Solute Partitioning in Aqueous Biphasic Systems Composed of

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Solute Partitioning in Aqueous Biphasic Systems Composed of Polyethylene Glycol and Salt: The Partitioning of Small Neutral Organic Species Heather D. Willauer, Jonathan G. Huddleston, and Robin D. Rogers*

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Department of Chemistry and Center for Green Manufacturing, The University of Alabama, Tuscaloosa, Alabama 35487

Aqueous biphasic systems (ABSs) represent wholly aqueous systems that are safe, nontoxic, and nonflammable, and thus, they represent relatively environmentally benign extraction media. Such systems could be employed as alternatives to traditional aqueous-organic systems for the separation of, inter alia, small organic molecules, thus it is important to develop a fundamental understanding of these systems and the variables that govern solute partitioning within them. The partitioning of a series of neutral, substituted benzene compounds and neutral, aliphatic compounds in ABSs composed of different molecular weights of poly(ethylene glycol) (PEG-1000, 2000, and 3400) and formed as a result of the addition of different salt types [K3PO4, K2CO3, (NH4)2SO4, Li2SO4, MnSO4, ZnSO4, and NaOH] has been examined. The results show that the distribution of organic solutes is a function only of the degree of phase divergence of the biphasic system as expressed by the difference in polymer concentration between the phases: ∆[poly(ethylene glycol)], ∆[ethylene oxide monomers], or tie line length (∆PEG, ∆EO, or TLL, respectively). Solute partitioning depends only on the composition of the phase-forming components, PEG and salt. Using ideas taken from the study of critical phenomena, it can be shown that the composition of the phases is the result of the salting-out ability of the salt and the number of ethylene oxide monomers comprising the PEG. The salting-out strength of the salt (measured by its lyotropic number or position in the Hofmeister series) is related to its ability to lower the cloud point of the PEG solution. Hence, cosmotropic salts salt-out PEG, producing a series of nearly identical ABSs that, although differing in their overall concentrations of PEG and salt, are identical in terms of their lyotropic properties. This is an extraordinary simplification of a complex array of different ABSs to a single series of ABSs of graded lyotropy. Further comparison of solute partitioning in PEG/salt ABSs to partitioning in 1-octanol/water systems is discussed, and a greater similarity of solute distribution was found between different PEG/salt ABSs than between PEG/salt ABSs and 1-octanol/water. 1. Introduction For over 40 years, aqueous biphasic systems (ABSs), formed when mixtures of water-soluble polymers are combined with one another or with certain inorganic salts above critical concentrations, have been used in the separation, concentration, and fractionation of biological solutes and particles.1-6 Several instances are known of the development of large-scale biorecovery processes operating at industrial scale, for example, the protein purification processes developed at the GBF, by Genentec, and by the Miles company.7,8 ABSs have also found utility as biocompatible phases for biotransformations,5,9 and they have been applied bioanalytically to macromolecular characterization in the study of protein surface properties (hydrophobicity,10 charge state,11,12 ligand binding,13,14 and protein conformational state15). Clinical applications have been suggested to determine disease conditions such as malignancies.16 These systems also find use in industrial biotechnology quality-control applications for the detection of misconformations and degradation of protein products.16 It has * Corresponding author: Robin D. Rogers, Department of Chemistry, Box 870336, The University of Alabama, Tuscaloosa, AL 35487. Phone: 205/348-4323. Fax: 205/348-0823. E-mail: [email protected].

also been suggested that ABS partitioning represents a viable and necessary alternative to log P determination for biotechnological products in QSAR (quantitative structure activity relationships).16,17 This is because log P cannot be used for labile biological species as higherorder conformations are not maintained in organic solvents. Thus, ultimately, because of the almost inevitable increase in molecular and conformational complexity of therapeutic agents, the application of ABSs to this end seems almost certain to increase. In recent years, ABSs have been studied for applications involving the selective distribution and separation of metal ion species18-25 and small organic molecules,26-28 for applications in biphasic catalysis,29-31 and in the delignification of cellulose during alkaline paper pulping.32,33 Workers active in the study of the partitioning of macromolecular biological solutes have examined the partitioning of small organic molecules in an effort to understand and predict the results of macromolecular partitioning in ABSs.34-36 For the most part, these studies have concentrated on the partitioning of amino acids and small peptides, although normal alcohols have also been used.37 An examination of the literature on phase partitioning in ABSs reveals some interesting features that have not, until recently, been exploited to extend their range of application to small molecular

10.1021/ie010598z CCC: $22.00 © 2002 American Chemical Society Published on Web 03/09/2002

Ind. Eng. Chem. Res., Vol. 41, No. 7, 2002 1893

species. For example, in a study of the partitioning of dipeptides, the strong effect of the aryl amino acid side chain of tryptophan was notable,38 and a role for surface tryptophan moieties in protein partitioning has been suggested.2 These observations were embodied in the successful development of “affinity handles” for the purification of recombinant proteins in ABSs by Veide and co-workers.39-41 Of similar interest was the observation of the strong tendency for biological pigments and other dyes to partition to the PEG-rich phase of PEG/ salt ABSs.42,43 These observations prompted the present examination of the partitioning of small organic species in these systems in more depth.26,27,33 Promising results would suggest some utility for these systems in the study of biological potency in an alternative to log P determination; in the recovery and purification of small organic molecules, in the treatment of wastewater, for example, in the removal of benzene from crude-oil desalting solutions arising in the petroleum industry;44 and in connection with the processing of a wide range of commercially important chemical species, such as pharmaceuticals, pesticides, herbicides, dyes, and pigments, as well as in the extraction of a wide range of structurally and functionally similar species from natural sources. In a recent review,45 we suggested that there are strong similarities at the level of implementation and also at the molecular level between biphasic systems formed with a variety of polymers in aqueous solution that, until recently, have tended to receive separate treatment. These systems are variously known and applied in extractive separations as cloud-point extraction; thermoseparating polymers; and, of course, aqueous biphasic systems. Such systems, in particular PEG/ salt ABSs, can be formed with a wide range of molecular weights and types of polymer and in the presence of a bewildering array of salts. Thus, it is often far from clear how the knowledge of solute distribution in one system can be transferred to a different system. The development of a rational molecular-level understanding of solute distribution in these systems is clearly important for their successful application and utilization. Here, we examine the partitioning of low-molecular-weight organic species in a range of ABSs composed of different molecular weights of PEG and formed with a wide variety of different salts. The information presented should be of considerable utility in advancing our understanding of partitioning behavior in these systems and should greatly aid in efforts toward practical implementation. 2. Experimental Section The chemicals used in the study, (NH4)2SO4, K3PO4, K2CO3, Li2SO4, MnSO4, ZnSO4, benzene, and poly(ethylene glycol) of molecular weights 1000, 2000, and 3400, were obtained from Aldrich (Milwaukee, WI) and were of reagent grade. Carbon-14-labeled acetonitrile, ethanol, caffeine, 1,3-dinitrobenzene, aniline, benzene, acetophenone, toluene, chlorobenzene, 1,4-dichlorobenzene, 1,2,4-trichlorobenzene, 4,4-dichlorobiphenyl, npropanol, methyl iodide, and phthalic acid were purchased from Sigma (St. Louis, MO). Upon receipt, the tracers were diluted to an activity of 0.06-0.08 µCi/µL for use as the “spike” in the partitioning experiments. For standard liquid scintillation analyses, Ultima Gold scintillation cocktail (Packard Instruments, Downers

Figure 1. Experimentally determined phase diagrams for a PEG2000/K3PO4 ABS: (- • -) binodal curve ADC, (- - -) tie lines ABC, (b) overall system composition B, (9) critical point D.

