Surfactant-Mediated Cloud Point Extractions: An Environmentally

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Surfactant-Mediated Cloud Point Extractions: An Environmentally Benign Alternative Separation Approach Frank H. Quina† and Willie L. Hinze* Department of Chemistry, Wake Forest University, P.O. Box 7486, Winston-Salem, North Carolina 27109

Aqueous solutions of certain surfactant micelles exhibit phase separation behavior upon temperature alteration. This phenomenon can be exploited in separation science for the development of extraction, purification, and preconcentration schemes for desired analytes. Since the addition of just a small amount of an appropriate nonionic or zwitterionic surfactant to the aqueous sample solution is required, this approach is convenient and fairly benign, eliminating the need for the use of organic solvents as in conventional liquid-liquid or solid-liquid extraction. The basic features, experimental protocols, and selected recent applications of this alternative extraction approach, termed cloud point extraction (CPE) or micelle-mediated extraction (ME), are briefly reviewed. In addition, the advantages, limitations, and anticipated future directions of this methodology are discussed. Introduction The purpose of this article is to provide a review and update on the utilization of the phase behavior of surfactant solutions for the purpose of analyte extraction, isolation and/or preconcentration. Since there are several comprehensive reviews on different aspects of the cloud point extraction (CPE) technique,1-11 only the basic features, experimental protocols, recent (since 1995) applications, and future trends will be discussed here following a brief overview of micelles in general. It is hoped that the basic background information and key references cited will lead to an understanding and appreciation of the CPE approach, principally for what it has to offer to the separation scientist as a viable and benign alternative to the current classical extraction methods. CPE is considered by some to be a particular case of aqueous-two phase systems (ATPS),12-15 more specifically aqueous micellar (or detergent or surfactant) two-phase systems (AMTPS).11,16 Traditional ATPS is based upon the phase behavior of polymeric systems and will not be further discussed in this work. Micelles: Formation and Solute Solubilization/ Binding. Surface-active agents (surfactants, detergents) can aggregate in aqueous solution to form colloidal-sized clusters referred to as micelles (normal micelles). The minimum concentration of surfactant required for this phenomenon to occur is called the critical micelle concentration (cmc). Some of the more subtle structural aspects (degree of surface roughness or irregularity, water penetration, etc.) of the micellar aggregates are still the subject of some debate. However, depending upon the specific surfactant and solution conditions, micelles can adopt a variety of shapes, ranging from roughly spherical to ellipsoidal (oblate or prolate). In either case, the interior region of the micelle contains the hydrophobic moieties of the surfactant molecules and the outer surface consists of the hydrated hydrophilic groups along with any bound water molecules.7-9,17-21 In the case of polyoxyethylenated nonionic * Corresponding author. E-mail: [email protected]. † Present address: Instituto de Quimica, Universidad de Sao Paulo, C.P. 26077, Sao Paulo 05599-970, Brazil.

Figure 1. Artistic representation of the rough shape of a typical nonionic surfactant micelle. In the specific case of Brij-35 (polyoxyethylene(23)dodecanol), the micellar aggregate is composed of about 40 individual Brij-35 molecules and is somewhat compact and rod-shaped. [Adapted with permission from Kalyanasundaram, K.; Thomas, J. K. In Micellization, Solubilization and Microemulsions; Mittal, K. L., Ed.; Plenum Press: New York, 1977; Vol. 2, p 571.]

surfactant micelles, this outer region is comprised of coils of hydrated polyoxyethylene chains.21 An artistic representation (cartoon) of a typical nonionic surfactant micelle aggregate is shown in Figure 1.22 Many nonionic surfactants, such as C10E4 and C12E5, and the zwitterionic C8-lecithin, form long, cylindrical, highly polydisperse micelles.5 A recent book chapter also focuses on the microstructure of nonionic surfactants.23 Blankschtein has stressed the importance of the fact that micellar shape can be tuned, sometimes dramatically, by altering the solution conditions (concentration of surfactant and additives, nature of additive, temperature, etc.).5,24-26 The critical micelle parameters, i.e., cmc and aggregation number (the number of surfactant molecules per micelle), for different nonionic and zwitterionic surfactants that have been utilized in surfactant-mediated phase separations are summarized in Table 1.2,7,19,27,28 Details of the salient features of the micellization process and the factors influencing it are provided in a number of monographs on the topic.7,8,17-21 It should be emphasized that changes in temperature, presence, and concentration of ionic or polar additives, etc., can dramatically alter such micellar parameters (and even preclude the formation of micelles).19-21,26 Molecularthermodynamic theories of nonionic surfactant solution

10.1021/ie980389n CCC: $18.00 © 1999 American Chemical Society Published on Web 09/11/1999

Ind. Eng. Chem. Res., Vol. 38, No. 11, 1999 4151 Table 1. Summary of the Aqueous Solution Properties of Some of the Nonionic and Zwitterionic Surfactant Systems That Have Been Employed in Cloud Point Extractionsa surfactant (abbreviation)

cmc,b mM

Nc

cloud point,d °C

Triton X-100 (TX-100) Triton X-114 (TX-114) PONPE-7.5e PONPE-10e C8H17C6H4(OCH2CH2)7.5OH, Igepal CO-630 CH3(CH2)7(OCH2CH2)3OH (C8E3) (CH3)2CH(CH2)10(OCH2CH2)15OH, Genapol X-150 CH3(CH2)9(OCH2CH2)4OH (C10E4) (CH3)2CH(CH2)10(OCH2CH2)8OH, Genapol X-80 (TridecylE8) CH3(CH2)11(OCH2CH2)4OH (C12E4; Brij-30) 1:1 C12E4.2:C12E8 CH3(CH2)11(OCH2CH2)5OH (C12E5) CH3(CH2)13(OCH2CH2)6OH (C14E6) C16H31(OCH2CH2)10OH (C16E10; Brij-56) C18H35(OCH2CH2)10OH (Brij-97) octyl β-D-thioglucoside (OTG) octyl β-D-glucoside (OG) C9-APSO4h Triton X-114-Cibacron Blue affinity surfactant Pluronic L61 (EO2PO31EO2)j Pluronic P105 (EO68PO69EO34)j C8-lecithinl

0.17-0.30 0.20-0.35 0.085 0.07-0.085 5.9-7.5 0.6-0.8 0.05

120-140 100 30 -

64-65 23-25 5-20 62-65 48-52 10.6 19-21 42

40 160 127 84 -

2-7 32-38 23-32 35-42 64-69 85 10-20f 2-20g 65 30-40i

500

25 45

0.02-0.06 0.049-0.065 0.010 0.0006 9.0 20.0-25.0 45.0 0.55i 3.0k -

a Structures and more information on these and other surfactant micelles can be found in refs 2, 4, 7, 8, 19-21, and 37. Typically, the surfactant-rich phase formed from the aromatic-containing surfactants (Tritons, PONPEs) is more dense than the bulk aqueous phase and forms the lower layer, while the less dense alkyl-containing surfactants (Genapol X-80, C8E3, C10E4, C12E5, C12E4.2/C12E8 and C14E6) form an upper (top) phase. b Critical micelle concentration; values taken from refs 2, 7, 8, and 19-21 unless otherwise specified. c N refers to the micelle aggregation number (number of surfactant molecules per micellar aggregate); values taken from refs 2 and 19-21. d Cloud point temperature depends on the surfactant concentration; values taken from refs 2, 37, and 38. [For a recent critical compilation of cloud point temperatures, see ref 27]. e PONPE-7.5 refers to polyoxyethylene(7.5)nonylphenyl ether while PONPE-10 refers to polyoxyethylene(10)nonylphenyl ether. f In the presence of 1.5-3.0% (w/v) dextran. g In the presence of 6.0-10% (w/v) PEG 6000. h Refers to 3-(nonyldimethylammonio)propyl sulfate; marketed under the name N,N-dimethyl-N-[3-(sulfooxy)propyl]-1-nonanaminium hydroxide, inner salt. i Value depends on surfactant concentration and salt/buffer concentrations. j EO refers to ethylene oxide and PO to propylene oxide. k Taken from ref 39. l Refers to dioctanoylphosphatidylcholine.

properties have been developed by Blankschtein and coworkers.29-31 Given only the surfactant molecular structure and solution conditions, properties such as the cmc, optimal micellar shape, size, and size distribution, surface tension, etc. can be predicted. In fact, this work culminated in the development of user-friendly computer programs, dubbed PREDICT and MIX, that are capable of quantitatively predicting many solution properties of single- or multicomponent surfactant (both neutral and charged) systems.31-34 One of the most important properties of surfactants, directly related to micelle formation, is solubilization.18-21 Both bulk solvent-soluble and solvent-insoluble species can reversibly interact with and bind to the micellar assembly. Sparingly-soluble or non-water-soluble materials can be solubilized in water due to their binding to the micelles in solution. The equilibrium constant, Kb, for incorporation of a solute with a micelle:

solute + micelle T micelle-bound solute

(1)

is conveniently defined as

Kb ) [Smic]/([Saq]Cd)

(2)

where [Smic] and [Saq] are the stoichiometric concentrations of the solute in the micellar and aqueous phases, respectively, and Cd is the analytical concentration of micellized detergent (which is equal to the total surfactant concentration minus the critical micelle concentration).35 This solute:micelle binding constant can be related to the partition (or distribution) coefficient, P, i.e., distribution of the solute between the micellar and

bulk aqueous phases, by

Kb ) (P - 1)v

(3)

where v is the partial molar volume of the surfactant.3 A linear solvation free energy relationship analysis approach has been developed which allows for fairly accurate estimation of Kb based upon medium-independent parameters for solute hydrogen bond acidity (∑R2) and basicity (∑β2), excess molar refraction (R2), dipolarity (π2), and volume (Vx).35,36 For a nonionic micelle formed from the surfactant Brij-35 [polyoxyethylene(23) dodecyl ether, C12E23], the following best-fit equation was found to describe solute binding:36

log Kb ) -0.31 + 1.06∑R2 - 3.58∑β2 - 0.15π2 + 0.88R2 + 2.83(Vx/100) (4) This equation allows the estimation of binding constants for incorporation of nonionic solutes in Brij-35 micelles to within better than a factor of 2 if the requisite solute parameters are available (or can be reliably estimated). As with the other charge-type surfactant micelles examined, “the solute incorporation constant [for binding to Brij-35] is dominated by the Vx term (positive, reflecting the hydrophobic effect) and the ∑β2 term (negative, implying that bulk water is a much better hydrogen bond donor compared to the micellar solubilization binding site)”.35 In addition, in contrast to cationic or anionic surfactant micelles, the coefficient of the R2 term is appreciable in the case of the Brij-35, presumably reflecting the contribution of the polyoxy-

