United States
Environmental Protection
Agency
National Exposure
Research Laboratory
Research Triangle Park, NC 27711
Research and Development
EPA/600/S-96/005 August 1995
ENVIRONMENTAL
RESEARCH BRIEF
Measuring Octanol/Water Partition
Coefficients by the "Slow-Stirring" Method
J. Jackson Ellington and Terry L. Floyd1
Abstract
A method for measuring the octanol/water partition coefficients
(K^s) is described. The "slow-stirring" method minimizes the
formation of emulsions and enables the laboratory measure-
ment of chemicals with a log K^ greater than 8. The tech-
niques and equipment are described.
Introduction
The octanol/water partition coefficient (K^) is the physico-
chemical parameter most commonly measured in the labora-
tory and used to characterize quantitatively the hydrophobic
nature of organic compounds. The currently accepted EPA
method for measuring K^ is the flask-shaking procedure de-
scribed in the Federal Register [40 CFR Part 796, 50 (188)
Friday, September 27, 1985, pp 39252-39255]. The EPA method
is applicable for the range of K^s from 101 to 106. Significant
variability appears, however, among measurements for com-
pounds having K^s greater than 106. This variability has been
attributed to emulsion formation during the shaking step that
can cause wide disparities in the concentrations of the solute in
the water phases. The slow-stirring method (1-3) avoids the
formation of emulsions, and the upper range of K^ measure-
ment is limited only by the ability to determine the water phase
concentration accurately. In this document, the slow-stirring
methodology is described in detail and the use of Empore™
disks to extract hydrophobic chemicals from water volumes up
to 8 liters is illustrated as a replacement for extraction of the
test chemicals with solvents.
Definitions and Units
The octanol/water partition coefficient (K^) is defined as the
ratio of the equilibrium concentration of a chemical in the
octanol phase to that in the aqueous phase. The concentra-
tions are expressed in terms of the mass or moles of chemical
per unit volume of liquid.
K —
/O
tanor water
(1)
1 Ecosystems Research Division, National Exposure Research Laboratory, U.S.
Environmental Protection Agency, Athens GA 30605-2700
Log K^ is the value most often reported in the literature. Log
K^ is used in the calculation of bioaccumulation factors, and
for estimating the organic carbon-normalized sediment/water
partition coefficient (Koc) and for calculating water solubility and
other properties using property reactivity correlations.
Experimental
Equipment
An Erlenmeyer flask or cylindrical jar of appropriate size to
permit withdrawal of sufficient sample for precise measurement
is modified and used for the equilibration of the test solute
between octanol and water. One-liter Erlenmeyer flasks are
used if the expected log K^ is less than 3. Larger vessels (6-
or 9-liters) are needed if the log K^ is greater than 3. Figure 1
shows a cylindrical jar that has been modified for use with this
method by installing a 2-mm straight bore stopcock with a
Teflon plug through the container side wall at a point near the
bottom. The stopcock is inserted near the bottom so that the
maximum volume of water layer can be withdrawn. The 2-mm
glass tube on the side of the Teflon plug away from the flask is
turned down to form a tap for withdrawing the aqueous layer. A
magnetic stirrer with stir bar is needed for each flask. The
stirrers should be of appropriate size with the speed adjusted
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Cover
1/4" Spacer
Stirring Bar
Octanol
Water
Stopcock
Stirrer
Figure 1. Equilibration vessel equipped with Teflon stopcock.
such that a < 2-cm deep vortex is generated at the octanol/
water interface.
Temperature Control
The temperature of the measurement vessel and contained
liquids should be maintained at 25(+1)°C. The vessel can be
either placed in a temperature-controlled room or the vessel
can be fabricated such that water of a constant 25°C flows
continuously across the exterior wall of the vessel.
Chemicals
It is important that the octanol used be of 99+% purity. The
water used should be American Society for Testing and Materi-
als Type I, produced by laboratory water purification systems.
Solvents used in the preparation of calibration solutions and for
extraction of the test chemical from the water layer must be of
the highest quality commonly used for trace analysis of envi-
ronmental samples.