Grove, IL) and a Packard Tri-Carb 1900 TR liquid scintillation analyzer (Packard Instruments) were used. All water was purified using a Barnsted (Dubuque, IA) commercial deionization system. Stock solutions of polymer and salt were prepared on a molar or mass percent basis. The conversion of salt concentraions from a molar to a mass percent basis was done by calculation with reference to measured solution densities. Salt and polymer compositions expressed in terms of molal concentrations were calculated from compositions determined on a mass percent basis. Phase diagrams were determined by the cloud-point method1 and described using an empirical expression derived by Merchuk.46

a exp(bXA0.5 - cXA3) ) YA

(1)

The constants a, b, and c were obtained by least-squares regression of the turbidometric data. Tie lines were assigned from the relationship between the mass phase ratio and system composition using mathematical methods46 that can be briefly described as follows. In Figure 1, a system represented by point B was selected in the phase diagram through which the tie line ABC would pass. The mass ratio of the resulting ABS was determined as

R ) (mass of the top phase)/(mass of the mixture) (2) Mass balance over the tie line ABC can be constructed such that the mass of any point B on the tie line ABC is equal to the mass of PEG and salt at node A plus the mass of PEG and salt at node C, that is

M B ) MA + MC

(3)

If YM and XM represent the overall percent compositions of PEG and salt, respectively, at point B on the tie line ABC and YA and YC represent the overall percent compositions of PEG at nodes A and C, respectively, then the following mass balance equation46 can be derived

YA ) (YM/R) - [(1 - R)/R]YC

(4)

A similar equation can be constructed for the composition of salt at nodes A and C of the tie line ABC.44

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Because the compositions at nodes A and C lie on the binodal and are thus a function of eq 1, a system of simultaneous equations can be established to find the salt compositions at the nodes. This method of tie line determination was used for each phase diagram unless otherwise indicated. Phase diagrams were also prepared at various temperatures by equilibration of defined ABSs at the required temperature, and the phase compositions were determined by gel permeation chromatography (GPC).47 Each biphase was equilibrated at the experimental temperature in a Neslab RTE-110 water bath (Neslab Instruments, Inc., Newington, NH). GPC was performed using a HPLC Shimadzu LC 10 (Shimadzu Corporation, Kyoto, Japan) size-exclusion chromatography column, Pharmacia Peptide HR 10/30 (Amersham Pharmacia Biotech, Inc., Piscataway, NJ) coupled to a refractive index detector (Shimadzu RID10A, Shimadzu Corporation). The running buffer was composed of 250 mM NaCl in deionized water. Chromatography was performed isochratically at a flow rate of 1 mL/min. Liquid/liquid distribution ratios were determined for each solute by mixing 1 mL of a 40% (w/w) PEG solution with 1 mL of a salt stock solution of known concentration. The systems prepared were vortex-mixed for 2 min and allowed to equilibrate. Tracer quantities (1-4 µCi) of the radionuclide of interest were added, and the system was centrifuged (2 min, 2000g) to pull the sample spike into the solution and then vortex-mixed for 2 min to ensure complete mixing. The contact time for the solute in these systems was experimentally determined to be sufficient for solute distribution to reach equilibrium. The phases were disengaged by centrifugation (2000g) for 2 min. Equal aliquots of each phase were then removed for standard liquid scintillation analysis. Because equal aliquots of each phase were analyzed, the activity of the tracers was directly proportional to their concentration. The distribution ratios were defined as the total concentration of solute in the upper PEG-rich phase divided by the concentration in the lower salt-rich phase, i.e. D) concentration in counts per minute in the PEG-rich phase concentration in counts per minute in the salt-rich phase (5)

All measurements were carried out at least in duplicate. A detailed account of these radiochemical methods is given elsewhere.18 3. Results and Discussion Phase Diagrams. Complete characterization of the phase diagram is crucial to the understanding of system variables that govern solute partitioning within ABSs. Important variables are the polymer type and molecular weight, the choice of phase-forming salt, the relative concentration of each component, the pH of the biphase, and the temperature at which the biphase is formed. To begin to develop an understanding of the interplay of these variables and their effect on the solute distribution in different systems, we extensively characterized the phase diagrams of several ABSs differing in molecular weight of polymer, type of salt, and equilibrium temperature at which phase formation took place. Figure 1 is an example of the level of detail required for minimal satisfactory characterization of PEG/salt

ABS phase diagrams. The figure shows the binodal curve for the system composed of PEG-2000 as the phase-forming polymer and K3PO4 as the phase-forming salt, with the concentration of the phase-forming components expressed in terms of molality. In the literature, it has become conventional to express the equilibrium component concentrations in terms of mass/mass (w/w) % polymer salt.16,48 This appears to be a result of the perceived difficulty in manipulating the ABS solutions by volume.48 It might seem preferable to use a molar, molal, or mole fraction basis to emphasize the molecular effects of the salt on the salting-out of the polymer. The binodal curve (ADC in Figure 1) represents the critical concentration at which two phases form. Thus, to the left of and below the binodal curve, polymer and salt exist as a single homogeneous phase. To the right of and above the binodal, two distinct phases form, one enriched in polymer and the other in salt. Systems having, for example, the overall compositions denoted by B on the tie lines ABC form two phases having the overall compositions denoted by the nodes at A and C. The phase composition represented by the node A is rich in PEG, and that denoted by node C is rich in K3PO4. The PEG-rich phase is normally the upper phase because it is less dense then the salt-rich phase; however, this is not always the case and can vary with temperature and salt concentration.49 Any system lying along a single tie line will separate into two phases having an overall composition given by the compositions at A and C. Although all the systems along a single tie line ABC have identical compositions, the relative volumes (or masses on a w/w % basis) of these systems will change, as indicated in Figure 1.5 This is the basis for the method of determining the tie lines outlined in the Experimental Section. At the critical point in the phase diagram, the compositions of the coexisting phases are theoretically identical, and the mass ratio is 1. The critical point is typically determined by drawing a curve through the midpoints (R ) 1) of a series of tie lines and extrapolating this curve to the point where it intersects the binodal curve.16 Moving from the critical point into the phase diagram traverses systems whose phases become increasingly divergent in composition (i.e., moving from D in the general direction of the tie lines ABC in Figure 1). Two important relationships have been established48 that express this degree of phase divergence, the difference in PEG composition between the phases (∆PEG), and the tie line length (TLL), eqs 6 and 7.

TLL ) (∆PEG2 + ∆salt2)1/2

(6)

In eq 6, TLL represents the orthogonal sum of the difference in polymer (∆PEG) and salt (∆salt) concentrations between the phases. ∆PEG (and similarly ∆salt) can be defined as in eq 7

∆PEG ) [(PEG)T - (PEG)B]

(7)

where the subscript T refers to the concentration of polymer in the top phase and B refers to the concentration of polymer in the bottom phase. Thus, as the concentrations of polymer and salt used to form the biphase increases, the TLL becomes longer, and the top and bottom phases become increasingly different in composition.

Ind. Eng. Chem. Res., Vol. 41, No. 7, 2002 1895 Table 1. Gibbs Free Energies of Hydration slope ∆Ghyda ∆Ghyda ∆Ghyda ∆PEG/∆salt (kJ molcation-1) (kJ molanion-1) (kJ mol-1)

salt type K3PO4 K2CO3 (NH4)2SO4 NaOH Li2SO4 MnSO4 ZnSO4 a

Figure 2. Experimentally determined phase diagrams for PEG2000/salt ABSs at 25 °C formed with the following salts: (- b -) K2CO3, (- 2 -) ZnSO4, (- O -) Li2SO4, (- 0 -) MnSO4, (- 9 -) NaOH.

13.80 11.86 7.43 7.30 10.11 7.42 8.60

-305 -305 -285 -385 -510 -1740 -1880

-2835 -1300 -1145 -345 -1145 -1145 -1145

-3750 -1910 -1715 -730 -2165 -2885 -3025

Values obtained from ref 51.

Figure 2, the binodal curves for several ABSs composed of CO32- anions (∆Ghyd ) -1300 kJ mol-1) form at lower concentrations of polymer and salt than systems composed of OH- anions (∆Ghyd ) -345 kJ mol-1). Similarly, in Figure 3, systems composed of PO43- anions (∆Ghyd ) -2835 kJ mol-1) form at lower concentrations of phase-forming components than systems formed with SO42- anions (∆Ghyd ) -1145 kJ mol-1). The free energies of hydration reported in Table 1 are those calculated by Marcus using the model51

∆G*hyd ) ∆G′neut + ∆Ge1+2 + ∆Gunsym

(8)

This equation is derived from the series of equations

∆G′neut/(kJ mol-1) ) 41 - 87(r/nm) + 1200(r/nm)2 (9) ∆Ge1+2/(kJ mol-1) ) -64.5z2[0.44(∆r/r) + 0.987]/(r + ∆r) (10) ∆Gunsym ) 120(r/nm)z3

Figure 3. Experimentally determined phase diagrams for PEG/ salt ABSs formed with different molecular weights of PEG, different salt types, at different temperatures: (- b -) PEG-3400/ K3PO4 at 25 °C, (- 9 -) PEG-2000/K3PO4 at 25 °C, (- 2 -) PEG1000/K3PO4 at 25 °C, (- ] -) PEG-2000/(NH4)2SO4 at 55 °C, (- O -) PEG-2000/(NH4)2SO4 at 40 °C, (- 0 -) PEG-2000/(NH4)2SO4 at 25 °C.