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ethylene headgroups in the nonionic surfactant molecule to the polarizability of the micelle solubilization site(s).35 It was concluded that, among the different chargetype micelles, the nonionic surfactant micelles (such as those of Brij-35) should provide the best general solubilization medium for the widest variety of solutes.35 This is fortunate since most surfactant-mediated phase separations reported to date have utilized nonionic surfactant micellar media and indicates that they are indeed “best” in terms of versatility of the solutes that can be preconcentrated and extracted using this approach. In addition to the solute’s structural features, other factors that influence the extent of solubilization include type and structure of the surfactant, presence of electrolytes and other monomeric or polymeric organic additives, and temperature.18,21,35,36 Because of their lower critical micelle concentrations, nonionic surfactants are better solubilizing agents compared to ionic micelles in dilute solutions.18 Many analytical and other applications of surfactant micellar media have been based upon their solute solubilization ability.19,21,40 Phase Separation Behavior of Aqueous Solutions of Neutral Surfactants. Upon heating, aqueous solutions of many nonionic surfactants become turbid at a temperature known as the cloud point (or the lower consulate temperature), above which there is a separation of the solution into two phases.18,19,21,27,37 Phase separation results from the competition between entropy, which favors miscibility of micelles in water, and enthalpy, which favors separation of micelles from water.5,24 Depending on the variation of these two contributions with temperature, either a lower (as for nonionic surfactants) or an upper (in the case of zwitterionic surfactants) consolute point can result.5,24,41 For nonionic systems, the temperature-induced dehydration of the polyoxyethylene headgroups promotes micellar growth and demixing.5,21,23,24,42 The turbidity of the system stems from the presence of very large surfactant aggregates that scatter the visible light passing through the solution.18 Phase separation typically occurs over a narrow temperature range.21 The phases consist of a surfactant-depleted (or dilute) phase and a surfactantrich aggregate (or concentrated) phase,43 sometimes also referred to as the coacervate phase, that appears only in the vicinity of the cloud point temperature.21 It is important to note that the surfactant concentration in the surfactant-depleted phase (typically referred to as the dilute aqueous phase in CPE literature) typically equals or exceeds the cmc, so that micelles or other surfactant aggregate species are also present.5,44 The actual physical separation of the phases is facilitated by the difference in density between the two (dilute aqueous and surfactant-rich) phases. The phase separation process is reversible and, upon cooling the mixture to a temperature below the cloud point, the two phases again merge to form an isotropic, homogeneous solution.19 Several recent studies have utilized fluorescence spectroscopic techniques to study the clouding phenomenon.45-49 Table 1 summarizes the cloud point temperature for some of the surfactant systems discussed in this article.2,27,37 For a homologous series of polyoxyethylated nonionic surfactants, the cloud point increases with decreasing length of the hydrocarbon chain or increasing length of the oxyethylene moiety.19 It should be noted that the presence of other surfactants, acids or bases,

Figure 2. (A) Typical phase diagram exhibited by an aqueous nonionic surfactant micellar solution. L refers to the single isotropic solution region, while 2L indicates the region in which two isotropic phases coexist. [Reprinted with permission from Saitoh, T.; Hinze, W. L. Anal. Chem. 1991, 63, 2521.] (B) Phase diagram exhibited by zwitterionic surfactant micellar solutions, such as that for C9-APSO4 [3-(nonyldimethylammonio)propyl sulfate] (40); L and 2L are as defined in (A). [Adapted with permission from Saitoh, T.; Hinze, W. L. Anal. Chem. 1991, 63, 2521.]

salts, and/or organic additives can alter the critical temperature of such aqueous surfactant solutions, sometimes dramatically.21,26,50-59 Several empirical, statistically based phenomenological theories, models, and mathematical formulations of the clouding process and for estimation/prediction of the cloud point temperature and its dependence on surfactant concentration have appeared in the literature.60-64 The cloud point temperature vs surfactant concentration phase diagrams for many nonionic surfactants are concave-upward, bellshaped curves (Figure 2A, coexistence or consolution curve) separating the one-phase region (L) from the twophase region (2L). The minimum in the curve, referred to as the lower critical point, is defined by a critical temperature and critical surfactant concentration.37 A surfactant-molecular-structure-based theoretical approach has been developed that describes the phase behavior and allows accurate prediction of the expected coexistence curves for nonionic, zwitterionic, and mixed

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Figure 3. Pictorial representation of steps involved in the phase separation (A, top) and cloud point extraction of an analyte (B, bottom). (A) Upon heating above the cloud point temperature, a nonionic surfactant micelle solution will become turbid and after a certain time (which can be accelerated by centrifugation) separate into two phases: the compact micelle or surfactant-rich layer (which, depending on its density, can be the lower or upper phase) and a bulk aqueous phase. (B) Addition of a nonionic surfactant above its cmc will form micelles (represented by the large circles) that can interact with and bind analytes (represented by naphthalene). After temperature alteration and centrifugation, the micellar-bound analyte will be separated from the original bulk aqueous phase and become concentrated in the micelle/surfactant-rich phase, which can be analyzed or subjected to further treatment as required.

micellar systems.29-31,34,41,65 In addition, the effect of additives, such as salt and urea, upon the phase separation process can be taken into account via this approach.26,34,58 In contrast, the phase diagram for zwitterionic surfactants, such as 3-(nonyldimethylammonio)propyl sulfate, C9-APSO4, (Figure 2B), and dioctanoylphosphatidylcholine (C8-lecithin), exhibits a concave-downward shaped coexistence curve, indicating that phase separation occurs upon decreasing temperature.38,41,58,66 Such zwitterionic surfactant micelle systems are said to have an upper critical point. The cloud point behavior exhibited by alkyl glucoside or alkyl thioglucoside surfactants in the presence of water-soluble polymers (such as dextrans or poly(ethylene glycol)) is similar to that of the zwitterionic surfactants in that an upper consolute boundary is observed.67,68 The cloud point extraction (CPE) method (sometimes also called micellar [or micelle-mediated] extraction, ME [or MEX], or liquid-coacervate extraction, LCE69) is based upon this unique phase separation behavior exhibited by aqueous solutions of certain neutral (nonionic and zwitterionic) surfactant micelles. The phase volume ratio, Vs/Vaq, i.e., volume of the surfactant-rich phase (Vs) to that of the dilute aqueous phase (Vaq),70 following the phase separation step, can be very low in such systems, on the order of 0.007-0.04.3,71,72 Consequently, any desired analyte that is solubilized by or bound to the micellar aggregate entity can be separated and extracted (and preconcentrated) into the small volume element of the surfactant-rich phase in a manner akin to a conventional liquid-liquid extraction step. This “new” methodology offers several advantages over conventional liquid-liquid (or liquid-solid) extraction techniques in terms of experimental convenience, lower cost, and the possibility of using relatively nontoxic and

less-dangerous nonionic or zwitterionic surfactants/ detergents in lieu of organic solvents.2,4,7,73-75 Some additional advantages accrue with respect to sample/ analyte storage and analyte detection as will be discussed.2,74,75 Experimental Considerations General Protocol. Experimentally, CPE is quite simple, as illustrated in Figure 3. First, the nonionic or zwitterionic surfactant is added to the aqueous solution containing the analyte(s) to be extracted and analyzed. The final surfactant concentration must be greater than its cmc value so that micelles are present in solution. Since the cmc values of neutral surfactants are quite low (Table 1), only a small amount of the pure surfactant or a small aliquot of a concentrated surfactant solution need be added to the sample solution in question. The pertinent partition equilibrium is established and the analyte(s) incorporated into the micellar aggregates in solution. Next, the temperature is altered (raised to above the cloud point temperature of nonionic surfactants or lowered to below the cloud point temperature in the case of zwitterionic surfactants) so that phase separation occurs. When extracting some biological fluids (serum, blood), the presence of salt is necessary to induce phase separation.76 After demixing of the biphasic system, either by gravity settling or more rapidly by centrifugation, the analytes are concentrated in the small volume (typically in range of 50-400 µL) of the surfactant-rich phase.71,72,77-82 Depending upon the density of this surfactant-rich phase in relation to that of the aqueous phase, it can be either the bottom or top layer. By addition of salt to the system, the density of the aqueous phase can be adjusted to some degree.2,83 In some cases, it is found

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to be easier and more desirable to work with (collect) an upper (rather than lower) surfactant-rich layer, thereby minimizing the possibility of cross-contamination of components from the corresponding aqueous phase.84 In quite a few reported CPEs using Triton X-114 for bioseparations, a cushion of intermediate density, typically a 6% (w/v) sucrose solution, is introduced into the separation mixture.4,6,85,86 Upon phase separation in such a system, the surfactant-rich phase appears below the cushion while the aqueous phase remains above this cushion.3,4 If needed, the CPE procedure can be repeated by addition of more surfactant to the aqueous phase, etc., in order to achieve higher extraction efficiency.6,71,87-89 The surfactant-rich phase is typically in the liquid state and has a very high viscosity; i.e., η ≈ 20 cP.78,90 Thus, in some applications, use of a relatively small amount of an appropriate solvent (dilution agent) such as water, methanol, ethanol, acetonitrile, aqueous solution of another surfactant, etc., has been employed to dilute the surfactant-rich phase 59,66,73,78,80,91,92 in order to decrease the viscosity prior to subsequent volumetric manipulations and/or to facilitate sample introduction prior to an instrumental measurement. In situations where one wants a clear aqueous-based final phase to work with, the addition of a concentrated solution of another appropriate surfactant to the surfactant-rich phase raises the cloud point temperature,6,93 so that the resulting final solution exists as a single phase. Surfactant-rich phases (original or diluted) are compatible with the usual carriers employed in flow injection analysis77and with the hydroorganic or micellar mobile phases employed in high-performance liquid chromatography. Although not yet demonstrated, after suitable dilution, the surfactant-rich phase should also be compatible with the run buffers typically employed in free zone capillary electrophoresis or micellar electrokinetic capillary chromatography. However, problems with sorption of the surfactant on the capillary wall (or stationary phase packing) might cause reproducibility problems, since migration time shifts in capillary electrochromatography (CEC) attributed to dynamic coating of the octadecylsilane stationary phase by the residual surfactant and/or co-extractants have been reported.92 In addition to liquid chromatography,66,77,94,95 different techniques that have been utilized to analyze the surfactant-rich phase include two-dimensional gel electrophoresis,96 fluorography,96 sodium dodecyl sulfate/ polyacrylamide gel electrophoresis (SDSPAGE),86,88,95,97-101 capillary electrochromatography (CEC),92 autoradiography,97 liquid scintillation spectrometry,100 Western or other immunoblotting analysis,86,88,101-103 enzyme linked immunosorbent assay, (ELISA),104,105 and radioactivity,104 among others.106,107 Generally, one should select a surfactant system that has a reasonable (not too high) cloud point temperature. Use of surfactants with high cloud points in the CPE technique could result in stability problems for thermally labile analytes/reagents. More importantly, during the centrifugation step, any temperature drop might result in a decrease in the observed extraction efficiency if the surfactant-rich phase redissolves in the bulk aqueous phase.73,93 However, results of a recent study revealed that centrifugation at different temperatures did not have a significant effect on analyte recovery as long as the phases remained separated.108 Although one could employ strict temperature control during the

