A.8 PARTITION COEFFICIENT

P le as e no tic e th at o nl y E ur op ea n C om m un ity ’s le gi sl at io n pu bl is he d in t he p ap er e di tio ns o f th e O ffi ci al J ou rn al o f th e E ur op ea n C om m un iti es is d ee m ed a ut he nt ic . W he n pr ep ar in g th is d oc um en t, ca re h as b ee n ta ke n to e ns ur e co rr ec tn es s of t he t ex t; ne v er th el es s po ss ib ili ty o f er ro rs c an no t be c om pl et el y ex cl ud ed , so d iff er en ce s m ay e xi st b et w ee n th is v er si on a nd t he o ne a gr ee d an d pu bl is he d in t he p ap er e di tio n of t he O ffi ci al J ou rn al . In c as e of d ou bt t he r ea de r is a dv is ed t o co ns ul t th e O ffi ci al Jo ur na l. T hi s m et ho d ca n be fo un d in D ir 92 /6 9/ E E C (O .J . L 38 3 A ) A c om pl et e lis t of A nn ex V T es tin g M et ho ds a nd t he c or re sp on di ng O J ca n be d ow nl oa de d fr om a p re vi ou s pa ge in t hi s si te . A.8. PARTITION COEFFICIENT 1. METHOD The 'shake flask' method described is based on the OECD Test Guideline (1). 1.1. INTRODUCTION It is useful to have preliminary information on structural formula, dissociation constant, water solubility, hydrolysis, n-octanol solubility and surface tension of the substance to perform this test. Measurements should be made on ionizable substances only in their non-ionized form (free acid or free base) produced by the use of an appropriate buffer with a pH of at least one pH unit below (free acid) or above (free base) the pK. This test method includes two separate procedures: the shake flask method and high performance liquid chromatography (HPLC). The former is applicable when the log Pow value (see below for definitions) falls within the range -2 to 4 and the latter within the range 0 to 6. Before carrying out either of the experimental procedures a preliminary estimate of the partition coefficient should first be obtained. The shake-flask method applies only to essentially pure substances soluble in water and n-octanol. It is not applicable to surface active materials (for which a calculated value or an estimate based on the individual n-octanol and water solubilities should be provided). The HPLC method is not applicable to strong acids and bases, metal complexes, surface-active materials or substances which react with the eluent. For these materials, a calculated value or an estimate based on individual n-octanol and water solubilities should be provided. The HPLC method is less sensitive to the presence of impurities in the test compound than is the shake- flask method. Nevertheless, in some cases impurities can make the interpretation of the results difficult because peak assignment becomes uncertain. For mixtures which give an unresolved band, upper and lower limits of log P should be stated. 1.2. DEFINITION AND UNITS The partition coefficient (P) is defined as the ratio of t he equilibrium concentrations (ci) of a dissolved substance in a two-phase system consisting of two largely immiscible solvents. In the case n-octanol and water: water oltanocn ow c c P -= The partition coefficient (P) therefore is the quotient of two concentrations and is usually given in the form of its logarithm to base 10 (log P). 1.3. REFERENCE SUBSTANCES Shake-flask method Reference substances do not need to be employed in all cases when investigating a new substance. They should primarily serve to check the performance of the method from time to time and to allow comparison with results from other methods. HPLC method In order to correlate the measured HPLC data of a compound with its P value, a calibration graph of log P vs. chromatographic data using at least 6 reference points has to be established. It is for the user to select the appropriate reference substances. Whenever possible, at least one reference compound should have a Pow above that of the test substance, and another a Pow below that of the test substance. For log P values less than 4, the calibration can be based on data obtained by the shake-flask method. For log P values greater than 4, the calibration can be based on validated literature values if these are in agreement with calculated values. For better accuracy, it is preferable to choose reference compounds which are structurally related to the test substance. P le as e no tic e th at o nl y E ur op ea n C om m un ity ’s le gi sl at io n pu bl is he d in t he p ap er e di tio ns o f th e O ffi ci al J ou rn al o f th e E ur op ea n C