partition coeff

Journal of Chromatography A, 1037 (2004) 314

Review

Determination of liquidliquid partition coefficientsby separation methods

A. Berthod a,, S. Carda-Broch ba Laboratoire des Sciences Analytiques, CNRS, Universit de Lyon 1, Bat CPE-308, 43 Boulevard du 11 November 1918,

69622 Villeurbanne Cedex, Franceb Area de Qumica Analtica, Universidad Jaime I, Cra. Borriol s/n, 12080 Castelln de la Plana, Spain

Abstract

By essence, all kinds of chromatographic methods use the partitioning of solutes between a stationary and a mobile phase to separate them.Not surprisingly, separation methods are useful to determine accurately the liquidliquid distribution constants, commonly called partitioncoefficient. After briefly recalling the thermodynamics of the partitioning of solutes between two liquid phases, the review lists the differentmethods of measurement in which chromatography is involved. The shake-flask method is described. The ease of the HPLC method is pointedout with its drawback: the correlation is very sensitive to congeneric effect. Microemulsion electrokinetic capillary electrophoresis has becomea fast and reliable method commonly used in industry. Counter-current chromatography (CCC) is a liquid chromatography method that usesa liquid stationary phase. Since the CCC solute retention volumes are only depending on their partition coefficients, it is the method of choicefor partition coefficient determination with any liquid system. It is shown that Ko/w, the octanolwater partition coefficients, are obtained byCCC within the 1 < logKo/w < 4 range, without any correlation or standardization using octanol as the stationary phase. Examples ofapplications of the knowledge of liquidliquid partition coefficient in the vast world of solvent extraction and hydrophobicity estimation arepresented. 2004 Elsevier B.V. All rights reserved.

Keywords: Reviews; Partition coefficients; Octanolwater partition coefficients; Thermodynamic parameters; Distribution constants; Hydrophobicity;Counter-current chromatography; Alkylbenzenes; Quinolines

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42. Partitioning of solutes between two liquid phases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

2.1. Nomenclature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42.1.1. Distribution ratio (D) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42.1.2. Distribution constant or partition ratio (KD) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

2.2. Thermodynamics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52.3. Effect of temperature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52.4. Effect of chemical reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

3. Measurement of partition coefficients . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63.1. The shake-flask method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

3.1.1. Small-scale spectroscopic methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63.1.2. Separation methods for phase analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63.1.3. Modern variations on the shake-flask method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

Corresponding author. Tel.: +33-472431434; fax: +33-472431078.E-mail address: [email protected] (A. Berthod).

0021-9673/$ see front matter 2004 Elsevier B.V. All rights reserved.doi:10.1016/j.chroma.2004.01.001

4 A. Berthod, S. Carda-Broch / J. Chromatogr. A 1037 (2004) 314

3.2. Liquid chromatography and correlations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73.2.1. A simple and easy method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73.2.2. Congeneric effect . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73.2.3. Variations on the method to improve the correlation quality . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

3.3. Capillary electrophoresis with ordered media . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83.4. The counter-current chromatography method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

3.4.1. Direct partition coefficient measurement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93.4.1.1. Method description . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93.4.1.2. Method range and accuracy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93.4.1.3. Method variation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

3.4.2. Using the liquid nature of the stationary phase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103.4.2.1. Dual-mode CCC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103.4.2.2. Cocurrent CCC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

4. Application: homologues and partition coefficients . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114.1. Alkylbenzene partition coefficient in the heptanemethanolwater system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 124.2. Petroleum components and waterless biphasic liquid systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 124.3. Quinoline homologues in heptaneacetonitrilemethanol systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

5. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

1. Introduction

If a third substance is added to a system of two immis-cible liquids in equilibrium, the added component will dis-tribute itself between the two liquid phases until the ratio ofits concentrations in each phase attain a certain value: thedistribution constant or partition coefficient.

Studied as soon as the end of the 19th century [1], the dis-tribution of solutes in biphasic liquid systems has become anessential field of study. The octanolwater distribution ratio,Ko/w, is the accepted physicochemical property measuringthe hydrophobicity of chemicals [2]. Ko/w is the most widelyemployed descriptor for quantitative structureactivity rela-tionship (QSAR) studies [3,4].