centrifugation step, this is an unnecessary complication in view of the many surfactant systems having low or reasonable cloud point temperatures. When the CPE technique is used as a prelude to liquid chromatographic analysis, cognizance must be taken of the fact that the elution of the surfactant could interfere with the detection of the analyte(s) of interest. This is particularly true when using the polyoxyethylenated alkylphenol nonionic surfactants and ultraviolet (or luminescence) detection,77,79,109,110 since the large absorbance (or fluorescence) peak due to the surfactant can obscure the smaller analyte signal. Use of the nonaromatic polyoxyethylenated alcohols71,111 or zwitterionic surfactants (C9-APSO4)66 eliminates this problem since they do not absorb appreciably above 210 nm. In addition, the reduced (nonaromatic) form of Triton X-114 can be employed with UV-absorption detection.106 Electrochemical detection is an ideal alternative since the typical surfactants employed in the CPE technique are relatively electroinactive.77,79,110,112 In many applications, the surfactant can be removed from the surfactant-rich phase via a variety of techniques. For surfactants with a relatively high cmc (>1 mM), the surfactant can be efficiently removed by dilution and dialysis99,113,114 (usually within ≈24 h; thus, for instance, ca. 90% of Trition X-114 was removed after 12 h by dialysis).99 For surfactants with low cmc values, contact with synthetic beads such as Bio-Beads SM2, a copolymeric material with an affinity for Triton-type surfactants,102,106 or Amberlite XAD-2 can be utilized to remove the surfactant.113 The detergent can also be removed by passing the surfactant-rich phase through a column of materials such as Bio-Gel HTP, hydroxyapatite,114 silica gel, or Florisil,109,115 as well as combinations of these. A recent report indicates that the addition of acetonitrile results in the precipitation of the surfactant in the surfactant-rich phase with good recovery of the analytes in the resulting supernatant.76 When injecting the surfactant-rich phase (neat or diluted) into a HPLC system, it is often necessary to wash the nonionic surfactant from the analytical column with a strong hydroorganic mobile phase between the chromatographic runs.116 Prior to GC analysis, the surfactant from the surfactant-rich phase must be physically removed using either column chromatography115 or a GC precolumn (C-18 containing guard column) in order to protect the analytical GC column from becoming coated with the surfactant.74,117 The use of gel permeation chromatography in this regard has also been suggested.108 It should be noted that, for some weakly acidic or basic analytes, it is possible to recover the analyte from the surfactant-rich phase via a back extraction (second CPE step) after pH alteration (addition of a stripping solution containing an appropriate amount of base or acid to the original surfactant-rich phase containing the analyte) to convert the analyte into its charged/ionized form.118 Using such back extraction, Akita and Takeuchi reported up to 100% recovery of phenol from the surfactant-rich phase.118 The nonionic surfactants119employed in CPE to date are all commercially available. Of these, the polyoxyethylenated alkylphenols (or alkylphenol ethoxylates) are the most widely employed. The polyoxyethylene linkages of these surfactants are very stable (toward hot dilute acid or base, as well as oxidizing agents).21 There are no apparent dermatological or toxicological problems associated with their use21 (for a good overview of


aspects of surfactant toxicity, see Chapter 10 of ref 19). The nonionic surfactants employed in CPE are nonvolatile and are classified as either relatively nontoxic or harmless reagents. In particular, those without branched aliphatic chains or aromatic moieties are considered nontoxic and classified as edible by the U.S. FDA.120 The toxicity and biodegradability screening of nonionic surfactants are the subject of a recent report.121 Nonaromatic polyoxyethylenated straight-chain alcohols (alcohol ethoxylates) can also be employed in the CPE technique.2,71 These are more readily biodegraded than the alkylphenol ethoxylate type surfactants.21 In addition, some 3-[alkyldimethylammonio]propylsulfate surfactants, which are zwitterionic at all usual pHs, have also been employed in this extraction approach.38,66 Recently, this class of surfactants has also become commercially available,122 which should facilitate their use in CPE. Currently, the zwitterionic and nonaromatic poly(oxyethylene glycol) monoether surfactants are more expensive compared to the aromatic nonionics (Tritons, Igepals, PONPE) that have been primarily employed in CPE to date. Some commercial surfactants, particularly those of technical grade, can contain impurities, such as peroxide or other contaminants,71,72,83 that might react with or interfere with detection, particularly at trace levels, of the analyte(s) of interest.76,92 In this case, the surfactant must be purified prior to use in a CPE step.72,83 Some highly purified nonionic surfactants are commercially available. It should be noted that nonionic surfactants do not contain a specific fixed number of ethylene oxide units, but instead consist of a statistical distribution of homologues (ethoxamer distribution), with the average degree of ethoxylation (n) reported in its name (CEn). Recently, newer techniques, such as capillary electrophoresis (CE) and solid-phase microextraction coupled with high-performance liquid chromatography (HPLC) have been utilized for determination of the specific ethoxamer distribution.123,124 Experimental Factors That Impact Extraction Parameters. Many factors can impact the concentration factors that can be achieved with the CPE approach. The concentration factor (CF, defined as the ratio of analyte concentration in the surfactant-rich phase to that in the original sample medium) is dictated by Vaq/ Vs, that is, the ratio of volumes of the dilute aqueous phase70 compared to that of the surfactant-rich phase following the phase separation step.72,79,80,118,125,126 This ratio increases as the alkyl chain length of the nonionic surfactant decreases or as the number of ethylene oxide units increases.3,71 This ratio also increases as the concentration of surfactant employed in the CPE step decreases.71,73,81,90,126-129 Typically, a compromise must be struck between the surfactant concentration required to achieve a maximum CF yet provide an adequate volume Vs for subsequent volume manipulation and extraction efficiency.90,108,126 The extraction efficiency typically increases with surfactant concentration up to a maximum value, with essentially quantitative recovery often being observed.81,89,108,110,112,118 In most studies to date, it was found that increases in the ionic strength do not appreciably affect the extracted volume of the surfactant-rich phase (Vs);118,129,130 however, a few notable exceptions have been reported.128 On the other hand, the ionic strength can alter the cloud point temperature, as well as facilitate the separation of the two phases by altering the density of the bulk aqueous phase.118,128-130

The salt concentration does appear to influence recovery, the percent recovery increasing with added salt up to saturation of the solution with the salt.71,108,118 The addition of electrolytes enhanced the extraction efficiency and preconcentration factors (the lower the lyotropic number of the electrolyte, the greater the effect) observed in the CPE of hydroxyaromatic compounds in dyestuffs.131 It has been reported that the analyte preconcentration factor and percent recovery in the CPE process increase as the equilibration temperature employed for phase separation is progressively increased above the cloud point temperature of the system.73,80,108,116 Of course, for thermally labile analytes, the use of elevated temperatures could result in decreased recovery due to decomposition of the solute.83 In addition, only very slight increases in the preconcentration factor (or percent extraction) were observed upon increasing the time of the centrifugation step.71,73,89,132 However, the concentration factor does increase (due to a decrease in the surfactant-rich phase volume) with time when the phases are allowed to separate via gravity settling.118 The equilibration time at elevated temperature (above the cloud point) can also influence the analyte distribution and recovery.89,130 The specific behavior observed seems to depend on the analyte class and surfactant employed in the CPE step.116,129,130 The initial analyte concentration does not appreciably affect the observed recovery, very good efficiency typically being observed over a wide range of analyte levels.71,108,129,130 A recent report indicates that the presence of additives (e.g., ethanol) prior to the extraction step led to an increase in the preconcentration factor and favorable extraction kinetics (i.e., significant decrease in the required equilibration time) in CPE.73,132 Sukhan and co-workers have reported the influence of concentration, time, acidity, and additives upon the properties (including volume) of the phases formed upon heating aqueous solutions of nonionic surfactants to their cloud point temperature.133 Likewise, several studies have focused on the effect that the variation of these experimental parameters has on the analyte distribution between the surfactant-rich and bulk aqueous phases,71,134 as well as the percent extraction in the CPE step.71 Several groups have examined the effect of pH on the extraction of ionizable organics.71,134,135 The neutral (uncharged) forms of such organics partition to the coacervate phase much more strongly than the ionized forms.71,82,83,116,118,127,134 Thus, the pH should be adjusted to ensure that the neutral molecular form of the analyte(s) is present prior to conducting the CPE step. There is also a linear relationship between the distribution ratio and the lowering of the cloud point temperature of the surfactant solution resulting from the presence of the organic compounds.134 As previously noted, the presence of additives, including the target analyte(s), can alter the cloud point temperature.11,136,137 Solutes that bind to the micelle are extracted into the phase-separated surfactant-rich layer to differing extents depending upon the specific micelle-solute binding interaction (Kb value).118 Although based on somewhat limited data sets, it would appear that, independent of the specific micellar system employed in a CPE, essentially quantitative extraction of neutral organic solutes is achieved when that solute’s binding constant, Kb, for interaction with the micelle (eq 1) is greater than about 1000-2000 M-1.1,125,135 This guideline,1,125 to-

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Table 2. Tabulation of Some Extraction Parameters for the Cloud Point Extraction of Metal Ions metal ion extracted/ complexing agent

surfactant employed

detection mode

extraction parametersa

ref

Pd(II)/coproporphyrin III Er(III)/CMAPc Gd(III)/CMAPc Cd(II)/PANe Ni(II) and Zn(II)/PANe U(IV)/PANe Cu(II)/LIX 54h Ru(III)/SCN- k Au(III)/HCl Ga(III)/HCl Ag(I) and Au(III)/DDTPm