om m un iti es is d ee m ed a ut he nt ic . W he n pr ep ar in g th is d oc um en t, ca re h as b ee n ta ke n to e ns ur e co rr ec tn es s of t he t ex t; ne v er th el es s po ss ib ili ty o f er ro rs c an no t be c om pl et el y ex cl ud ed , so d iff er en ce s m ay e xi st b et w ee n th is v er si on a nd t he o ne a gr ee d an d pu bl is he d in t he p ap er e di tio n of t he O ffi ci al J ou rn al . In c as e of d ou bt t he r ea de r is a dv is ed t o co ns ul t th e O ffi ci al Jo ur na l. T hi s m et ho d ca n be fo un d in D ir 92 /6 9/ E E C (O .J . L 38 3 A ) A c om pl et e lis t of A nn ex V T es tin g M et ho ds a nd t he c or re sp on di ng O J ca n be d ow nl oa de d fr om a p re vi ou s pa ge in t hi s si te . Extensive lists of values of log Pow for many groups of chemicals are available (2)(3). If data on the partition coefficients of structurally related compounds are not available, then a more general calibration, established with other reference compounds, may be used. A list of recommended reference substances and their Pow values is given in Appendix 2. 1.4. PRINCIPLE OF THE METHOD 1.4.1. Shake-flask method In order to determine a partition coefficient, equilibrium between all interacting components of the system must be achieved, and the concentrations of the substances dissolved in the two phases must be determined. A study of the literature on this subject indicates that several different techniques can be used to solve this problem, i.e. the thorough mixing of the two phases followed by their separation in order to determine the equilibrium concentration for the substance being examined. 1.4.2. HPLC method HPLC is performed on analytical columns packed with a commercially available solid phase containing long hydrocarbon chains (e.g. C8, C18) chemically bound onto silica. Chemicals injected onto such a column move along it at different rates because of the different degrees of partitioning between the mobile phase and the hydrocarbon stationary phase. Mixtures of chemicals are eluted in order of their hydrophobicity , with water-soluble chemicals eluted first and oil-soluble chemicals last, in proportion to their hydrocarbon-water partition coefficient. This enables the relationship between the retention time on such a (reverse phase) column and the n-octanol/water partition coefficient to be established. The partition coefficient is deduced from the capacity factor k, given by the expression: o or t tt k - = in which, tr = retention time of the test substance, and to = average time a solvent molecule needs to pass through the column (dead-time). Quantitative analytical methods are not required and only the determination of elution times is necessary. 1.5. QUALITY CRITERIA 1.5.1. Repeatability Shake-flask method In order to assure the accuracy of the partition coefficient, duplicate determinations are to be made under three different test conditions, whereby the quantity of substance specified as well as the ratio of the solvent volumes may be varied. The determined values of the partition coefficient expressed as their common logarithms should fall within a range of ± 0,3 log units. HPLC method In order to increase the confidence in the measurement, duplicate determinations must be made. The values of log P derived from individual measurements should fall within a range of ± 0,1 log units. 1.5.2. Sensitivity Shake-flask method The measuring range of the method is determined by the limit of detect ion of the analytical procedure. This should permit the assessment of values of log Pow in the range of -2 to 4 ( occasionally when conditions apply, this range may be extended to log Pow up to 5) when the concentration of the solute in either phase is not more than 0,01 mol per litre. HPLC method The HPLC method enables partition coefficients to be estimated in the log P ow range 0 to 6. P le as e no tic e th at o nl y E ur op ea n C om m un ity ’s le gi sl at io n pu bl is he d in t he p ap er e di tio ns o f th e O ffi ci al J ou rn al o f th e E ur op ea n C om m un iti es is d ee m ed a ut he nt ic . W he n pr ep ar in g th is d oc um en t, ca re h as b ee n ta ke n to e ns ur e co rr ec tn es s of t he t ex t; ne v er th el es s po ss ib ili ty o f er ro rs c an no t be c om pl et el y ex cl ud ed , so d iff er en ce s m ay e xi st b et w ee n th is v er si on a nd t he o ne a gr ee d an d pu bl is he d in t he p ap er e di tio n of t he O ffi ci al J ou rn al . In c as e of d ou bt t he r ea de r is a dv is ed t o co ns ul t th e O ffi ci al Jo ur na l. T hi s m et ho d ca n be fo un d in D ir 92 /6 9/ E E C (O .J . L 38 3 A ) A c om pl et e lis t of A nn ex V T es tin g M et ho ds a nd t he c or re sp on di ng O J ca n be d ow nl oa de d fr om a p re vi ou s pa ge in t hi s si te . Normally, the partition coefficient of a compound can be estimated to within ± l log unit of the shake- flask value. Typical correlations can be found in the literature (4)(5)(6)(7)(8). Higher accuracy can usually be achieved when correlation plots are based on structurally-related reference compounds (9). 