Figures 2 and 3 indicate the complex array of ABS phase diagrams that can be obtained at different temperatures, with different molecular weights of PEG, and with a wide variety of phase-forming salts. Each of these systems has been characterized in the same detail as the ABS shown in Figure 1; however, the tie line relationships for each ABS have been omitted from the figures in the interest of clarity. These phase diagrams illustrate the complexity involved in arriving at a developed understanding of the interplay of the system variables that govern solute partitioning in ABSs, which hinders attempts to predict solute distribution for any particular ABS. It can be seen that ABSs composed of PEG-2000 in the presence of different salts at 25 °C bring about phase separation at different concentrations of polymer and salt.50 Reference to Table 1 indicates that ABSs formed of anions having highly negative free energies of hydration display phase separation at lower concentrations of polymer and salt than salts of anions having less negative free energies of hydration.22,26,27 Referring to

(11)

In these equations, ∆r is the thickness of the hydration shell that surrounds the ion that has a radius r. ∆Ge1+2 is the Gibbs free energy of the electrostatic interactions in the hydration shell and beyond. ∆G′neut represents the Gibbs free energy of hydration without the contribution of charge. The effect of the ion charge, z, is taken into account in ∆Gunsym. Because ∆Ghyd is a relatively straightforward function of size and charge, it is evident that no distinction can be developed for particular ions that goes beyond these simple features. Although the anion appears to dominate the process of phase separation,52 the cation clearly plays an important role. Note, for example, in Figure 2, the relationships between the binodal curves of MnSO4, Li2SO4, and ZnSO4. The cations, particularly Mn2+ and Zn2+, have large negative Gibbs free energies of hydration, but this does not promote a greater increase in phase separation over the other sulfate salts. The role of the salts in bringing about phase separation and its relationship to the Hofmeister or lyotropic series has been pointed out.52 When the molecular weight of the polymer is increased from 1000 to 3400, as shown in Figure 3, biphase formation occurs at lower concentrations of polymer and salt. Similar effects are also observed when the equilibrium temperature of biphase formation increases. Thus, Figure 3 shows binodals for a PEG-2000/ (NH4)2SO4 ABS formed at 25, 40, and 55 °C. As the temperature of the ABS is increased, biphase formation occurs at lower concentrations of phase-forming polymer and salt. Although it is possible, in this way, to make generalized statements about the effect of salt type and concentration, polymer molecular weight, and temper-

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later empirically extended to34,53

ln K ) A(w1′′ - w1′) + b(w1′′ - w1′)2

Figure 4. Distribution ratios vs ∆EO for 4,4-dichlorobiphenyl (b), 1,2,4-trichlorobenzene (9), 1,4-dichlorobenzene (2), chlorobenzene (1), toluene ([), acetophenone (O), benzene (0), aniline (4), 1,3dinitrobenzene (3), caffeine (]), ethanol (‚ ‚ b ‚ ‚), and acetonitrile (‚ ‚ 9 ‚ ‚) in ABSs prepared by mixing 40% (w/w) PEG-2000 with salt solutions of increasing concentration of K3PO4.

ature on phase separation in ABSs, little progress has been made in developing a comprehensive understanding of the interplay of all the system variables and their effects on solute partitioning in ABSs. Solute Partitioning. The partitioning of a number of neutral solutes has been examined in various ABSs formed with PEG in conjunction with different phaseforming salts and at different temperatures. Figure 4 shows the distribution ratios of 12 neutral solutes in ABSs formed with 40% (w/w) PEG-2000 and increasing concentrations of K3PO4. The distribution ratio is shown relative to the PEG concentration difference between the phases (∆PEG of eq 7) expressed as a function of ethylene oxide monomer concentration. The partitioning of the solutes is strongly correlated with the degree of phase divergence, expressed by the difference in PEG composition between the phases measured in molality with respect to ethylene oxide monomers as in eq 12

ln K ) k∆w2

(12)

where K is the partition coefficient, k is an equilibrium constant, and ∆w2 is the concentration difference between the phases of one of the phase-forming components. In terms of the figure, ∆w2 ) ∆EO, and the partitioning of each of these solutes confirms the expectation that the observed distribution for solutes should be a linear function of distance from the critical point, where the partition coefficient is, theoretically, unity.37,53,54 This relationship has been derived from theoretical considerations a number of times since Bro¨nsted formulated his famous relationship55

ln k )

λM(C - Co) KT

(13)

where K is the Boltzmann constant, T is the absolute temperature, and M is the molecular mass of the solute. C - Co relates the composition at the critical point to the change in composition of the system, and λ is a potential energy term. Diamond and Hsu derived a similar relationship using a Flory-Huggins approach applied to polymer/polymer systems, which they also used in the context of polymer/salt systems. This was

(14)

The percent concentration of polymer w is expressed in (w/w) % and the subscripts refer to PEG, and the single- and double-prime superscripts refer to the top and bottom phases, respectively, so that the terms in parentheses represent the difference in the polymer concentration between the phases. It is clear from the distribution data in Figure 4 that solute partitioning at longer tie line lengths is less than would be expected for a strictly linear relationship (eqs 12 and 13) and would be better described by a relationship having the form of eq 14. Qualitatively, it appears that the partition coefficient at longer tie line lengths begins to saturate. It is also possible that certain experimental shortcomings could contribute to the observation of this effect. Saturation could be due to difficulties associated with the quantitative measurement of solute concentration in the bottom phase for solutes whose partition coefficients approach 104. This difficulty is well-known in the 1-octanol/water system.56 Saturation could also result from sample disengagement from the scintillant in the radiochemical analysis as the concentration of polymer and salt approaches a maximal value. The apparent relationship between the partitioning of small organics in ABSs and other solvent/water systems, particularly in the widely used 1-octanol/water system, has often been alluded to.26,27,37,54,57-59 Eiteman summarized this relationship between the solute distributions in ABSs and in the 1-octanol/water system in studies on the partitioning of normal alcohols26,37,54 as

ln K ) b + m log P

(15)

The relative solute hydrophobicity, as conventionally measured by the distribution in the 1-octanol/water system, log P, is related to its distribution in the ABS, ln K, through the constants b and m. These constants have been related to the intrinsic hydrophobicity of the phase system,37 log Po, by

log Po ) -B/m

(16)

This expression attempts to relate the intrinsic hydrophobicity of a given ABS to that of the 1-octanol/water system for a hypothetical solute whose 1-octanol/water distribution ratio is equal to 1. Taken at face value, this relationship indicates the value of solute log P above which solutes will partition to the upper PEG-rich phase of the ABS and below which they will partition to the lower salt-rich phase.37 Figure 5 shows the distribution ratios (log D) of 12 neutral solutes in ABSs, formed with 40% (w/w) PEG2000 and increasing concentrations of K3PO4, in relation to the published values of their 1-octanol/water partition coefficients (P). The values of log P used were the socalled best values indicated in the published version of the Pamona School’s database.60 The figure shows the solute distributions at three different values of ∆EO (polymer concentration difference between the phases), and as also indicated in Figure 4, their distribution increases with increase in this parameter. At a defined polymer concentration difference between the phases (∆EO, molal), the partition coefficients of the solutes


Figure 5. Logarithm of the distribution ratios for (1) acetonitrile, (2) ethanol, (3) caffeine, (4) aniline, (5) 1,3-dinitrobenzene, (6) acetophenone, (7) benzene, (8) toluene, (9) chlorobenzene, (10) 1,4dichlorobenzene, (11) 1,2,4-trichlorobenzene, and (12) 4,4-dichlorobiphenyl in ABSs formed with 40% (w/w) PEG-2000 and increasing concentrations of K3PO4 [(b) 1 M K3PO4, (0) 2 M K3PO4, (2) 3.0 M K3PO4] vs logarithm 1-octanol/water partition coefficient (P).

follow the series acetonitrile < ethanol < caffeine < 1,3dinitrobenzene < aniline < benzene < acetophenone < toluene < chlorobenzene < 1,4-dichlorobenzene < 1,2,4trichlorobenzene < 4,4-dichlorobiphenyl. The increase in distribution ratios for these solutes appears to follow their overall increase in hydrophobicity (log P) as shown by the regressions placed on the data in Figure 5. However, Figure 5 also illustrates the very real difficulty and complexity involved in comparing solute partitioning in ABSs with that of 1-octanol/water. For solutes of similar chemical natures having minimal possibilities of strong electrostatic interactions (e.g., hydrogen bonding with solutes or solvents, such as trichlorobenezene, dichlorobenzene, chlorobenzene, toluene, benzene, dinitrobenzene), a strong correlation between log D of the ABS and log P exists and can be expected.26,27,37,54,57-59 Distribution ratios of the solutes 4,4-dichlorobiphenyl, acetophenone, aniline, and caffeine show considerable differences in partitioning in these two systems compared to the former solutes. This contrasting behavior can be attributed to the difference in solute/solvent interactions in the two systems and, in particular, to differences in hydrogen-bonding interactions. The relationship emphasized by Eiteman is yet another limited example of the application of the Collander equation to the comparison of solute partitioning between different solvent systems

log P2 ) a log P1 + b

(17)

where a and b are constants and the subscripts 2 and 1 denote the systems (in this case, 1-octanol/water and ABS) under comparison. 56 The limitations of this relationship are, by now, wellknown. In essence, the Collander equation can be true only when solvent systems having no differences in hydrogen-bonding interactions with the solutes or when solutes having insignificant differences in chemical nature are compared. Under other circumstances, the occurrence of an absolute minimum of three different Collander-type relationships has been suggested (donor, acceptor, and neutral species), but the reality is likely