Triton X-100 PONPE 7.5 PONPE 7.5 Triton X-114 Triton X-114 Triton X-114 Igepal CO-630i Triton X-110 PONPE-10 PONPE-7.5 Triton X-114

RTPb Visd Visd FAASf FAASf Visd/FIAg FAASf or ISEj Visd FAASf ICPl FAASf

CF ≈ 10; DL ) 2.0 nM CF ) 20; DL ) 0.15 µM CF ) 3.3 to 25; DL ) 5.8 nM; %E ) 99.88 DL < 4 pp; SEF ) 120; CF ) 60 DL ) 6 ppb (Ni); 8 ppb (Zn) DL ) 1.1 ppb; %E ) 98; CF ) 100 %E ) 95-98.6 CF ) 5-10 %E g 90 %E ≈ 90 CF ) 9-130

90 73 132 78 80 141 127 91 81, 142 82 59

a Abbreviations: C ) concentration factor; D ) detection limit; %E ) extraction efficiency; SEF ) signal enhancement factor. b Refers F L to room temperature phosphorescence. c 2-(3,5-Dichloro-2-pyridylazo)-5-dimethylaminophenol. d Visible spectroscopy. e 1-(2-Pyridylazo)2-naphthol. f Flame atomic absorption spectrometry. g Flow injection analysis. h Refers to dodecylbenzoylacetone. i Refers to nonylphenoxypoly(ethylenoxy)ethanol. j Ion (copper) selective electrode potentiometric measurement. k Extracted as an ion pair with zephiramine (tetradecyldimethylbenzylammonium ion). l Inductively coupled plasma spectroscopy. m O,O-Diethyldithiophosphoric acid.

gether with estimation of solute-micelle binding from eq 4,35 thus allows a rough prediction of the CPE efficiency. For highly hydrophobic analytes, only very little optimization of the experimental extraction conditions is required in order to achieve essentially quantitative extraction of the analyte. Applications Historically, the CPE procedure was introduced and pioneered by Hiroto Watanabe and co-workers in Japan,93,138 who utilized it for the effective extraction of metal ions as hydrophobic metal chelate complexes. Subsequently, Bordier extended the scope of the approach to include the extraction of hydrophobic biomolecules.139 In most applications, CPE serves as a primary isolation step in the purification of proteins. In fact, the greatest number of publications involving CPE concern the purification of membrane proteins.4,10,11 For instance, in 1996 alone, the basic or modified Bordier CPE technique was employed in over 120 literature articles140 (and a cursory examination reveals a similar number for 1998). Most recently, CPE has been extended to the extraction, preconcentration, and analysis of environmental organics as well. This section will focus on some of the more recent interesting applications of CPE in these areas, as well as touch on current trends and required developments in this field. Cloud Point Extraction of Metal Ions. The published results clearly demonstrate the usefulness of the CPE technique for quantitatively extracting and concentrating a variety of metal ions (for a more detailed overview, refer to refs 3, 6, and 7). Using the CPE approach, concentration factors and extraction efficiencies comparable to those obtained using classical liquidliquid or liquid-solid extraction methods with organic solvents can be achieved. The CPE technique offers a simple, safe, inexpensive, and nonpolluting approach for the enrichment and analysis of samples containing trace metals. Relatively small amounts of the nonionic (or zwitterionic) surfactant are required, eliminating the need to use relatively large amounts of the volatile and flammable organic solvents that are routinely employed in liquid-liquid extraction schemes for metal ions. At the same time, compared to conventional solvent extraction approaches (which often require up to 1 L of the aqueous sample), CPE requires smaller volumes of the aqueous sample (typically 50-100 mL) in order to obtain the same sample concentration factor.6 In addi-

tion, many of the CPE-based analytical methods offer enhanced sensitivity due to the fact that concentration factors in the range of 10-100 are easily obtained with good recoveries. Table 2 summarizes some of the most recent literature in this area. As can be seen, use of the CPE method has allowed the development of several ultratrace methods for the determination of metal ions. For example, Igarashi and Endo reported a detection limit of 2.0 × 10-9 M for palladium(II) in a procedure based on room-temperature phosphorescence (RTP) of the palladium(II) coproporphyrin III complex in a Triton X-100 surfactant-rich phase following CPE.90 Advantages of using the phase-separated Triton X-100 as a medium for RTP included facile means of concentrating the phosphor, cumbersome deoxygenation step not required, and high light permeability of the TX-100 surfactant-rich phase, among others.90 Olsina and coworkers have reported an optimized CPE procedure for determining and monitoring total and free gadolinium(III) ion in urine following administration of gadolinium-based pharmaceuticals.132 Using their microscale CPE protocol, the limit of detection for Gd(III) is 5.80 × 10-9 M. A 120-fold increase in the flame atomic absorption analyte signal was observed following CPE of the Cd(II)/PAN complex with Triton X-114.78 The CPE of chromium(III) ion employing derivatives of 8-hydroxyquinoline has been reported.144 A sensitive method for the CPE determination of microamounts of ruthenium(III) ion has been reported using Trition X-100 as the nonionic surfactant.91 CPE of gold(III) using PONPE10 has been successfully applied to recover gold from printed substrates.81,142 A downstream processing scheme following CPE allowed the recovery of gold (with purity as high as 99.8%) from printed substrates.142 Several studies have reported very good recoveries of metal ions from synthetic samples via the CPE approach.73,78,90,132 All of these applications clearly demonstrate that the CPE method constitutes a simple, rapid and inexpensive alternative to other preconcentration techniques. It should be noted that the kinetics, thermodynamics, and stoichiometry of the interaction of metal ions with chelating agents is often different in the presence of surfactant micelles from that observed in bulk aqueous or mixed solvent systems.3,145 In addition, the spectroscopic properties (λmax and ; fluorescence intensity, etc.) of the metal chelates in the micelle or surfactant-rich phase can be different from those observed in bulk water alone.7-9,91,146 Likewise, the presence of surfactants/

Ind. Eng. Chem. Res., Vol. 38, No. 11, 1999 4157 Table 3. Examples of Recent CPE of Biomaterials/Clinical Analytes biomaterial/analyte separated/extracted vitamins K, E, and A extracted from aq soln proteinase 3 (Pr3) from neutrophil azurophilic granules tyrosinase from mushroom pileus

surfactant system employed/conditions

CF ) 40-75 purity > 90%; tield ca. 50%

77 85

Triton X-114, 37 °C

5.5-fold purification achieved with a recovery of 84% PEX 12 recovered in surfactant-rich phase

154

Triton X-114, 37 °C Triton X-114, 37 °C

extraction of VSP4A1 from Giardia trophozoites

Triton X-114, 37 °C

cholesterol oxidase extracted from Nocardia rhodochrous isolation of gangliosides extraction of steroid hormonese

C12E5,b 30 °C

cytochrome b5 extraction of 6-aminopenicillanic acid from a penicillin-G hydrolysate extraction of vitamins A and E in serum and blood

ref


extraction of PEX12 fractionation of membrane-bound tissue factor (mTF) and tissue factor (TF) extraction of cell adhesion molecule, C-CAM from rat placenta separation of R- and β-tubulin submits

extraction of vitamin E separation of bacteriorhodopsin from cytochrome c avidin from aqueous solution hexokinase from aqueous solution lactate dehydrogenase from aqueous solution

commentsa


C-CAM partitioned into the surfactant-rich phase 90% R-tubulin extracted into surfactant-rich phase; β-tubulin remained in bulk aqueous phase 14-fold purification of the protein achieved with neglible loss PF ) 5; CF ) 4; with >90% recovery

155 156 101 99 94 126

23 °C PONPE-7.5, 35 °C C9-APSO4, 35 °C C9-APSO4, 35 °C C9-APSO4, 35 °C C9-APSO4/R-biotin,f 4 °C C9-APSO4/OG,g 4 °C Triton X-114-Cibacron Blue conjugate, 45 °C OTGh/PEG 6000,i 0 °C Triton X-114-dextran sulfate C10E4, 30 °C

quantitative recovery; K > CF ) 12-19; with 67-82% recoveries CF ) 26-35; with 88 to >96% recoveries CF ) 45 ≈90% extraction of bacteriorhodpsin %E ) 88 %E ) 50 %E ) 83; PF ) 2.7

84 66 66 66 66 157 157 158

CF ) 21.3; %E ) 94 Pr ) 10; %R ) 91

6 159

SF ) 200

160

Genapol X-80, 40-60 °C

%R ) 85.6 and 82.6, respectively

76

C14E6,c

60d

CF ) concentration factor; PF ) purification factor (typically defined as the ratio of the activity per mg of total protein in the surfactantrich phase to the activity per mg of total protein in the total volume); %E ) extraction efficiency; SF ) separation factor for the extractive separation step; %R ) recovery factor. b Refers to the nonionic surfactant pentaethyleneglycol mono n-dodecyl ether. c Refers to the nonionic surfactant hexaethyleneglycol mono n-tetradecyl ether. d Refers to the partition coefficient for the ganglioside for the nonionic surfactant phase. e Steroids extracted included estriol, β-estradiol, estrone, and progesterone. f Refers to an affinity ligand, N-(biotinoyl)dipalmitoylL-R-phosphatidylethanolamine. g Refers to an affinity ligand, octyl glucoside. h Refers to the surfactant octyl β-D-thioglucoside. i Refers to poly(ethylene glycol) 6000. a

micelles can impact the nebulization process and sensitivity observed in flame atomic absorption spectroscopy.7,9,147 Thus, preliminary studies of such effects caused by the extraction system are an essential step in developing any practical CPE application.3 It is also notable that the partition coefficients of metal chelates in the surfactant-rich phase appear to depend on the nature of the metal ion, in contrast to the behavior observed in organic solvents or predicted by regular solution theory.6 This effect can potentially be utilized to enhance the inherent selectivity via the CPE approach.148 Molar volume effects and thermodynamics of distribution of different metal complexes in micelles of several nonionic surfactants have recently been reported.149,150 CPE in Bioseparation and Purification Schemes. The CPE technique is an effective means by which to isolate and purify membrane proteins in conjunction with chromatographic separations.4-6,10,11,139 Most of the applications to date employ CPE to separate hydrophobic from hydrophilic proteins (for reviews, see refs 2, 4, 6, 10, 11, and 106); the hydrophobic proteins partition preferably into the surfactant-rich phase, while the hydrophilic proteins remain in the dilute aqueous phase.70,87,139 Since biomaterials partition between the two phases according to their hydrophobicity, the CPE technique can also be employed to gauge the degree of hydrophobicity (or hydrophilicity) of biomaterial frac-

tions before and after biomolecular modifications.88,95,103,151 Numerous other enzymes, receptors, and biomaterials have been separated/purified by the CPE technique.4,6,10,11 It has been concluded that the CPE technique with Triton X-114 is the most sensitive, rapid, and convenient method for routinely separating the hydrophilic and hydrophobic forms of proteins and other biomaterials, particularily when screening multiple samples.152 Several standard biological/biochemical purification methods have incorporated the CPE technique into the overall recommended protocols.10,106,114,153 In addition to a few representative examples (Table 3), only the most recent advances in this area will be discussed. Of particular interest are alternative surfactant or surfactant-like systems that (i) exhibit lower cloud point temperatures, making it is possible to conduct the phase separation at 0-4 °C,4,6,10,11 (ii) have a greater degree of extraction selectivity, and (iii) incorporate affinity ligands/surfactants for hydrophilic proteins that permit separation of these by CPE. In addition, development of closed systems that permit downstream recovery and recycling of the surfactant represent an important advance. A particularly impressive example is the purification of the membrane-bound cholesterol oxidase of Nocardia rhodochrous from an unclarified broth by CPE with C12E5 reported by Kula and co-workers.111,126 A two-step scheme was developed for recycling the surfactant, and