Besides pharmaceutical and biochemical industry thatuses QSAR in drug design and toxicology, the measurementof liquidliquid partition coefficients is extremely impor-tant in: (1) fundamental chemistry for studying inorganicand/or organic complex equilibria; (2) industrial chemistryfor optimization of production and waste treatment; and (3)food chemistry for purification and extraction of sugars, fator caffeine [5]. This field is so important that many univer-sities offer a special course in liquidliquid partitioning andsolvent extraction.

Combinatorial chemistry is able to produce large num-bers of new compounds that could be potential drugs[6,7]. A drug has to cross four barriers associated withabsorption, distribution, metabolism and excretion referredas ADME interface [8]. One of the core properties re-quired to assess ADME characteristics is hydrophobicitythat is Ko/w. If the Ko/w determination is not to be a bot-tleneck in the combinatorial discovery process, it mustbe at least as easy as it is to synthesize the compound[9].

Partitioning of solutes between a stationary and a mobilephase is the fundamental principle of all kinds of chromato-graphic methods. Separation methods are the tools of choiceto determine liquidliquid partition coefficients. This articlewill describe the different ways offered by separation meth-ods focusing on accuracy, ease and speed of the differentmethods: classical methods, high-performance liquid chro-matography (HPLC), capillary electrophoretic (CE) meth-ods and counter-current chromatography (CCC).

2. Partitioning of solutes between two liquid phases

2.1. Nomenclature

The term liquidliquid partition coefficient is notrecommended by IUPAC in its 1993 nomenclature forliquidliquid distribution [10]. Since this term was widelyused, not recommended is read as not forbidden and itcontinues to be preferred to the recommended terms thatare distribution constant or partition ratio whose symbolshould be KD. To be understood by chromatographers, theterm partition coefficient will be used throughout this arti-cle with the correct symbol, KD, but the IUPAC definitionsare recalled here. IUPAC points the common confusionmade between distribution ratio and distribution constant(or partition coefficient).

2.1.1. Distribution ratio (D)The ratio of the total analytical concentration of a solute

in the liquid phase 1, regardless of its chemical form, toits total analytical concentration in the other phase. As de-fined, the distribution ratio varies with experimental condi-tions (chemical reaction, precipitation, ionization). It should

A. Berthod, S. Carda-Broch / J. Chromatogr. A 1037 (2004) 314 5

not be confused with the distribution constant (or partitioncoefficient). The distribution ratio is an experimental param-eter and its value does not necessarily imply that distributionequilibrium between phases has been achieved [10].

2.1.2. Distribution constant or partition ratio (KD)The ratio of the concentration of a substance in a single

definite form, A, in the liquid phase 1 to its concentrationin the same form in the other phase (liquid phase 2) atequilibrium:

KD = [A]1[A]2 (1)

In equations relating to aqueous/organic systems, the organicphase concentration is, by convention, the numerator andthe aqueous phase concentration the denominator. This def-inition clearly states that the substance should have a singledefinite form. It means that the distribution constant (parti-tion coefficient) is a true constant. If the substance evolves,the distribution ratio (D) will change; the distribution con-stant (KD) will not since it is, by definition, invariant. Ofcourse, if the solutes are not ionizable and do not changedue to chemical reaction or complexation, then D = KD.

2.2. Thermodynamics

If we consider two practically immiscible solvents, 1 and2, they form two liquid phases of one solvent saturated in theother. When a solute A is introduced in such a biphasic liquidsystem, it distributes between the two phases. Assumingideal mixtures, in the solvent 1 phase, the Gibbs free energyof A, or chemical potential, 1A, is expressed by:

1A = 01A + RT ln x1A (2)where 01A is the standard chemical potential of A at infinitedilution in liquid phase 1. Similarly, in the other phase, thechemical potential, 2A, is:

2A = 02A + RT ln x2A (3)If the chemical potential is not identical in the two phases,mass transfer of A occurs, the mole fractions x change so thatthe chemical potential of A becomes equal in both phases,i.e. the equilibrium is reached. Then:

01A 02A = RT ln(x2Ax1A

)(4)

in which x2A/x1A is the distribution constant, KD, expressedby:

x2Ax1A

= KD2/1 = exp[01A 02A

RT

](5)

In case of non-ideal mixtures, the mole fractions, x, shouldbe replaced by activities, a = xf, in which f is the activity co-efficient. The distribution constant or partition ratio is con-stant only if the activity coefficients are constant which is

not true in concentrated solutions [2]. Partition coefficientsare usually expressed as molarity ratio. Molar solubilities,[A], and mole fractions, xA, are, in first approximation anddiluted solutions, proportional and related as follows:

[A]1 = xAV1

(6)

where V1 is the solvent 1 molar volume (M1).

