United States Prevention, Pesticides EPA712-C-96-040
Environmental Protection and Toxic Substances August 1996
Agency (7101)
&EPA Product Properties
Test Guidelines
OPPTS 830.7570
Partition Coefficient
(n-Octanol/Water),
Estimation By Liquid
Chromatography
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INTRODUCTION
This guideline is one of a series of test guidelines that have been
developed by the Office of Prevention, Pesticides and Toxic Substances,
United States Environmental Protection Agency for use in the testing of
pesticides and toxic substances, and the development of test data that must
be submitted to the Agency for review under Federal regulations.
The Office of Prevention, Pesticides and Toxic Substances (OPPTS)
has developed this guideline through a process of harmonization that
blended the testing guidance and requirements that existed in the Office
of Pollution Prevention and Toxics (OPPT) and appeared in Title 40,
Chapter I, Subchapter R of the Code of Federal Regulations (CFR), the
Office of Pesticide Programs (OPP) which appeared in publications of the
National Technical Information Service (NTIS) and the guidelines pub-
lished by the Organization for Economic Cooperation and Development
(OECD).
The purpose of harmonizing these guidelines into a single set of
OPPTS guidelines is to minimize variations among the testing procedures
that must be performed to meet the data requirements of the U. S. Environ-
mental Protection Agency under the Toxic Substances Control Act (15
U.S.C. 2601) and the Federal Insecticide, Fungicide and Rodenticide Act
(7U.S.C. I36,etseq.).
Final Guideline Release: This document is available from the U.S.
Government Printing Office, Washington, DC 20402 on The Federal Bul-
letin Board. By modem dial 202-512-1387, telnet and ftp:
fedbbs.access.gpo.gov (IP 162.140.64.19), internet: http://
fedbbs.access.gpo.gov, or call 202-512-0132 for disks or paper copies.
This guideline is available in ASCII and PDF (portable document format)
from the EPA Public Access Gopher (gopher.epa.gov) under the heading
"Environmental Test Methods and Guidelines."
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OPPTS 830.7570 Partition coefficient (n-octanol/water), estimation
by liquid chromatography.
(a) Scope—(1) Applicability. This guideline is intended to meet test-
ing requirements of both the Federal Insecticide, Fungicide, and
Rodenticide Act (FIFRA) (7 U.S.C. 136, et seq.) and the Toxic Substances
Control Act (TSCA) (15 U.S.C. 2601).
(2) Background. The source materials used in developing this har-
monized OPPTS test guideline are the OPPT guideline under 40 CFR
796.1570 Partition Coefficient (n-Octanol/water)—Estimation by liquid
chromatography, OPP guideline 63-11 OctanoI/water partition coefficient
(Pesticide Assessment Guidelines, Subdivision D: Product Chemistry, EPA
Report 540/9-82-018, October 1982), and OECD guideline 117 Partition
Coefficient (n-octanol/water), High Performance Liquid Chromatography
(HPLC) Method.
(b) Introduction. (1) The partition coefficient (P) is defined as the
ratio of the equilibrium concentrations of a dissolved substance in a two-
phase system consisting of two largely immiscible solvents. The partition
coefficient being the quotient of two concentrations, is dimensionless and
is usually given in the form of its logarithm to base ten. In the case of
w-octanol and water,
"ow ~~ L-n-octanol/L-water
(2) Pow is a key parameter in studies of the environmental fate of
chemical substances. A highly-significant relationship between the Pow of
substances and their bioaccumulation in fish has been shown. It has also
been shown that Pow is a useful parameter in the prediction of adsorption
on soil and sediments and for establishing quantitative structure-activity
relationships for a wide range of biological effects.
(3) POW values in the range log Pow between -2 and 4 can be experi-
mentally determined by the shake-flask method (OPPTS 830.7550). Pow
values in the range log Pow between 0 and 6 can be estimated using high
performance liquid chromatography (HPLC) under paragraphs (f)(l)
through (f)(4) of this guideline. The HPLC method requires a preliminary
estimation of Pow, generally done through calculation. Calculation methods
are briefly discussed under paragraph (d)(l)(i) of this guideline.
(c) Principle of the method. (l)(i) HPLC is performed on analytical
columns packed with a commercially available solid phase containing long
hydrocarbon chains (e.g. Cg, Cig) chemically bound onto silica. Chemicals
injected onto such a column move along it by partitioning between the
mobile solvent phase and the hydrocarbon stationary phase. The chemicals
are retained in proportion to their hydrocarbon-water partition coefficient,
with water-soluble chemicals eluted first and oil-soluble chemicals last.
