2837
Anal. Chem. 1985, 57, 2837-2843
Influence of Micelles on Partitioning Equilibria of Ionizable Species in Liquid Chromatography: pH and Ionic Strength Effects Manop Arunyanart and L. J. Cline Love* Department of Chemistry, Seton Hall University, South Orange, New Jersey 07079
The effects of prolotropic equlllbria on the assoclation of weak organlc acids and bases with micellar aggregates were investlgated theoretically and experlmentaily, and a model was developed to explain the effects of pH and mlceiie concentration on partitlonlng of soiublllzates into or onto the micelle amphiphatic surface. The model accurately predicts retention behavior, wlth devlatlons from predicted values explalned in terms of electrostatic contributions to the blnding of anionic species to the micelles. The dependencies of chromatographic capaclty factors on pH (sigmoidal) and concentration of micelles (parabolic) are In excellent agreement with predicted values for anionlc, neutral, and cationic solubiilrates. Ocladecyidimethylchiorosllane (ODs)-bonded and cyanobonded slllca columns, whlch interact qulte differently wlth surfactant monomers, allowed assessment of changes In partitioning equllibria resulting from surfactant-modified substrates. Addltlon of salt to the micellar moblie phase causes a reduction of electrical potentlal at the micellar surface that alters solute-micelle Interactions. The model also explains the changes In the sign of the slwe (posillve, zero, or negative) of k’vs. micelle concentration in terms of changes in the sign of the free energy of association resulting from changes In the form of the solute. Apparent pK, values of acids and conjugate acids obtalned chromatographically are reported.
The properties of the heterogeneous micellar microenvironment that provide uniqueness in partitioning of solubilizates include solubilizing power (hydrophobic effects), electrostatic interactions, surface tension changes, and microviscosity changes ( I ) . The effects of micellar systems on solubilization have been found useful in wide variety of techniques, such as separation of biological macromolecules (Z), triplet state photophysics (3-5), drug adsorption (6),and reaction kinetics (7), and they have been employed as micellar mobile phases to perform chromatographicseparations (8-12). Several theoretical models for the liquid chromatographic behavior of micellar systems have been described (12-14). These theoretical models are similar and allow evaluation of partition coefficients, K,,, or solute-micelle equilibrium constants, Kq,from the measured elution parameters. They are useful in determining the strength of micelle-solute interactions for neutral species. For charged solutes, electrostatic attraction or repulsion with the charged head groups of the micelle and/or with the head groups of surfactant monomers adsorbed on the stationary phase can occur, and experimental results that deviate from predictions are not unpommon. Present models cannot accommodate the additional equilibria observed for weak organic acids and bases. For ionizable species, the ratio of undissociated-to-dissociated forms is a function of mobile phase conditions, e.g., pH, ionic strength, buffer type, etc. Large shifts in the acid dissociation constant, pK,, of organic acids occur which generally increase with increasing surfactant concentration (15, 16). Clearly, so0003-2700/85/0357-2837$01.50/0
lute-micelle partition coefficients, K,, of the dissociated and undissociated forms are different. Small changes in pK, can significantly alter chromatographic retention, particularly when the mobile phase pH is near the 50% ionization point of the species. Therefore, in order to calculate solute-micelle equilibrium constants from chromatography experiments, the pH of the micellar mobile phase must be specified. The competing partitioning of molecules to the stationary phase is also influenced by micellar hydrophobic effects and electrostatic interactions. Modification of the stationary phase by adsorption of surfactant can significantly alter the elution behavior of charged solutes. Consequently, the elution behavior of charged solutes is dictated both by the form of the prototropic species and by the type of modified stationary phase. This paper describes a model for micelle-solubilizatemodified stationary phase partitioning behavior of weak organic acids and bases using a straightforward equilibrium treatment. The equations predict sigmoidal dependence of chromatographic capacity factors, k’, on mobile phase pH, and parabolic dependence of k’on micelle concentration. For weak organic bases, good agreement between predicted values of retention vs. micellar-phase pH and the experimental data is obtained. For weak organic acids, predictions of k’could not be made using cyano-bonded silica columns, but the effects of the two competing equilibria were clearly discernible and the acid dissociation constants were obtained. However, by using ODs-bonded silica columns, the predicted k ‘values of the weak organic acids at various mobile phase pH values agreed well with the experimental k’ values. Experimental values of apparent pKa from the chromatographic data for both the acids and protonated bases were in good agreement with literature values. The negative and zero slopes in plots of retention parameters vs. micelle concentrationare predicted and explained by the model in terms of changes in the form of the species (change in sign of the free energy of association of solubilizate-micelle). This is observed experimentally by the presence of an isoeluting point (pH), about which the change in retention with surfactant concentration reverses direction depending on which conjugate species predominates.