2.3. Effect of temperature

Eqs. (4) and (5) show that the distribution constant, thepartition coefficient, is sensitive to temperature. Eq. (4) ex-presses the free energy of transfer, G2/1:

G2/1 = RT lnKD2/1 (7)Assuming the standard molar enthalpy is constant in a lim-ited temperature range, the plot of ln KD2/1 versus 1/T (clas-sical Van t Hoff plots) should produce a straight line withslope G2/1/R. Unfortunately, this is not that simple. Themutual solubility of the two solvents is also temperature de-pendent. At the critical solution temperature, the biphasicsystem becomes monophasic [5]. As a general rule, it is pos-sible to consider that the effect of temperature on the KDvalue is not great if the solvents are not very miscible andthe temperature change is not dramatic. An average changeof 0.009 log KD unit per degree, either positive or negative,was found for a variety of biphasic systems including theoctanolwater system [2,3,11].

2.4. Effect of chemical reactions

As clearly defined in the IUPAC definition, when anychemical reaction occurs, the concentration of a particularspecies will change, the distribution ratio will change, but thedistribution constant (partition ratio or partition coefficient)of this particular species does not change. This implies thatthe concentration of this species will change in the otherphase to maintain the chemical potentials equal in the twophases. The distribution ratio, D, can change dramatically.

The case of a compound that has a carboxylic group willbe used as an example. This compound can be representedas AH and can ionize:

AH A + H+ (8)Defining, respectively, KD, K

D and D as the distribution

constants of the molecular and ionized form of AH, and itsdistribution ratio, it comes:

D = [AH]2 + [A]2

[AH]1 + [A]1 (9)

Using the expression of Ka, the dissociation constant of AH,D can be trivially formulated as:

D = KD +KD (Ka/[H+])1+ (Ka/[H+]) (10)


0

2500

5000

7500

10000

1 3 5 7 9 11pH

D

pK a= 3 5 7 9

Fig. 1. Change in the distribution ration, D, of ionizable compounds ofthe AHA type with different acidity strength. KD of all molecularforms is 10 000, KD of all anionic form is 1. The dissociation constantsare indicated as pKa = logKa.

Fig. 1 shows the change in the partitioning of a hydropho-bic AH compound in a biphasic liquid system, e.g. theoctanolwater system, when the pH changes. At low pHvalues, AH is essentially located in the organic phase withvery high D values. Because the ionized form of AH, A,is hydrophilic, upon increase of pH, most of the solute isextracted in the aqueous phase in the ionized form. The pHzone corresponding to the change of phases depends on thesolute acidity (Ka value, Fig. 1). The distribution ratio is di-vided by two when pH = pKa. The case of the partitioningof ionizable solutes was fully studied recently [12]. Appli-cations of the equations developed can also be found in theliterature [13].

3. Measurement of partition coefficients

Several review articles describing the various methodsused to determine liquidliquid partition coefficients andespecially Ko/w appeared recently [2,9,14,15]. The methodsare briefly described focusing on accuracy and speed.

3.1. The shake-flask method

KD values of many solutes are directly determined us-ing the so-called shake-flask method. The solute is simplypartitioned between the two liquid phases of the proposedsolvent system in a test tube. After equilibrium and centrifu-gation, the relative concentration in each layer is determinedusing a variety of techniques. These include spectroscopicmethods, HPLC, GC, TLC among others.

3.1.1. Small-scale spectroscopic methodsThe two phases of the liquid system should be mutu-

ally well saturated before use. A suitable quantity, typ-ically 100g, of solute is deposited in a test tube. Onemilliliter of each saturated layer is added to the sample,and the tube is capped and equilibrated for several hours

on a wrist-action shaker. When needed, a blank solutionis also prepared. The absorbance values of the equili-brated layers and the blanks are read at a suitable wave-length, and the partition coefficient is calculated with theequation:

KD =[A]org [A]blank[A]aq [A]blank (11)

The experiment is repeated using a lower quantity, say 50g,of solute to increase the method precision and accuracy.

