C
A useful formula for calculating the % ionisation of a compound at a particular pH from
its pKa is
Acid
with
pKa
=
8.0
Base
with
pKa
=
8.0
Partition Coefficient
Partition Coefficient, P = [Organic] / [Aqueous] Where [] = concentration
Log P= log10 (Partition Coefficient)
NOTE:
Log P = 1 means 10:1 Organic:Aqueous
Log P = 0 means 1:1 Organic:Aqueous
Log P = -1 means 1:10 Organic:Aqueous
Log D is the log distribution coefficient at a particular pH. This is not constant and will
vary according to the protogenic nature of the molecule. Log D at pH 7.4 is often quoted
to give an indication of the lipophilicity of a drug at the pH of blood plasma.
Distribution Coefficient
Distribution Coefficient, D = [Unionised] (o) / [Unionised] (aq) + [Ionised] (aq)
Log D = log10 (Distribution Coefficient )
for acids
for bases
The graphs below show the distribution plots of an acid a base and a zwitterion
Acid pKa = 8
Base pKa =8
The spectroscopic measurements of Log P are measured at a much lower ionic strength,
hence comparisons will be invalid.
The question arises how valid is the use of a background electrolyte? Typically 0.1M of a
background electrolyte is used. This is very close to the biological level of 0.16M. The
type of electrolyte is also called into question. 0.15 M KCl is generally used due to its
similarity with NaCl. NaCl cannot be used because of the "sodium effect" on the
electrode at high pH. Measurements in KCl have been found to match those in NaCl
almost exactly. Initially the Sirius Instruments used KNO3, as used in the development of
Metal Ligand binding titrations, from which the titrimetric method was developed. KNO3
is obviously alien to most biological systems.
amphiprotic
(Hbonding)
Chloroform
proton
donor (Hbonding)
PGDP
proton
acceptor
(Hbonding)
Alkane
inert
Phospholipid
Phospholipid
Model: (ref 8)
Which solvent to use is debatable; however delta log P values have been found to be
useful in several QSAR studies.
log P(octanol-water) - logP(PGDP-water)
Liposomes.
Recently partitioning experiments have been carried out with Liposomes. Liposomes are
self assembling model membranes composed of phopholipid groups such as
phosphatadylcholine. The lipid molecule is dissolved in chloroform and deposited by
evaporation onto a large surface such as a large round bottomed flask. The liposome is
then hydrated by adding water and agitated. The lipids then self assemble to form lipid
bilayers which form spheres, often concentric (multilammellar). For partitioning
experiments it has been found that Unilamellar (single layer) liposomes are required.
These can be formed by a a combination of freeze-thawing and extrusion through a fine
filter or french press under pressure.
Neutral LogP values from liposomes tend to be very similar to those measured in octanol
but the ion-pair LogP values differ. The "Surface Ion Pair" log P is found to be much
higher in bases, zwitterions and amphophiles. The values for acids tend to be similar to
the octanol values. This reflects the increased potential for partitioning of molecules with
basic groups into membranes.
QSAR studies have found improved correlations with liposome derived "Surface Ion
Pair" LogP values.
It should be realised that for some compounds it is not possible to make measurements
due to insolubility, impurity or instability reasons. It is practically impossible to make
measurements on highly insoluble compounds, although pKa values may sometimes be
measurable by aqueous-methanol titrations. In practical terms results become
meaningless for compounds with extreme insolubility.
are generated using regression analysis to correlate observed biological data with
measured partition coefficients.
The best way of relating LogP, pKa and other physico-chemical data to biological
activity is using Multivariate techniques such as Principal Components Analysis and
Partial Least Squares Regression. To understand these techniques and for software to
do this please visit Umetrics at www.umetrics.co.uk
It must be remembered that measured log P values only correlate with activity in certain
instances. The use of organic solvents to model complex biolipids is very simplistic
and cannot explain phenomena such as the large difference in activity between
molecules of wildly different structures or between enantiomers. In these cases it is
very useful to combine physical measurements with molecular modelling, molecular
property and spectroscopic data and use multivariate analysis.
