CS-1420
October, 1985
PARTITION COEFFICIENT (_n-OCTANOL/WATER)
GENERATOR COLUMN METHOD
OFFICE OF TOXIC SUBSTANCES
OFFICE OF PESTICIDES AND TOXIC SUBSTANCES
U.S. ENVIRONMENTAL PROTECTION AGENCY
WASHINGTON, DC 20460

-------
CS-1420 (October, 1985)
TABLE OF CONTENTS
Page
I.	NEED FOR THE TEST......	 1
II.	SCIENTIFIC ASPECTS	 2
A.	Rationale For the Test Method	 2
B.	Other Methods For Determining Kow	 3
1.	The Conventional Shake-Flask
Method	 3
2.	Reverse-Phase High-Pressure Liquid
Chromatography as a Method of Estimating Kow.... 4
3.	Estimation from Water Solubility	 6
4.	Estimation Using the Fragment Constant Method... 8
5.	Estimation Using Thin-Layer Chromatography	10
C.	Rationale for the Test Conditions	10
1.	Special Laboratory Equipment	10
2.	Temperature Control	11
3.	Purity of Octanol, Water and Other Solvents	11
D.	Test Data and Reporting	12
1.	Test Report	12
2.	Analytical Procedures					13
III.	REFERENCES 	14

-------
CS-1420 (October, 1985)
I. NEED FOR THE TEST
The octanol/water partition coefficient (Kow) is one of the
most frequently measured and most widely used parameters in
assessing the environmental fate of organic chemicals. Its
importance is due primarily to the fact that Kow is used to
predict the general lipophilicity or hydrophobicity of a
chemical. Of greater significance on the quantitative level,
however, is the fact that KQW has been found to correlate with
the water solubility, the soil/sediment sorption coefficient and
the bioconcentration factor.
Although all of the above mentioned properties are
important in predicting the fate and residence time of a chemical
in the environment, the bioconcentration factor (BCF) carries
with it the greatest consequences. This is because the BCF is
used to predict the potential for a chemical to bioconcentrate in
living tissue. When applied to the aquatic environment, the BCF
is indicative of the potential for a chemical to bioconcentrate
in the tissue of fish and other lower aquatic organisms. In
terms of the Kow, chemicals with a value less than 10 will not
bioconcentrate while those with a value greater than 104 will.
Thus, although a chemical may be present in a stream or lake at
subtoxic concentrations, if its Kow is greater than 104 it could
bioconcentrate and accumulate to levels that may be toxic to not
only the organism itself, but also to the consumers of that
organism, not the least important of which are Homo sapiens.
1

-------
CS-1420 (October, 1985)
The direct determination of Kow, using the conventional
shake-flask method, CS-1400 (EPA, 1983), is subject to numerous
experimental difficulties. These include the formation of
colloidal dispersions (emulsions) during the shaking step,
incomplete separation of the octanol and water phases, difficulty
in analyzing very hydrophobic chemicals in the water phase, the
adsorption of the solute onto the surfaces of transfer vessels,
and the loss of volatile solute into the atmosphere. In
addition, the method is time consuming.
II. SCIENTIFIC ASPECTS
A. Rationale for the Test Method
This test method uses a chromatographic technique for
determining the octanol/water partition coefficient. The method,
called the generator column method, was developed and validated
by Wasik et al (1981) and Tewari et al (1982); using this method
KQW is calculated Kow as the ratio of the molar concentration of
a 1.0% (w/w) solution of the test substance in octanol to the
molar concentration of the test substance in water, at
equilibrium. The advantages of the generator column method
include: (1) all the difficulties of the shake-flask method are
avoided; (2) equilibrium is achieved rapidly; (3) easy to carry
out and gives precise and accurate results (±3%), especially for
very hydrophobic chemicals and (4) is applicable to a wide range
2

-------
CS-1420 (October, 1985)
of chemicals with different functional groups and over a wide
range of log Kow (1 to >6).
Wasik et al (1981) and Tewari et al. (1982) have also
provided a thermodynamic basis for their method which was
validated on 62 organic test substances falling into seven
general chemical classes. Their validation studies showed
excellent agreement between the values of KQW obtained using the
generator column method and those obtained using the standard
shake flask method. Their results established that the data
obtained using the generator column are precise and accurate to
±3% and that their method is the best currently available method
for determining Kow.
B. Other Methods for Determining
1. The Conventional Shake Flask Method
The conventional method for determining the octanol/water
coefficient involves shaking a solute with two immiscible
solvents and then measuring the solute concentration in the two
solvents after equilibration (Leo, et al. 1971). The
distribution of the solute between the two solvents is a direct
consequence of the thermodynamic requirements for equilibrium
that apply only to dilute solutions. The resulting ratio of the
two solute concentrations is the partition coefficient which is
constant at a given temperature. Numerous researchers have used
this method and published their results (Fujita et al. 1964;
3