1.5.3. Specificity Shake-flask method The Nernst Partition Law applies only at constant temperature, pressure and pH for dilute solutions. It strictly applies to a pure substance dispersed between two pure solvents. If several different solutes occur in one or both phases at the same time, this may affect the results. Dissociation or association of the dissolved molecules result in deviations from the Nernst Partition Law. Such deviations are indicated by the fact that the partition coefficient becomes dependent upon the concentration of the solution. Because of the multiple equilibria involved, this test method should not be applied to ionizable compounds without applying a correction. The use of buffer solutions in place of water should be considered for such compounds; the pH of the buffer should be at least 1 pH unit from the pKa of the substance and bearing in mind the relevance of this pH for the environment. 1.6. DESCRIPTION OF THE METHOD 1.6.1. Preliminary estimate of the partition coefficient The partition coefficient is estimated preferably by using a calculation method (see Appendix 1), or where appropriate, from the ratio of the solubilities of the test substance ill the pure solvents (10). 1.6.2. Shake-flask method 1.6.2.1. Preparation n-Octanol: The determination of the partition coefficient should be carried out with high purity analytical grade reagent. Water: water distilled or double distilled in glass or quartz apparatus should be employed. For ionizable compounds, buffer solutions in place of water should be used if justified. Note: Water taken directly from an ion exchanger should not be used. 1.6.2.1.1. Pre-saturation of the solvents Before a partition coefficient is determined, the phases of the solvent system are mutually saturated by shaking at the temperature of the experiment. To do this, it is practical to shake two large stock bottles of high purity analytical grade n-octanol or water each with a sufficient quantity of the other solvent for 24 hours on a mechanical shaker and then to let them stand long enough to allow the phases to separate and to achieve a saturation state. 1.6.2.1.2. Preparation for the test The entire volume of the two-phase system should nearly fill the test vessel. This will help prevent loss of material due to volatilization. The volume ratio and quantities of substance to be used are fixed by the following: -the preliminary assessment of the partition coefficient (see above), -the minimum quantity of test substance required for the analytical procedure, and -the limitation of a maximum concentration in either phase of 0,01 mol per litre. Three tests are carried out. In the first, the calculated volume ratio of n-octanol to water is used; in the second, this ratio is divided by two; and in the third, this ratio is multiplied by two (e.g. 1:1, 1:2,2:1). P le as e no tic e th at o nl y E ur op ea n C om m un ity ’s le gi sl at io n pu bl is he d in t he p ap er e di tio ns o f th e O ffi ci al J ou rn al o f th e E ur op ea n C om m un iti es is d ee m ed a ut he nt ic . W he n pr ep ar in g th is d oc um en t, ca re h as b ee n ta ke n to e ns ur e co rr ec tn es s of t he t ex t; ne v er th el es s po ss ib ili ty o f er ro rs c an no t be c om pl et el y ex cl ud ed , so d iff er en ce s m ay e xi st b et w ee n th is v er si on a nd t he o ne a gr ee d an d pu bl is he d in t he p ap er e di tio n of t he O ffi ci al J ou rn al . In c as e of d ou bt t he r ea de r is a dv is ed t o co ns ul t th e O ffi ci al Jo ur na l. T hi s m et ho d ca n be fo un d in D ir 92 /6 9/ E E C (O .J . L 38 3 A ) A c om pl et e lis t of A nn ex V T es tin g M et ho ds a nd t he c or re sp on di ng O J ca n be d ow nl oa de d fr om a p re vi ou s pa ge in t hi s si te . 