4158


a conventional anion-exchange chromatographic step was successfully coupled to the CPE step to form a closed purification procedure. This overall process resulted in a 160-fold purification of the cholesterol oxidase (specific activity ) 16 units/mg, which was suitable for analytical applications) with an overall yield of 80%.111 The general scheme that was developed for recycling the surfactant used in the CPE step should prove very beneficial in other CPE applications as well.111 A three-cycle Triton X-114 CPE procedure was successfully applied for the removal of as much as 99% of the original amount of endotoxin from recombinant protein preparations isolated from E. coli lysate on a very large scale (200-500 mg protein preparations).87 The recovery of proteins after endotoxin removal was greater than 90% in all cases. This CPE technique was found to be more effective than existing chromatographic methods in terms of recovery, retention of biological activity of the proteins, simplicity, and costeffectiveness, particularly in large-scale applications.10,87 In fact, pilot-scale processing levels employing CPE have been reported.161 Schwarz and co-workers have reported the quantitative isolation of labeled gangliosides from cultured rat cerebellum neurons and gangliosides via the CPE technique with C14E6 as the nonionic surfactant.84 The CPE approach was as efficient, but much less timeconsuming, than previous methods used for gangliosides, which required long extraction times, and employed toxic solvents such as pyridine or chloroform/ methanol.84 Heegaard and co-workers have described a simple, rapid, essentially one-step CPE method using Triton X-114 for the extraction and purification of proteinase 3 (Pr3) from azurophilic granules.85 Guina and Oliver have reported that the borrelia haemolysin gene, blyA, can be obtained in high purity from E. coli in a single step utilizing the Triton X-114 CPE technique.162 CPE using Genapol X-80 has been employed for extraction and quantifiying two clinically important vitamins (A and E) from human serum and blood.76 It is anticipated that a comprehensive validation of the CPE method for these vitamins will be developed. Numerous other examples (Table 3) using Triton X-114 (or other surfactants) and the CPE technique have been reported in the recent literature.86,87,96-102,113,155,156,163 In particular, CPE has become an important alternative technique in the purification of plant proteins.10 Phenolic compounds and chlorophylls which interfere in the usual conventional extraction techniques and analysis of proteins, such as polyphenol oxidase, can be successfully removed by CPE.11 Consequently, problems due to the binding of secondary species such as phenols to the target enzyme (which can alter the enzyme’s characteristics), chlorophyll interference with absorption methods for monitoring the isolated enzyme’s activity, and potential modification of enzyme activity during the purification/isolation steps, encountered with traditional strategies, such as ammonium sulfate fractionation or use of acetone powders, have largely been overcome via use of CPE with Triton X-114 as the nonionic surfactant.10 Garcia-Carmona and co-workers154 successfully removed phenols from a crude mushroom extract and isolated tyrosinase with the Triton X-114 CPE approach. This optimized CPE method was much faster than the most recently published “improved” method for purification of mushroom tyrosinase (6 h compared to 3 days) and eliminated the need for an acetone powder step

(acetone precipitated protein).154 The partial purification (5-fold purification, 50% recovery) of a banana polyphenol oxidase using CPE with Triton X-114 and poly(ethylene glycol) (PEG 8000) for the removal of polyphenols was recently reported.164 Other specific examples of such applications utilizing CPE in plant biotechnology have been presented in reviews by Garcia-Carmona10 and Tani11 and co-workers. Zwitterionic surfactants, such as C9-APSO4 and C10APSO4, have also been successfully employed for the CPE of steroidal hormones, vitamin E, and bacteriorhodopsin from aqueous solution.66 A comparative study revealed that greater preconcentration factors and percent recoveries were achieved via use of zwitterionic surfactants in the CPE procedure rather than the nonionic surfactant PONPE-7.5.66 In addition, the zwitterionics do not absorb in the ultraviolet region, an additional advantage relative to Triton X-100 or X-114 when employing spectral detection. Zwitterionic surfactant systems also undergo phase separation upon decreasing the temperature; i.e., their two-phase region occurs at lower temperature than the one-phase region (Figure 2B). This means that one does not have to heat the solution to cause the phase separation and can conduct the extraction at low temperatures (at or below 0 °C). After extraction, it is also much easier to separate and recover the biomaterials from the zwitterionic surfactant-rich phase by dialysis. This is a consequence of the fact that dialysis removes surfactant from protein molecules much more efficiently when the surfactant has a relatively high cmc value.66 To increase the specificity of interactions and, hence, extraction selectivity in such CPE systems, affinity ligands or affinity-derivatized surfactants have been suggested5 and successfully employed in a few cases.157,158,165 In addition, the use of an appropriate hydrophobic affinity ligand should permit the separation of hydrophilic proteins, as illustrated in Figure 4. To date, only a few publications have been concerned with such applications. A Triton X-114-Cibacron Blue conjugate was synthesized and utilized in a three-phase system as the first step of the purification of lactate dehydrogenase.158 The three-phase system was comprised of an upper poly(ethylene glycol) (PEG) phase, the surfactant conjugate-rich middle phase, and a lower hydroxypropyl-starch (HPS) phase. Lactate dehydrogenase concentrated in the middle labeled Triton X-114 conjugate phase with an extraction efficiency of 83%. Subsequent recovery of the lactate dehydrogenase from the surfactant affinity conjugate was achieved by addition of buffer to the surfactant-rich phase followed by another CPE step at 45 °C, in which the enzyme partitioned into the bulk aqueous phase. The recovery achieved was about 60-70%.158 The CPE extraction of vancomycin via use of C10E4 in conjunction with an alanylalanine derivatized cholesterol affinity label has been reported.165 CPE employing the zwitterionic surfactant C9-APSO4 in conjunction with the affinity ligand N-(biotinoyl)dipalmitoyl-L-R-phosphatidylethanolamine allowed the extraction of the hydrophilic protein avidin; similarly, the use of octyl β-D-glucoside (OG) as the affinity ligand allowed extraction of hexokinase.157 In contrast, it was not possible to extract hexokinase via the use of Triton X-114 as surfactant in conjunction with OG as the affinity ligand.


Figure 4. Schematic representation of the surfactant-mediated phase separation process and affinity ligand partitioning in neutral micellar systems. If the hydrophobic affinity ligand strongly binds to both the target protein and the micellar system, then it is possible to extract hydrophilic proteins from an aqueous sample solution as shown. It should be noted that the surfactant-rich phase in the final phase separated system does not necessarily consist of micellar aggregates (21). [Reprinted with permission from Saitoh, T.; Hinze, W. L. Talanta 1995, 42, 119.]

Another development in this field concerns the use of alkyl glucoside surfactants instead of the usual polyoxyethylene alkanol surfactants in conjunction with a water-soluble polymer, such as dextran (Dx) or poly(ethylene glycol) (PEG), for inducing phase separation.4,6,11,67,68 The usual cloud point temperature of alkyl glucoside surfactants is relatively high;166 however, the addition of water-soluble polymers can induce phase separation of such systems into an aqueous phase (which contains all of the water-soluble polymer) and a surfactant-rich phase at temperatures around 0 °C.67,68 These CPE systems also allow the separation of hydrophobic from hydrophilic proteins.11,68 Use of OG and the polymer in this type of CPE application seems to result in better protein stability as compared to systems employing TX-100 or Triton X-114 in the CPE step.4,6,11 Protein inactivation in the presence of nonionic Tritons has been ascribed to their very hydrophobic nature and/ or the presence of impurities in the surfactant.6 The use of octyl β-D-thioglucoside (OTG) in combination with PEG or Dx allowed near quantitative extraction of cytochrome P450 and cytochrome b5.67 However, when the water-soluble polymer in the system was changed to diethylaminoethyl-dextran (DEAE-Dx), cytochrome P450 was still essentially quantitatively extracted into the surfactant-rich phase while extraction of cyctochrome b5 was reduced to ca. 20%. Use of Triton X-114 in conjunction with anionic dextran sulfate (Dx-S) resulted in concentration of cytochrome b5 in the surfactant-rich phase upon CPE from microsomal proteins (with a 10-fold purification and ca. 90% recovery).159 Thus, manipulation of the functionality of the watersoluble polymer can be used to control the selectivity and degree of extraction.11,67 Other advantages of this CPE approach using alkyl glucosides in combination with water-soluble polymers include the ability to tune the two-phase separation to occur at almost any temperature (by controlling the