-------
CS-1420 (October, 1985)
Hansch and Anderson 1967; Leo et al. 1971; Chiou et al. 1977).
Both EPA (1983) and OECD (1981) specify the use of this method in
their protocols (see CG-1400). Although there is no ASTM
Standard Test Method for Kow, the shake-flask method is the
"standard method" used in industry when Kow is to be determined.
As noted earlier, the shake flask method is not without
problems, the most important of which are the formation of
emulsions, surface adsorption of the solute, and the time
consuming nature of the test procedure. However, with suitable
care and patience these problems are minimized and basic
experimental data are obtained that can be correlated with other
important environmental fate parameters.
2. Reverse-Phase High-Pressure Liquid Chromatography
as a Method of Estimating
A rapid method based on reverse-phase high-pressure liquid
chromatography has been developed by Veith (Veith et al. 1979) to
estimate the octanol/water partition coefficient of organic
chemicals. This method has been incorporated into test guideline
CG-1410: Partition Coefficient (jv-Octanol/Water) Estimation by
Liquid Chromatography (EPA, 1983). A summary of the method
follows. Using the solvent mixture water/methanol (15/85 v/v) as
the elutant, the log of the retention time [log (tR)] of organic
chemicals on a permanently bonded (C-18) reverse-phase high-
4

-------
CS-1420 (October, 1985)
pressure liquid chromatographic system has been found to be
linearly related to log KQW. This relationship has been
expressed by the equation
log KQW = A log (tR) - B,	(1)
where A and B are constants determined from the experimental data
for some organic chemicals. Using a mixture of the chemicals
benzene, bromobenzene, biphenyl,	-DDE [2,2-bis(p-chloro-
phenyl) -1,1-dichloroethylene] and 2,4,5,2•,51-pentachloro-
biphenyl, A and B were found to be 5.106 and 1.258, respectively,
with a coefficient of determination of 0.975. It must be
emphasized that this correlation is limited with respect to being
representative of the organic chemicals encountered. This
calibration mixture was selected largely on the basis of the log
Kow values reported in the literature, and the correlation is
linear over five orders of magnitude of Kow. To determine the
accuracy of this method of estimating log Kow by comparision with
data reported in the literature, Veith and coworkers measured the
retention time of 18 chemicals, and the standards and log Kow
values were calculated from the regression equation (1). More
recently Garst and Wilson (1984) have used the HPLC method to
determine the partition coefficient for 66 structurally diverse
chemicals ranging in log Kow from 0 to near 8. Veith's results
indicated that log Kow can be estimated to within (22.8 4^20.0)
5

-------
CS-1420 (October, 1985)
percent when compared with the values reported in the literature
from measurements using other methods. The percent error was
calculated assuming the literature value is the correct log Kow;
these researchers had some reservations about this assumption.
It should be noted that some of the greatest relative errors were
observed with polar chemicals that dissociate in water (e.g.,
nr-chlorobenzoic acid, 2, 4, 5-trichloro-phenol, and
diphenylamine). This method has a definite advantage, since the
estimation of Kow can be made rapidly and relatively easily in
comparison to the determination of Kow by the conventional
method. Furthermore, Kow can be estimated for individual
chemicals in complex mixtures (e.g., solid wastes) without
knowing the specific chemical structure of each chemical.
Other researchers have developed high-pressure liquid
chromatographic methods to determine Kow (Mirrless et al. 1976;
Carlson et al. 1975; Hulshoff and Perrin 1976; McCall 1975).
However, these methods are based on a very limited number of
experiments and considerably more work is needed to develop them.
3. Estimation from Water Solubility
By definition, the partition coefficient expresses the
equilibrium ratio of an organic chemical partitioned between an
organic liquid (i.e., j^-octanol) and water. This partitioning
is, in essence, equivalent to partitioning of the organic
6

-------
CS-1420 (October, 1985)
chemical between itself and water. Thus, one would expect that a
correlation might exist between the partition coefficient (KQW)
and water solubility(s). Indeed, as shown by Mackay (1977), both
of these properties are a function of the aqueous phase activity
coefficient of the compound, and the correlation between the KQW
and S is based on the ratio of the activity coefficients in water
and octanol.
Chiou et al. (1977) studied the relationship between K ,
o w
and the water solubility, S, and found that, for 34 organic
chemicals, an excellent linear correlation was observed between
log KQW and log S that extended to more than eight orders of
magnitude in water solubility (10~^ to 10^ ppm), and six orders
of magnitude in Kow (10 to 107). From their data the following
regression equation was derived:
log KQW = 5.00 - 0.670 log S, r2 = 0.970
where S is expressed in ymol/L, and r2 is the coefficient of
determination.
To date a total of 18 different regression equations have
been derived that correlate water solubility with the octanol
water partition coefficient. This large number of equations
results from the fact that solubility varies with the functional
group of the molecule. Lyman et al. ( 1982) have summarized these
7