This method is a popular method well suited for com-pounds that distribute in the liquid system used, i.e. when0.1 < KD < 10. In this case it provides a precision higherthan 1% using 1 ml phase volumes and, and can be slightlyimproved by using 2 ml phase volumes. The method wasused with UV spectrometers [16,17] and fluorimeters [18].Its principal drawback is that pure compounds are needed. Ifimpurities are present, they will distribute also in the liquidphases but with a different KD value, biasing the measuredabsorbance in each phase.

3.1.2. Separation methods for phase analysisThe solute purity problem can easily be solved using a

method of separation to quantitate the amount of solutepresent in each phase. It is often not possible to simply sub-stitute a chromatographic method for spectroscopy. Indeed,the technique restrictions apply. It is not possible to use GCif the solute is not volatile or if the liquid phase is aque-ous. The organic solvent of the biphasic liquid system maynot be compatible with RPLC. However, the major advan-tage of chromatographic methods is the ability to deal withmixtures and to determine partition coefficients of severalsolutes in a single assay. This is partly offset by the effortnecessary to develop a reliable chromatographic assay forthe mixture of interest. HPLC and GC were used in the de-termination of pesticide partition coefficients [1921]. TLCwas also used [22].

The major point of the shake-flask method is that itis a direct method measuring without approximation theliquidliquid partition coefficient of the solute in the bipha-sic liquid system in the flask. The weak point is its limitedrange that is depending on the method used to analyze thephases. It can be roughly given as 3 < logKD < +3with chromatographic phase analysis. Partition coefficientsas big as 100 000, logKD = 5, need to be known accu-rately. In this case, if 100g of the compound is introducedin a test tube containing 2 ml of each phase of a liquidsystem, only 1 ng will pass in one phase while 99.999gwill stay in the other phase. The very low concentration,1 ng in 2 ml or 0.5g/l, obtained at equilibrium in onephase may be below the limit of detection of many an-alytical methods. This level was accurately determinedusing radiochemical methods working with radioactive iso-topes [23]. This method cannot be easily used by mostlaboratories.


3.1.3. Modern variations on the shake-flask methodFlow injection analysis (FIA) was used to determine Ko/w

coefficients. The manifold comprised three pumps: one forthe aqueous phase, one for the octanol phase and one forthe sample that could be dissolved either in the aqueousor in the octanol phase. The octanol and aqueous phasesform segments in which the injected compound of interestcan partition. A spectrophotometric detector was used tomonitor the aqueous phase absorbance. The recorded peakareas obtained at different flow ratios allowed to extrapolatethe Ko/w solute value [24]. A modified FIA system wasdesigned for rapid Ko/w determination adapted for com-binatorial chemistry. The result was obtained in less than4 min consuming less than 1l of sample [25]. The FIAmethod cannot determine accurately Ko/w values higherthan 2000 (logKo/w < 3.3).

A dialysis tubing was used to contain the aqueous phaseand to separate it from the octanol phase. Ultrasonic agita-tion shortened the equilibration time and HPLC was usedto measure the solute concentration in each phase. Ko/wvalues as high as 2.0106 (logKo/w = 6.3, perylene) wereaccurately determined in less than 6 h [26].

3.2. Liquid chromatography and correlations

3.2.1. A simple and easy methodThe use of liquid chromatographic methods using stan-

dards and correlations is the most widespread way tomeasure rapidly liquidliquid partition coefficients. It is ex-tremely simple: the logarithms of the retention factor of thesolutes are linearly correlated with the logarithm of theirpartition coefficients as first described by Collander [27]:logKD = a log k + b (12)The a and b correlation coefficients are determined mea-suring the retention factors of a set of solutes on a RPLCcolumn using a mobile phase that differs from the biphasicliquid system in which the KD values of the test solutes areknown. This method is fast and easy (the solute concentra-tion does not need to be known). Several compounds can bemeasured simultaneously. Unfortunately, the quality of theKD determination dramatically depends on the test solutesused to determine the a and b correlation coefficients.