1.6.2.1.3. Test substance A stock solution is prepared in n-octanol pre-saturated with water. The concentration of this stock solution should be precisely determined before it is employed in the determination of the partition coefficient. This solution should be stored under conditions which ensure its stability. 1.6.2.2. Test conditions The test temperature should be kept constant ( ± 1 °C) and lie in the range of 20 to 25 °C. 1.6.2.3. Measurement procedure 1.6.2.3.1. Establishment of the partition equilibrium Duplicate test vessels containing the required, accurately measured amounts of the two solvents together with the necessary quantity of the stock solution should be prepared for each of the test conditions. The n-octanol phases should be measured by volume. The test vessels should either be placed in a suitable shaker or shaken by hand. When using a centrifuge tube, a recommended method is to rotate the tube quickly through 180° about its transverse axis so that any trapped air rises through the two phases. Experience has shown that 50 such rotations are usually sufficient for the establishment of the partition equilibrium. To be certain, 100 rotations in five minutes are recommended. 1.6.2.3.2. Phase separation When necessary, in order to separate the phases, centrifugation of the mixture should be carried out. This should be done in a laboratory centrifuge maintained at room temperature, or, if a non- temperature controlled centrifuge is used, the centrifuge tubes should be kept for equilibration at the test temperature for at least one hour before analysis. 1.6.2.4. Analysis For the determination of the partition coefficient, it is necessary to determine the concentrations of the test substance in both phases. This may be done by taking an aliquot of each of the two phases from each tube for each test condition and analyzing them by the chosen procedure. The total quantity of substance present in both phases should be calculated and compared with the quantity of the substance originally introduced. The aqueous phase should be sampled by a procedure that minimizes the risk of including traces of n- octanol: a glass syringe with a removable needle can be used to sample the water phase. The syringe should initially be partially filled with air. Air should be gently expelled while inserting the needle through the n-octanol layer. An adequate volume of aqueous phase is withdrawn into the syringe. The syringe is quickly removed from the solution and the needle detached. The contents of the syringe may then be used as the aqueous sample. The concentration in the two separated phases should preferably be determined by a substance-specific method. Examples of analytical methods which may be appropriate are: -photometric methods, -gas chromatography, -high-performance liquid chromatography. 1.6.3. HPLC method 1.6.3.1. Preparation Apparatus A liquid chromatograph, fitted with a pulse-free pump and a suitable detection device, is required. The use of an injection valve with injection loops is recommended. The presence of polar groups in the P le as e no tic e th at o nl y E ur op ea n C om m un ity ’s le gi sl at io n pu bl is he d in t he p ap er e di tio ns o f th e O ffi ci al J ou rn al o f th e E ur op ea n C om m un iti es is d ee m ed a ut he nt ic . W he n pr ep ar in g th is d oc um en t, ca re h as b ee n ta ke n to e ns ur e co rr ec tn es s of t he t ex t; ne v er th el es s po ss ib ili ty o f er ro rs c an no t be c om pl et el y ex cl ud ed , so d iff er en ce s m ay e xi st b et w ee n th is v er si on a nd t he o ne a gr ee d an d pu bl is he d in t he p ap er e di tio n of t he O ffi ci al J ou rn al . In c as e of d ou bt t he r ea de r is a dv is ed t o co ns ul t th e O ffi ci al Jo ur na l. T hi s m et ho d ca n be fo un d in D ir 92 /6 9/ E E C (O .J . L 38 3 A ) A c om pl et e lis t of A nn ex V T es tin g M et ho ds a nd t he c or re sp on di ng O J ca n be d ow nl oa de d fr om a p re vi ou s pa ge in t hi s si te . stationary phase may seriously impair the performance of the HPLC column. Therefore, stationary phases should have the minimal percentage of polar groups (11). Commercial 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. Mobile phase HPLC grade methanol and HPLC grade 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% should be used. Typically a 3:1 (v/v) methanol-water mixture is satisfactory for eluting compounds of log P 6 within an hour, at a flow rate of 1 ml/min. For compounds of high log P it may be necessary to shorten the elution time