Octanol-water partition coefficient shake-flask method

Han, S. D., Park, S. J., and Park, S.-J. Determination of octanol/water partition coefficients by nsing a shake-flask method,... 

Paschke, A., Neitzel, P.E., Walther, W., and Schiirmann, G. Octanol/water partition coefficient of selected herbicides determination using shake-flask method and reversed-phase high-performance liquid chromatography, J. Chem. Eng. Data, 49(6) 1639-1642, 2004. 

Microemulsion electrokinetic chromatography, MEEKC (or MECC), is a relatively new technique which holds some promise of delivering octanol/water partition coefficients much more conveniently than the shake-flask method (Gluck, 1996 Ishihama, 1994). MEEKC is claimed to have all the advantages of an HPLC method but it is not suitable for solutes with pKas much below 7.0 (Adlard, 1995). It has been used over a log P range of -1.0 to +4.0. 

Mirrlees MS, Moulton SJ, Murphy CT et al. (1976) Direct measurement of octanol-water partition coefficients by high-pressure liquid chromatography. J Med Chem 19 615-619 Pagliara A, Khamis E, Thrinh A et al. (1995) Structural properties governing retention mechanisms on RP-HPLC stationary phases used for lipophilicity measurements. J Liquid Chromatography 18 1721-1745 Slater B, McCormack A, Avdeef A et al. (1994) pH-Metric log P. 4. Comparison of Partition Coefficients Determined by Shake-Flask, HPLC and Potentiometric Methods. J Phar-maceut Sci 83 1280-1283... 

The octanol-water partition coefficient for surfactants can not be determined using the shake-flask or slow stirring method because of the formation of emulsions. In addition, the surfactant molecules will exist in the water phase almost exclusively as ions, whereas they will have to pair with a counter-ion in order to be dissolved in octanol. Therefore, experimental determination of K w does not characterize the partition of ionic surfactants (Tolls, 1998). On the other hand, it has been shown that the bioconcentration of anionic and non-ionic surfactants increases with increasing lipophilicity (Tolls, 1998). Tolls (1998) showed that for some surfactants, an estimated log Kow value using LOGKOW could represent the bioaccumulation potential however, for other surfactants some correction to the estimated log Kow value using the method of Roberts (1989) was required. These results illustrate that the quality of the relationship between log Kow estimates and bioconcentration depends on the class and specific type of surfactants involved. Therefore, the classification of the bioconcentration potential based on log Kow values should be used with caution. 

The octanol-water-partition coefficient (Kow) is the most frequently cited measure of environmental partitioning behavior. Partition coefficients traditionally have been determined by some variation on the shake-flask method, however, over the past several years high-pressure liquid chromatography has been shown to measure octanol-water partition coefficients accurately over a wide range of values with greater ease. 

Figure 4. Distribution of simulated octanol-water partition coefficient from the shake-flask method (true value 1.00).

In QSAR studies the lipophilic character of a compound is usually expressed in terms of the logarithm of the octanol/water partition coefficient (i.e., log P). Experimentally, the octanol/water partition coefficient is determined by the shake-flask method. Unfortunately, it is often difficult to obtain precise values for the octanol/water partition coefficient because solute impurities will affect the measured distribution coefficient. For example, reported log P values for benzene (1-5) vary from 1.56 to as high as 2.34. 

Technical details, including solid support, coating and column filling techniques, eluents, and factors affecting the reproducibility have been reviewed [173, 235, 236]. Lipophilicity values from HPLC measurements are not on a unique scale, but the log k values can be converted to -octanol/water partition coefficients with the help of a set of HPLC calibration standard compounds for which classical shake-flask partition coefficients are known. Experience shows once the HPLC method is successfully established in a laboratory, it will remain the method of choice for lipophilicity determinations. 

The n-octanol/water partition coefficient logP in Table 1.8 was determined by the shake-flask method, as described in detail (70). 

Groves, F., Jr. Solubility of cycloparaffins in distilled water and saltwater, J. Chem. Eng. Data, 33 (2) 136-138, 1988. Grunewald, G.L., Pleiss, M.A., Gatchell, C.L., Pazhenchevsky, R., and Rafferty, M.F. Gas chromatographic quantitation of underivatized amines in the determination of their octanol-0.1 Msodium hydroxide partition coefficients by the shake-flask method, J. Chromatogr. A, 292(2) 319-331, 1984. 

The partition coefficient logP or the distribution coefficient logD between water and n-octanol are determined traditionally by the shake flask method (see chapter about physicochemical parameters). The shake flask method is a rather labour intensive method and not suited for high throughput analysis needed in pharmaceutical research. 