This enables the relationship between the retention time on a reverse-phase
column and the w-octanol/water partition coefficient to be established. The
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partition coefficient is deduced from the capacity factor k, given by the
expression
k = (tR - t0)/to
where tR is the retention time of the test substance, and t0 is the dead-
time, i.e. the average time a solvent molecule needs to pass the column.
Quantitative analytical methods are not required and only the determina-
tion of retention times is necessary. If standard reference compounds are
available and standard experimental conditions are used, the HLPC method
can be performed faster than shake-flask method under OPPTS 830.7550.
(ii) The HPLC method enables partition coefficients to be estimated
in the log Pow range between 0 and 6. The method is not applicable to
strong acids and bases, metal complexes, substances which react with the
eluent, or surface-active agents. Measurements should be made on
ionizable substances in their nonionized form (free acid or free base) only
by using an appropriate buffer with a pH below the pK for a free acid
or above the pK for a free base (e.g. phosphoric acid for pH = 2 and
0.01-0.02 M phosphate buffer for pH = 7.5).
(iii) The HPLC method is less sensitive to the presence of impurities
in the test substance than the shake-flask method. Nevertheless, in some
cases impurities can make the interpretation of the results difficult due
to uncertainty in peak assignments. For mixtures which result in an unre-
solved band, upper and lower limits of log P should be stated (see para-
graph (f)(3) of this guideline).
(2) Information on the test substance. The structural formula and
the dissociation constant should be known before using the method. Infor-
mation on solubility and hydrolysis characteristics is useful.
(3) Repeatability and accuracy. In order to increase the confidence
in the measurement, duplicate determinations must be made. The values
of log POW derived from the different measurements should fall within a
range of ±0.1 log units. An interlaboratory comparison test has shown
that with the HPLC method log Pow values can be obtained to within ±0.5
units of the shake-flask values (under paragraph (f)(5) of this guideline).
Other comparisons can be found under paragraphs (f)(3), (f)(4), (f)(6),
(f)(7), and (f)(8) of this guideline. Correlation graphs based on structurally
related reference compounds give the most accurate results (under para-
graph (f)(9) of this guideline)
(4) Reference compounds, (i) In order to correlate the measured ca-
pacity factor k of a compound with its Pow, a calibration graph using at
least 6 points has to be established. It is up to the user to select the appro-
priate reference compounds. It is preferable that these should be struc-
turally related to the test substance. Whenever possible, at least one ref-
erence compound should have a Pow above that of the test substance, and
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another a Pow below that of the test substance. For log Pow values below
4, the calibration can be based on data obtained by the shake flask method.
For log POW values above 4, the calibration can be based on literature
values if they correspond to calculated values.
(ii) Extensive lists of log Pow values for many groups of chemicals
are available (under paragraphs (f)(10) and (f)(ll) of this guideline). If
data on the partition coefficients of structurally related compounds are not
available, a more general calibration, established with other reference com-
pounds, may be used. Recommended reference compounds and their Pow
values are listed in the following table 1. For ionizable substances the
values given apply to the nonionized form. The values were checked for
plausibility and quality during an interlaboratory comparison test.
