Shake-flask method, partition coefficients

In the traditional shake-flask method, the apparent partition coefficient, log D, is measured, usually at pH 7.4 (sometimes at pH 6.5). Different buffers are used to control each pH used in the determinations [70]. Usually, in a comprehensive study, several pH measurements are made, and values of log are plotted against the pH. This plot is often called the lipophilicity profile . One can determine the true partition coefficients (log P ) and the ionization constants from the features in such a curve. 

However, as stated above, the partition coefficients measured by the shake-flask method or by potenhometric titration can be influenced by the potenhal difference between the two phases, and are therefore apparent values which depend on the experimental condihons (phase volume ratio, nature and concentrahons of all ions in the solutions). In particular, it has been shown that the difference between the apparent and the standard log Pi depends on the phase volume raho and that this relationship itself depends on the lipophilicity of the ion [80]. In theory, the most relevant case for in vivo extrapolation is when V /V 1 as it corresponds to the phase ratio encountered by a drug as it distributes within the body. The measurement of apparent log Pi values does not allow to differentiate between ion-pairing effect and partihoning of the ions due to the Galvani potential difference, and it has been shown that the apparent lipophilicity of a number of quaternary ion drugs is not due to ion-pair partitioning as inihally thought [80]. 

Niflumic acid, which has two pKa values, was studied both pH-metrically and spectroscopically using the shake-flask method [224]. The monoprotonated species can exist in two forms (1) zwitterion, XH 1 and (2) ordinary (uncharged) ampholyte, XH°. The ratio between the two forms (tautomeric ratio) was measured spectroscopically to be 17.4. On assuming that a negligible amount of zwitterion XH partitions into octanol, the calculated micro-log/1 for XH° was 5.1, quite a bit higher than the macro-log/1 3.9 determined pH-metrically in 0.15 M NaCl. It is noteworthy that the distribution coefficient D is the same regardless of whether the species are described with microconstants or macroconstants [275]. 

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. 

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. 

While applications and analysis of ILs may provide some guidance on potential applications of SCIL-based phases in EC, these phases may also provide useful information about ILs. As Poole points out [16], a key requirement for the successful integration of ILs in industrial processes is the ability of being applied to rapid liquid-liquid phase separation systems. Shake-flask methods are commonly used to measure IL/water partition coefficients. However, the high viscosity and cost of these materials coupled with the time and effort required for traditional shake-flask methods render this... 

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. 

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 classical measurement of LogP is the shake flask method [17]. A known amount of drug is dissolved in a flask containing both octanol phase and aqueous buffer at controlled pH to ensure the existence of only nonionic form (at least two units from the drug pA) ). The flask is shaken to equilibrate the sample between two phases. There must be no undissolved substance present in both phases. After the system reaches its equilibrium, which is time- and temperature-dependent, the concentration of drug is analyzed by HPLC in both phases. Partitioning coefficient is calculated as... 

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... 

Further analysis of the physical nature of the partition coefficient reveals that it is a composite property depending on size, shape, dipole moment, and hydrogen-bonding ability. Although many researchers have attempted to replace logP with other simple parameters, only limited success has been achieved for some, but not all, molecules. It appears that for entirely new complex molecules, it still would be necessary to measure the partition coefficient, preferably validated by the conventional 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). 

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).

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. 

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