EXPERIMENTAL SECTION Apparatus. The high-performance liquid chromatography (HPLC) system consisted of a Technicon FAST.LC high pressure liquid chromatographic pump, a LDC UV monitor detector (254 nm) (Laboratory Data Control, Riviera Beach, FL), and a Rheodyne sample injector equipped with a 20-pL injection loop. The two different type columns (both 25 cm X 4.6 mm i.d.) employed contained 5-pm Ultrasphere ODs-bonded silica (Altex Scientific,Jnc.) or 10-wm cyano-bonded silica (Kratos Instrunients, Ramsey, NJ). A precolumn (12.5 cm X 4.6 mm i.d.) packed with silica gel (25-40 Fm) was located between the pump and sample injector to saturate the mobile phase with silica to minimize dissolution of the analytical column packing. Column temperature was controlled by immersing both columns in a water bath where the temperature was maintained at 25 OC by a Model 73 constant temperature circulating bath (Fisher Scientific). A Model 5000 Fisher Recordall strip chart recorder was used to record the 0 1985 American Chemical Society
2838
ANALYTICAL CHEMISTRY, VOL. 57, NO. 14, DECEMBER 1985
\%qK
HA
K2
d M,
i
K y 65 3
Ls Flgure 1. Schematic representation of equilibria of neutral weak organic acid, HA, and conjugate base, A-, in micellar chromatography with the micelle, M,, and the hydrocarbonaceous bonded stationary phase, L,. K, toK, are equilibrium constants, and K,, is the apparent acid dissociation constant of the acid in micellar solution.
chromatograms,and a Model 7410 Leeds and Northrup pH meter and Fisher combination electrode were used for all pH measurements. Reagents. Electrophoresis grade sodium dodecyl sulfate surfactant (SDS), obtained from Bio-Rad, Inc., was used as received. Sodium chloride, aniline, phenol, bromocresol green (sodium salt) (all from Fisher), bromophenol blue (sodium salt), 2-naphthalenesulfonicacid, phosphoric acid, disodium hydrogen phosphate (all from J. T. Baker), pyridine (Aldrich), p-chloroaniline (Eastman), phenylacetic acid, benzoic acid (both from MC/B), and l-pyrenesulfonicacid (Frinton)were used as received. Procedure. Appropriate concentrations of aqueous SDS containing 0.01 M disodium hydrogen phosphate (or 0.01 M disodium hydrogen phosphate with 0.1 M NaCl) were prepared in single batch quantities sufficient for a complete pH variation study. The mobile phase was filtered through a 0.45-wm Nylon-66 membrane filter (Rainin) and degassed under vacuum prior to use. The pH of the micellar mobile phase was adjusted to the desired pH with phosphoric acid and the column equilibrated by purging with the mobile phase until the pH before and after the column was identical. Methanolic stock solutions of the eluates were diluted to the desired concentrations with distilled water. Unless otherwise specified, mobile phase flow rates of 1.0 and 2.0 mL/min were used for the ODS and cyano columns, respectively. Retention times were measured from the injection point and the chromatographicpeak maxima. The time equivalent of the void volume (1.9 and 1.0 min for ODS and cyano columns, respectively) was measured as described previously (14).