By far, this method is used with C18 or C8 columns andmethanolwater mobile phases to determine the Ko/w par-tition coefficients in the octanolwater system [28]. Indeed,the hydrophobicity of a compound is often quantitativelyexpressed by the logarithm of its octanolwater partitioncoefficient, log Ko/w. The prediction of log Ko/w of a com-pound is essential in various fields such as pharmacology,toxicology, environmental chemistry and food chemistry.With the development of reversed phase HPLC in manylaboratories, the readily accessible retention factor of a newcompound, compared to the standard but time consumingshake-flask method, made the method extremely popularand widely used.

The method was extensively evaluated in 1988 in an in-terlaboratory study involving a large set of solutes and dif-ferent C18 columns. The conclusions of the interlaboratorytest were [29]:(1) At least six substances should be used to prepare the

correlation line.(2) The substances should belong to the validated list of

reference compounds.(3) The mobile phase should contain at least 25% (v/v)

water.(4) Extrapolation beyond the calibration range obtained

with the selected substances should only be carried outfor very hydrophobic substances (logKo/w > 6.

(5) When the conditions are fulfilled, the reliability of themethod is, in the range 0 < logKo/w < 6, 0.5 log unitor less of deviation from the shake-flask value.

This test was used by the Organization for Economic Co-operation and Development (OECD) to prepare a guidelinethat should be followed to produce reliable Ko/w values us-ing HPLC [30]. The guideline used the five points listedabove adding to the second point that the reference com-pounds should be structurally related to the test substance.The proposed list of reference compounds are summarizedin Table 1. Three main classes of reference compounds aredistinguished: neutral, basic and acidic compounds. This listcan be adapted for particular cases [31].

3.2.2. Congeneric effectIt is interesting to comment on the five points of the con-

clusion of the interlaboratory test and the OECD text addi-tion to the second point. The first point proposes that a min-imum of six solutes be used to prepare a correlation line.This comes from statistical analysis (and common sense).The second point proposes that the six substances belong toa validated list and the OECD addition says that the sub-stances should be structurally related. This deals with thecongeneric effect that is always observed with the HPLCindirect method. The correlation line is very good and themeasured log Ko/w values are very accurate when the com-pounds used to prepare the correlation lines belong all to thesame family as well as the unknown compounds. A counterexample is: trying to estimate log Ko/w of an amine using acorrelation line prepared with aromatic acids gives disastrousresults. Alternatively, the correlation line prepared using aset of solutes with widely differing functionalities will havea poor regression coefficient and poor predictive capability.

The third point: the mobile phase should contain at least25% (v/v) water, is due to the residual silanols that werealways present in 1988 C18 columns. Also it makes sensethat some water be present in the mobile phase when thepartitioning stationary phase octadecyl/mobile phase is cor-related to octanolwater. The last point said in clear that ifthe log Ko/w value of a compound is 2, or Ko/w = 100, themethod can give logKo/w = 1.5, or Ko/w = 32 as well aslogKo/w = 2.5, or Ko/w = 320. It means that the estimated


Table 1OECD recommended reference compounds

Reference compound log Ko/w Class pKa2-Butanone 0.3 n4-Acetylpyridine 0.5 BAniline 0.9 B 4.63Acetanilide 1.0 nBenzyl alcohol 1.1 n4-Methoxyphenol 1.3 A 10.26Phenoxyacetic acid 1.4 A 3.12Phenol 1.5 A 9.922,4-Dinitrophenol 1.5 A 3.96Benzonitrile 1.6 nPhenylacetonitrile 1.6 n4-Methylbenzyl alcohol 1.6 nAcetophenone 1.7 n2-Nitrophenol 1.8 A 7.173-Nitrobenzoic acid 1.8 A 3.474-Chloraniline 1.8 B 4.15Nitrobenzene 1.9 nCinnamic alcohol 1.9 nBenzoic acid 1.9 A 4.19p-Cresol 1.9 A 10.17cis-Cinnamic acid 2.1 A 3.89trans-Cinnamic acid 2.1 A 4.44Anisole 2.1 nMethyl benzoate 2.1 nBenzene 2.1 n3-Methylbenzoic acid 2.4 A 4.274-Chlorophenol 2.4 A 9.10Trichloroethene 2.4 nAtrazine 2.6 BEthyl benzoate 2.6 n2,6-Dichlorobenzonitrile 2.6 n3-Chlorobenzoic acid 2.7 A 3.82Toluene 2.7 n1-Naphthol 2.7 A 9.342,3-Dichloroaniline 2.8 B 2.05Chlorobenzene 2.8 nAllyl phenyl ether 2.9 nBromobenzene 3.0 nEthylbenzene 3.2 nBenzophenone 3.2 n4-Phenylphenol 3.2 A 9.54Thymol 3.3 n1,4-Dichlorobenzene 3.4 nDiphenylamine 3.4 B 0.79Naphthalene 3.6 nPhenyl benzoate 3.6 nIsopropylbenzene 3.7 n2,4,6-Trichlorophenol 3.7 A 6.0Biphenyl 4.0 nBenzyl benzoate 4.0 n2,4-Nitro-6-sec-butyl phenol 4.1 n1,2,4-Trichlorobenzene 4.2 nDodecanoic acid 4.2 A 4.8Diphenyl ether 4.2 nPhenanthrene 4.5 nN-Butylbenzene 4.6 nFluoranthene 4.7 nDibenzyl 4.8 n2,6-Diphenylpyridine 4.9 nTriphenylamine 5.7 nDDT 6.2 n