For both CNS penetration and gastric absorption many studies show a parabolic
relationship with an optimum Log P value of around 2 1. Evidence for this comes from
a wide variety of experiments in the literature from brain concentration of radiolabelled
compounds to CNS behavioural studies.
Recently more sophisticated analysis of molecular properties such as "Partial Charged
Surface Area" (PSA) and the hydrogen bonding properties of molecules have lead to
better predictions of oral absorption.
Although lipophilicity is just one of many factors involved in biological activity it is
often one of the most influential. In PLS regression of molecular properties vs biological
activity measurements of LogP almost always features in the more important coefficients.
It is also a good idea to add a LogP squared to any regression analysis to take account of
the non linearity mentioned above.
It is therefore possible to have compounds with high Log P values which are still soluble
on account of their low melting point. Similarly it is possible to have a low Log P
compound with a high melting point, which is very insoluble.
In cases of precipitation when titrating a basic compound, the solubility of the free base
may be calculated using the equation:
Where:
= solubility at
= solubility of free base
Increasing LogD 7.4 above 0 will decrease renal clearance and increase metabolic
clearance.
High Log D7.4 compounds will tend to be metabolised by P450 enzymes in the
liver.
A high degree of ionisation keeps drugs out of cells and decreases systemic
toxicity.
pKa in range 6 to 8 is advantageous for membrane penetration.
Drugs should be designed with the lowest possible Log P, to reduce toxicity,
non-specific binding, increase ease of formulation and bioavailability. Drugs
should also be as low mw as possible to lower the risk of allergic reactions. (See
principle of minimum hydrophobicity)
Physiological pH values:
Stomach 2
Kidneys 4.2 (variable)
Small Intestine Fed 5.0 Fasted 6.8
Duodenal Mucus 5.5
Plasma 7.4
Chris Lipinski of Pfizer derived an easy to use 'rule of thumb' for drug likeness in
molecules after surveying the worlds marketed drugs.
The rule states that for reasonable absorption
Like all rules they are there to be broken and a number of exceptions exist. I have
personally worked on a couple of well-absorbed drugs which broke this rule but as a
general guide it works well. Remember that you may have charge in your molecule so
that LogD(7.4) or LogD(5.5) is really the important parameter rather than Log P. Keeping
LogD(7.4) around 2 seem generally good advice. Manipulating the pKa can be a way of
improving a molecule.
Mw 200-400
Mpt <200
LogPoct <2
pKa (base) 7+/- 2
Log Sw 2+/-1
Stability alerts <2
For other attempts at rules for agrochemicals see references 19 and 20 Refs
to compare and contrast the properties of a closely related series, using directly
comparable techniques.
to find a common measurement strategy for all the compounds in a series
to identify experimental problems common to the series
to prevent unnecessary measurements, only key members of the series should be
chosen
to ensure reagents with short shelf lives, and apparatus can be prepared
Measures
Advantage
Disadvantage
Conc required
Sample
size
Sirius Potentiometric
pKa/Log P
pKa, Log P,
Rapid, Convenient
Insoluble or neutral
samples cannot be
measured
0.0001M
(0.1mM)
1-5 mg
Log Papp
Sirius YesudaShedlovsky
pKa
0.0005M
(0.5mM)
5mg
0.0001M
(0.1mM)
3-15 mg
Manual potentiometric
pKa
pKa
Simple, rapid
>0.0025M(2.5mM)
50 mg
pKa by UV
pKa
Slow
0.000025M(25uM)
6 mg
pKa by Solubility
pKa
Below
0.0005M
(0.5mM)
10 mg
Log P
0.000025M
(25uM)
6 mg
0.000025M
(25uM)
6 mg
Log P, Log D at
chosen pH
0.000025M
(25uM)
6mg
Log P by HPLC *
Log D at pH 7
Inaccurate, generally
only carried out at pH 7
~ 2.5mM
0.5 mg
(* NB: This table is rather out of date. See Sirius Analytical's new high throughput
instruments)
GlpKa (TM)
PCA101
This more recent technique gives an unprecedented amount of information about the
ionisation and partition behaviour of a molecule; however this is accompanied by more
attention to detail in the calculation and interpretation stages. If a sample is soluble and
well behaved, then it is possible to determine all its pKa values, its Log P and the
apparent log P at every pH. In addition log P values of ionised species where they occur
may be calculated.
