Octanol/aqueous shake flask

Distribution of benzodiazepines in I-octanol - water system was investigated by a direct shake flask method at the presence of the compounds used in HPLC mobile phases the phosphate buffer with pH 6,87 (substances (I) - (II)), acetic and phosphate buffer, perchloric acid at pH 3 (substances (III) - (VI)). Concentrations of substances in an aqueous phase after distribution controlled by HPLC (chromatograph Hewlett Packard, column Nucleosil 100-5 C, mobile phase acetonitrile - phosphate buffer solution with pH 2,5, 30 70 (v/v)). 

A detailed descriphon of octanol-water distribuhon coefficient measurements by shake-flask can be found in publications by Dearden [2] and Hansch [24], The method usually involves the following solubilization of the compound in a mixture of mutually presaturated buffered water and octanol, agitation unhl equilibrium has been reached, careful separation of octanol and aqueous phases, and direct measurement of the solute concentration in both phases. Although seemingly simple, the method has a number of caveats making it inappropriate for some compounds. 

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... 

Fig. 12.3. An aulo-sampicr needle sampling from the lower aqueous pha.se through the coloured octanol phase without cniss-contamination during the high-throughput micro-shake-flask method for the octanol-waler partition coelTicient determination.

Different procedures provide comparable values for /fow with log Aiow < 5. However, there is some indication that lower values can result from the shake flask procedure probably resulting from higher concentrations in the aqueous phase due to the formation of emulsions. Some octanol-water partition coefficients are compiled in Tables 2.17 and 2.18. 

Especially when calibrated with standard compounds relative to the classical shake-flask method, rp-HPLC is the method of choice for the experimental determination of partition coefficients. The liability to large variation in measured log for the highly lipophilic compounds (log > 5) subjects the experimental data to major uncertainties and for such molecules calculation is generally preferable (Taylor, 1990). Reported log P >1 (e.g. for some chlorinated dioxins up to 11), corresponding to concentration differences between the aqueous and the 1-octanol phase of about 10 and more are not exact values, but reflect the accuracy of the experimental analytical techniques used they may be subject to major uncertainties of 2 log units. The respective data have to be treated with great caution in QS AR studies, because at best they indicate the order of magnitude of the compounds lipophilicity. 

Few measurements of important parameters are as simple to make as the partition coefficient of a solute between water and octanol. The principles of the shake-flask measurements remain the same as in the Berthelot and Jungfleisch reports of 1872 and the further analysis by Nemst. Yet the interplay of solvation forces in both the water and octanol phases are so involved and complex that current molecular mechanics and quantum chemical calculations can dispel very little of the empiricism that now dominates logP estimation. It is difficult to predict how soon ab initio calculations will give us dependable information regarding the conformation, the tautomeric form, and the electron distribution of complex solutes, not just as they exist in a vacuum or crystal, but also as they exist in the aqueous phase as well as the water-saturated octanol phase. At present the quantum chemical methods that look the most promising for the complex solutes seem to fail when applied to the simplest hydrophobic organic structures of all the aliphatic hydrocarbons. 

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