THEORY The previously described methematical model relating micellar liquid chromatography capacity factor to micellar mobile phase concentration and for calculation of solutemicelle equilibrium constants of neutral species ( 1 4 ) can be extended to ionizable solutes such as weak organic acids. The additional prototropic equilibria affecting micelle-solute association and chromatographic partitioning must be considered (Figure 1). The interaction of undissociated HA, and the dissociated form, A-, with the stationary phase, L,, and the surfactant in the micelle, M,, forming complexes HAL,, HAM,, ALL, and AMm-,are represented by eq 1-7 where the species molar concentrations in the two phases are denoted by subscripts s and m. The equilibrium constants corre-
+
HA L, HAL, HA + M, + HAM,
+ L, + AL,A- + M, + AM,HAM, + L, + HAL, + M, AM,- + L, + ALL + M, HA + A- + H+ A-
(1) (2) (3)
(4) (5) (6)
(7) sponding to eq 1-6 are Kl to Ks, and K,, represents the apparent acid dissociation constant of HA in micellar solution (eq 7). Equations 5 and 6, representing the direct transfer of solute in the micelle to the stationary phase, may be ne-
glected (K& = K1,and KJC4= K3). [M,] is calculated by [M,] = [surfactant] - cmc, where cmc is the critical micelle concentration. This model calculates equilibrium constants, not partition coefficients, and has the advantages over other models that the volume of the stationary phase, us, and the partial specific volume of the surfactant, 8, need not be known. Significantly, if micelle-solubilizate equilibrium constants are available from independent sources, the model can predict chromatographic behavior of neutral species at any concentration of micelles and a t any pH. The magnitude of solute retention expressed as the capacity factor k‘, is defined conventionally as k‘ =
r([HAL,I + [ALL]) .~ [HA] + [A-] + [HAM,] [AM,-]
+
(8)
where y is the phase ratio of the volume of column stationary phase, V,, to that of the mobile phase, V,. Concentrations of protonated and neutral forms of acids depend on pH, and a t low pH only the neutral form exists. Combining eq 1, 2, and 8, one obtains where Iz{ is the limiting capacity factor for neutral forms. Similarly, at high pH where only the dissociated form is present, combination of eq 3, 4, and 8 results in
(10) kl’ = r[LsIK,/(1 + Kd[MrnI) where 12,’ is the limiting capacity factor for the dissociated form. Equations 9 and 10 predict parabolic curve dependence of k‘on [M,]. For intermediate pH values, combination of eq 7-10 yields an equation relating capacity factor to pH
h’=
k,’(l
+ K,[MmI) + kl’(1 + K~[MmI)K,m/[H+l
(11) 1 + K2[MmI + (1 + K4[MmI)Kam/[H+I A similar equation can be derived for the interaction of weak organic bases by replacing HA and A- with the protonated base BH+, and free base form, B, in the model in Figure 1and into eq 1-10. The equation relating capacity factor to pH for weak organic bases follows
where k,,’ is the limiting capacity factor of neutral base, B, k< is the limiting capacity factor of protonated BH+ form, and K,, is the apparent acid dissociation constant of the protonated weak organic base, BH+. If [M,] is constant, eq 11 and 1 2 predict a sigmoidal-type dependence of k’on pH, and if the pH is constant, they predict parabolic curve dependence of k’on [M,]. For each solute, the three parameters of this model (k,,’, kl’, and K,,) can be estimated from experimental data obtained by measuring solute capacity factor vs. mobile phase pH, and these values can be used to calculate Fz’values for organic acids and bases at different pHs. Note that all equations given above do not contain an electrostatic repulsion term (14, 17). Therefore, for ionic species of the same charge as the micellar aggregate, deviation from predicted k’ values would be expected if electrostatic effects are a major factor in the micelle-solute-stationary phase separation mechanism. Also, changes in the cmc caused by electrolytes and solubilizates can occur ( 1 8 ) . However, if the surfactant concentration is above the cmc, plots of l / k ’ vs. [M,] or [SDS] (Figure 2) should show the same general behavior independent of the absolute cmc value. Thus, the elution behavior of solubilizates can be explained without knowing the exact cmc. It should be noted that the derivations described above were based on earlier work by Knox and Hartwick (19) and Horvath
ANALYTICAL CHEMISTRY, VOL. 57, NO. 14, DECEMBER 1985
0
0.01
0.02
0.03
2839
0.04
1SDSI.M
Flgure 2. Dependence of llk’vs. [SDS] for benzene: column, 5 pm Supelco C18, 15 cm X 4.6 mm; flow rate, 1.9 mLlmln; mobile phase, aqueous SDS.
Flgure 3. Dependence of (A) k’and (B) Ilk’on [SDS] for phenol: column, 10 pm cyano-bonded silica; flow rate, 2.0 mL/min; mobile phase, SDS in 0.01 M phosphate buffer. Each k’is the average of the values obtained at several pH settings (k’essentially constant vs.
PHI.
et al. (17) and are similar to other equations in the literature. For example, Sybilska developed an equation similar to eq 11 above to describe the elution behavior of solute-cyclodextrin complexes using cyclodextrin mobile phases (20). Also, eq 9 above is the same as one developed previously if the substitutions k’ = (v, - u,)/v, and K2 = b(K,, - 1) are made (21). However, eq 10 in this report is used to describe the elution behavior of anionic species.