to be more complex as a result of departures from strict additivity through intramolecular and positional effects, etc.56 This can be illustrated by reference to Figure 6 in which the relative difference in the partition coefficient for the present, rather limited, set of neutral solutes between ABSs and the 1-octanol/water system (∆ log P) are compared for three different PEG/salt ABSs. The figure compares ∆ log P found for several solutes on a particular tie line in one PEG/salt ABS to that found for the same solutes in a different PEG/salt ABS. Thus, partition in a PEG/K3PO4 ABS (∆EO ) 18.4) is compared to partition in a PEG/(NH4)2SO4 ABS (∆EO ) 16.0) and is shown by the solid symbols in Figure 6. Similarly, the open circles show the same PEG/K3PO4 ABS (∆EO ) 18.4) compared to a different ABS, PEG/ K2CO3, (∆EO ) 16.8). A strong correlation exists between log D of one ABS and log D of another ABS formed by the addition of a different salt. Solutes such as methyl iodide and 4,4-dichlorobiphenyl exhibit differences in partitioning between the systems; however, these differences appear similar in comparing partitioning in one ABS to that in another ABS. They appear to represent differences in the behavior of these solutes relative to others when partitioned in a 1-octanol/water system and an ABS. This feature of the data might indicate a difference in molecular form (association) in the ABSs and the 1-octanol/water systems or a problem with the nature (purity) of our radiochemical, which was not assessed independently of this partitioning study. Nevertheless, the data strongly indicate that each PEG/ salt ABS is governed by similar solute-solvent interactions regardless of salt type and concentration, as even the anomalous solutes partition in a consistent way in the different ABSs. The importance of the relative hydrophobicities of the phases in solute distribution in ABSs has been recognized as one of its most fundamental features.58 A scale of hydrophobicity based on the free energy of transfer of a methylene group between the phases has been proposed57

ln K ) C + Enc

(18)

where K is the partition coefficient, E is a constant related to the free energy of transfer of a methylene group between the phases, and C is a constant related to the hydration properties of the phases. The term nc is the number of carbon atoms in the side chain of the solutes partitioned. Figure 6 demonstrates that the hydration properties of different PEG/salt ABSs can be very similar and suitable for the measurement of the relative solute hydrophobicity in an environment characterized by strong electrostatic interactions (high salt concentrations). Such measurements should be of considerable interest in QSAR analysis,16,17 for example, in the characterization of drugs61 or studies of environmental fate.62,63 Figure 7 shows the distribution of a neutral aromatic solute, benzene, in seven different PEG-2000/salt ABSs. The data are shown relative to the salt concentration used to form a biphase with 40% (w/w) PEG-2000. We have previously shown similar data for more limited numbers of salts and solutes.26,27 The figure shows that, in common with macromolecular and charged solutes, the distribution of this small aromatic molecule brought about by the increasing divergence of the composition of the two coexisting phases becomes increasingly onesided.6,22 The molar concentration of salt required to

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Figure 6. Relative difference in the distribution ratios for (1) acetonitrile, (2) ethanol, (3) caffeine, (4) aniline, (5) 1,3-dinitrobenzene, (6) acetophenone, (7) benzene, (8) toluene, (9) chlorobenzene, (10) 1,4-dichlorobenzene, (11) 1,2,4-trichlorobenzene, (12) 4,4dichlorobiphenyl, (13) n-propanol, and (14) methyl iodide between ABSs and the 1-octanol/water system (∆ log P) compared for three different PEG/salt ABSs of defined tie line length: (b) PEG/K3PO4 ABS (∆EO ) 18.4) compared to PEG/(NH4)2SO4 ABS (∆EO ) 16.0), (O) PEG/K3PO4 ABS (∆EO ) 18.4) compared to PEG/K2CO3 ABS (∆EO ) 16.8).

Figure 7. Distribution ratios for benzene in ABSs formed with 40% (w/w) PEG-2000 and increasing concentrations of salt: (b) K3PO4, (9) K2CO3, (2) (NH4)2SO4, ([) Li2SO4, (0) MnSO4, (3) ZnSO4, (1) NaOH.

achieve a given distribution decreases in the following anion order: PO43- < CO32- < SO42- < OH-. Thus, the distribution of neutral solutes follows the Hofmeister series, that is, the more negative the ∆Ghyd of the salt (Table 1), the greater the salting-out effect of the salt and the greater the distribution coefficient of the solutes.22,64,65 The effect of salt type seems to be largely dependent on the anion, which makes the dominant contribution, and this is reflected by its ∆Ghyd.22 The contribution of the cation to phase separation and solute distribution seems not to reflect the magnitude of the cationic Gibbs free energy of hydration, which is very large for cations such as Zn2+ and Mn.2+ For a series of ABSs composed of (NH4)2SO4 and several different molecular weights of PEG (300, 600, 6000, and 20000), Zaslavsky showed that the parameter E, the free energy of transfer of a methylene group (eq 18) is dependent only on the difference in PEG composition between the phases, ∆PEG.59 Here, we have ef-

Figure 8. Distribution ratios for benzene and 1,4-dichlorobenzene vs ∆EO in ABSs formed with 40% (w/w) PEG-2000 and increasing concentrations of salt: (b) K3PO4, (9) K2CO3, (2) (NH4)2SO4, (1) NaOH, ([) Li2SO4, (0) MnSO4, (3) ZnSO4. Distribution ratios for benzene vs ∆EO molality in ABSs formed with K3PO4 and (O) 40% (w/w) PEG-1000 and (]) 40% (w/w) PEG-3400.

fectively extended the availability of neutral solute partitioning data in ABSs to include ABSs formed in the presence of different types of salts. Figure 8 shows examples of the distribution of the neutral solutes benzene and 1,4-dichlorobenzene in several PEG/salt ABSs differing in type of salt and molecular weight of polymer. The regression placed on the data follows the general form of the empirical relationship in eq 14 established by Diamond and Hsu.34,53 Clearly, if the data collected at longer TLL were excluded, there would be a linear relationship between solute distribution and ∆EO having a 0 intercept at the critical point as predicted by eqs 12 and 1337,53,54 and fulfilling the expectations of the Bro¨nsted relationship (eq 13).55 The distribution ratios of neutral solutes in these systems are approximately the same regardless of polymer molecular weight, salt type, or salt concentration used to form the biphase, provided that system composition is expressed in terms of its degree of phase divergence (∆PEG, ∆EO, or TLL). The salting-out strength of the salt and the molecular weight of the polymer determine not only the relative concentration of PEG and salt at which phase separation takes place but also the relative rate of phase divergence with respect to salt concentration. The distribution ratios of added solutes in PEG/salt ABSs shown in Figure 8 seem to depend only on the degree of phase divergence, i.e., the difference in PEG concentration between the phases, expressed as molality with respect to ethylene oxide monomers. Because there is an orthoganol relationship between TLL and ∆PEG as shown in eq 6, a relationship between D (solute distribution) and ∆PEG or TLL seems equally plausible (eq 12). If the difference in PEG composition is measured in (w/w) %, then there is a similar relationship between ∆PEG and TLL

ln D ) k∆PEG or ln D ) kTLL

(19)

However, if eq 19 is expressed in terms of moles of PEG polymer and moles of salt, the scaling of the orthoganol relationship given by eq 6 is completely transformed simply because of the very great compression of the PEG scale caused by the relatively high molar mass of the polymer. Under these circumstances,


Figure 9. Distribution ratios for benzene vs ∆salt in ABSs formed with 40% (w/w) PEG-2000 and increasing concentrations of salt: (b) K3PO4, (9) K2CO3, (2) (NH4)2SO4, (1) NaOH, ([) Li2SO4, (0) MnSO4, (3) ZnSO4.

