(i) The measurement and estimation of the n-octanol/water partition coefficient (K_ow), has become the cornerstone of a myriad of structure-activity relationships (SAR) property. The coefficient has been used extensively for correlating structural changes in drugs with changes observed in biological, biochemical, or toxic effects. These correlations are then used to predict the effect of a new drug for which a K_ow could be measured.

(ii) In the study of the environmental fate of organic chemicals, the K_ow has become a key parameter. K_ow is correlated to water solubility, soil/sediment sorption coefficient, and bioconcentration and is important to SAR.

(iii) Of the three properties that can be estimated from K_ow, water solubility is the most important because it affects both the fate and transport of chemicals. For example, highly soluble chemicals become quickly distributed by the hydrologic cycle, have low-sorption coefficients for soils and sediments, and tend to be more easily degraded by microorganisms. In addition, chemical transformation processes such as hydrolysis, direct photolysis, and indirect photolysis (oxidation) tend to occur more readily if a compound is soluble.

(iv) Direct correlations between K_ow and both the soil/sediment sorption coefficient and the bioconcentration factor are to be expected. In these cases, compounds that are more soluble in n-octanol (more hydrophobic and lipophilic) would be expected to partition out of the water and into the organic portion of soils/sediments and into lipophilic tissue. The relationship between K_ow and the bioconcentration factor, are the principal means of estimating bioconcentration factors. This relationship is discussed in the reference listed in paragraph (e)(14) of this section. These factors are then used to predict the potential for a chemical to accumulate in living tissue.

(v) This section describes a method for determining the K_ow based on the dynamic coupled column liquid chromatographic (DCCLC) technique, a technique commonly referred to as the generator column method. The method described herein can be used in place of the standard shake-flask method specified in § 799.6755 for compounds with a log₁₀K_ow greater than 1.0.

Extractor column is used to extract the solute from the aqueous solution produced by the generator column. After extraction onto a bonded chromatographic support, the solute is eluted with a solvent/water mixture and subsequently analyzed by high-performance liquid chromatography (HPLC), gas chromatography (GC), or any other analytical procedure. A detailed description of the preparation of the extractor column is given in paragraph (c)(1)(i) of this section.

Generator column is used to partition the test substance between the n-octanol and water phases. The column in figure 1 in paragraph (c)(1)(i)(A)(2) of this section is packed with a solid support and is coated with the test substance at a fixed concentration in n-octanol. The test substance is eluted from the column with water and the aqueous solution leaving the column represents the equilibrium concentration of the test substance that has partitioned from the n-octanol phase into the water phase. Preparation of the generator column is described in paragraph (c)(1)(i) of this section.

n-Octanol/water partition coefficient (K_ow) is defined as the ratio of the molar concentrations of a chemical in n-octanol and water, in dilute solution. The coefficient K_ow is a constant for a given chemical at a given temperature. Since K_ow is the ratio of two molar concentrations, it is a dimensionless quantity. Sometimes K_ow is reported as the decadic logarithm (log₁₀K_ow). In this equation, C_octanol and C_water are the molar concentration of the solute in n-octanol and water, respectively, at a given temperature. This test procedure determines K_ow at 25 ±0.05 °C. The mathematical statement of K_ow is:

Response factor (RF) is the solute concentration required to give a one unit area chromatographic peak or one unit output from the HPLC recording integrator at a particular recorder and detector attenuation. The factor is required to convert from units of area to units of concentration. The determination of the RF is given in paragraph (c)(3)(iii)(C)(2) of this section.

Sample loop is a 1⁄16 inch (in) outside diameter (O.D.) (1.6 millimeter (mm)) stainless steel tube with an internal volume between 20 and 50 µL. The loop is attached to the sample injection valve of the HPLC and is used to inject standard solutions into the mobile phase of the HPLC when determining the RF for the recording integrator. The exact volume of the loop must be determined as described in paragraph (c)(3)(iii)(C)(1) of this section when the HPLC method is used.

