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Another question is the mobile-phase composition. The pH of the hydroorganic mobile phase can be very important for ionizable compounds. The appearance of more than one species of a compound (ionized and neutral) often results in nonsymmetric peak shapes, which might also influence an accurate retention determination. Therefore, it is even more important to use a mobile-phase pH in which the solute is in neutral form, than it is with the shake-flask method. As the practical log k' values range between -1 and 2, compounds with only a narrow range of hydrophobicity could be determined by using water as the mobile phase. The application of a water-miscible organic modifier in the mobile phase makes it possible to use RP-HPLC for hydrophobicity determination of compounds with much wider range of hydrophobicity. When only the log k' values are applied for characterizing the hydrophobicity of the compounds, the parameters of Eq. (5) should be determined for each concentration of the organic phase in the mobile phase by using standard reference compounds with known log P values.
When the mobile-phase composition has to be changed for the measurement of a compound series, the log k' versus organic phase concentration ô should also be taken into consideration. Let us suppose that the log k' versus ô plot is linear. Then by measuring the log k' values at various mobile-phase compositions the linearity of the plot can be tested. It was suggested  that the extrapolated log k' value to the zero concentration of the organic modifier (log k'w; i.e., log k' values referring to only water as the mobile phase) can then be
correlated with the log P values, it can be considered a measure of hydrophobicity. However, the log ê^ values are different if they are obtained from acetonitrile-water or methanol-water mobile-phase mixtures [23,24]. Schoenmakers et al.  describe the solute retention over the full range of mobile-phase composition in RP-HPLC, with special emphasis on mobile phases with a high water content. A quadratic relation between the logarithm of the capacity factor and the volume fraction of organic modifier is generally valid for mobile phases containing less than 90% water; however, when more water is added to the mobile phase, a quadratic equation is insufficient.
Good correlations were generally found between log k^ and log P values only for structurally related compounds [26-28]. Dissimilar compounds (noncongeners) usually showed poorer correlations between the log P and log k'w values [29-33]. Figure 1 shows the plot of the log k'w (obtained in a regular RP-HPLC system using acetonitrile as an organic modifier in the mobile phase) and log P values of structurally unrelated compounds. When the compounds are subdivided into congeneric groups, the correlation can be improved. The explanation for this is that the limitation of the Collander-type relation revealed by Leo , is a differential hydrogen-bonding effect that is qualitatively similar to that observed when 1-octanol-water partition coefficients are correlated with alkane-water systems. The usual RP-HPLC system, as well as the alkane-water systems, exhibit greater hydrogen-bonding selectivity than does the octanol-water system.
1.6 - Õ6 x10 14
1.4 - X *22
X D x 13
1.0 - CO xl8
> 00 X **3 x 9 X20
î 17 *
tji 0.6 - x 21
Ï . 7 X 12 1 ! 1 ³ * ³ i .. t 1 ........-
-0. 5 0 0.5 1.0 1.5 2.0 2.5 3.0 3.5
Figure 1 The plot of the log k’w values against the log P values of structurally unrelated drug molecules. (From Ref. 33.)
D. Practical Aspects of Using HPLC for Hydrophobicity Determination
As early as 1976, Mirrlees et al.  described a thin-layer chromatographic method for modeling the log P values of noncongeneric compounds by using octanol-saturated Kieselguhr as the stationary phase and octanol-saturated water as the eluant. Unger et al.  obtained very good correlations between log P values of noncongeneric compounds and their log k' values by applying octadecyl silica gel dynamically coated by 1-octanol and octanol-saturated water as the mobile phase. This approach can be considered to be the most straightforward to produce a chromatographic partition system that is most similar to that of 1-octanol-water. However, this approach suffers from practical disadvantages. The stationary phase reproducibility is very poor. We cannot define exactly the adsorbed volume of octanol on the stationary phase and, therefore, the reproducibility of the measurements is rather poor. To increase the reproducibility, the measurements of log k' values of several reference compounds with known log P values are necessary. The more difficult problem in this approach is that we cannot adjust these chromatographic systems for a wide range of compounds (exerting a wide range of hydrophobicity), because the mobile-phase composition cannot be varied.