Figure 10. ∆EO vs ∆salt for ABSs formed with 40% (w/w) PEG2000 and increasing concentrations of salt: (b) K3PO4, (9) K2CO3, (2) (NH4)2SO4, (1) NaOH, ([) Li2SO4, (0) MnSO4, (3) ZnSO4.

it can be easily comprehended that the tie line length then scales with ∆salt. Expressing eq 19 in terms of moles of EO, or (w/w) %, restores the scaling of eq 6 so that both forms of eq 19 are approximately obeyed. Zaslavsky found, in his study59 of ABSs formed with ammonium sulfate and several different molecular weights of PEG, that there appears to be no relationship between the difference in salt concentration between the coexisting phases and the parameters C or E of eq 18. Thus, as shown in Figure 9, there is no apparent relationship between the distribution of benzene in various PEG/salt ABSs and the difference in salt concentration between the phases. This can be understood in terms of eq 6 and by reference to Figure 10, which shows the relationship between ∆PEG and ∆salt for all ABSs represented in Figures 2 and 3. In this figure, all data are experimental points except that the point ∆EO ) 0, ∆salt ) 0 has been included in the regression as it legitimately represents the critical point of the phase diagram where there is theoretically no difference in PEG and salt concentration between the phases. From the figure, it can be understood that the salting-out strength of the salt, its type, and its concentration determine both the onset of phase separation

in the presence of PEG (the critical point) and the degree of phase divergence of the resulting biphasic system. Just as it requires different amounts of each salt type to produce phase separation with PEG, it requires different amounts of each type of salt to produce approximately equal extensions in TLL or differences in PEG composition between the phases, ∆PEG. Thus, to reiterate a point already made, Figure 9 illustrates that there is no simple relationship between solute partition coefficient (ln D) and the difference in salt concentration between the coexisting phases. It is worth emphasizing that the clouding of a PEG solution in the presence of salts52,66 results from the lowering of its lower critical solution temperature (LCST). It is thus physically identical to the clouding of nonionic surfactants and polymers as employed, for example, in cloud-point extraction67 or as exemplified by so-called thermoseparating polymers.68 Such solutes also exhibit solubility gaps, exemplified by exhibiting both a LCST and a UCST (upper critical solution temperature) in aqueous and other H-bonded solvents. Such behavior is not confined to polymeric systems but is also shown by relatively low-molecular-weight solutes such as the famous nicotine/water system, or glycerol/ guiacol, or lutidine/water, among many others.69 The essential characteristics of all of these systems seem to be similar to those existing for pairs of liquids having dissimilar properties such as polarity, polarizability, or intermolecular bonding, which frequently form incompletely miscible solutions.70,71 On the other hand, the situation with regard to bifunctional molecules is also well-known. These can be partially miscible with liquids of different functionality and display amphiphilic properties, for example, alcohols, fatty acid salts, and surfactants.70 With such materials, self-agregation, surface excess, and micellization can occur, along with the formation of mesomorphic structural phases.70 However, such materials, and, in particular, the nonionic surfactants, also often exhibt closed-loop coexistence curves, especially in mixtures with water or another hydrogenbonding component.70,72 In the present context, such materials are exemplified by the nonionic surfactants used in cloud-point extraction.67 Closed-loop coexistence curves of this type are characterized by the existence of both a LCST and a UCST. However, it is entirely possible that this solubility gap might be obscured in the region of the UCST if it falls above the liquid/gas critical point. Equally, the LCST might be missing if it falls below a crystal boundary. Water can often be replaced for hydrogen-bonding solutes with another hydrogen-bonding solvent without loss of the miscibility gap (although it will differ greatly in form), for instance, by glycerol70 or formamide.72 The molecular-level explanation of this closed-loop miscibiltiy behavior poses less of a conceptual problem for the existence of a UCST than it does for the existence of the LCST.73-75 In the case of the UCST, simple molecular models involving entropic contributions to the free energy of mixing overcoming unfavorable interaction enthalpies will suffice.73-75 A number of explanations of closed-loop coexistence have been proposed over the years. The earliest appears to be that of Hirschfelder et al.,73 who proposed that directionality of hydrogen bonding between solute and solvent and the increase in rotational free energy at higher temperatures represented a mechanism whereby a LCST could occur. This conceptual suggestion was developed in the form

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of a decorated lattice model69,74,76 and simplified by Goldstein to conform with a Flory-Huggins formalism.77-79 It is envisaged that the forces that promote miscibility (directionally oriented hydrogen bonding) decrease with increasing temperature, resulting in phase separation at the critical point of the lower consolute boundary. Ultimately, further increases in temperature result in entropic contributions to the free energy of mixing reestablishing miscibility again as the upper consolute boundary is crossed.69,73-79 Somewhat similar models have been developed to describe the miscibility gap of nonionic polymers.80-84 That of Kjellander and Florin80,81 was based on the ordered structuring of hydrogen-bonded water around the PEO chains in aqueous solution, which becomes increasingly disordered at higher temperature, leading to phase separation. The model might be considered somewhat solute- and solvent-specific, given that PEO can show solvent incompatibility in solvents other than water.72 Karlstrom,83 in the context of polyoxyalkylenes and other nonionic polymers, proposed a conformational model in which it was suggested (from molecular modeling studies) that PEO chains exist in different conformational forms depending on the temperature, with polar conformations being preferred in a polar environment and at lower temperature. A change in the overall population to more hydrophobic conformations with increasing temperature leads to unfavorable interactions with the solvent and to phase separation.83 Although slightly different conceptually, the models of Karlstrom and Goldstein are essentially mathematically isomorphic.83 It is instructive to consider the simple physics of these critical clouding systems that display, close to the critical point, scaling relationships similar to those of other self-organized critical systems such as supercritical fluids, superconductors, ferromagnets, etc.85,86 In performing these studies of solute partitioning, we have worked in density-density space [i.e., under conditions determined by the concentrations of added poly(ethylene glycol) and salt]. However, as has been demonstrated by de Belval et al.,47 it would have been perfectly possible to perform identical experiments in fielddensity space by selection of a polymer/salt system composition close to the critical composition at the experimental temperature. The applied field, the temperature, could then have been varied, and identical changes in system composition would have resulted. Admittedly, in practical terms, it might have proved difficult to elevate the temperature and achieve meaningful measurements of the partition coefficients of these relatively volatile small organics at the extended tie line lengths actually used in the study, but this does not detract from the general principal. Under these circumstances (operation in T-$ space), a diagram of system composition can be constructed in terms of the polymer concentration difference between the phases. This is shown schematically in Figure 11a as the concentration ($) of the polymer in the system in relation to the temperature field (T). It can be seen that this is exactly analogous to the phase diagram for a cloud-point polymer or thermoseparating polymer.45,67,68 This condition is exactly reproduced in PEG/salt systems at zero salt concentration and elevated temperature (ca. 180 °C for PEG-2000). Phase diagrams in T-$ space (a field and a density) can be transformed using a Legendre transformation70 into field space with a loss

Figure 11. (a) Schematic representation of system composition constructed in terms of the polymer concentration difference between the phases of the polymer in the system in relation to the temperature field (T). Polymer concentration in one phase ($1), polymer concentration in the other phase ($2). (b) Schematic representation where the temperature field (T) remains unchanged, but the polymer concentration is transformed into ∆µ, the chemical potential difference between the phases.

of dimensionality such that an area becomes a line and so on. This is shown in Figure 11b, where the field, T, remains unchanged but the density (polymer concentration) is transformed into ∆µ (the chemical potential difference), another field variable, and the phase diagram of Figure 11a is transformed into the line ∆µ of Figure 11b. Consider a system constructed at defined polymer concentration, salt concentration, and temperature. If this system lies above the critical point, it will separate into two phases defined by the polymer concentrations $1 and $2 shown in Figure 11a. It is obvious that these concentrations can be expressed in terms of the polymer concentration difference between the phases or the tie line length. Tie line length and ∆PEG are thus seen to be direct measures of the chemical potential difference between the phases of the system, and the relationship between these measures of system composition and the solute partition coefficient can be understood directly in these terms. From the above considerations and by analogy with other critical phenomena,85,86 recourse can be made to the use of the reduced or dimensionless salt concentration as a correlate for the partition coefficient, in place


Figure 12. Distribution ratios for chlorobenzene vs the reduced salt concentration [SR] in ABSs formed with 40% (w/w) PEG-2000 and increasing concentrations of salt: (b) K3PO4, (9) K2CO3, (2) (NH4)2SO4, (1) NaOH, ([) Li2SO4, (0) MnSO4, (3) ZnSO4.

of the direct use of the salt concentration

[SR] )

[SP] - [SC] [SC]

