Chemistry Reference
In-Depth Information
Experimental measurement of the partition coefficient
There are three convenient ways in which
P
can be determined in the chem-
istry laboratory. These are the original
shake flask method
, the use of
thin-
layer chromatography
or the use of
reversed-phase
,
high-performance
liquid chromatography
.
Shake flask method
In the shake flask method, the drug whose
P
is to be determined is tradition-
ally added to a separating funnel containing the two immiscible phases,
although it works just as well to use a centrifuge tube (and requires less
sample). The two immiscible phases chosen are usually 1-octanol and pH
7.4 buffer. Octanol is used in partition coefficient work because the answers
obtained from octanol seem to correlate best with biological data obtained
in vivo
. This may be because the eight carbon atoms are essentially
hydrophobic
(or water-hating) and the one hydroxyl group is
hydrophilic
(water-loving) and together they give the closest balance to that found in
human cell membranes. The aqueous buffer at pH 7.4 represents aqueous
compartments within the body, e.g. blood plasma.
The two phases are thoroughly mixed to give buffer-saturated octanol
in the top phase and octanol-saturated buffer in the bottom. Once the two
phases have separated (this can take a while), the drug is added and the
whole flask is shaken mechanically for at least an hour. The two phases are
allowed to separate (or centrifuged, if you are in a hurry) and the concen-
tration of drug in the aqueous phase is then determined. This may be done
by titration if the drug is sufficiently acidic or basic or, more usually, spec-
trophotometrically. The concentration in the octanol phase is found by
subtraction and the value of
P
is calculated. This method works perfectly
well if there is sufficient sample and the drug possesses a chromophore to
allow spectroscopic assay of the aqueous phase.
What is important in liquid-liquid extractions of this type is not the
volume of the organic phase but rather the number of times the extraction is
carried out. Five extractions of 10 mL organic phase will remove more
compound than one extraction of 50 mL, even though the total volume of
organic solvent used is the same. Similarly, ten extractions of 5 mL will be more
efficient still, and so on. This effect (which is general to all extractions) is
obvious when thought about. Each time one phase is removed and replaced by
fresh solvent, the equilibrium for the partitioning process must re-establish
according to the partition coefficient ratio and drug must leave the aqueous
phase to enter the organic phase and restore the equilibrium ratio.
