# Cromatography Handbook of HPLC - Rizzi A.

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A combined approach was developed by Compudrug Ltd. (Budapest, Hungary) for the calculation of log P values by their ProLogP [16] expert systems. The possibilities of the calculation of log P values by personal computers have been summarized [17]. These calculation methods are mentioned here because they were used in the correlation studies with the chromatographic hydrophobicity values.

Hydrophobicity Determination

As early as 1941 Martin and Synge [18] showed that the thin-layer chromatographic (TLC) R{ values can be related to P according to Eq. (2):

P = constant • ^ yj (2)

In 1963 Green and Marcinkiewicz [19] pointed out that the RM values, which can be defined by Eq. (3) are linearly related to the log P values and, therefore, RM values are analogous to these values.

The first exploitation of this relation in biological context was by Boyce and Milbarrow [20], who showed a relation between the molluscicidal activity of some TV-alkyltritylamines and their RM values on TLC plates.

The applicability of chromatographic methods for characterizing the hydrophobicity of the compounds can be supported by two facts. First, the chromatographic retention (log k' values in HPLC or RM values in TLC) is proportional to the solute distribution in the mobile and the stationary phases and, therefore, analogous to the distribution or partition in two nonmiscible solvents. Second, Collander [7] in 1951, found linear correlations between partition coefficients obtained in two different partition systems, described by Eq. (4).

log P, = a log P7 + b (41

where 1 and 2 refer to two liquid-liquid partition systems, a and b are constant.

Leo [21], however, demonstrated the limitations of the Collander type [Eq. (4)] relation by investigating the partition coefficients of more than 300 compounds in 12 various liquid-liquid partition systems. He found that the linear relation between log P values obtained in two different partition systems was valid only when the partitioning solvents in the two systems were chemically related. When the partition coefficients obtained in octanol-water and in hexane-water were compared, the linear correlation was valid only when he divided the compounds into proton donor and proton acceptor groups. The values of the a and b parameters were different in the two cases. The deviation was explained by the fact that,

Physicochemical Measurements

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although 1-octanol is able to form hydrogen bonds, the hexane is not; therefore, different weak interactions dominate the partition process in the two systems. So, he stated that Eq. (4) can be valid only when either the compounds or the partitioning liquids are similar in character. These limitations are valid also when the partition coefficients (or analogous retention parameters) obtained by chromatographic and shake-flask method are compared. We can expect linear correlations for only structurally related compounds, or when directly 1 -octanol and water is applied in the chromatographic partition system.

Chromatographic methods can be applied for characterizing the molecular hydrophobicity only when the chromatographic retention (i.e., distribution) is governed mostly by hydrophobic forces. This means that the chromatographic system (the mobile and the stationary phases) should contain aqueous and nonpolar phases. Therefore, only reversed-phase HPLC can be used when the stationary phase is usually a chemically bonded n-paraffin hydrocarbon silica and the mobile phase is water or a mixture of water and organic solvent. (In TLC the silica plates can be impregnated by paraffin or octanol; also, a hydroorganic solvent can be used for the development.)

In HPLC, the most frequently used chromatographic retention parameter for characterizing hydrophobicity is the logarithmic value of the retention factor (log k' = log (tR - tM)/ tM, were tR and ?M are the retention times of the compound and an unretained compound, respectively), which can be related to the chromatographic partition coefficient (Kchr) according to Eq. (5).

log k' = log Kclu + log(jpj (5)

where Vs and Vm are the volume of the stationary and the mobile phases, respectively, and Vs/Vm is the so-called phase ratio. The phase ratio can be considered as constant, but it is difficult to determine its exact value. Therefore, it is necessary to determine the log k' values of a few standard compounds with known log P values, and by plotting their log k' values against their log P values, the parameters a and b of Eq. (4) can be determined. It might cause differences that, whereas the shake-flask method refers to a static equilibrium, chromatography provides a “dynamic” equilibrium system. This means that during the chromat-ographic process, the compound actually is never in equilibrium, it always goes through numerous local equilibrium processes.

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