(i) This test method is based on the DCCLC technique for determining the aqueous solubility of organic compounds. The development of this test method is described in the references listed in paragraphs (e)(6), (e)(12), and (e)(19) of this section. The DCCLC technique utilizes a generator column, extractor column, and HPLC coupled or interconnected to provide a continuous closed-flow system. Aqueous solutions of the test compound are produced by pumping water through the generator column that is packed with a solid support coated with an approximately 1.0% weight/weight (w/w) solution of the compound in n-octanol. The aqueous solution leaving the column represents the equilibrium concentration of the test chemical which has partitioned from the n-octanol phase into the water phase. The compound is extracted from the aqueous solution onto an extractor column, then eluted from the extractor column with a solvent/water mixture and subsequently analyzed by HPLC using a variable wavelength ultraviolet (UV) absorption detector operating at a suitable wavelength. Chromatogram peaks are recorded and integrated using a recording integrator. The concentration of the compound in the effluent from the generator column is determined from the mass of the compound (solute) extracted from a measured volume of water (solvent). The K_ow is calculated from the ratio of the molar concentration of the solute in the 1.0% (w/w) n-octanol and molar concentration of the solute in water as determined using the generator column technique.

(ii) Since the HPLC method is only applicable to compounds that absorb in the UV, an alternate GC method, or any other reliable quantitative procedure must be used for those compounds that do not absorb in the UV. In the GC method the saturated solutions produced in the generator column are extracted using an appropriate organic solvent that is subsequently injected into the GC, or any other suitable analytical device, for analysis of the test compound.

(i) Columns 2, 3, 4, and 5 of table 1 in paragraph (b)(4)(ii) of this section list the experimental values of the decadic logarithm of the n-octanol/water partition coefficient (log₁₀K_ow) at 25 °C for a number of organic chemicals as obtained from the scientific literature. These values were obtained by any one of the following experimental methods: Shake-flask; generator column; reverse-phase HPLC; or reverse-phase thin-layer chromatography, as indicated in the footnotes following each literature citation. The estimation method of Hawker and Connell as described in paragraph (e)(8) of this section, correlates log₁₀K_ow with the total surface area of the molecule and was used to estimate log₁₀K_ow for biphenyl and the chlorinated biphenyls. These estimated values are listed in column 7 of table 1 in paragraph (b)(4)(ii) of this section. Recommended values of log₁₀K_ow were obtained by critically analyzing the available experimental and estimated values and averaging the best data. These recommended values are listed in column 8 of table 1 in paragraph (b)(4)(ii) of this section.

(ii) The recommended values listed in table 1 of this section have been provided primarily so that the generator column method can be calibrated and to allow the chemical laboratory the opportunity to compare its results with these values. The testing laboratory has the option of choosing its reference chemicals, but references must be given to establish the validity of the measured values of log₁₀K_ow.

(i) Special laboratory equipment -

(A)

(1) Generator column. Either of two different methods for connecting to the generator column shall be used depending on whether the eluted aqueous phase is analyzed by HPLC (Procedure A, as described in paragraph (c)(3)(iii) of this section) or by solvent extraction followed by GC analysis, or any other reliable method of solvent extract (Procedure B, as described in paragraph (c)(3)(iv) of this section).

(2)

(i) The design of the generator column is shown in the following figure 1:

(ii) The column consists of a 6 mm (1⁄4 in) O.D. pyrex tube joined to a short enlarged section of 9 mm pyrex tubing which in turn is connected to another section of 6 mm (1⁄4 in) O.D. pyrex tubing. Connections to the inlet teflon tubing (1⁄8 in O.D.) and to the outlet stainless steel tubing (1⁄16 in O.D.) are made by means of stainless steel fittings with teflon ferrules. The column is enclosed in a water jacket for temperature control as shown in the following figure 2:

Figure 2 - Setup Showing Generator Column Enclosed in a Water Jacket and Overall Arrangement of the Apparatus Used in GC Method

(ii) Purity of n-octanol and water. Purified n-octanol, described in paragraph (c)(2)(i) of this section, and water meeting appropriate American Society for Testing and Materials Type II standards, or an equivalent grade, are recommended to minimize the effects of dissolved salts and other impurities. An ASTM Type II water standard is presented in the reference listed in paragraph (e)(20) of this section).

