1

ENVIRONMENTAL

RESEARCH BRIEF

J. Jackson Ellington and Terry L. Floyd1

E AP

United States

Environmental Protection

Agency

National Exposure

Research Laboratory

Research Triangle Park, NC 27711

Research and Development EPA/600/S-96/005 August 1995

Abstract

A method for measuring the octanol/water partition coefficients

(Kows) is described. The “slow-stirring” method minimizes the

formation of emulsions and enables the laboratory measurement of chemicals with a log Kow greater than 8. The techniques and equipment are described.

Introduction

The octanol/water partition coefficient (Kow) is the physicochemical parameter most commonly measured in the laboratory and used to characterize quantitatively the hydrophobic

nature of organic compounds. The currently accepted EPA

method for measuring Kow is the flask-shaking procedure described 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 Kows from 101 to 106. Significant

variability appears, however, among measurements for compounds having Kows 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 Kow measurement 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 EmporeTM

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.

Measuring Octanol/Water Partition

Coefficients by the “Slow-Stirring” Method

Definitions and Units

The octanol/water partition coefficient (Kow) is defined as the

ratio of the equilibrium concentration of a chemical in the

octanol phase to that in the aqueous phase. The concentrations are expressed in terms of the mass or moles of chemical

per unit volume of liquid.

Kow = Coctanol/Cwater (1)

Log Kow is the value most often reported in the literature. Log

Kow 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 Kow is less than 3. Larger vessels (6-

or 9-liters) are needed if the log Kow 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

1 Ecosystems Research Division, National Exposure Research Laboratory, U.S.

Environmental Protection Agency, Athens GA 30605-2700
2

Figure 1. Equilibration vessel equipped with Teflon stopcock.

Octanol

Water

Stopcock

Cover

1/4" Spacer

Stirring Bar

Stirrer

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 Materials 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 environmental 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 confirmed by comparison of their ultraviolet, infrared or other

appropriate spectra to library reference spectra. Nonvolatile

test compounds are analyzed by high performance liquid chromatography/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 chromatography (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 properties of the solute. Commonly used detectors are flame ionization, electron capture and nitrogen-phosphorus for GCs.

Ultraviolet and fluorescence detectors are used for HPLCs.

The concentration of a >106 Kow solute in the water layer

extract will be in the low parts per billion. Therefore, analysis of

these extracts will require efficient chromatographic columns
3

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 deviation. The minimum acceptable detection level of the chromatographic 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 concentration 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 Kow, 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 Kow

is within the linear range of the detector. The calibration standards are not used to determine the absolute test solute concentration in the octanol and water phases.

An error in the measured Kow 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 distinguished from the test solute. If nonchromatographic methods

are used, the error introduced by the water-soluble contaminants can be minimized by subjecting the test solute-containing octanol phase to consecutive equilibrations with water. The

equilibrations should be repeated with new water phases until

the Kow is unchanged for two consecutive equilibrations.

Calculation of Kow

It is not necessary to determine the absolute concentration of

the test solute in either phase. The Kow can be calculated

directly by dividing the linear detector response to equal volumes of the octanol and water phases, respectively. In theory,

the Kow of a test solute analyzed by GC can be calculated by

dividing the solute detector response to a 1-μl injection of the

octanol layer by the solute detector response to a 1-μl injection

of the water layer. However, for solutes with log Kow greater

than 1, the concentrations in the two phases differ by orders of

magnitude and usually exceed the dynamic range of the detector. 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 technique. 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 Kow for test solutes with Kow greater than 1 is

then calculated by the following equation:

(Detector response)water ÷ Concentration Factor

(If unequal volumes of the final octanol dilution or water concentration are analyzed, the unequal volumes must be taken

into consideration when determining the detector response.)

Procedure

At the beginning of the measurement procedure, the appropriate 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 temperature 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 contents 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 coworkers (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 Kow is less than 3, samples are taken on

successive days (after the first 24 hours) until the Kow becomes

constant. The sampling regime is at 2 to 3 day intervals for

compounds with log Kow 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 Kow if an

estimate of the Kow 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 calculated by substituting the estimated Kow and the starting Coctanol

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 withdrawing 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 methylene chloride or use of EmporeTM extraction disks. The extraction solvents are usually concentrated to a final volume of 1 ml.

K = (Detector response) x Dilution Factor (2) ow octanol
4

The dilution and concentration factors are calculated and used

in equation 2 to calculate the Kow.

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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BULK RATE

POSTAGE & FEES PAID

EPA

PERMIT No. G-35

United States

Environmental Protection Agency

National Risk Management Research Laboratory (G-72)

Cincinnati, OH 45268

Official Business

Penalty for Private Use

$300

EPA/600/S-96/005