(20)

where [SR] is the reduced salt concentration, [SC] is the critical salt concentration, and [SP] is the salt concentration of a given biphasic systems. This is shown in relation to solute distribution in Figure 12, where we have assumed, despite the fact that the partitioning was performed a long way from critical conditions, that the relationship should be of the form

ln D ) a[SR]γ

(21)

where γ is an exponent, which, by least-squares fitting of the data, we determined to be 1.45 for the data shown in Figure 12. [SR] is the reduced salt concentration, and a is the critical amplitude of the partition coefficient, which should be solute-specific. Because this expression closely unifies solute distribution data from different systems, it seems that the salt concentration acts as a field-strength-dependent temperature in inducing phase separation. Salt concentration in terms of the reduced salt concentration [SR] seems to represent the field strength of the salt. The reduced salt concentration seems to be a measure of the strength of the field that produces a given difference in chemical potential difference between the resulting phases of the system. This results in a nearly identical series of ABSs for different salt species, as shown in Figure 10. The effectiveness of a given salt in lowering the cloud point of PEG solutions is related to its “salting-out strength”, that is, to its lyotropic number or position in the Hofmeister series. Thus, cosmotropic salts salt-out PEG and, in doing so, produce an identical series of ABSs, which, though differing in absolute concentrations of PEG and salt, are identical in terms of their lyotropic properties. This represents a remarkable simplification of an otherwise complex array of different ABSs into a single series of biphases of graded lyotropy, as shown in Table 1. This field strength analogy can be taken further as the cloud point of typical cloud-point (LCST) systems is reduced progressively by increasing salt concentration

and the rate of reduction is dependent on the saltingout strength of the salt. How does this work? In the models briefly described above, temperature can be understood to increase the translational entropy (or rotational entropy) of the molecules in the system. How then can the addition of a salt, typically cosmotropic water structure makers that are expected to promote the hydrational order present in the system, act to achieve the same result as an increase in the temperature that results in an increase in entropic disorder. Taken at face value, the addition of a cosmotropic salt would appear to increase the amounts of electrostricted and structured water, leading to a decrease in rotational entropy. One striking similarity with the temperature/salt effect that can be noted from the literature concerns the effect of ions on the infrared spectra of water.87 In the past, the effects of various solutes, particularly electrolytes, on the rotation of water as revealed by infrared spectral/studies were compared using the concept of the “structure temperature”. The concept was defined as the temperature at which pure water would have the same (spectral) properties as a solution of a given electrolyte. Thus, water structure makers were found to have high structure temperatures and vice versa, reflecting the degree to which water was adduced to become more, or less, ordered under the influence of the ions. As the concept was based on the rate of change in frequency of various spectral bands, it was entirely possible for salt solutions to have structure temperatures in excess of 100 °C. The concept seems not to have seen further recent development. However, more recently, neutron diffraction studies have shown that high salt concentrations in aqueous solutions can disrupt the tetrahedral structure of water.88 This change in structure has been considered to be equivalent to that produced by the application of high pressure.89,90 Concentrations of a few moles per liter were considered to produce water structural changes equivalent to the effect of pressures of around 1000 atm.88 Finally, it has also recently been noted that the observed water structural changes that occur upon an increase in pressure and the addition of high concentrations of salts can also be regarded as equivalent to those that occur upon increasing the temperature of pure water at constant density.88,90 In each case, the effect is to increase the orientational disorder of the water structure.88 There seem to be only a few possibilities by which the presence of salts could affect the lowering of the LCST in light of the models of the clouding phenomenon outlined above. Perhaps electrolytes could act by reducing the free water available or increasing the activation energy of hydrogen-bond formation or by imposing an orientational order on the water that is incompatible with that of the directional hydrogen bonds of the solutes displaying a miscibility gap. Recent molecular dynamics simulations91 of the effect of salts on the hydration of hydrophobic solutes seem to illustrate the mechanism by which the apparent contradiction of salt-induced ordering of water structure could lead to effects on the LCST in the same general direction as an increase in temperature. It has been suggested88,91 that water molecules in the region of ionic hydration spheres must have strong orientational preferences, which could considerably restrict their ability to reorient and form hydration shells around nearby

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nonpolar solutes. Such ideas seem pleasantly compatible with the previously mentioned models of LCST occurrence in bond-directional solvents. It is obvious from the work presented here, as well as from numerous other studies of clouding52,66 and salting out,92,93 that the effect of aqueous salt solutions is highly salt-specific as reflected in the Hofmeister94 or lyotropic series. The specific physical-chemical properties of the individual salts that produce this effect of lowering the cloud point are less easy to quantify precisely. To this day, the magnitude of the effect of different salts on solute solution behavior, for example, in reducing cloud points or bringing about the salting-out or salting-in of particular solutes seems not to be completely understood. Early electrostatic theories based on DebyeHu¨ckel95 and Kirkwood96 theories were successful in predicting the overall form of solute behavior as influenced by the presence of electrolytes (descriptions of salting-out and salting-in). Predictions at the level of the atom of the detailed effects of particular salts (e.g., the Hofmeister series)94 have proved more elusive.97 It has often appeared that short-range forces were as important in determining the effect of particular salts on nonelectrolyte solution behavior as long-range electrostatic forces. For Sinanoglu98 and later Melander and Horvath,92 forces important in effecting cavity formation by the solute were stressed and correlated with the effect of the salt on the microscopic surface tension of aqueous solutions. Although to some extent successful, for instance, in partially predicting the order in which electrolytes would salt-out proteins, numerous exceptions remained. Earlier work by Long and McDevitt99 took a similar cavity-based approach but stressed, as the macroscopic physical correlate, the effect of the salts on the isothermal compressibility of the aqueous solutions. This is clearly related to the partial molar volume of the salt at infinite dilution, which has long been considered to result in an increase in “internal pressure” of the solution. It is interesting to reflect that hydration numbers of ions can be obtained from isothermal compressibility data100 (among other methods). In Table 1, ∆Ghyd, ∆Shyd, and hydration numbers derived from isothermal compressibility data indicate that each can aid our understanding of the effects of salts on the clouding of PEG solutions, yet none seems overwhelmingly convincing. Most recently, these ideas have been revived in the context of the neutron diffraction studies of aqueous salt solutions88 mentioned earlier. In this case, it is considered that ions having significantly smaller partial molar volumes than water exert a substantial electrostrictive effect and thereby introduce disorder into the tetrahedrally coordinated water structure. Such studies have led to the hope being expressed that a quantitative and detailed description of the effect of salts on neutral solutes in aqueous solution, in other words a molecular-level explanation of the details of the Hofmeister series, will emerge soon.101 Experimental studies based on the clouding properties of different polymers in aqueous solution recommend themselves as a safe, molecularly simplified, yet realistic systems for the analytical development and testing of such hypotheses. In discussing the data presented here, strong comparisons have been made between various cloud-point systems and ABSs. However, it should be stressed that solute behavior is likely to be different for systems composed of different polymers. For instance, in this

Figure 13. Logarithm of the distribution ratios vs the 1-octanol/ water partition coefficient for (1) acetonitrile, (2) ethanol, (3) acetic acid, (4) caffeine, (5) phthalic acid, (6) aniline, (7) 4-hydroxybenzoic acid, (8) 1,3-dinitrobenzene, (9) acetophenone, (10) benzoic acid, (11) benzene, (12) p-toluic acid, (13) salicylic acid, (14) toluene, (15) chlorobenzene, (16) 1,4-dichlorobenzene, (17) 1,2,4-trichlorobenzene, and (18) 4,4-dichlorobiphenyl in ABSs formed with 40% (w/w) PEG-2000 and 1.5 M K3PO4.

paper, the behavior of various small neutral solutes seems little influenced by the molecular weight of the phase-forming polymer, and yet, it is known that, for macromolecular solutes, this parameter strongly influences the resulting distribution.102 Equally, cloud-point systems formed from different polymers or clouding solutes67,68 certainly exhibit solute distributions that are very different from those shown here for PEG/salt ABSs. Nevertheless, it seems likely that an understanding of solute distribution in those systems can be approached in a similar way. Furthermore, the data presented here have been confined to the partitioning of neutral solutes or charged solutes under conditions guaranteeing their neutrality, and thus, the influence of charged state on smallmolecule partitioning has not been considered. It is obvious that PEG/salt ABSs formed with different salts differ widely in pH. Systems used in this study range from pH 4.5 [(NH4)2SO4] to pH 14 (NaOH). For some systems, the influence of pH and salt composition is irrevocably intertwined because the salt is also a buffer ion species (e.g., potassium phosphate). For other systems, such as Na2SO4 and (NH4)2SO4, pH can be controlled to a large extent independently of the salt composition of the system by inclusion of an appropriate buffering salt at a concentration much lower than that of the phase-forming salt. Under all such conditions, the partitioning of charged solutes can differ from system to system, as illustrated in Figures 13 and 14. These figures are examples of neutral and charged solute partitioning in PEG/K3PO4 and PEG/NaOH ABSs in comparison to that in the 1-octanol/water system. Intriguingly, in the PEG/K3PO4 ABS, the charged solute partitioning is very similar to the neutral solute partitioning. Remembering that, in the 1-octanol/water system, the partition coefficients of solutes are always reported for the neutral form, the distribution of the charged solutes in the PEG/K3PO4 ABS is therefore higher than might be expected.103 By contrast, Figure 14 shows that, in the PEG/NaOH ABS, the charged solutes show a reduction in the distribution value more clearly reminiscent of that seen in aqueous/organic systems. A more detailed account of the partitioning of


Literature Cited

Figure 14. Logarithm of the distribution ratios vs the 1-octanol/ water partition coefficient for (5) phthalic acid, (6) aniline, (7) 4-hydroxybenzoic acid, (8) 1,3-dinitrobenzene, (10) benzoic acid, (11) benzene, (12) p-toluic acid, (13) salicylic acid, (14) toluene, (15) chlorobenzene, (16) 1,4-dichlorobenzene, and (17) 1,2,4trichlorobenzene in ABSs formed with 40% (w/w) PEG-2000 and increasing concentrations of NaOH: (b) 4 M NaOH and (9) 6 M NaOH.