polymer type and concentration) and the possibility of manipulating the extractability of the desired hydrophobic protein by introduction of an appropriate functional group on the polymer (which remains in the bulk aqueous phase). This CPE system is particularly useful for very labile biomaterials given the low cloud points, which allow one to conduct the entire separation process at 0-4 °C, and the mildness of the alkyl glucoside detergents.6,11 Most important, the cmc of alkyl glucoside surfactants is relatively high, which facilitates the use of dialysis for the subsequent recovery of the separated membrane proteins from the surfactant.67,68 Surfactants such as Triton X-114, which have much smaller cmc values, cannot be removed as rapidly by dialysis.4 It should be noted that polymers such as the dextrans can also be added in conjunction with the alkylpolyoxyethlene type surfactants. For instance, the partitioning of proteins between the phases of a dextran-pentaethylene glycol mono-n-dodecyl ether (C12E5) system has been reported.167 The use of water-soluble polymers in this manner should extend the scope of the CPE technique, particularly with respect to the choice of nonionic surfactants that can be employed.6 It appears that the nature of the water-soluble polymer can help to control the degree of extractability of analytes as well.6,167 In the general Bordier-type CPE approach, the separations are governed by the extent of partitioning to the surfactant-rich phase, which is in turn dependent on factors such as solubilization, hydrophobic effects, affinity interactions, etc., that determine the overall magnitude of the interaction between the solute of interest and the nonionic surfactant micelles. An interesting alternative CPE approach, which is fundamentally different and of demonstrated practical utility, has been pioneered by Blankschtein and co-workers. This latter approach is based upon preferential partitioning of the desired hydrophilic biomolecule into the dilute aqueous168 (i.e., surfactant-depleted or micelle-poor) phase70 as a consequence of repulsive, excludedvolume interactions between the micelles and the solutes.5,16,25,168-171 A theoretical model for hydrophilic protein partitioning based upon such excluded-volume interactions has been developed;5,16,25,169 close agreement is observed between the theoretical results and experimental data for protein partitioning when the neutral surfactants C10E4 and C8-lecithin are employed in the CPE step.5,16,169 By exploiting the stronger excluded-volume interactions between micelles and viruses, as compared to those between micelles and proteins, the Blankschtein CPE approach has been successfully utilized to effect the simultaneous separation of viruses (bacteriophages) from water-soluble proteins in meaningful yields.168,170 The continued development of this approach will serve to expand the scope of CPE, not only in the area of bioseparations but also for separations involving metal ions and organic compounds as well. It should be pointed out that, in many of the published cloud point bioextractions, additional proteins/biomaterials are also co-extracted into the respective phases. Thus, CPE represents only one of several steps involved in the overall purification scheme; subsequent steps typically include column chromatography or electrophoretic procedures. However, the use of CPE in such systems has proven to be very effective at eliminating

4160


Table 4. Use of the CPE Technique in the Extraction and/or Preconcentration of Organic and Environmental Compounds compound(s) extracted phenol lutidine pyridine anthracene benzo(a)pyrene parathion fenitrothion PCBsd PCBse PCDDsh PAHsi PAHsj PAHsl

PAHsm atrazinen napropamide (NP) napropamide (NP) and thiabendazole (TB) fungicideso organophosphorus pesticidesp chlorophenolsq 4-chloroaniline 2,4-D and 2,4,5-Tr fulvic acid hydroxyaromatics aromatic amines PCDFs phthalates and PCDBDt

matrixa

surfactant system/conditionsb

A A A A A A A A and NW SW SW HS HS HS WS WS/S

PONPE-10, 70 °C; GC/FID PONPE-10, 70 °C; GC/FID PONPE-10, 70 °C; GC/FID Triton X-114, 40 °C; HPLC-F Triton X-114, 40 °C; HPLC-F Triton X-114, 40 °C; HPLC-EC Triton X114, 40 °C; HPLC-EC Triton X-100, 71 °C; GC-ECD C12E4, 100 °C; HPLC-F C18E10, 100 °C; HPLC-F Triton X-100, 50 °C; HPLC-UV Triton X-100, 60 °C; HPLC-UV Genapol X-080, 46 °C; CEC Triton X-114, 40 °C; HPLC-WPF Triton X-114, 40 °C; HPLC-F

AS/SS A A A and S

Genapol X-80, 75 °C; SF Triton X-100, 72.5 °C; ELISA Genapol X-150, F Genapol X-80, 75 °C; F

RW RW

Triton X-114, 40 °C; HPLC-EC Triton X-114, 40 °C; HPLC-EC

A A A A RW DS DS SW HS

C8E3, 55 °C; HPLC-UV C12E4.2/C12E8, 45 °C C8E3, 36 °C C12E8/C12E4.2, 45 °C Triton X-100, 90 °C; ion pair HPLC-UV Triton X-114, 40 °C; HPLC-UV Triton X-114, 40 °C; HPLC-UV Genapol X-80 or Brij-56, 95 °C; HPLC-F Genapol X-080, 46 °C; CEC

commentsc %R ) 70-90 %R ) 70 %R ) 40 CF ) 40 CF ) 30 CF ) 75 CF ) 200 %R ) 94-116 from pure water %R ) 82-97,f 88-98;g CF ≈ 35 %R ) 85-96,f 76-93;g CF ≈ 8.5 %R ) 85-98 %R ≈ 100 %R ) 30-109k CF ) 15-70; %R ) 94-100 (from wood ash samples), 81-104 (for river water samples) %R ) 79-104; CF ) 135 CF ) 42.1 CF ) 86; %R ) 78 CF ) 56 (NP), 17 (TB); %R ) 76 (NP) and 22 (TB) CF ) 75; %R g 75 CF ) 40; %R ) 85-l00; %E ) 99.6 CF ) 25-50; %R ) 87.1-99.9 CF ) 20-50; %R ) 88.0 to g99.9 %R ) 70 %R ) 85 and 98 CF ) 13.5; %R ) 78 %R ) 86-106 CF ) 14-135; %R ) 24-125 %R ) 88-100

ref 118 118 118 77 77 77 77 115 129 129 108 108 92 109 75

72 105 128 83 79 110, 112 71 125 71 125 172 131 143 130 92

a A ) extraction of component(s) from an aqueous solution; NW ) natural waters, refers to seepage water of a municipal landfill and seepage water from a hazardous waste dump; SW ) seawater samples; DS ) commercial dyestuffs; HS ) human serum; WS ) water samples (both bottled and network supply); WS/S ) bottled and river water samples as well as solid samples (smoke particulate and wood ash materials); AS/SS ) aqueous solutions and soil suspensions; soil samples (sandy, loamy, and humic soils); RW ) river water. b GC-FID ) gas chromatographic analysis with flame ionization detection (of the bulk aqueous phase); HPLC-F ) HPLC analysis with fluorescence detection; HPLC-EC ) HPLC analysis with electrochemical detection; CEC ) capillary electrochromatography with ultraviolet absorption detection; GC-ECD ) gas chromatographic separation with electron capture detection; HPLC-UV ) HPLC analysis with ultraviolet spectroscopic detection; HPLC-WPF ) HPLC analysis with wavelength programming fluorescence detection; SF ) analysis using synchronous fluorescence; ELISA ) enzyme-linked immunosorbent assays; F ) fluorescence analysis; UV ) ultraviolet spectroscopic analysis. c CF ) concentration factor; %R ) percent extraction recovery; SF ) separation factor (R) for indicated extractive separation. d Polychlorinated biphenyls extracted included PCB 28, PCB 52, PCB 101, PCB 138, PCB 153, PCB 180, and PCB 209. e Biphenyls extracted included biphenyl, 4-chlorobiphenyl, 4,4′-dichlorobiphenyl, 3,4,4′-trichlorobiphenyl, 3,3′,4,4′-tetrachlorobiphenyl, 3,3′,4,4′,5-pentachlorobiphenyl, and 3,3′,4,4′,5,5′-hexachlorobiphenyl. f Initial PCB concentrations in the range of 3-30 ppm. g Initial PCB concentrations in the range of 0.3-3.0 ppm. h Polychlorinated dibenzo-p-dioxins extracted included 1,4,7,8-TCDD, 1,2,3,4-TCDD, 1,2,4,6,9-PCDD, and 1,2,3,4,7-PCDD. i Polycyclic aromatic hydrocarbons extracted included naphthalene, fluorene and acenaphthene, phenanthrene, anthracene, fluoranthene, pyrene, benzo[a]pyrene, chrysene, benzo[b]fluoranthene, benzo[k]fluoranthene, benzo[a]pyrene, dibenz[a,h]anthracene, benzo[g,h,i]perylene and indeno[1,2,3-cd]pyrene. j Polycyclic aromatic hydrocarbons extracted included acenaphthene, anthracene, benzo[a]anthracene, benzo[b]fluoranthene, benzo[k]fluoranthene, benzo[g,h,i]perylene, benzo[a]pyrene, chrysene, dibenz[a,h]anthracene, fluoranthene, fluorene, indene[1,2,3-cd]pyrene, naphthalene, phenanthrene, and pyrene. k Recoveries from spiked drinking water; initial PAH concentrations were in the range of 10.7-25 ng/L. l Polycyclic aromatics extracted included fluoranthene, benzo[b]fluoranthene, benzo[k]fluoranthene, benzo[a]pyrene, benzo[g,h,i]perylene, indeno[1,2,3-cd]pyrene, and pyrene. m Polycyclic aromatics extracted included fluoranthene, benzo[b]fluoranthene, benzo[k]fluoranthene, benzo[a]pyrene, benzo[g,h,i]perylene, and indeno[1,2,3-cd]pyrene. n 6-ChloroN2-ethyl-N4-isopropyl-1,3,5-triazine-2,4-diamine. o Fungicides extracted included flopet, captan, and captafol. p Pesticides extracted included paraoxon, methylparathion, fenitrothion, and ethylparathion. q Chlorophenols extracted included 4-chlorophenol, 2,4-dichlorophenol, 2,4,5trichlorophenol, 2,3,5,6-tetrachlorophenol, and pentachlorophenol. r Refers to 2,4-dichlorophenoxyacetic acid and 2,4,5-trichlorophenoxyacetic acid, respectively. s Polychlorinated dibenzofurans. t Polychlorinated dibenzodioxins.