B
RESULTS AND DISCUSSION Cyano-bonded and ODs-bonded silica columns interact very differently with surfactant monomers resulting in different elution behavior of organic acids and bases as a function of micelle mobile phase concentrationand pH. The ODS column is modified by adsorption of SDS monomers with the negative head groups in contact with the mobile phase, such that the surface is charged and will repulse anionic species. Cyano packing does not appear to adsorb SDS monomers and generally has the capability to retain various ionic species. The aqueous solution pKa values for all solutes are within the pH range examined (pH 3-7) except phenol (pK, = 9.98) and two sulfonic acid derivatives. The pKa of 2-naphthalenesulfonic acid and 1-pyrenesulfonicacid were not available, but the pKa of 1-naphthalenesulfonic acid is 0.68 (22). These two compounds most probably are in their anionic form, and phenol is in its neutral form under the conditions of the experiment. Weak Organic Acids on Cyano Columns. Micelle Concentration Effects. Figure 3 shows the behavior of phenol on the cyano column plotted as k’ vs. [SDS] and l / k ’ vs. [SDS], where the capacity factor decreases with increasing [SDS] and is invariant over the pH range 3-7 at SDS concentrations from 0.02 to 0.10 M. The separation process is mainly controlled by hydrophobic interactions of the neutral species with the micellar assembly and stationary phase, and the experimental behavior follows the prediction of eq 9. The relatively small curvature in Figure 3A indicates that the phenol-micelle equilibrium constant, K2,is small, resulting in a slow change in k ‘with micielle concentration and, in this case, a small k’ (see Figure 3 in ref 14 for comparison). Anionic 2-naphthalenesulfonate should experience electrostatic repulsion from the micelles and be retained longer with increasing micelle concentration. In Figure 4,the capacity factor is directly related to [SDS] rather than l/k’, and the anion elution behavior plotted as k’ and l / k ’ vs. [SDS] is exactly the opposite of that seen for neutral phenol. This opposed behavior results most probably because more micellar aggregates would more effectively solubilize a neutral species (phenol) causing it to elute faster but would increase electrostatic repulsion of a like-charge anionic species (2-
0.41
0
I oL 0.02
0.05
0.08 0.10
[SDS], M
0.02
0.05
0.08 0.10
[SDS], M
Figure 4. Chromatogrpahlc retention values for 2-naphthalenesulfonic acid: (A) k‘vs. [SDS] at (0)pH 3.0, (A)pH 4.5, (0)pH 5.5, and (U) pH 6.5; (B) l/k‘vs. [SDS] at (0)pH 3.0, (A)pH 4.5, (0)pH 5.5, and (U) pH 6.5. Chromatographic conditions are the same as those given
In Flgure 3. naphthalenesulfonate) causing it to elute more slowly. The additional surfactant also decreases the free water content of the solution, thereby increasing the electrostatic density. Furthermore, the K,[M,] term in eq 10 for anionic species must be very small compared to 1. Consequently, eq 10 predicts that k’for these types of species should be directly related to a constant term, y[L,]K,. However, if an excluded volume effect is present, resulting from the unavailability of the micelles to the solute, this would increase y, and thereby increase k’, with increasing [SDS]. Because no electrostatic or excluded volume terms are included or adjusted in the equation, it does not predict the experimentally observed increase in k’ with [micelle] resulting from electrostatic repulsion or expulsion of the anion from the micellar aggregate. Thus, for species exhibiting this type of behavior, the equilibrium constant for the solute-micelle interaction cannot be accurately calculated from the slope/intercept ratio. A hydrophobic component influencing micelle-solute interaction of solutes with like charge centers can be seen by comparing retention data of 2-naphthalene- and l-pyrenesulfonates (Figures 4 and 5). Because the strength of the micelle-solute association increases with the hydrophobicity of the solubilizate, the larger pyrene moiety should produce a counterbalancingof the electrostatic repulsion and associate more strongly with the micelle. This would produce a larger
2840
ANALYTICAL CHEMISTRY, VOL. 57, NO. 14, DECEMBER 1985
1.2
0.9
-
0
0.02
0.05
-
0.080.10
[SDSI, M
0
0.02
0.05
0.08 0.10
WSI, M
k’ 0.6
Figure 5. Chromatographic retention values for 1-pyrenesulfonicacid:
(A) k’vs. [SDS] at (0)pH 3, (A)pH 5, (e)pH 6, and (D) pH 7;(B) llk’ vs. [SDS] at (0)pH 3, (A)pH 5, (e)pH 6, and (m) pH 7. Chromatographic conditions are the same as those given in Figure 3.