Table 1.—Recommended Reference Compounds
Reference substance
2-Butanone
4-Acetylpyridine
Aniline
Acetanilide
Benzyl alcohol
4-Methoxyphenol
Phenoxyacetic acid
Phenol
2 4-Dinitrophenol
Benzonitrile
Phenylacetonitrile
4-Methylbenzyl alcohol
Acetophenone
2-Nitrophenol
3-Nitrobenzoic acid
4-Chloraniline
Nitrobenzene
Cinnamic alcohol
Benzole acid
p-Cresol
c/s-Cinnamic acid
frans-Cinnamic acid
Anisole
Methyl benzoate
Benzene
3-Methylbenzoic acid
4-Chlorophenol
Trichloroethene
Atrazine
Ethyl benzoate
2.6-Dichlorobenzonitrile
log
r ow
0.3
05
0.9
1 0
1 1
1.3
1 4
1.5
1 5
1.6
1 6
1.6
1 7
1.8
1 8
1.8
1.9
1 9
1.9
1 9
2.1
2 1
2.1
2 1
2.1
24
2.4
24
26
2.6
2.6
pKa
10.26
3 12
9.92
396
7.17
347
4.15
4.19
10 17
3.89
444
427
9.1
Reference substance
3-Chlorobenzoic acid
Toluene
1-Naphthol
2 3-Dichloroaniline
Chlorobenzene
Allyl phenyl ether
Bromobenzene
Ethylbenzene
Benzophenone
4-Phenyl phenol
Thymol
1 ,4-Dichlorobenzene
Diphenylamine
Naphthalene
Phenyl benzoate
Isopropylbenzene
2,4,6-Trichlorophenol
Biphenyl
Benzyl benzoate
2 4-Dinitro-6-sec-butyl phenol
1 ,2,4-Trichlorobenzene
Dodecanoic acid
Diphenyl ether
Phenanthrene
n-Butylbenzene
Fluoranthene
Dibenzyl
2 6-Diphenylpyridine
Triphenylamine
DDT
log
r ow
2.7
27
2.7
28
28
2.9
30
3.2
32
3.2
33
3.4
34
3.6
36
3.7
3.7
40
4.0
4 1
4.2
42
4.2
45
4.6
47
4.8
49
57
6.2
pKa
3.82
9.34
9.54
079
6.0
(d) Description of the method—(1) Preliminary estimate of the
partition coefficient. The partition coefficient of the test substance is esti-
mated preferably by using a calculation method or, where appropriate, by
using the ratio of the solubilities of the test substance in the pure solvents
(under paragraph (f)(12) of this guideline).
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(i) POW calculation methods. This discussion provides a short intro-
duction to the calculation of Pow. For further information the reader is
referred to paragraphs (f)(15) and (f)(16) of this guideline. Calculated val-
ues of POW are used for:
(A) Deciding which experimental method to use: Shake flask method
for log POW between -2 and 4 and HPLC method for log Pow between
0 and 6.
(B) Selecting conditions to be used in HPLC (reference compounds,
methanol/water ratio).
(C) Checking the plausibility of values obtained through experimental
methods.
(D) Providing an estimate when experimental methods cannot be ap-
plied.
(ii) Principle of calculation methods. Calculation methods are based
on the theoretical fragmentation of the molecule into suitable substructures
for which reliable log Pow increments are known. The log Pow is obtained
by summing the fragment values and the correction terms for
intramolecular interactions. Lists of fragment constants and correction
terms are available under paragraphs (f)(15) through (f)(20) of this guide-
line; some are regularly updated (under paragraph (f)(17) of this guide-
line).
(iii) Reliability of calculated values. In general, the reliability of cal-
culation methods decreases as the complexity of the compound under study
increases. In the case of simple molecules of low molecular weight and
with one or two functional groups, a deviation of 0.1 to 0.3 log Pow units
between the results of the different fragmentation methods and the meas-
ured value can be expected. The margin of error will depend on the reli-
ability of the fragment constants used, the ability to recognize
intramolecular interactions (e.g. hydrogen bonds) and the correct use of
correction terms. In the case of ionizing compounds the charge and degree
of ionization must be taken into consideration (under paragraph (f)(24)
of this guideline).
(iv) Fujita-Hansch 7i-method. The hydrophobic substituent constant,
71, originally introduced under paragraph (f)(21) of this guideline, is de-
fined as:
TCX = log Pow(PhX) - log Pow(PhH),
where PhX is an aromatic derivative and PhH the parent compound. For
example
= log P0w(C6H5Cl) - log P0w(C6H6) = 2.84 - 2.13 = 0.71
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The 7i-method is primarily of interest for aromatic compounds. Values of
7i for a large number of substituents are available (see paragraphs (f)(18)
and (f)(19) of this guideline).
(v) Rekker method. Using the Rekker method (under paragraph
(f)(22) of this guideline) the log Pow value is calculated as:
log Pow =2_,aifi + /1(interactionterms}
i
where ai is the number of a given fragment present in the molecule and
fi is the log POW increment of the fragment. The interaction terms can
be expressed as an integral multiple of one single constant Cm (so-called
"magic constant"). The fragment constants fi and Cm have been deter-
mined from a list of 1,054 experimental Pow values of 825 compounds
using multiple regression analysis (under paragraphs (f)(20) and (f)(22)
of this guideline). The determination of the interaction terms is carried
out according to set rules (under paragraphs (f)(20), (f)(22), and (f)(23)
of this guideline).