A check on the purity of the octanol and other solvents used
and the identification and measurement of volatile impurities
should be made by gas chromatography/mass spectrometry
(GC/MS). The identities of nonvolatile compounds are con-
firmed by comparison of their ultraviolet, infrared or other
appropriate spectra to library reference spectra. Nonvolatile
test compounds are analyzed by high performance liquid chro-
matography/mass spectrometry (HPLC/MS) when available.
Octanol/water volume ratio
At least 20 ml of octanol should be used for each liter of water
(1:50).
Chemical concentration in octanol
The solute should be <0.01 M in octanol and not exceed 50%
of the solute solubility in either phase.
Analysis
Analysis systems based on chromatography, such as gas chro-
matography (GC) and HPLC, are preferred for test solute
measurement in the octanol and water phases. The choice of
the detector is determined by the physical and chemical prop-
erties of the solute. Commonly used detectors are flame ion-
ization, electron capture and nitrogen-phosphorus for GCs.
Ultraviolet and fluorescence detectors are used for HPLCs.
The concentration of a >106 K^ solute in the water layer
extract will be in the low parts per billion. Therefore, analysis of
these extracts will require efficient chromatographic columns
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and sensitive detectors capable of a reproducible response to
nanogram/microliter injections of the solute. GCs equipped
with autosamplers, and LCs equipped with loop injectors yield
the highest precision injections and should be used when
available. Detector response of triplicate manual injections
should not deviate by more than +3% relative standard devia-
tion. The minimum acceptable detection level of the chromato-
graphic analysis is established by triplicate injections of serial
dilutions of the stock solution of the solute. The minimum
acceptable detection level is defined as the minimum concen-
tration or mass flux of analyte (in a solvent) that gives a
detector signal that can be discerned from the noise with
reasonable certainty, generally recognized to be twice the
peak-to-peak noise (4). As explained in more detail in the
section on calculation of K^, it is not necessary to determine
the absolute concentration of the solute in the two phases.
Analysis of calibration standards is performed only to ensure
that the amount of solute analyzed for determination of the K^
is within the linear range of the detector. The calibration stan-
dards are not used to determine the absolute test solute con-
centration in the octanol and water phases.
An error in the measured K^ can occur if a nonchromatographic
method, such as radiolabeling or UV absorption spectroscopy,
is used to determine response of the test solute in the two
layers. These errors result from the presence of traces of
more-water-soluble contaminants that are not analytically dis-
tinguished from the test solute. If nonchromatographic methods
are used, the error introduced by the water-soluble contami-
nants can be minimized by subjecting the test solute-contain-
ing octanol phase to consecutive equilibrations with water. The
equilibrations should be repeated with new water phases until
the K^ is unchanged for two consecutive equilibrations.
Calculation ofK,
ow
It is not necessary to determine the absolute concentration of
the test solute in either phase. The K^ can be calculated
directly by dividing the linear detector response to equal vol-
umes of the octanol and water phases, respectively. In theory,
the K^ of a test solute analyzed by GC can be calculated by
dividing the solute detector response to a 1-^.1 injection of the
octanol layer by the solute detector response to a 1-^.1 injection
of the water layer. However, for solutes with log K^ greater
than 1, the concentrations in the two phases differ by orders of
magnitude and usually exceed the dynamic range of the detec-
tor. To minimize this difference in concentrations and to obtain
samples from the two phases that will give approximately the
same detector response when equal volumes are analyzed, an
aliquot of the octanol layer is diluted and the test solute in the
water layer is concentrated by an appropriate extraction tech-
nique. Extraction of the test solute with a solvent is used for
water samples <1 L; solid sorbent extraction is used for water
samples >1 L. The K^ for test solutes with K^ greater than 1 is
then calculated by the following equation:
Kow = (Detector response)octanol x Dilution Factor
(Detector response)water •*• Concentration Factor
(2)
(If unequal volumes of the final octanol dilution or water con-
centration are analyzed, the unequal volumes must be taken
into consideration when determining the detector response.)
