Ion pair partitioning

It is useful to conclude with several examples from our laboratory. We have applied the procedure of ion pair partition chromatography to the separation of biochemically important mixtures (47,48). In this method of liquid-liquid partition, the basic distribution process can be written as... 

Figure 7. Separation of eight biogenic amines using ion pair partition liquid chromatography. Conditions 30 cm column with 4 fim silica stationary phase, 0.1M HClO /0.9M NaClO mobile phase, ethylacetate/tributyl phosphate/hexane (72.5/10/17.5) velocity, 0.8 cm/sec (47).

For the parhtioning of the latter ion, the ion-pair partition coefficient may be defined as ... 

The lipophilicity profile of an acid is a mirror image of the shape in Fig. 3.1, i.e. the maximum (neutral-species) partitioning occurs at low pH and the minimum (ion-pair) partitioning at high pH. Other shapes are described in the review by Avdeef [18]. 

The experimental approaches used to characterize ion-pair partitioning are cyclic voltammetry and potentiometric titration. Cyclic voltammetry is overall more powerful, but requires special instrumentation which is not commercially available as a ready-to-use set-up. For this reason the potentiometric titration technique has been more widely used. 

However, as stated above, the partition coefficients measured by the shake-flask method or by potenhometric titration can be influenced by the potenhal difference between the two phases, and are therefore apparent values which depend on the experimental condihons (phase volume ratio, nature and concentrahons of all ions in the solutions). In particular, it has been shown that the difference between the apparent and the standard log Pi depends on the phase volume raho and that this relationship itself depends on the lipophilicity of the ion [80]. In theory, the most relevant case for in vivo extrapolation is when V /V 1 as it corresponds to the phase ratio encountered by a drug as it distributes within the body. The measurement of apparent log Pi values does not allow to differentiate between ion-pairing effect and partihoning of the ions due to the Galvani potential difference, and it has been shown that the apparent lipophilicity of a number of quaternary ion drugs is not due to ion-pair partitioning as inihally thought [80]. 

Takacs-Novak, K., Szasz, G. Ion-pair partition of quartemary ammonium... 

Takacs-Novak, K. Szasz, G., Ion-pair partition of quaternary ammonium drugs The influence of counter ions of different lipophilicity, size, and flexibility, Pharm. Res. 16, 1633-1638 (1999). 

All of the methods described and referenced here are capable of producing high-quality, accurate results, but only if the raw data are correctly interpreted and analyzed to detect (and correct) for issues arising from issues such as low solubility, ion pair partition or multiple ionizable groups with overlapping pKas. It is not... 

Two types of system are used for ion-pair liquid chromatography. When polar stationary phase materials, such as silica gel, are used an ion-pair partition mechanism is applied. When non-polar stationary phase materials, such as octadecyl-bonded silica gel and polystyrene gel, are employed a paired-ion adsorption mechanism is involved. The former is called normal-phase ion-pair partition liquid chromatography, and the latter is called reversed-phase ion-pair liquid chromatography. 

Normal-phase Ion-pair Partition Liquid Chromatography... 

The molecular absorption intensity of polar compounds is usually small, but highly sensitive detection can be obtained after pre- or post-column derivatiza-tions. The use of ultraviolet absorption or fluorescence-active counter-ions makes it possible to achieve highly sensitive detection of polar compounds and enhance the capability of ion-pair liquid chromatography. For example, N,N-dimethylprotriptyline has been used as a counter-ion for carboxylic acids12 and picric acid for quaternary amines13 in normal-phase ion-pair partition liquid chromatography. Phenethylammonium, cetylpyridinium, l-phenethyl-2-pyco-linium, and naphthalene-2-sulfonic acid have been used for sulfonic acid and alkyl amines detection.14,15 Ion-pair post-column extraction was applied on-line for fluorescence detection.16... 