-------
CS-1420 (October, 1985)
equations along with the class and number of chemicals that apply
to each equation, the respective correlation coefficient, the
applicable range of S and Kow values and the temperature at which
the solubility data were obtained. Recent developments in the
correlation between S and Kow indicate that many of the above
equations can be combined into a single general relationship
(Lyman, 1984). This would greatly simplify the estimation
procedure, but introduces greater error, particularly for certain
classes of compounds.
This method has a definite advantage in that KQW can be
estimated from an experimentally determined parameter - water
solubility. Although this approach also has a cost advantage in
that two parameters are determined for the cost of one, it must
be kept in mind that the Kow is an estimated value subject to the
limitations usually associated with approximations. Thus, while
the estimation of Kow from water solubility is definitely a valid
method, the values obtained cannot be construed as being
equivalent to those obtained from the conventional shake-flask
method, the HPLC method, or the generator column method.
4. Estimation Using the Fragment Constant Method
Hansch and Leo (1979) have developed a method to estimate
Kow from empirically derived atomic or group fragment constants
8

-------
CS-1420 (October, 1985)
(f) and structural factors (F). Using these, the log Kow is
calculated using the following equation:
log Kow = Sum of fragments (f) + structural factors (F)
Of course, the critical piece of information required to apply
this method is the structure of the chemical in question—which
is not always known. However, if the structure is known then
there are a total of over 200 fragments (f and F) available that
take into account such structural factors as molecular
flexibility, unsaturation, multiple halogenation, branching, and
h-polar fragment interactions. These and the method of
calculation, with examples, are reviewed extensively by Lyman et
al. ( 1982). In addition, a large collection (about 15,000) of
both measured and calculated Kow values has been compiled by
Hansch and Leo (1979); the method is also available for use on
computer (Chou and Jurs 1979).
Estimation by the fragment constant is probably the best
initial step in determining Kow since it can be done without
experimentation and is quite reliable for a large number of
common organic chemicals. However, for some functional groups
and complex or highly substituted molecules the fragment constant
method can give erroneous, misleading results.
9

-------
CS-1420 (October, 1985)
5. Estimation Using Thin-Layer Chromatography
It has been reported that thin-layer chromatography can be
used to estimate Kow (Mirrless et al. 1976; Hulshoff and Perrin
1976). However, the generator column method is far superior to
thin-layer chromatography (TLC) because of its accuracy, i.e.,
definition of the peak, reproducibility; ease of detection in
many cases; and above all the range of applicability: the
generator column method is applicable over 5 orders of magnitude
of Kow while TLC is only applicable over 1.5 orders of magnitude
of Kow (Mirrless et al. 1976).
C. Rationale for the Test Conditions
1. Special Laboratory Equipment
For the determination of the molar concentration of the
test substance in water, several pieces of special laboratory
equipment are required. This equipment is the same as that used
in the generator column method for water solubility, CG-1510
(EPA, 1983) and includes: 1) a specially designed generator
column; 2) a constant temperature bath; 3) a high pressure liquid
chroniatograph with detector and integrator; 4) a specially
designed extractor column; 5) two high pressure rotary switching
valves; 6) specially designed collection vessels; and 7) a gas
chromatograph with detector. In addition, particular types or
brand names of column packing are specified. All of the special
10

-------
CS-1420 (October, 1985)
laboratory equipment is same as that used by Tewari et al (1982)
who developed and validated the test method. Thus, in order to
maintain the integrity of the test method the procedure should be
conducted using the special equipment as described. Any changes
in or modification to the equipment should be reported in detail.
2.	Temperature Control
From the theory of the distribution law as outlined in the
test support document for determining Kow by the conventional
shake flask method, CG-1400 (EPA, 1983), the distribution
coefficient Kow is a function of the temperature, and is a
constant at a fixed temperature. Hence, in performing the
octanol water partition coefficient measurements using the
generator column, the temperature should be controlled.
Controlling the temperature to ±0.05 °C is easy to carry out and
is inexpensive.
3.	Purity of Octanol, Water and Other Solvents
Dissolved salts and other impurities can affect the
solubility of a compound in octanol, water or any solvent. Thus,
the test guideline specifies the use of purified _rv-octanol and
reagent grade water. Trace amounts of impurities present in
jv-octanol tend to produce emulsions and must be removed. The
purified _rv-octanol may either be prepared according to the
procedure given in the guideline or purchased from Fisher
11