The basic principle of the method is to measure the dissolution of the substance in two different phases, water and -octanol. In order to determine the partition coefficient, equilibrium between all interacting components of the system must be achieved after which the concentration of the substances dissolved in the two phases is determined. The shake-flask method is applicable when the log Kow value falls within the range from -2 to 4 (OECD 107, 1995). The shake-flask method applies only to essential pure substances soluble in water and -octanol and should be performed at a constant temperature ( 1°C) in the range 20-25 °C. 

OECD Test Guideline 107, 1995. OECD Guidelines for testing of chemicals. Partition Coefficient (n-octanol/water) Shake Flask Method... 

The partition coefficient between n-octanol and water, Po/w> can be measured experimentally using the shake-flask method or can be calculated from structural fragments. The direct measurement of log values by equilibration between n-octanol and water faces some difficulties, such as the necessary high purity of the substance that must be available in an adequate quantity, and the method is time-consuming too. In addition, it is not applicable to very lipophilic or very hydrophilic compounds. 

If octanol is the stationary phase and water the mobile phase, Pq/w is the oetanol-water partition eoeffieient without any assumption. Correlations of the or log Pq/ values obtained with the same liquid system by the shake flask method and by CCC produce straight lines with a slope unity and a negligible intercept. The validity and solidity of the method was assessed by Gluck and Martin for P / coefficients [2]. The P / range that can be obtained directly by CCC is 0.05-200 [1]. It is limited on the high side by the experiment duration. A P / value of 200 corresponds to a Vr retention volume of 6 L with a Vs value of only 30 mL [Eq. (1)]. This is 1200 min or 20 h with a 5-mL/min flow rate. The lower-side limitation is due to experimental precision. The difference between the retention volume Vr and the dead volume Vm is equal to PVs [Eq. (1)]. With a 30-mL Vs volume, the P / value of 0.05 corresponds to a - Vm value of only 1.5 mL. Such a low value may be difficult to evaluate with an acceptable accuracy. To increase the measurable Pq/w range, the fact that the CCC stationary phase is a liquid can be used. This led to the dual-mode use of CCC and the cocurrent operation. 

Accurate values of Pow are clearly necessary for the ultimate calibration of all surrogate systems, but, in practice, direct measurements of Powby the traditional shake-flask method are seldom used. Particularly for compounds with low water solubility, experimental difficulties may arise from problems in phase separation without carryover, sorption to glass surfaces, or from formation of emulsions. All of these introduce serious uncertainties into the concentrations in the appropriate phases, and may consequently lead to substantial errors in the estimates of partition coefficients. The problem is particularly acute for compounds with extremely low solubility in water such as the chlorinated dibenzo[l,4]dioxins for which widely varying values have been reported (Marple et al. 1986 Shiu et al. 1988). For such compounds, use of a generator column has been advocated (De Voe et al. 1981 Woodburn et al. 1984). In essence, the following steps are carried out (1) a solution of the test substance in octanol is equilibrated with water and the concentration in the octanol phase is determined, (2) the octanol phase is... 

The most common method for determining partition and distribution coefficients is the shake flask method. In this technique, the candidate drug is shaken between octanol (previously shaken together to presaturate each phase with the other) and water layers, from which an aliquot is taken and analyzed using UV absorption, HPLC or titration. In terms of experimental conditions, the value of the partition coefficient obtained from this type of experiment is affected by such factors as temperature, insufficient mutual phase saturation, pH and buffer ions and their concentration, as well as the nature of the solvents used and solute examined (Dearden and Bresnen 1988). 

Log P values can be measured experimentally or predicted from molecular structure. Experimental approaches run the gamut from the highly reliable and traditional shake-flask method to newer methodologies such as electrokinetic chromatographic methods that are rapid, accurate and precise, and can be automated. However, for validation purposes, partition coefficient data of large, structurally diverse datasets obtained from such systems should be compared with that from the standard octanol/water system. 

The HPLC technique has certain advantages that allow partition coefficients to be determined relatively rapidly in comparison to the conventional shaking-flask method. The elution process also makes it feasible to run mixture of solutes and yield results relatively free of solute-solute or solute-impurity interactions with a column giving good separation. Basically, the precision of the method depends on whether the value of yw/7o solute in the octanol-water system [Eq. (5)] can be effectively simulated by the column composition. It is therefore important that efforts be expanded to ensure that the properties of the column environment accurately reflect the bulk solvent-water (e.g., octanol-water) characteristics. If the solute can truly be equilibrated between mobile and stationary phases during elution, its retention time is determined by the ratio of the activity coefficients (i.e., partition constant) in the mobile and stationary phases. When octanol and... 

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