By mass balance, the equilibrium amount of A in octanol is given as the difference between the initial amount of A in water and its equilibrium amount in water. If the volume of the octanol phase equals that of the aqueous phase, then a corresponding statement holds for the concentrations; the equilibrium concentration of A in octanol is the difference between its initial concentration in water and its equilibrium concentration in the aqueous phase 2 where c W init is the initial concentration of A in water.
Because the peak area of an 1 H NMR signal integration is proportional to the concentration of the protons causing it and so to the concentration of the compound of interest , we can replace the concentrations by 1 H signal integrations 3. Absolute integrations, however, are not usually displayed by NMR instruments. Moreover, integrations before and after equilibration may differ because of magnetic-field drift.
Such a change in the signal integration, if any, should be the same for both the signal of interest and the water signal. Of course, I water is proportional to the water concentration in the aqueous phase which should be nearly constant before and after equilibration, as long as the concentrations of the analyte and 1-octanol in the water phase are low.
This means that water is used as the internal standard. The experiment thus consists of dissolving an amount of compound A in water in an NMR tube, taking an 1 H NMR spectrum, adding an equal volume of 1-octanol, mixing the phases intensely in the NMR tube, allowing for phase separation, and again taking an NMR spectrum of the aqueous lower phase Figure 1. A characteristic peak of compound A is integrated against the water peak in both spectra Figure 2. High Resolution Image. Representative Procedure.
It is advisable to repeat the steps of phase mixing, phase separation, and spectrum recording to ensure that partition equilibrium was achieved. Figure 3 shows that in an experiment with methanol 10 inversions were not enough to approach the partition equilibrium, while 20 inversions were sufficient.
As in this experiment, we usually performed five parallel side-by-side runs. The results obtained by this method are shown in Table 1 , columns 1—4. Table 1. In most cases, after equilibration, the volume of the octanol phase does not equal that of the aqueous phase, and therefore, the assumption of equal volumes used in derivation of eqs 2 — 4 is not justified.
This is due to the following two effects: i The mutual solubility of water and 1-octanol: while the solubility of octanol in water is very low, the equilibrium solubility of water in octanol at room temperature is reported to be about 5 mass percent. Both effects are corrected for by taking the phase volume ratio into account as a factor in K OW determination or its logarithm as an additive term for log K OW , eqs 5a and 5b.
The phase volume ratio is easily measured using a ruler as the ratio of the final phase column heights in the NMR tube. The results obtained according to eq 5b are shown in Table 1 , column 7.
The fundamental reason for preferring log K OW over K OW itself is the linear dependence of log K OW on an energy variable eq 6b that in turn is the sum of energy contributions from structural motifs present in a molecular structure. Thus, log K OW increases linearly with the number of hydrophobic structural elements such as methylene groups, benzene rings, and chlorine atoms in a molecule. Then, replacing n O equil by n W init — n W equil and replacing each n by RI as above directly lead to eq 5a.
Because the phase volume ratio in an NMR tube is easily measured using a ruler and is explicitly taken into account in this method, it is not critical to set the phase volume ratio to a predetermined value, for example, This obviates the need to mutually pre-equilibrate water and octanol and renders the exact amounts of water and octanol used uncritical, while the exact amount of analyte A within its limit of solubility does not matter either.
The method requires the analyte to be sufficiently soluble in water. An NMR signal has to be obtained that can be integrated against the strong water peak with sufficient accuracy. In our experiments, also methyl acetate, n -propanol, and butanone, compounds fitting in this log K OW range, provided good results. To extend the amenable log K OW range to the lower side, we subjected 1,4-dioxane, methanol, DMF, acetamide a solid , and DMSO to the procedure; the results were close to the respective literature values for the former three compounds, whereas acetamide and DMSO showed some deviations.
Larger deviations from the literature were observed for an acid acetic acid and a base pyridine. In such cases of partially ionized compounds, the quantity measured is called log D , describing the overall distribution of the neutral and the ionized species, in contrast to log P that describes the partition of a single species.
Ionization depends on both pH and concentration. For these exploratory experiments, we used unbuffered solutions.
A corresponding association may also play a role for acetamide. In all previous variations of the shake-flask method that analyze one phase only, mutually pre-equilibrated samples of water and 1-octanol are used to avoid uncontrolled volume changes. Initially, in our experiment, we waived this precaution for experimental ease, which was justified by this effect being small compared to the volume effect of the analyte in most cases.
Theoretically, the analyte effect on the phase volume ratio could be minimized by using a far lower amount of the analyte. Therefore, in our final procedure, we adjust for both effects by explicitly taking into account the phase volume ratio eq 5 , easily measured in an NMR tube.