small charged solutes in PEG/salt ABSs will be the focus of a future publication. 4. Conclusions We have examined neutral solute partitioning in a wide range of different PEG/salt ABSs formed with different salts and polymer molecular weights and shown that the solute distribution can be understood in terms of the degree of phase divergence of the system, provided that the phase diagram is well characterized for each ABS. Using simple techniques derived from the study of critical phenomena, we have shown that it requires different amounts of salt to produce the same increase in ∆PEG for different salt species. This is significant because, once the PEG is salted-out, the identity of the salt is insignificant for solute partitioning as long as the system composition is expressed in terms of the degree of phase divergence. As a result, we are able to simplify a wide range of PEG/salt ABSs, including PEG/salt ABSs formed with salts whose positions in the Hofmeister series do not predict their abilities to salt-out PEG (Li2SO4, MnSO4, ZnSO4), to a series of closely similar biphases of graded lyotropy. The relative molecular simplicity of these aqueous systems recommends them as ideal vehicles for the analytical study of the molecular nature of aqueous lyotropic behavior, which is of considerable importance in for example functional biochemistry. For small neutral solutes, partitioning in ABSs composed of PEG of differing molecular weights and differing salt types is more closely similar than that in comparative solvent/water systems. These results should greatly aid the implementation of PEG/salt ABSs in practical applications such as analytical molecular characterization or the extraction and separation of macromolecules, small organics, or metal ion species. Acknowledgment This research was supported by the Division of Chemical Sciences, Office of Basic Energy Sciences, Office of Energy Research, U.S. Department of Energy (Grant DE-FG02-96ER14673).

(1) Partitioning in Aqueous Two-Phase Systems: Theory, Methods, Uses, and Applications to Biotechnology; Walter, H., Brooks, D. E., Fisher, D., Eds.; Academic Press: New York, 1985. (2) Huddleston, J. G.; Veide, A.; Ko¨hler, K.; Flanagan, J.; Enfors, S.-O.; Lyddiatt, A. Trends Biotechnol. 1991, 9, 381. (3) Aqueous Two-Phase Systems; Walter, H., Johansson, G., Eds.; Methods in Enzymology Series, Vol. 228; Academic Press: New York, 1994. (4) Aqueous Biphasic Separations: Biomolecules to Metal Ions; Rogers, R. D., Eiteman, M. A., Eds.; Plenum Press: New York, 1995. (5) Albertsson, P.-A. Partition of Cell Particles and Macromolecules; Wiley & Sons: New York, 1986. (6) Huddleston, J. G.; Lyddiat, A. Appl. Biochem. Biotechnol. 1990, 26, 249-279. (7) Builder, S.; Hart, R.; Lester, P.; Ogez, J.; Reifsnyder, D. Aqueous Multiple-Phase Isolation of Polypeptide. U.S. Patent 5,407,810, Apr 18, 1995. (8) Kula, M.-R.; Kroner, K.-H.; Stach, W.; Hustedt, H.; Durekovic, A.; Grandja, S. Process for the Separation of Enzymes. U.S. Patent 4,144,130, Mar 13, 1979. (9) Hustedt, H.; Kroner, K. H.; Kula, M.-R. In Partitioning in Aqueous Two-Phase Systems: Theory, Methods, Uses, and Applications to Biotechnology; Walter, H., Brooks, D. E., Fisher, D., Eds.; Academic Press: New York, 1985; pp 529-587. (10) Berggren, K.; Johansson, H.-O.; Tjerneld, F. J. Chromatogr. A 1995, 718, 67-79. (11) Franco, T. T.; Andrews, A. T.; Asenjo, J. A. Biotechnol. Bioeng. 1996, 49, 309-315. (12) Fan, W.; Bakir, U.; Glatz, C. E. Biotechnol. Bioeng. 1998, 59, 461-470. (13) Giuliano, K. A. Anal. Biochem. 1991, 197, 333-339. (14) Cheng, L.; Joelsson, M.; Johansson, G. J. Chromatogr. 1990, 523, 119-130. (15) Jensen, P. E. H.; Stigbrand, T.; Shanbhag, V. P. J. Chromatogr. A 1994, 668, 101-106. (16) Zaslavsky, B. Y. Aqueous Two-Phase Partitioning: Physical Chemistry and Bioanalytical Applications; Marcel Dekker: New York, 1994. (17) Zaslavsky, B. Y.; Mestechkina, N. M.; Miheeva, L. M.; Rogozhin, S. V. Biochem. Pharmacol. 1982, 31, 3757-3762. (18) Huddleston, J. G.; Griffin, S. T.; Zhang, J.; Willauer, H. D.; Rogers, R. D. In Aqueous Two-Phase Systems: Methods and Protocols; Kaul, R. H., Ed.; Methods in Biotechnology 11; Humana Press: Totowa, NJ, 2000; pp 77-94. (19) Huddleston, J. G.; Griffin, S. T.; Willauer, H. D.; Roger, R. D. In Metal-Ion Separation and Preconcentration: Progress and Opportunities; Bond, A. H., Dietz, M. L., Rogers, R. D., Eds.; ACS Symposium Series 716; American Chemical Society: Washington, DC, 1999; pp 79-100. (20) Rogers, R. D.; Bond, A. H.; Bauer, C. B. Sep. Sci. Technol. 1993, 28, 139-153. (21) Rogers, R. D.; Bond, A. H.; Bauer, C. B.; Zhang, J.; Jezl, M. L.; Roden, D. M.; Rein, S. D.; Chomko, R. R. In Aqueous Biphasic Systems: Biomolecules to Metal Ions; Rogers, R. D., Eiteman, M. A., Eds.; Plenum Press: New York, 1995; pp 1-20. (22) Rogers, R. D.; Bond, A. H.; Bauer, C. B.; Zhang, J.; Griffin, S. T. J. Chromatogr. B 1996, 680, 221-229. (23) Rogers, R. D.; Zhang, J. In Ion Exchange and Solvent Extraction; Marinsky, J. A., Marcus, Y., Eds.; Marcel Dekker: New York, 1997; pp 141-193. (24) Rogers, R. D.; Horwitz, E. P.; Bond, A. H. Process for Recovering Chaotropic Ions from an Aqueous Solution Also Containing Other Ions. U.S. Patent 5,888,397, Mar 30, 1999. (25) Chaiko, D. J. Methods for Extracting Metals from Aqueous Waste Streams for Long-Term Storage. U.S. Patent 5,395,532, Mar 7, 1995. (26) Rogers, R. D.; Willauer, H. D.; Griffin, S. T.; Huddleston, J. G. J. Chromatogr. B 1998, 711, 255-263. (27) Willauer, H. D.; Huddleston, J. G.; Griffin, S. T.; Rogers, R. D. Sep. Sci. Technol. 1999, 34, 1069-1090. (28) Chaiko, D. J.; Mego, W. A. Method for Separating Water Soluble Organics From a Process Stream by Aqueous Biphasic Extraction. U.S. Patent 5,948,263, Sep 7, 1999. (29) C Ä erma´k, J.; Kvı´cˇalova´, M.; Bletchta, V. Collect. Czech. Chem. Commun. 1997, 62, 355-363. (30) Cornils, B. J. Mol. Catal. A 1999, 143, 1-10.