-------
CS-1420 (October, 1985)
Scientific. The water is prepared according to ASTM 01193-77:
Standard Specification for Water. All other solvents used in the
test method should be reagent or HPLC grade and contain no
impurities that could interfere with the determination of the
test compound.
D. Test Data and Reporting
1. Test Report
As a matter of standard laboratory practice, three
determinations of the KQW for each test substance are required
along with the mean and standard deviation. In addition, the
weights of test substance and octanol used in the preparation of
the test solution are required, as is the molar concentration of
the test solution. Other ancillary data required for a complete
assessment of the results include: test temperature, and if
the HPLC method is employed, the method used to determine the
sample loop volume and its average and standard deviation from
three runs, and the response factor and its mean and standard
deviation. For the GC method, data on the calibration procedure,
the resulting regression equation, and results from the replicate
GC runs should be reported in order that the results can be
vai idated.
12

-------
CS-1420 (October, 1985)
2. Analytical Procedures
In those cases where a separate analytical procedure is
used to determine the concentration of the test substance in n-
octanol a description of the analytical method should be given in
the test report. This will enable the accuracy and precision of
the gravimetric method for preparing the test substance in
n-octanol to be assessed. If any changes are made or problems
encountered in the test method, they should be reported so that
any necessary revisions can be incorporated in the test method.
13

-------
CS-1420 (October, 1985)
III. REFERENCES
ASTM. 1978. Annual Book of ASTM Standards, American Society for
Testing and Materials, Philadelphia, PA., Part 31, Method D 1193.
Carlson RM, Carlson RE, Kopperman HL. 1975. Determination of
partition coefficients by liquid chromatography. J Chromatogr
107:219.
Chou JT, Jurs PC. 1979. Computer assisted computation of
partition coefficients from molecular structure using fragment
constants. J Chem Inf Comput Sci 19:172.
Chiou CT, Freed VH, Schmedding DW,
coefficient and bioaccumulation of
Environ Sci Technol 11:475.
Kohnert RL. 1977. Partition
selected organic chemicals.
EPA. 1983. U.S. Environmental Protection Agency. Office of
Toxic Substances. Chemical Fate Test Guidelines. PB 83-257717.
Fujita T, Iwasa J, Hansch C. 1964. A new substituent constant,
derived from partition coefficients. J An Chem Soc 86:5175.
Garst JE, Wilson WC. 1985. An Accurate, Wide Range, Automated
HPLC Method for Determination of Octanol:Water Partition
Coefficients. To be published.
Hansch C, Anderson SM. 1967. The effect of intramolecular
hydrophobic bonding on partition coefficients. J Org Chem
23:2583.
Hansch C, Leo A. 1979. Substituent constants for correlation
analysis in chemistry and biology. New York: J. Wiley & Sons.
Hulshoff A, Perrin JH. 1976. A comparison of the determination
of partition coefficients of 1,4-benzodiazepines by high-
performance liquid chromatography and thin-layer
chromatography. J Chromatogr 129:263.
Leo A, Hansch C, Elkins D. 1971. Partition coefficients and
their uses. Chem Rev 71:525.
Lyman WJ. 1984. Personal communication.
Lyman WJ, Reehl WF, Rosenblatt DH. 1982. Handbook of Chemical
Property Estimation Methods. Environmental Behavior of Organic
Compounds. McGraw Hill Book Company. New York.
14

-------
CS-1420 (October, 1985)
Mackay D. 1977. Environ Sci Technol 11:1219.
McCall JM. 1975. Liquid-liquid partition coefficients by high-
pressure liquid chromatography. J Med Chem 18:549.
Mirrless MS, Moulton SJ, Murphy CT, Taylor PJ. 1976. Direct
measurement of octanol-water partition coefficients by high-
pressure liquid chromatography. J Med Chem 19:615.
OECD. 1981. Organization for Economic Cooperation and
Development (OECD). Guidelines for Testing Chemicals: No. 107-
Partition Coefficient (jv-Octanol/Water). Director of
Information, OECD; 2 Rue Andre-Pascal, 75775 PARIS CEDEX 16,
France.
Veith GD, Austin NM, Morris RT. (1979) A rapid method for
estimating log P for organic chemicals. Water Res 13:43.
Wasik SP, Tewari YB, Miller MM, Martire DE. 1981. Octanol/Water
partition coefficient and aqueous solubilities of organic
compounds. National Bureau of Standard (NBS), U.S. Department of
Commerce, Washington, DC. NBS Report NBSIR 81-2406.
Tewari YB, Miller MM, Wasik SP, Martire DE. 1982. Aqueous
Solubility and Octanol/Water Partition Coefficient of Organic
Compounds at 25.0° C. J Chem Eng Data 27:451-454.
15

-------