The technique can be performed on samples at a concentration of 0.0001M or above, the
ideal concentration being 0.0005 M. Using the PCA101 for a well behaved molecule the
analysis time would be 0.5 Day, including calculation time. The newer GLpKa (TM) has an
autosampler and can also do multiple titrations on each sample and recently has much
improved software for semi-automated refinement.
If the sample is very insoluble then the Log P cannot be measured. The pKa however
may be measurable by either partial titration or by a Yesuda Shedlovsky experiment.
Ion pair LogPs may be determined by at least two, preferably three titrations in different
ratios of octanol to water. The apparent pKa in the presence of octanol , the poKa, can be
used to determine the presence of ion pair partitioning according to the equations:
The main advantage of the HPLC method is that a range of compounds may be
determined at the same time. A new rapid technique has been reported where all
compounds and standards are simultaneously injected and the identity of each peak is
determined by mass spectroscopy. Although this means a lot of work for the
spectroscopist, the amount of chromatography is dramatically reduced.
A refinement of this teqnique is the determination of logk'0 This is achieved by measuring
logk' in several different concentrations of aqeous methanol mobile phases and
extrapolating back to 0% methanol. The resultant l logk'0 values have been correlated to
log P values more sucsessfully. The concerns about polar interactions and the charge
present on the analytes still remain.
References
1.
A.Albert and E.P.Seargent " The Determination of Ionisation Constants - A laboratory Manual", 3rd Edition, Chapman and Hall
1984 ISBN 0-412-24290-7
2.
Avdeef A "Weighting Scheme for Regression Analysis Using pH data from Acid Base Titrations" Anal.Chim.Acta 1983 148
pp237-244
3.
Avdeef A "pH-Metric LogP 1 Difference plots for determining Ion-Pair Octanol-Water Partition Coefficients of Multiprotic
Substances" Quant.Struct-Act. Relationships 1992 11 pp510-517
4.
Avdeef A, Comer J.E.A, Thomson, S.J. "pH-Metric Logp 3. Glass Electrode Calibration in Methanol-Water Applied to pKa
Determination of Water Insoluble Subatances by Potentiometric Titration "Anal.Chem 1993 65 pp42-49
5.
6.
Leahy D.E et al "QSAR: Rational Approaches to the design of Bioactive Compounds" Elsevier 1991 pp75-82
7.
Ganellin C.R. "Uses of partition coefficients by brain penetration applied to the design of H2-receptor histamine antagonists "
Elsevier 1991 pp103-110
8.
H Heller, M Schaeffer, K Schulten, "Molecular Dynamics simulation of a bilayer of 200 lipids in the gel and in the Liquid-crystal
phases", J Phys Chem 97 1993 pp8343-60,,
9.
Tomlinson E. "Filter Probe Extractor: A tool for the rapid Determination of Oil-Water Partition Coefficients" J.Pharm.Sci 1982 71
602-604
El Tayar et al "Partition of solutes in different solvent systems: the contribution of hydrogen bonding, capacity and polarity
J.Pharm.Sci 80 590-598 & 744-749.1991
10. Clarke F.H. "Ionisation constants by Curve Fitting. Application to the determination of partition coefficients"
J.Pharm.Sci 1984
73 226-230
11. Leo A. Hansch C. Elkins D. Partition Coefficients and their uses Chemical Reviews 71 No.6 December 1971
12. W.Dunn III, J.H.Block, R.S.Pearlman Partition Coefficient Determination and estimation. Pergamon 1986 ISBN 0-08-033649-3
13. Perrin D.D. Dissociation Constants of Organic Bases in Organic Solution Butterworths London 1965
14. Kortum G. Vogel W. Andrussow K. Dissociation Constants of Organic Acids in Aqueous Solution Butterworths 1961 (Reprint of
Pure and Applied Chemistry Vol1 No. 2-3 1961
15. Dissociation Constants of Inorganic Acids Butterworths 1969 ISBN 408-70015-7 (Reprint of Pure and Applied Chemistry Vol 20
No.2 1969)
16. Sirius Analytical Instruments Ltd STAN Sirius Technical Application notes Volume 1 1994
17. Sirius Analytical Instruments Ltd Applications and Theory Guide to pH-Metric pKa and logP determination 1993
18. Christopher A. Lipinski, Franco Lombardo, Beryl W. Dominy, Paul J. Feeney "Experimental and computational approaches to
estimate solubility and permeability in drug discovery and development settings", Adv. Drug Delivery Rev., 1997, 23(1-3), 3-25:
19. C.M.Tice Pest Management Science 2001,57,3-16. "Selecting the right compounds for screening: Does Lipinski's rule of 5 for
pharmaceuticals apply to agrochemicals?