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(31) Haggin, J. Chem. Eng. News 1994, 72, 28-36. (32) Li, M.; Willauer, H. D.; Huddleston, J. G.; Rogers, R. D. Sep. Sci. Technol. 2001, 36, 835. (33) Willauer, H. D.; Huddleston, J. G.; Li, M.; Rogers, R. D. J. Chromatogr. B 2000, 743, 127-135. (34) Diamond, A. D.; Hsu, J. T. Biotechnol. Bioeng. 1989, 34, 1000-1014. (35) Eiteman, M. A.; Gainer, J. L. Biochim. Biophys. Acta 1991, 1073, 451-455. (36) Eiteman, M. A.; Gainer, J. L. Bioseparation 1991, 2, 3141. (37) Eiteman, M. A.; Gainer, J. L. J. Chromatogr. 1991, 586, 341-346. (38) Diamond, A. D.; Xia, L.; Hsu, J. T. Biotechnol. Technol. 1989, 3, 271-274. (39) Hassinen, C.; Ko¨hler, K.; Veide, A. J. Chromatogr. A 1994, 668, 121-128. (40) Carlsson, M.; Berggren, K.; Linse, P.; Veide, A.; Tjerneld, F. J. Chromatogr. A 1996, 756, 107-117. (41) Berggren, K.; Veide, A.; Nygren, P-Å.; Tjerneld, F. Biotechnol. Bioeng. 1999, 62, 135-144. (42) Huddleston, J. G.; Willauer, H. D.; Boaz, K. R.; Rogers, R. D. J. Chromatogr. B 1998, 711, 237-244. (43) Rogers, R. D.; Horwitz, E. P.; Bond, A. H. Process for Separating and Recovering an Anionic Dye from an Aqueous Solution. U.S. Patent 5,707,525, Jan 13, 1998. (44) Maughan, M.; Dion, M.; Hart, P. Benzene Reduction through Chemical Addition Alone. Presented at the 3rd International Conference on Refining Processing Topical Conference, AIChE Spring National Meeting, Atlanta, GA, Mar 5-9, 2000. (45) Huddleston, J. G.; Willauer, H. D.; Griffin, S. T.; Rogers, R. D. Ind. Eng. Chem. Res. 1999, 38, 2523-2539. (46) Merchuk, J. C.; Andrews, B. A.; Asenjo, J. A. J. Chromatogr. B 1998, 711, 285-293. (47) de Belval, S.; le Breton, B.; Huddleston, J. G.; Lyddiat, A. J. Chromatogr. B 1998, 711, 19-29. (48) Bamberger, S.; Brooks, D. E.; Sharp, K. A.; Van Alstine, J. M.; Webber, T. J. In Partitioning in Aqueous Two-Phase Systems: Theory, Methods, Uses, and Applications to Biotechnology; Walter, H., Brooks, D. E., Fisher, D., Eds.; Academic Press: New York, 1995; pp 86-126. (49) Eiteman, M. A. J. Chromatogr. A 1994, 668, 13-19. (50) Rogers, R. D.; Bauer, C. B. J. Chromatogr. B 1996, 680, 237-241. (51) Marcus, Y. J. Chem. Soc., Faraday Trans. 1991, 87, 29952999. (52) Ananthapadmanabhan, K. P.; Goddard, E. D. Langmuir 1987, 3, 25-31. (53) Diamond, A. D.; Hsu, J. T. J. Chromatogr. 1990, 513, 137143. (54) Eiteman, M. A.; Gainer, J. L. Sep. Sci. Technol. 1992, 27, 313-324. (55) Bro¨nsted, J. N.; Volqvartz, K. Z. Phys. Chem. 1931, 155A, 211-224. (56) Taylor, P. J. In Comprehensive Medicinal Chemistry; Hansch, C., Ed.; The Rational Design, Mechanistic Study & Therapeutic Application of Chemical Compounds Series; Elsevier Science: New York, 1990; pp 241-294. (57) Zaslavsky, B. Y.; Miheeva, L. M.; Rogozhin, S. V. J. Chromatogr. 1981, 212, 13-22. (58) Zaslavsky, B. Y.; Miheeva, L. M.; Mestechkina, N. M.; Rogozhin, S. V. J. Chromatogr. 1982, 253, 139-148. (59) Zaslavsky, B. Y.; Gulaeva, N. D.; Djafarov, E. A.; Masimov, E. A.; Miheeva, L. M. J. Colloid Interface Sci. 1990, 137, 147156. (60) Hansch, C.; Leo, A.; Hoekman, D. Exploring QSAR: Hydrophobic, Electronic, and Steric Constants; American Chemical Society: Washington, DC, 1995. (61) Zaslavsky, B. Y.; Gulaeva, N.D.; Rogozhin, S. V.; Gasano, A. A.; Masimov, A. A. Mol. Cell. Biochem. 1985, 65, 125-130. (62) Brooke, D.; Nielsen, I.; de Bruijn, J.; Hermens, J. Chemosphere 1990, 21, 119-133. (63) Krop, H. B.; van Velzen, M. J. M.; Parsons, J. R.; Govers, H. A. J. Chemosphere 1997, 34, 107-119. (64) Rogers, R. D.; Bond, A. H.; Bauer, C. B.; Zhang, J.; Rein, S. D.; Chomko, R. R.; Roden, D. M. Solvent Extr. Ion Exch. 1995, 13, 689-713. (65) Rogers, R. D.; Zhang, J. J. Chromatogr. B 1996, 680, 231236.

(66) Ananthapadmanabhan, K. P.; Goddard, E. D. J. Colloid Interface Sci. 1986, 113, 294-296. (67) Quina, F. H.; Hinze, W. L. Ind. Eng. Chem. Res. 1999, 38, 4150-4168. (68) Persson, J.; Johansson, H.-O.; Tjerneld, F. Ind. Eng. Chem. Res. 2000, 39, 2788-2796. (69) Andersen, G. R.; Wheeler, J. C. J. Chem. Phys. 1978, 69, 3403-3413. (70) Lang, J. C.; Morgan, R. D. J. Chem. Phys. 1980, 73, 58495861. (71) Rowlinson, J. S. Liquids and Liquid Mixtures; Butterworth: London, 1969. (72) Waernheim, T.; Bokstrom, J.; Williams, Y. Colloid Polym. Sci. 1988, 266, 562-565. (73) Hirschfelder, J.; Stevenson, D.; Eyring, H. J. Chem. Phys. 1937, 5, 896-912. (74) Wheeler, J. C. J. Chem. Phys. 1975, 62, 433-439. (75) Chelguet, E. L.; Weber, M. E.; Vera, J. H. Chem. Eng. Sci. 1993, 48, 1415-1426. (76) Andersen, G. R.; Wheeler, J. C. J. Chem. Phys. 1978, 69, 2082-2088. (77) Goldstein, R. E. J. Chem. Phys. 1984, 80, 5340-5341. (78) Goldstein, R. E. J. Chem. Phys. 1985, 83, 1246-1254. (79) Goldstein R. E. J. Chem. Phys. 1986, 84, 3367-3378. (80) Kjellander, R.; Florin, E. J. Chem. Soc., Faraday Trans. 1 1981, 77, 2053-2077. (81) Kjellander, R. J. Chem. Soc., Faraday Trans. 2 1982, 77, 2025-2042. (82) Carlsson, M.; Hallen, D.; Linse, P. J. Chem. Soc., Faraday Trans. 1995, 91, 2081-2085. (83) Karlstro¨m, G. J. Phys. Chem. 1985, 89, 4962-4964. (84) Johansson, H.-O.; Karlstrom, G.; Tjerneld, F.; Haynes, C. A. J. Chromatogr. B 1998, 711, 3-17. (85) Domb, C. The Critical Point; Taylor & Francis: Bristol, PA, 1996. (86) Anisimov, M. A. Critical Phenomena in Liquids and Liquid Crystals; Gordon & Breach: Langhorne, PA, 1991. (87) Verrall, R. E. In Water: A Comprehensive Treatise; Franks, F., Ed.; Perseus Publishing: Cambridge, MA, 1973; pp 211-264. (88) Leberman, R.; Soper, A. K. Nature 1995, 378, 364-366. (89) Tromp, R. H.; Postorino, P.; Neilson, G. W.; Ricci, M. A.; Soper, A. K. J. Chem. Phys. 1994, 101, 6210-6215. (90) Postorino, P.; Ricci, M. A.; Soper, A. K. J. Chem. Phys. 1994, 101, 4123-4132. (91) Mancera, R. L. J. Chem. Soc., Faraday Trans. 1998, 94 (24), 3549-3559. (92) Melander, W.; Horvath, C. Arch. Biochem. Biophys. 1977, 183, 200-215. (93) Shih, Y. C.; Prausnitz, J. M.; Blanch, H. W. Biotechnol. Bioeng. 1992, 40, 1155-1164. (94) Hofmeister, F. Arch. Exp. Path. Pharmakol. 1888, 24, 247260. (95) Tanford, C. Physical Chemistry of Macromolecules; Wiley & Sons: New York, 1961. (96) Edsall, J. T.; Wymann, J. Biophysical Chemistry: Thermodynamics, Electrostatics & the Biological Significance of the Properties of Matter; Academic Press: New York, 1958; Vol. 1. (97) Parsegian, V. A. Nature 1995, 378, 335-336. (98) Sinanogˇlu, O.; Abdulnur, S. Fed. Proc. 1965, 24, S12-S23. (99) Long, F. A.; McDevit, W. F. Chem. Rev. 1952, 51, 119169. (100) Marcus, Y. Ion Properties; Marcel Dekker: New York, 1997. (101) Teixeira, J.; Luzar, A. In Hydration Processes in Biology: Theoretical & Experimental Approaches; Bellisent-Funel, M.C., Ed.; Nato Science Series Vol. 305; IOS Press: Amsterdam, 1905; pp 35-65. (102) Huddleston, J. G.; Ottomar, D. M.; Ngonyani, D. M.; Lyddiatt, A. Enzyme Microb. Technol. 1991, 13, 24-32. (103) Schaper, K. J. J. Chem. Res., Synop. 1979, No. 11, 357.

Received for review July 13, 2001 Revised manuscript received January 25, 2002 Accepted January 27, 2002 IE010598Z

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