many of the tedious, time-consuming steps previously required in conventional purification schemes (such as ammonium sulfate precipitation and/or column chromatographic techniques) for bioanalytes.6,10,11 It appears that the CPE technique has achieved rather widespread acceptance in the realm of protein/biomaterial isolation and purification schemes. Cloud Point Extraction of Environmental Organic Compounds. Most recently, the use of the CPE technique as a sample preparation and preconcentration step prior to liquid (or gas) chromatographic analysis

of organic and environmental compounds has become the focus of considerable interest (Table 4). This stems from the quest to develop rapid, simple, sensitive and efficient sample preparation procedures for trace environmental assays. In addition to the safety and cost benefits associated with avoiding the use of large amounts of toxic and flammable organic solvents, the CPE technique offers other potential advantages over conventional liquid-liquid extraction procedures in terms of enhanced detection due to the large preconcentration factors, the elimination of analyte losses


during the evaporation of solvents used in liquid-liquid extraction step(s), and enhanced analyte sample storage (the surfactant eliminates or slows the rate at which analytes sorb onto the container walls thereby increasing analyte recoveries),74,80,75 among others.80,108 Both HPLC separation with UV-visible absorption, fluorescence, and/or electrochemical detection and gas chromatographic separation with electron capture and flame ionization detection have been successfully employed for the quantification of environmentally important compounds following their CPE. For example, the results obtained for the CPE of polychlorinated biphenyls (200 ng/L) from both ultrapure and natural waters (seepage waters from municipal and hazardous waste landfills) were compared to those of the standard German liquid-liquid extraction procedure (DIN 38407) using hexane as the extraction solvent.115 While both methods gave comparable results when extracting ultrapure water samples, with natural waters the performance of CPE was superior to that of the hexane liquid-liquid extracton method. For example, for the extraction of PCB 209 from municipal and hazardous waste landfill water, the recoveries were 103.2% and 81.3%, respectively, when using the CPE method and only 50.7% and 32.4% when using the standard liquid-liquid extraction procedure.115 Similar recovery percentages were also obtained using HPLC analysis with fluorescence detection following the CPE of PCBs from seawater using Brij-30 or Brij-97 as the nonionic surfactant.129 The cloud point extraction of 3-chlorobiphenyl, 3,3′-dichlorobiphenyl, and DDT (1,1,1trichloro-2,2-bis(p-chlorophenyl)ethane) resulted in g99.9% extraction efficiency and a concentration factor of ca. 20 when using a mixed nonionic surfactant system composed of C12E4.2/C12E8.125 The CPE method was also more convenient, with none of the emulsion formation that frequently occurs during liquid-liquid extraction of complex, highly contaminated natural water samples.115 It was concluded that the CPE method is a competitive alternative for the extraction of PCBs from difficult/complex matrixes such as landfill leachates.115 The CPE of polychlorinated dibenzo-p-dioxins in human serum using Triton X-100 under optimized conditions (12% (w/v) TX-100, 4.5 M NaCl at 50 °C) followed by HPLC analysis with UV detection yielded an average extraction recovery of 98%.108 Polychlorinated dibenzofurans (PCDF) have also been extracted from seawater and analyzed via HPLC following CPE.130 The CPE extraction of 15 polycyclic aromatic hydrocarbons (PAH) with the same general system (8% TX-100, 5 M NaCl at 60 °C) was quantitative.108,173 In another study, the CPE of 16 PAHs from bottled and river water samples using Triton X-114 (1% TX-114 at 40 °C) in conjunction with a cleanup procedure to remove the surfactant from the resulting surfactant-rich phase followed by HPLC analysis using fluorescence detection resulted in recoveries of about 70% (with the exception of naphthalene and anthracene).109 Triton X-114 was also employed by another group to extract PAHs (at sub-ppb levels) from bottled water and river water with recoveries in the range of 75-110% and 81-104%, respectively.75 Recoveries approaching 100% were observed for the CPE extraction of PAHs from humic acid solutions as well. In fact, excellent recoveries were achieved after the CPE of PAHs from spiked wood ash samples.75 A comparative study also indicated that CPE of PAHs from smoke particulate samples was as effective and precise as

conventional extraction with organic solvents.75 The detection limits of these different CPE methods for extraction of PAHs from water-based samples were much lower than by alternative methods, with detection limits in the ng/L or sub-ng/L range.72,109 Taken together, the results clearly indicate that the CPE approach with micellar solutions of several different nonionic surfactants is as effective, if not more so, than the traditional organic solvent extraction of PAHs from either aqueous or solid matrixes, and also eliminate the need to use organic solvents for sample pretreatment and preconcentration. Thus, CPE offers “an impressive alternative to conventional solvent extraction” for the preconcentration and cleanup of trace toxicants prior to analysis “because of its environmentally friendly properties and its greater extraction efficiencies.”108 An additional benefit of the CPE approach is that the surfactant employed for the CPE step can itself have beneficial effects during sample storage. In particular, two of the more serious problems that often lead to low recoveries when working with real-world environmental samples, i.e., analyte loss due to its adsorption onto the surfaces of the container or to its interaction with organic particulate matter (humic materials, etc.) or soil sediments (including bentonite), can be greatly diminished or eliminated by the presence of the surfactant.1,74,75,174 A comparative study revealed that the presence of Triton X-114 prevented the loss of analyte due to sorption and that recoveries from humic acid solutions were very high (g97%).75 The adsorption of chlorophenols onto hydrous montmorillonite was significantly decreased in the presence of nonionic surfactants as Triton X-100 or Brij-35.175 In another study, it was found that the presence of surfactants, such as nonionic Brij-35, in the sample was as effective as 40% (v/v) acetonitrile in preventing loss of PAHs due to adsorption on the surface of borosilicate glass, white polyethylene, or PTFE containers.176 Thus, the same surfactants required for CPE can have very beneficial effects with respect to storage and recovery of analytes from environmental samples with no detrimental influence of dissolved organic matter or suspended solids in such samples. An ELISA method for determination of atrazine has been improved via incorporation of a CPE step.105 Use of a Triton X-100 CPE step results in an enrichment (concentration factor) of 42.1 and an improvement of the detection limit by a factor of 10 (detection limit ) 8.0 ng/L). In addition, the presence of the surfactant enhanced the activity of horseradish peroxidase and modified the selectivity by altering the cross-reactivity of other triazine herbicides, allowing confirmation of analyte identity without the need to use different antibodies in the ELISA procedure.105 The plant growth regulator napropamide and the fungicide thiabendazole have also been successfully extracted from both aqueous (tap and mineral water128) and soil samples using the CPE approach with Genapol X-80 (or Genapol X-150) as the nonionic surfactant.83 Napropamide could be detected at concentration levels of 51, 84, and 14 µg/kg soil for sandy, humic, and loamy soils, respectively. The CPE concentration factors (using Genapol X-80 as surfactant) ranged from 57 to 74 (for napropamide) and 17 to 39 (for thiabendazole) depending upon the extraction temperature employed.83 CPE has also been successfully employed for the extraction of aromatic amines

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and hydroxyaromatic compounds from commercial dyestuffs.131,143 The CPE with Triton X-114 as the nonionic surfactant permitted the isolation and enrichment of fungicides in river water samples.79 The preconcentration factor attained was ca. 75 and the detection limits were in the range of 4-6 µg/L, as determined by HPLC with electrochemical detection following the CPE step. The addition of Triton X-114 also improved sample storage since it stabilized the fungicides by preventing their hydrolysis in aqueous solution; this in turn led to more reliable and precise determination of these analytes.79 Likewise, CPE of pesticides using Triton X-114 resulted in quantitative extraction of methylparathion, ethylparathion, and fenitrothion and 85% recovery of paraoxon, with detection limits of 0.04, 0.06, 0.03, and 0.08 µg/L, respectively.110 The CPE of five chlorinated phenols from water using the nonionic surfactant C8E3 yielded good extraction efficiency and concentrating ability, with recoveries in the range of 88-99%.71 These recoveries were slightly better than those obtained using a conventional liquidliquid extraction procedure with hexane as the organic solvent. Similar results were reported for the CPE of these same chlorophenols using a mixed nonionic surfactant system composed of C12E4.2 and C12E8.125 Fulvic and humic acids were also successfully cloud point extracted from river water samples using Triton X-100 at low pH.89,116,172 Under optimized conditions, ca. 80% fulvic acid and 96% humic acid were extracted into the surfactant-rich phase and a preconcentration factor of 13.5 was achieved.89,172 In the CPE of some organic compounds (PCBs, PAHs, amino and hydroxy aromatics; Table 4), recoveries exceeding 100% from spiked samples were observed.115,129,131,143 Although no experimental evidence for this was provided, it was speculated that this might be due to (i) difficulty in the manipulation of the surfactant-rich extractant phase due to its high viscosity129 or (ii) modifications in the microenvironment about the analyte due to the presence of the surfactant which might alter spectroscopic properties (absorbance or luminescence intensity) when employing UV-visible or fluorescence detection.131,135 However, the latter explanation would imply that the chromatographic stationary phase did not separate the analytes from the surfactant-rich phase, which does not seem plausible. The utilization of an internal standard, the standard addition method, or preparation of calibration curves using standards that were subjected to CPE and chromatographed under the same conditions should serve to eliminate such matrix effects upon detection. Current/Future Trends In the realm of metal ion extractions, the continued development of so-called specific chelating surfactants/ micelles can be expected to lead to superior CPE methods for target metal ions.3,177,178 In this context, the synthesis of specially designed hydrophobic ligands containing a chelating moiety (specific for a class of metal ions) and a tuned alkyl chain (for optimal hydrophobic interaction with the surfactant micelle employed in the CPE scheme) should prove to be valuable and result in enhanced extraction efficiencies. Several such surfactant-like chelating reagents have already been reported in the literature, including alkyl derivatized PANs (1-(2-pyridylazo)-2-naphthols),177 long-

chain 5-alkoxypicolinic acids,179 6-(alkylamino)methyl2-hydroxymethyl)pyridines (CnNHMePy),180 and longchain alkoxylmethyl crown ethers,181 among others. Thus far, not very many of these functional chelating surfactants have been utilized for the CPE of metal ions. However, a series of acyl derivatives of 4-aminosalicylic acids (PAS-Cn) has been employed for the CPE of ferric ion in a mixed nonionic surfactant micelle system composed of Triton X-100 and polyoxyethylene(4.2)dodecyl ether, C12E4.2.3 Whereas only 55% of the iron(III) was cloud point extracted when PAS-C4 was the chelating surfactant in this system, 95% was extracted by the more hydrophobic PAS-C8 chelating surfactant. Essentially quantitative extraction of iron(III) from water was achieved when PAS-C10 was employed with Triton X-100/C12E4.2 in the CPE procedure.3 More recently, the hydrophobic chelating ligand, dodecylbenzoylacetone, has been utilized to extract copper(II) ion from aqueous solution using Igepal CO-630 as the nonionic surfactant.127 This CPE procedure was suggested to be suitable for the extractive removal of copper(II) from etching wastewater and related samples.127 These results indicate that the hydrophobicity of the surfactant ligand and that of the metal:ligand complex are the essential factors which regulate the extraction efficiency.3,127 In addition, mixed micelle systems composed of a neutral surfactant and a surfactant-type affinity ligand that target a desired analyte should be pursued as an approach for enhancing the selectivity and specificity of the CPE method, as suggested by Blankschtein.5 In this regard, utilization of a rhamnolipid biosurfactant as an affinity cosurfactant in conjunction with nonionic surfactants might prove beneficial for CPE of such metal ions as lead and cadmium.182 Likewise, utilization of alkyl crown ether compounds for CPE extraction of metal ions has not been investigated.183 A new system, which appears to be particularly useful for the separation of biomaterials, is based on the use of aqueous triblock copolymer surfactant systems.11 The series of polymeric nonionic surfactants referred to as the Pluronics consist of triblock copolymers containing two relatively hydrophilic poly(ethylene oxide) (PEO) moieties covalently united to either extremity of a more hydrophobic poly(propylene oxide) (PPO) segment. As with conventional nonionic surfactants, aqueous solutions of Pluronic surfactants separate into two phases upon heating above their cloud point temperature184 (Table 1). However, the clouding is due to the temperature induced or triggered aggregation behavior of these triblock polymeric materials. Using Pluronic L61 and the CPE technique, Tani et al.185 reported efficient extraction and separation of the hydrophobic protein cytochrome b5 from the hydrophilic cytochrome c. However, the observed extractability was strongly dependent on the specific structure of the Pluronic surfactant employed, in contrast to the behavior observed with the typical nonionic surfactants previously examined. The difference in the extractability of these two proteins with different Pluronic surfactants was attributed to structural differences between the surfactants employed. In addition, Svensson and co-workers186 have utilized a similar triblock copolymer surfactant, Pluronic P105, in conjunction with the polymer dextran, to examine the factors that impact the partitioning of hydrophobic amino acids and oligopeptides between the triblock surfactant-rich and bulk aqueous dextran phases. Thus, such triblock surfactant materials appear to have excel-