20. G.G.Briggs Agrevo UK. SCI Meeting Dec 1997. Uptake of Agrochemicals & Pharmaceuticals. Predicting uptake and movement of
agrochemicals from physical properties.
Trademarks: The registered trademarks GlPka (TM) and Four-Plus (TM) are used with kind
permission of Sirius Analytical Limited.
Question 1.
An Acid has a pKa of 5.2. What percentage of the acid is ionised at pH 6.0?
The % ionisation of an acid is given by the equation:
hence at pH 6;
% ionised = 100/(1+10(5.2 - 6))
= 100/(1+0.585)
= 86.3 % ionised
Question 2.
A 0.0049 M aqueous solution of Compound X precipitated out of solution at pH 6.3.
Estimate the solubility of the free base in pure water in g/litre. (pKa = 7.6, mw = 435.6)
Solubility of the free base may be calculated using the equation:
Where:
= solubility at
Question 3.
A compound has a Log P of 3.95 and a pKa of 7.3. Estimate the apparent Log P (or
Log D ) at pH 7.4.
The distribution constant may be calculated by the equations:
for acids
for bases
hence :
Log D(7.4) = 3.95 - log [ 1+10(7.3-7.4)]
Log D(7.4) =3.696
Question 4.
What strength Sodium Hydroxide is needed to form the sodium salt of a compound
with a weakly acidic group possessing a Ka of 3.7 x 1011 ?
pKa = -log Ka, hence pKa = -log (3.7x10-11) = 10.43
The compound will only be significantly ionised above its pKa, ideally > 2 units above.
Since Sodium Hydroxide is 100% dissociated, we can calculate the strength of NaOH to
get pH > pKa
pH
%ionised
M NaOH Solution
11
78%
0.001M
11.5
92%
0.003M
12
97%
0.01M
12.5
99%
0.03M
Question 6.
Weak acids with pKa's less than 16 will not be detectable as acids at all since the [H+]
they produce will be less than that produced by the autolysis of water. Similarly strong
acids are completely ionised in water and so appear to be the same strength. Suggest
ways in which (i) a weak acid (or strong base) and (ii) a strong acid (or weak base)
could be measured
Non Aqueous measurements can extend the range of pKa measurements:
For a weak acid must provide a stronger base as a solvent than water
For a strong acid must provide a weaker base (stronger acid) as a solvent than water.
Measurements are subject to large errors and involve lengthy and careful calibration.
Example: Urea pKa=0.1 (weak base) determined in Acetic Acid
Question 5.
Consider the following Compounds:
Compound
Type
pKa
Toluene-4-sulphonic acid
Acid
-1.3
Benzoic Acid
Acid
4.2
Thiopental
Acid
7.6
Codeine
Base
8.2
Atropine
Base
10
(c) At pH 7.3 Codeine is 11% in the molecular form, whereas Thiopental is 67% nonionised, and so will be absorbed the best. All the other compounds are too highly ionised
to penetrate.
(d) Re-adsorption of substances in the urine by the tubules in the kidneys will be greatest
for un-ionised molecules. Hence the weak acid Thiopental will be re-adsorbed the most
since it is non-ionised at pH4.2. Benzoic acid is only half-ionised, and all the rest are
ionised.