For a thorough discussion of the role of the phase volume ratio in K OW measurement, see ref In the case of an alcohol, the signal of all OH protons coincides with the water peak in the initial spectrum, whereas in the equilibrium spectrum, some OH protons escape measurement by the alcohol partially evading into the octanol.
This can be corrected for by diminishing, in the evaluation of both spectra, the water peak by the contribution of alcohol OH protons, as taken from the observed characteristic NMR signal of the analyte.
For methanol, this correction increased log K OW by 0. For these alcohols, their corresponding corrections are included in Table 1 , whereas for the other butanols, the corrections were less than 0. Being a variation of the shake-flask method, our procedure is in principle subject to potential errors as described for the shake-flask method in ref 6.
A problem specific to the present NMR method is the use of the water peak as the internal standard. After all, we know that the water concentration in the aqueous phase is not constant. Another source of error and a severe limitation of the method is the uncertainty in the NMR peak integration, particularly for peaks of very different intensities, such as the water peak and the analyte peak in cases of higher K OW. In addition, unfortunately eqs 4 , 5a , and 5b by their very form are highly sensitive to measurement errors because of RI W equil , a number often close to zero, appearing as the denominator.
Similarly, for compounds of very low K OW , the numerator becomes close to zero. Furthermore, coarseness of the integration display results in coarseness of log K OW. Had RI W equil been found as 0. However, in many such cases, one peak is more suitable than others. For example, in 2-propanol, the CH 3 doublet is integrated more reliably than the CH septet; therefore, the log K OW given in Table 1 for 2-propanol is derived from the methyl signal only.
Of course, the method is not restricted to using a low-field instrument. However, even with a compact NMR instrument as used here, improvement may be achieved if absolute intensities rather than relative to water intensities were displayed.
This would liberate us from using H 2 O as the internal standard, allowing treatment of less water-soluble compounds or allowing measurement in D 2 O rather than in H 2 O, both with the effect to broaden the range of amenable log K OW to higher values. Experimental Section. Water was Millipore water of resistivity For integration, the Mnova software which comes with the instrument was used with integrals displayed to three decimal places.
For the effect of temperature on log K OW , see the literature. Author Information. The authors declare no competing financial interest. Partition coefficients and their uses Chem. A review with refs. An extensive tabulation of partitioning data is presented. Determination of log K OW values for four drugs J. The octanol-water partition coefficient: Strengths and limitations Environ. Environmental Organic Chemistry , 3 rd ed. Google Scholar There is no corresponding record for this reference.
The measurement of partition coefficients Quant. A discussion and review with 48 refs. American Chemical Society. We present a new atom type classification system for use in atom-based calcn. The 68 at. A sep. Both calcns. Octanol-water partition coefficients of simple organic compounds J. Data , 18 , — DOI: Octanol-water partition coeffs. Recommendations are given. Pertinent thermodn. Setup and validation of shake-flask procedures for the determination of partition coefficients log D from low drug amounts Eur.
Elsevier B. Cs is the concentration of analyte in sample phase; Cg is the concentration of analyte in gas phase. Compounds that have low K values will tend to partition more readily into the gas phase and have relatively high responses and low limits of detection.
Compounds that have high K values will tend to partition less readily into the gas phase and have relatively low responses and high limits of detection.
Sensitivity is increased when K is minimised Sensitivity is increased as the partition coefficient is decreased and volatiles can more readily enter the gas phase. This is illustrated by the graph on the left. K can be lowered by changing the temperature at which the vial is equilibrated or by changing the composition of the sample matrix.
The partition coefficient may also be changed by adding salts or by changing the Phase Ratio. These will be examined in the next two sections. High salt concentrations in aqueous samples decrease the solubility of polar organic volatiles in the sample matrix and promote their transfer into the headspace, resulting in lower K values. The magnitude of the salting-out effect on K, however, is not the same for all compounds. Compounds with K values that are already relatively low will experience very little change in the partition coefficient after adding a salt to an aqueous sample matrix.
Generally, volatile polar compounds in polar matrices aqueous samples will experience the largest shifts in K and have higher responses after the addition of salt to the sample matrix. Common salts used to decrease matrix effects:. Partition coefficient determines how much of a given solute ends up in which phase and is of major importance to the cosmetic chemist.
De Paula and P. Atkins, Atkins' Physical Chemistry, 7th ed. J Sangster, Octanol-water partition coefficients: Fundamentals and physical chemistry, Vol. Partition Coefficient.
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