(iii) Purity of solvents. It is important that all solvents used in this method be reagent or HPLC grade and contain no impurities which could interfere with the determination of the test compound.

(iv) Reference compounds. In order to ensure that the HPLC system is working properly, at least two of the reference compounds listed in table 1 in paragraph (b)(4)(ii) of this section should be run. Reference compounds shall be reagent or HPLC grade to avoid interference by impurities.

(i) n-Octanol and water. Very pure n-octanol can be obtained as follows: Wash pure n-octanol (minimum 98% pure) sequentially with 0.1N H²SO₄, with 0.1N NaOH, then with distilled water until neutral. Dry the n-octanol with magnesium sulfate and distill twice in a good distillation column under reduced pressure [b.p. about 80 °C at 0.27 kPa (2 torr)]. The n-octanol produced should be at least 99.9% pure. Alternatively, a grade equivalent to Fisher Scientific Co. No. A-402 “Certified Octanol-1” can be used. Reagent-grade water shall be used throughout the test procedure, such as ASTM Type II water, or an equivalent grade, as described in paragraph (c)(1)(ii) of this section.

(ii) Presaturated water. Prepare presaturated water with n-octanol to minimize the depletion of n-octanol from the column when measuring the K_owof a test chemical. This is very important when the test chemical is lipophilic and the log₁₀K_ow ≤4.

(i) Test solution. The test solution consists of an approximately 1.0% (w/w) solution of the test substance in n-octanol. A sufficient quantity (about 10-20 milliliter (mL)) of the test solution should be prepared to coat the generator column. The solution is prepared by accurately weighing out, using a tared bottle, quantities of both the test substance and n-octanol required to make a 1.0% (w/w) solution. When the weights are measured precisely (to the nearest 0.1 milligram (mg)), knowing the density of n-octanol (0.827 gram (g)/mL at 25 °C), then the molar concentration of the test substance in the n-octanol is sufficiently accurate for the purposes of the test procedure. If desired, however, a separate analytical determination (e.g., by GC, or any other reliable analytical method) may be used to check the concentration in the test solution. If storage is required, the test solution should be kept stoppered to prevent volatilization of the test chemical.

(ii) Test procedures. Prior to the determination of the K_ow of the test chemical, two procedures shall be followed:

(iii) Procedure A - HPLC method.

(A) Procedure A covers the determination of the aqueous solubility of compounds which absorb in the UV. Two reciprocating piston pumps deliver the mobile phase (water or solvent/water mixture) through two 6-port high-pressure rotary valves and a 30 × 0.6 cm C₁₈ analytical column to a UV absorption detector operating at a suitable wavelength. Chromatogram peaks are recorded and integrated with a recording integrator. One of the 6-port valves is the sample injection valve used for injecting samples of standard solutions of the solute in an appropriate concentration for determining RFs or standard solutions of basic chromate for determining the sample-loop volume. The other 6-port valve in the system serves as a switching valve for the extractor column which is used to remove solute from the aqueous solutions. The HPLC analytical system is shown schematically in the following figure 3:

Figure 3 - Schematic of HPLC - Generator Column Flow System

(C)

(1) Determination of the sample-loop volume. Accurate measurement of the sample loop may be accomplished by using a spectrophotometric method such as the one described in the reference listed in paragraph (e)(6) of this section. For this method, measure absorbance, A_loop, at 373 nanometers (nm) for at least three solutions, each of which is prepared by collecting from the sample valve an appropriate number, n, of loopfuls of an aqueous stock solution of K₂CrO₄ (1.3% by weight) and diluting to 50 mL with 0.2% KOH. (For a 20 µL loop, use n = 5; for a 50 µL loop, use n = 2.) Also measure the absorbance, A_stock, of the same stock solution after diluting 1:500 with 0.2% KOH. Calculate the loop volume to the nearest 0.1 µL using the relation:

Equation 2:

(2) Determination of the RF.