Procedure
At the beginning of the measurement procedure, the appropri-
ate volume of Type I water is brought into the vessel together
with a Teflon-coated magnetic stirring bar. One-liter water
samples are stirred for 1 hour to equilibrate the temperature to
25°C; 5L and larger samples are stirred overnight. The surface
of the stirrer warms during the equilibration time; to minimize
heat transfer, three to four 1-inch-square by 1/4-inch-thick
pieces of wood are placed between the flask and the surface of
the stirrer. When the water has reached an equilibrium tem-
perature of 25°C, the stirring is stopped and pure octanol equal
in volume to 25% (10 ml for 1L and 25 ml for 6L flasks) of the
final volume is carefully added without mixing the phases. The
addition of octanol is accomplished by tilting the flask and
gently pouring the octanol down the interior surface of the
vessel so that the octanol flows slowly onto the surface of the
water.
Stirring is resumed and the stirring rate is adjusted until a
vortex of approximately 2 cm is formed at the octanol/water
interface. Stirring is continued overnight to achieve mutual
saturation of the phases. The stirring is again stopped and the
remaining 75% volume of octanol, containing the test solute of
interest, is carefully layered onto the surface of the flask con-
tents as described previously. Stirring is then resumed and is
stopped only for taking samples. The overnight prestirring of
the pure phases without test solute allows sufficient time for
mutual saturation of the two phases. If the two phases are first
contacted at the start of the test solute equilibration, the octanol
that initially migrates into the water phase can also transport
solvated solute molecules, thereby elevating the test solute
concentration in the water phase to levels higher than the final
equilibrium solute concentration in water. De Bruijn and co-
workers (1) recognized the problems of test solute equilibration
with unsaturated octanol and water phases. Mutual presaturation
of the octanol and water phases described as the preferred
method above is based on the work of Marple (3).
If the expected log K^ is less than 3, samples are taken on
successive days (after the first 24 hours) until the K^ becomes
constant. The sampling regime is at 2 to 3 day intervals for
compounds with log K^ greater than 3. Since the approximate
concentration of the solute at time zero in the octanol layer is
known, the estimated equilibrium water concentration can be
calculated from the equation (2) used to calculate K^ if an
estimate of the K^ of the test compound is also available. The
volume of water that must be withdrawn from the flask and
extracted is determined based upon the calculated equilibrium
water concentration and the analysis method sensitivity. The
volume of the water sample required for the analysis is calcu-
lated by substituting the estimated K^ and the starting Coctano|
into Equation 1 and solving for Cwater. The water sample taken
should be of sufficient volume that the final concentration of the
solute in the extraction solvent will be sufficient for analysis by
the detection systems used.
The water layer is sampled by stopping the stirring and with-
drawing a sample of water from the Teflon tap after discarding
the first 25 ml. The water is collected in a volumetric flask. A 1-
ml sample of the octanol is obtained by placing the tip of a 1-ml
pipette just below the surface of the octanol and applying
gentle suction. The octanol is diluted with the final solvent used
in the extraction of the test solute from the water layer. The
octanol is diluted until a detector response approximately equal
to the response of the water layer extract is obtained.
Nonpolar solutes are concentrated from the water layer by
conventional methods such as solvent extraction using methyl-
ene chloride or use of Empore™ extraction disks. The extrac-
tion solvents are usually concentrated to a final volume of 1 ml.
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The dilution and concentration factors are calculated and used
in equation 2 to calculate the K^.
References
1. De Bruijn, J., F. Busser, W. Seiner and J. Hermens.
1989. Determination of octanol/water partition coefficients
for hydrophobic organic chemicals with the "slow-stirring"
method, Environ. Toxicol. and Chem. 8:449512.
2. Brooke, D.N., A.J. Dobbs and N. Williams. 1986.
Octanol:water partition coefficients (P): measurement,
estimation, and interpretation, particularly for chemicals
with P>105, Ecotoxicol. Environ. Safety 11:251260.
3. Marple, L, B. Berridge, and L. Throop. 1986.
Measurement of the water-octanol partition coefficient of
2,3,7,8-tetrachlorodibenzo-p-dioxin, Environ. Sci. Technol.
20:397399.
4. Foley, J. P., and J. G. Dorsey. 1984. Clarification of the
limit of detection in chromatography. Chromatographia
18:503511.
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United States
Environmental Protection Agency
National Risk Management Research Laboratory (G-72)
Cincinnati, OH 45268
Official Business
Penalty for Private Use
$300
BULK RATE
POSTAGE & FEES PAID
EPA
PERMIT No. G-35
EPA/600/S-96/005
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