W. Santi, J.M. Huen, and R.W. Frei, High-speed ion-pair partition chromatography in pharmaceutical analysis , J. Chromatogr., 1975,115, 423. 

The use of distribution coefficients for the QSAR treatment of ionizable compounds has been extended to consideration of ion-pair partitioning into biolipid phases. Two experimental methods for determining ion-pair partition coefficients are described. One is a single-phase titration in water-saturated octanol, in which case (for acids) log Pj = log P + pKa - pKa. The other is a two-phase titration (octanol/water) from which the ratio (P + 1)/(Pj + 1) can be calculated. An example outcome is that the uncoupling activity of phenols can be represented by an equation in log instead of log D and pKa. 

Martin H) has written a perceptive analysis of the possible ways in which an ionized species may behave in various models and contribute to or be responsible for a given activity. QSAR studies that have dealt with ion-pair partitioning include a study of fibrinolytics ( ) and the effect of benzoic acids on the K ion flux in mollusk neurons ( ). Schaper (10) recently reanalyzed a large number of absorption studies to include terms for the absorption of ionized species. Because specific values were not available for log Pj, he let the relation between log Pi and log P be a parameter in a nonlinear regression analysis. In most cases he used the approximation that the difference between the two values is a constant in a given series. This same assumption was made in the earlier studies (, ) Our work suggests that the pKa of an acid can influence this differential (see below). The influence of structure on the log P of protonated bases or quaternary ammonium compounds is much more complex (11,12) and points out the desirability of being able to easily measure these values. 

When the ion-pair partitioning is indicated in the quadrant diagram (below) it becomes obvious that a circle of equilibria is present. Knowing the octanol pKa, the log P and the aqueous pKa should allow one to calculate the partition coefficient of the ion pair. From these equilibria one can write that the difference in log P between the acid and its salt is the same as the difference between the pKa s (Equation 9). The closer the pKa s, the more lipid soluble the ion pair will be, relative to the acid. Internal hydrogen bonding or chelation that stabilizes an ion pair will affect the octanol stability more than the aqueous stability, where it is less needed, and so will decrease the delta pKa. Chelation should therefore favor biolipid solubility of ion pairs. Ultimate examples are available in some ionophores. This is one of the properties of some of the herbicides I pointed out earlier. 

We decided to try a direct titration in water-saturated octanol. What we hoped to achieve, if not duplicating literature values, was to obtain partition coefficients proportional to "true" values so that regression analyses could be run and would be meaningful. Secondly, this should be a rapid method to assess structural features in a series for their effect on ion-pair partitioning. 

The reader who doesn t continue to the end of this chapter should be cautioned that Equations 18 and 19 hold only when ion-pair partitioning is not significant.]... 

Sometimes the log P from a two-phase titration using Equations 18 or 19 is low, compared with shake-flask values. We attribute this to ion-pair partitioning. The quadrant diagram. Figure 8, is helpful for developing the pertinent equations. The amount of each species in each phase is shown in the appropriate sector. 

If [X] is the concentration of AH in the aqueous phase (Figure 8), the concentration of AH in the octanol is P times this. The aqueous-phase concentration of the ion is [X] times the degree of dissociation. Multiplying this product by the ion-pair partition coefficient Pj (or P -) gives the concentration of the ion pair in the octanol. The actual amount of species in a phase is given by its concentration times the volume of the phase. At the pK, the amount of neutral species equals the amount of ionized species. Setting the sum of the two terms in the left quadrants equal to the sum of the two terms on the right, one can derive Equation 20. The equivalent e ression for bases is Equation 21. 

The recent work of Wang and Lien (29) illustrates that ion-pair partitioning occurs to a greater extent than previously realized. Partition coefficients calculated from measurements made on partially ionized compounds depend not only on the pH, but on the buffer used. They may vary by more than one log unit. The authors derived equations to correct log P to octanol/water values, but these can still be off by several tenths of a log unit. A preferable solution would be to know the log Pj and account for ion-pair partitioning. 

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