(i) For all determinations adjust the mobile phase solvent/water ratio and flow rate to obtain a reasonable retention time on the HPLC column. For example, typical concentrations of organic solvent in the mobile phase range from 50 to 100% while flow rates range from 1 to 3 mL/minutes (min); these conditions often give a 3 to 5 min retention time.

(ii) Prepare standard solutions of known concentrations of the solute in a suitable solvent. Concentrations must give a recorder response within the maximum response of the detector. Inject samples of each standard solution into the HPLC system using the calibrated sample loop. Obtain an average peak area from at least three injections of each standard sample at a set detector absorbance unit full scale (AUFS), i.e., at the same absorbance scale attenuation setting.

(iii) Calculate the RF from the following equation:

Equation 3:

(3) Loading of the generator column.

(i) The design of the generator column was described in paragraph (c)(1)(i) of this section and is shown in figure 1 in paragraph (c)(1)(i)(A)(2)(i) of this section. To pack the column, a plug of silanized glass wool is inserted into one end of the 6 mm pyrex tubing. Silanized diatomaceous silica support (about 0.5g of 100-120 mesh Chromosorb W chromatographic support material) is poured into the tube with tapping and retained with a second plug of silanized glass wool.

(ii) The column is loaded by pulling the test solution through the dry support with gentle suction and then allowing the excess solution to drain out. After loading the column, draw water up through the column to remove any entrapped air.

(4) Analysis of the solute. Use the following procedure to collect and analyze the solute:

(i) With the switching valve in figure 3 in paragraph (c)(3)(iii)(A) of this section in the inject position (i.e., water to waste), pump water through the generator column at a flow rate of approximately 1 mL/min for approximately 15 min to bring the system into equilibrium. Pump water to the generator column by means of a minipump or pressurized water reservoir as shown in the following figure 4:

Figure 4 - Water Reservoir for GC Method

(ii) Flush out the organic solvent that remains in the system from previous runs by changing the mobile phase to 100% H₂O and allowing the water to reach the HPLC detector, as indicated by a negative reading. As soon as this occurs, place a 25 mL weighing bottle (weighed to the nearest mg) at the waste position and immediately turn the switching valve to the load position.

(iii) Collect an amount of water from the generator column (as determined by trial and error) in the weighing bottle, corresponding to the amount of solute adsorbed by the extractor column that gives a reasonable detector response. During this extraction step, switch back to the original HPLC mobile phase composition, i.e., solvent/water mixture, to condition the HPLC analytical column.

(iv) After the desired volume of sample has been extracted, turn the switching valve back to the inject position in figure 3 in paragraph (c)(3)(iii)(A) of this section. As soon as the switching valve is turned to the inject position, remove the weighing bottle, cap it and replace it with the waste container; at the same time turn on the recording integrator. The solvent/water mobile phase will elute the solute from the extractor column and transfer the solute to the HPLC analytical column.

(v) Determine the weight of water collected to the nearest mg and record the corresponding peak area. Using the same AUFS setting repeat the analysis of the solute at least two more times and determine the average ratio of peak area to grams of water collected. In this equation, S = solubility (M), RF = response factor, V_loop = sample-loop volume (L), and R = ratio of area to grams of water. Calculate the solute solubility in water using the following equation:

Equation 4:

(iv) Procedure B - GC Method. In the GC method, or any other reliable quantitative method, aqueous solutions from the generator column enter a collecting vessel in figure 2 in paragraph (c)(1)(i)(A)(2)(ii) of this section containing a known weight of extracting solvent which is immiscible in water. The outlet of the generator column is positioned such that the aqueous phase always enters below the extracting solvent. After the aqueous phase is collected, the collecting vessel is stoppered and the quantity of aqueous phase is determined by weighing. The solvent and the aqueous phase are equilibrated by slowly rotating the collecting vessel. A small amount of the extracting solvent is then removed and injected into a GC equipped with an appropriate detector. The solute concentration in the aqueous phase is determined from a calibration curve constructed using known concentrations of the solute. The extraction efficiency of the solvent shall be determined in a separate set of experiments.