lent potential for designing more specific extractions via variation of the triblock polymer structure, the nature of the water-soluble polymer employed, and/or the specific experimental conditions (temperature, type, and concentration of added salt, etc.).11,185,186 The utilization of alkyl polyglycosides187 in lieu of alkyl glycosides11 in CPE might also prove interesting. In addition, the blending of the Bordier-type and Blankschtein excluded-volume CPE approaches170 via use of nonionic-ionic mixed surfactant micelles in order to combine the benefits of both appears promising and should facilitate optimization of the separation process.168 Further examination of the utilization and practical significance of the CPE technique for large-scale downstream processing of biomaterials and for environmental remediation should prove useful. The few studies conducted to date demonstrate the feasibility of CPE for such downstream processes.111,126,161 In addition, greater emphasis should be given to recovery of the biomaterial (or other extracted analyte) from the surfactant-rich phase following the CPE step,118,158 particularly in preparative and process level applications. In this regard, recent studies with zeolites specially prepared to adsorb nonionic surfactants have been shown to enhance the rate of surfactant adsorption.188 This implies shorter contact times for surfactant removal, which should be very beneficial in batch processes for the removal of nonionic detergents from proteincontaining solutions following CPE.188 Despite numerous reports of practical applications of CPE, some mechanistic aspects of the method remain unclear. There is a need to develop theories to describe and predict the “Watanabe and Bordier and related type separations” which are driven by attractive interactions between the micelles and target analytes,168 not only in the context of biological molecules but also for metal chelates and organic compounds as well. Such additional information and insight would, no doubt, lead to the design of superior, more selective cloud point extraction systems and increase the use of this technique. Further examination of the different experimental variables that influence the degree of extraction and the development of new (and improved) techniques for removal of the surfactant from the surfactant-rich phase are required; this is especially important for expanding the scope and applicability of the CPE technique so that it can be coupled to gas chromatographic and GC-mass spectrometric analysis.108 In this regard, an extraction procedure employed to remove surfactant from detergentsolubilized hydrophobic materials prior to electrospray ionization mass spectrometry might prove to be useful for surfactant removal following CPE as well.189 The potential of a new strategy, termed the cloud point foaming technique, should be investigated. As noted by Shen, the foaming behavior exhibited by nonionic surfactant micellar solutions at the cloud point is altered compared to that observed at lower temperatures and this effect can be utilized for foam separating nonionic surfactants from solution.190 This approach can be used for surfactant recovery from environmental sample solutions and potentially coupled to the CPE technique in order to recover/recycle the nonionic surfactant employed in the initial CPE step. This cloud point foaming technique could prove to be important for separations in general, especially when coupled to the CPE technique. However, basic studies are required to

determine its feasibility, benefits, and limitations in these areas of application. Another potential area of application for CPE is in the direct extraction of both nonvolatile and volatile organics from soil and other solid matrixes. The adsorption of hydrophobic analytes on soil, suspended soil particles, or sediments can be dramatically impacted by their contact with aqueous micellar surfactant solutions. The surfactant can also interact with such natural colloids by forming hemimicelles, admicelles, etc.1 on the surface of the colloidal particles. If the surfactant concentration is sufficiently high (much higher than the cmc for that surfactant), then a competition can occur between adsorption and solubilization in the surfactant aggregates. This can have practical applications with respect to sampling of the organic compounds present in such environmental matrixes. The presence of surfactant micelles can reduce the analyte adsorption on the container walls and on any suspended solids, improving storage ability as well as recovery efficiency in extractions, as previously noted.1,74,75,176 Surfactant also stabilizes colloidal suspensions of solids against flocculation. More important, detergents permit the direct extraction of organics from such soil/sediment samples. As previously mentioned, napropamide and PAHs were successfully extracted from spiked soils using Genapol X-80 or Triton X-114 in the CPE procedure.72,75,83 von Wandruszka and co-workers have used the CPE approach with Triton X-114 or Igepal CO-630 as the nonionic surfactant to decontaminate spiked oil- or DDT-polluted soils.191,192 In fact, micellar surfactant solutions, including solutions of nonionic surfactants, have been employed in soil remediation schemes;2,3,193 a subsequent CPE step can be employed to reduce the volume of the surfactant wash solution.2,3,191 In addition, the zwitterionic surfactant C9-APSO4 has been employed to extract anthracene from a natural coal sample.66 The fact that zwitterionic surfactants have their two-phase region on the lower temperature side should be particularly beneficial for extractions from solid or soil samples since extraction of hydrophobic compounds from solids often requires elevated temperatures to improve analyte mass transfer. Thus, with zwitterionic CPE systems, a clear homogeneous micellar surfactant solution is present at the elevated temperature and phase separation occurs only when the temperature is lowered to below ca. 65 °C.66 However, comparative studies of the CPE technique against conventional approaches (such as Soxhlet and supercritical fluid extractions) are required in order to determine the relative mertis of CPE, as well as the dominant factors influencing extraction parameters and performance. In addition, for all environmental applications, the CPE approach needs to be compared to standard regulatory extraction protocols and validated. Conclusions In view of these numerous successful applications of the CPE technique, which employs water as the predominant component along with small amounts of nonionic or zwitterionic surfactants, it should be evident that CPE represents an attractive alternative to conventional organic-solvent-based extractions. The CPE of metal ions, biomaterials, and environmental organic compounds with what is in essence soap water reduces costs associated with organic solvent purchase, storage,

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and disposal, as well as the associated worries regarding toxicity or hazards such as fire and explosion. CPE should therefore be seriously considered as a viable alternative separation method with a bright future, particularly in view of the excellent extraction efficiencies and preconcentration factors typically obtained with the technique. Continuing research emphasis should be placed on comparing the CPE technique to other commonly employed extraction procedures, with special emphasis on the efficiency (preconcentration and recovery), convenience (speed), and economy (solvent/surfactant usage) of the extraction process; the ease of interfacing extraction with subsequent instrumental analysis (particularly via CE or GC/GC-mass spectrometry); and validation of promising new CPE-based methods. Acknowledgment Grants to F.H.Q. from the Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP; Thematic Project 1994/3505-3) and PADCT-FINEP (Project No. 65-92-0063-00) and a fellowship from the CNPq (Conselho Nacional de Desenvolvimento Cientifico e Tecnologico) are gratefully acknowledged. The authors thank Dr. Shigendo Akita (Nagoya Municipal Industrial Research Institute, Japan), Prof. Daniel Blankschtein (Massachusetts Institute of Technology), Prof. Bernardo Moreno Cordero (University of Salamanca, Spain), Prof. Cheng-Kang Lee (National Taiwan University of Science & Technology, Taiwan), Prof. Robert Revia (Tbilisi State University, Georgia), Prof. Jose J. Santana Rodriguez (University of Las Palmas de Gran Canaria, Spain), Dr. Sarath R. Sirimanne (Centers for Disease Control & Prevention, Atlanta), Prof. Hirofumi Tani (Hokkaido University, Japan) and Prof. Ray von Wandruszka (University of Idaho) for providing reprints and/or preprints of articles (or posters) pertaining to their recent work in this field, as well as critical comments and suggestions for improvement of this paper. Julianne M. Braun (Wake Forest University), Prof. Jacinto Guiteras and colleagues (University of Barcelona, Spain), Prof. Roberto A. Olsina and colleagues (Universidad Nacional De San Luis, Argentina) and the five anonymous referees are also gratefully acknowledged for additional comments, criticisms and suggestions pertinent to the paper. Literature Cited (1) Pramauro, E.; Pelizzetti, E. The Effect of Surface Active Compounds on Chemical Processes Occurring in Aquatic Environments. Colloids Surfaces 1990, 48, 193. (2) Hinze, W. L.; Pramauro, E. A Critical Review of SurfactantMediated Phase Separations (Cloud-Point Extractions): Theory and Applications. Crit. Rev. Anal. Chem. 1993, 24, 133. (3) Pramauro, E.; Prevot, A. B. Solubilization in Micellar Systems. Analytical and Environmental Applications. Pure Appl. Chem. 1995, 67, 551. (4) Saitoh, T.; Tani, H.; Kamidate, T.; Watanabe, H. Phase Separation in Aqueous Micellar Solutions of Nonionic Surfactants for Protein Separation. Trends Anal. Chem. 1995, 14, 213. (5) Liu, C. L.; Nikas, Y. J.; Blankschtein, D. Novel Bioseparations Using Two-phase Aqueous Micellar Systems. Biotechnol. Bioeng. 1996, 52, 185. (6) Tani, H.; Kamidate, T.; Watanabe, H. Micelle-Mediated Extraction. J. Chromatogr. 1997, 780, 229. (7) Pramauro, E.; Pelizzetti, E. Surfactants in Analytical Chemistry: Applications of Organized Amphiphilic Media; Elsevier: Amsterdam, 1996.

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Received for review June 18, 1998 Revised manuscript received July 1, 1999 Accepted August 2, 1999 IE980389N

Surfactant-Mediated Cloud Point Extractions: An Environmentally

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