(vi) Hansch-Leo method. Using the Hansch and Leo method (under
paragraph (f)(18) of this guideline), the log Pow value is calculated as:
where fi is a fragment constant, Fj a correction term (factor), ai and bj
the corresponding frequency of occurence. Lists of atomic and group frag-
mental values and of correction terms Fj were derived by trial and error
from experimental Pow values. The correction terms have been divided
into several different classes (under paragraphs (f)(15) and (f)(18) of this
guideline). Software packages have been developed to take into account
all the rules and correction terms (under paragraph (f)(17) of this guide-
line).
(vii) Combined method. The calculation of log Pow of complex mol-
ecules can be considerably improved, if the molecule is dissected into larg-
er substructures for which reliable log Pow values are available, either from
tables under paragraphs (f)(3) and (f)(4) of this guideline or by existing
measurements. Such fragments (e.g. heterocycles, anthraquinone,
azobenzene) can then be combined with the Hansch-7i-values or with
Rekker or Leo fragment constants.
(viii) Remarks. (A) The calculation methods are only applicable to
partly or fully ionized compounds when the necessary correction factors
are taken into account.
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(B) If the existence of intramolecular hydrogen bonds can be as-
sumed, the corresponding correction terms (approximately +0.6 to +1.0
log POW units) must be added (under paragraph (f)(15) of this guideline).
Indications on the presence of such bonds can be obtained from stereo
models or spectroscopic data.
(C) If several tautomeric forms are possible, the most likely form
should be used as the basis of the calculation.
(D) The revisions of lists of fragment constants should be followed
carefully.
(2) Apparatus. A liquid-phase chromatograph, fitted with a pulse-
free pump and a suitable detection device is required. The use of an injec-
tion valve with injection loops is recommended. The presence of polar
groups in the stationary phase may seriously impair the performance of
the HPLC column. Therefore, stationary phases should have a minimal
percentage of polar groups (see paragraph (f)(13) of this guideline). Com-
mercial microparticulate reverse-phase packings or ready-packed columns
can be used. A guard column may be positioned between the injection
system and the analytical column.
(3) Mobile phase, (i) HPLC-grade methanol and distilled water are
used to prepare the eluting solvent, which is degassed before use. Isocratic
elution should be employed. Methanol/water ratios with a minimum water
content of 25 percent should be used. Typically a 3:1 (v/v) methanol/water
mixture is satisfactory for eluting compounds with a log P of 6 within
an hour, at a flow rate of 1 mL/min. For compounds with a log P above
6 it may be necessary to shorten the elution time (and those of the ref-
erence compounds) by decreasing the polarity of the mobile phase or the
column length.
(ii) The test substance and the reference compounds should be soluble
in the mobile phase in sufficient concentration to allow their detection.
Additives may be used with the methanol/water mixture in exceptional
cases only, since they will change the properties of the column. In these
cases a separate column of the same type should be used. If methanol/
water is not appropriate, other organic solvent/water mixtures can be used,
e.g. ethanol/water and acetonitrile/water.
(iii) The pH of the eluent is critical for ionizable compounds. It
should be within the operating pH range of the column, usually between
2 and 8. Buffering is recommended. Care must be taken to avoid salt pre-
cipitation and column deterioration which occur with some organic phase/
buffer mixtures. HPLC measurements with silica-based stationary phases
above pH 8 are not advisable since the use of an alkaline mobile phase
may cause rapid deterioration in the performance of the column.
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(4) Solutes. The test and reference compounds should be the purest
available. Compounds to be used for test or calibration purposes are dis-
solved in the mobile phase if possible.
(5) Test conditions. The temperature during the measurements should
not vary by more than + 2 K.
(6) Determination of dead time to. The dead time to can be meas-
ured by using unretained organic compounds (e.g. thiourea or formamide).
It can also be derived from the retention times measured for a set of ap-
proximately seven members of a homologous series (e.g. w-alkylmethyl
ketones) (under paragraph (f)(14) of this guideline). The retention times
tR(nc + i) are plotted against tR(nc), where nc is the number of carbon atoms.
A straight line,
tR(nc + 1) = AtR(nc) + (1 ~ A)to
is obtained, where A, representing k(nc + i)/k(nc), is constant. The dead
time to is obtained from the intercept (1 - A)to and the slope A.
(7) Calibration graph. The next step is to plot a correlation graph
of log k versus log P for appropriate reference compounds with log P
values near the value expected for the test substance. In practice, from
5 to 10 reference compounds are injected simultaneously. The retention
times are determined, preferably on a recording integrator linked to the
detection system. The corresponding logarithms of the capacity factors,
log k, are calculated and plotted as a function of log P. The calibration
is performed at regular intervals, at least once daily, so that account can
be taken of possible changes in column performance.