(A) Determination of calibration curve.

(1) Prepare solute standard solutions of concentrations covering the expected range of the solute solubility. Select a column and optimum GC operating conditions for resolution between the solute and solvent and the solute and extracting solvent. Inject a known volume of each standard solution into the injection port of the GC. For each standard solution determine the average of the ratio R of peak area to volume (in µL) for the chromatographic peak of interest from at least three separate injections.

(2) After running all the standard solutions, determine the coefficients, a and b, using linear regression analysis on the equation of concentration (C) vs. R in the form:

Equation 5:

(D) Analysis of the solute.

(1) After rotating, allow the collection vessel to stand for approximately 30 min; then remove a known volume of the extracting solvent from the vessel using a microliter syringe and inject it into the GC. Record the ratio of peak area to volume injected and, from the regression equation of the calibration line, determine the concentration of solute in the extracting solvent. If the extraction efficiency is not 100%, the measured extraction efficiency shall be used to obtain the correct concentration of solute extracted. In this equation, C_es is the molar concentration of solute in extracting solvent, d_H2_O and d_es are the densities in grams per milliliter of water and extracting solvent, respectively, and g_es and g_H2_O are the grams of extracting solvent and water, respectively, contained in the collection vessels. The molar concentration of solute in water C(M) is determined from the following equation:

Equation 6:

(2) Make replicate injections from each collecting vessel to determine the average solute concentration in water for each vessel. To make sure the generator column has reached equilibrium, run at least two additional (for a total of three) collection vessels and analyze the extracted solute as described in paragraph (c)(3)(iv)(D)(1) of this section. Calculate C(M) from the average solute concentration in the three vessels.

(3) If another analytical method is used in place of the GC, then Procedure B, as described in paragraph (c)(3)(iv) of this section, shall be modified and the new analytical procedure shall be used to determine quantitatively the amount of solute extracted in the extraction solvent.

(v) Analysis of reference compounds. Prior to analyzing the test solution, make duplicate runs on at least two of the reference compounds listed in table 1 in paragraph (b)(4)(ii) of this section. When using the reference compounds, follow the same procedure previously described for preparing the test solution and running the test. If the average value obtained for each compound is within 0.1 log unit of the reference value, then the test procedure and HPLC system are functioning properly; if not a thorough checking over of the HPLC and careful adherence to the test procedures should be done to correct the discrepancy.

(vi) Modification of procedures for potential problems - Decomposition of the test compound. If the test compound decomposes in one or more of the aqueous solvents required during the period of the test at a rate such that an accurate value for water solubility cannot be obtained, then it will be necessary to carry out detailed transformation studies, such as hydrolysis studies. If decomposition is due to aqueous photolysis, then it will be necessary to carry out the studies in the dark, under red or yellow lights, or by any other suitable method to eliminate this transformation process.

(i) For the test solution, report the weights to the nearest 0.1 mg of the test substance and n-octanol. Also report the weight percent and molar concentration of the test substance in the n-octanol; the density of n-octanol at 25 °C is 0.827 grams per milliliter (gm)/mL.

(ii) For each run provide the molar concentration of the test substance in water for each of three determinations, the mean value, and the standard deviation.

(iii) For each of the three determinations calculate the K_ow as the ratio of the molar concentration of the test substance in n-octanol to the molar concentration in water. Also calculate and report the mean K_ow and its standard deviation. Values of K_ow shall be reported as their logarithms (log₁₀K_ow).

(iv) Report the temperature (±0.05 °C) at which the generator column was controlled during the test.

(v) For each reference compound report the individual values of log₁₀K_ow and the average of the two runs.

(vi) For compounds that decompose at a rate such that a precise value for the solubility cannot be obtained, provide a statement to that effect.

(i) For the HPLC method describe and/or report:

(ii) For the GC method report:

(iii) If another approved analytical method is used to determine the concentration of the test chemical in water, then all the important test conditions shall be reported.

(iv) If the concentration of the test substance in n-octanol is determined by an independent analytical method such as GC, provide a complete description of the method.