From [30]. A: acidic compounds; B: basic compounds; n: neutral com-pounds.

Ko/w value may be three times two big or too low. A recentwork with deactivated modern C18 columns did not changemuch the 1988 conclusions. It did show that, with a 5050methanolwater mobile phase, the H-bonding atmosphereof the C18 deactivated phase (Capcell Pak C18, a siliconepolymer-coated silica gel chemically modified with C18) isvery similar to that of octanol [32].

3.2.3. Variations on the method to improve the correlationquality

Several parameters were added to the log KD versus log krelationship to improve the correlation. For example, Yam-agami et al. proposed the equation [33]:logKo/w = a log k + b+ I + sSHA (13)with a, b, and s are the constants for a column and a mobilephase, and I and SHA an inductive electronic constant andthe proton acceptor value of the studied solute. Of course,adding parameters does improve the correlation quality, butit decreases the method ease since the I and SHA values ofunknown compounds should be also estimated.

It was proposed to use log kw, the extrapolated solute re-tention factor in pure water [34,35]. log kw is obtained byplotting log k of the solute versus the organic modifier con-tent in the mobile phase and extrapolating the straight lineto 0% modifier (=pure water). Then, log kw can be used inEq. (12) rather than log k. The log kw parameter became awidely used chromatographic descriptors of hydrophobic-ity per se [3642]. It should be noted that this extrapolatedvalue can yield large errors. This is proved by the fact thatthe extrapolated log kw value, obtained with the same so-lute and column, may differ significantly depending on theorganic modifier used: methanol or acetonitrile.

It was also proposed to use gradient elution to increasethe speed of the partition coefficient estimation. It was nec-essary to use the Abraham linear solvation coefficients tocorrelate the solute retention factors with log Ko/w [43,44].Associating methanol gradient elution with a modern sta-tionary phase that can work within the 28 pH range showedthat log Ko/w values were measured in less than 45 min witha better than 0.3 log unit error in the 0.1 to 4.0 range [45].

The use of octanol- or glycerol-coated stationary phasescomplicated the method and did not fully suppress the con-generic effect observed with the HPLC methods [46,47].Micellar liquid chromatography is another HPLC variationthat can be used for hydrophobicity measurements [48].

Taking in account the congeneric effect and acceptingthe 0.5 log unit possible error on the determined log Ko/wvalues, HPLC is now one of the tools most often used toevaluate quickly the magnitude of the hydrophobicity of anew synthesized compound [4952].

3.3. Capillary electrophoresis with ordered media

Classical CE uses an electric field to sort charged solutesby differing electrophoretic mobilities. Micellar liquid chro-


matography had shown that the solute micelle binding con-stants were related to solute hydrophobicity and Ko/w co-efficients [48]. Micelles were introduced in CE to separateneutral solutes according to their affinity for the micelles[53]. Herbert and Dorsey showed that the retention factorsobtained with micellar electrokinetic capillary chromatogra-phy (MEKC) allowed to estimate the respective Ko/w solutevalues over 8.5 orders of magnitude (2.3 < logKo/w

partition coeff

Documents