(8) Determination of the Pow of the test substance. The test sub-
stance is injected in the smallest quantity possible. The retention time is
determined in duplicate. The partition coefficient of the test substance is
obtained by interpolation of the calculated capacity factor on the calibra-
tion graph. Extrapolation is necessary for very low and very high partition
coefficients. In these cases, special attention must be given to the con-
fidence limits of the regression line.
(e) Test report. The following should be included in the report:
(1) Test and reference substances, and their purity.
(2) Description of equipment and operating conditions: Analytical col-
umn, guard column.
(3) Mobile phase, means of detection, temperature range, pH.
(4) Elution profiles.
(5) Dead time and how it was measured.
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(6) Quantities of test and references substances introduced in the col-
umn.
(7) Retention data and literature log P values for reference compounds
used in calibration.
(8) Details on fitted regression line (log k versus log P).
(9) Preliminary estimate of the partition coefficient and the method
used, and, if a calculation method was used, its full description including
identification of the data base and detailed information on the choice of
fragments.
(10) Average retention data and interpolated log P value for the test
substance.
(f) References. The following references should be consulted for ad-
ditional background material on this test guideline.
(1) Eadsforth, C.V. and P. Moser. Chemosphere 12:1459 (1983).
(2) Eadsforth, C.V. Pesticide Science 17:311 (1986).
(3) Ellgehausen, H. et al. Pesticide Science 12:219 (1981).
(4) McDuffie, B. Chemosphere 10:73 (1981).
(5) Klein W. et al. Chemosphere 17:361 (1988).
(6) Renberg, L.O. et al. Chemosphere 9:683 (1980).
(7) Hammers, W.E. et al. Journal of Chromatography 247:1 (1982).
(8) Haky, J.E. and A.M. Young. Journal of Liquid Chromatography
7:675 (1984).
(9) Fujisawa, S. and E. Masuhara. Journal of Biomedical Materials
Research 15:787(1981).
(10) Hansch, C. and A.J. Leo. Substituent Constants for Correlation
Analysis in Chemistry and Biology, Wiley, New York (1979).
(11) Pomona College Medical Chemistry Project, C. Hansch, Chair-
man; A.J. Leo, Director. Log P and Parameter Database: A tool for the
quantitative prediction of bioactivity. Available from Pomona College,
Claremont, CA 91711 (1982).
(12) Jubermann, O. Verteilen und Extrahieren, in Methoden der
Organischen Chemie (Houben Weyl), Band I/I, Allgemeine
laboratoriumpraxis (edited by E. Miiller), pp. 223-239, Georg Thieme
Verlag, Stuttgart (1958).
8
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(13) Rekker, R.F. and H.M. de Kort, European Journal of Medicinal
Chemistry 14:479 (1979).
(14) Berendsen, G.E. et al. Journal of Liquid Chromatography 3:1669
(1980).
(15) Lyman, W.J. W.F. Reehl, and D.H. Rosenblatt (Eds.), Handbook
of Chemical Property Estimation Methods, McGraw-Hill, New York
(1982).
(16) Dunn, W.J., J.H. Block and R.S. Pearlman (Ed.), Partition Coef-
ficient, Determination and Estimation, Pergamon, Elmsford (New York)
and Oxford (1986).
(17) Pomona College, Medicinal Chemistry Project, Claremont, CA
91711, USA, Log P Database and Medical Chemistry Software (Program
CLOGP-3).
(18) Hansch, C. and A.J. Leo, Substituent Constants for Correlation
Analysis in Chemistry and Biology Wiley, New York (1979).
(19) Leo, A. et al. Chemical Reviews 71:525 (1971).
(20) Rekker, R.F. and H.M. de Kort. European Journal of Medicinal
Chemistry 14:479 (1979).
(21) Fujita, T. et al. Journal of the American Chemical Society
86:5175(1964).
(22) Rekker, R.F. The Hydrophobic Fragmental Constant,
Pharmacochemistry Library, vol. 1, Elsevier, New York (1977).
(23) Eadsforth, C.V. and P. Moser. Chemosphere 12:1459 (1983).
(24) Scherrer, R.A. American Chemical Society Symposium Series
no. 255, p. 225, American Chemical Society, Washington, DC (1984).
(25) Organization for Economic Cooperation and Development,
Guidelines for The Testing of Chemicals, OECD 107, Partition Coefficient
(n-octanol/water), High Performance Liquid Chromatography (HPLC)
Method, OECD, Paris, France.
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