rate of change in k’with [SDS]. Its equilibrium constants, K 3 and K 4 , should be larger than that of the naphthalenesulfonate anion, and its interactions should be stronger with both the micelle and the stationary phase. Thus, with increased solute hydrophobicity, a decrease in electrostatic repulsion with the micelle, concomitant with a increase in stationary phase influence, should occur. Figure 5 shows that the influence of the two opposing forces and the sign of the curvature is reversed for the more hydrophobic acid, with results which deviate somewhat from that predicted by eq 10. The rate of change of k’ with micelle content is greater for the more hydrophobic sulfonic acid, and it is more strongly retained by the stationary phase. The hydrophobic force overcomes the anion solute-micelle repulsion, producing elution behavior similar in direction of curvature to neutral phenol. The pyrenesulfonate anion is more strongly retained on the column at lower pH, most probably due to ionic strength effects and not to an increase in the fraction of neutral form present. Note, a t one extreme, if the electrostatic repulsion of the solubilizate with the micelle is much greater than the hydrophobic attraction, the micelle concentration becomes directly related to k’rather than its reciprocal, l/k‘(Figure 4). Although the equilibrium constant, K4,for anionic solutemicelle may be small (AG = -RT In [1], negative). Micelle Bulk p H Effects. The pH of the bulk micellar solution was found to be of fundamental importance in determining the sign of the slopes of the llk’vs. [SDS] plots (positive, negative, or zero). Plots of capacity factor vs. pH at varying concentrations of SDS for benzoic acid and bromocresol green are given in Figures 6 and 7. Comparable changes were observed for the other weak organic acids studied. Completely opposite behavior is observed depending on the pH, where at low pH the neutral acid k’values decrease, and at high pH the anionic conjugate bases k’values increase with increasing [SDS]. In the intermediate pH value range, there is an isoeluting point where the capacity factor is completely independent of SDS concentration. This isoeluting
0.3
0 1
2
3
4
5
6
7
PH Figure 6. Chromatographic retention variation for benzoic acid with pH at various concentrations of SDS: (0)0.02, (A)0.05, (e)0.08, and (B) 0.10 M. Chromatographic conditions are the same as those given in Figure 3.
1.!
1.c
k’
0.5
0 1
2
3
4
5
6
7
PH Figure 7. Chromatographic retention changes for bromocresol green with pH at various concentrations of SDS. Legend and conditions are given in Figure 6. point is the pH where two species (acid and conjugate base or base and conjugate acid) in equilibrium with each other have the same k’value. Thie is exactly analogous to isosbestic and isoemissive points in spectroscopy. The k ’value will not be a qualitative indicator of a particular species unless the pH and micelle concentration are maintained constant, or the pH corresponding to the isoeluting point is used. These results are consistent with the discussion above, Le., the separation process in micellar systems is controlled by a balancing of electrostatic and hydrophobic interactions. The predominate mechanism is not significant changes in the micelle structure but is based on changes in the charge form of the solubilizate. On the basis of these data, the pH value
’ANALYTICAL CHEMISTRY, VOL. 57, NO. 14, DECEMBER 1985
2841
Table I. Limiting Capacity Factors of Selected Organic Acids and Bases at Four Concentrations of SDS, pKamValues Estimated from Chromatographic Measurements on ODS and Cyano Columns, and Literature pK, Values ODS Column
compound benzoic acid phenylacetic acid bromocresol green
[SDS], 0.04 M
[SDS], 0.06 M
kd
kd
kl’ -0 -0
20.8 14.3 3.38
18.8 13.1 2.47
0.05
kl’ -0 -0
0.05
[SDS], 0.08 M kd kl‘ 16.2 12.2 1.79
[SDS], 0.10 M kd kl‘
-0 -0 0.05
pKamn
-0 -0
13.0 9.0 1.55
4.7 4.8 6.1
0.05
PK,~ 4.2 4.3 4.9
Cyano Column [SDS], 0.02 M
compound aniline p-chloroaniline pyridine benzoic acidc phenylacetic acid’ hydrocinnamic acidc bromocresol greenc bromophenol blueC
0.33 0.23 1.47
1.75 1.78 7.0
[SDS], 0.05 M 1.48 0.99 5.56
0.14 0.12 0.57
[SDS], 0.08 M 1.16 0.69 4.50
[SDS], 0.10 M
0.12 0.11 0.42
p ~ , , ~
0.10 0.11 0.37
1.09 0.58 4.33
6.0 5.8 6.0 4.7 4.7 5.3 6.3 5.2
p ~ , b
4.6 4.0 5.2 4.2 4.3 4.7 4.9 4.2
Apparent acid dissociation constant in micellar solution obtained from measured capacity factors vs. pH, and calculated according to ref Values are average from four different SDS concentrations. *Aqueousacid dissociation constants from ref 22 and 24. CTheapparent pK,, values determined from the isoeluting points. a
23.
13
3.5
12 10 8
2.5
6
k’
4
1.5
2 2
3
4
5
6
7
PH Figure 9. Dependence of anillne k’on pH with 0.05 M SDS mobile phase in 0.01 M phsophate buffer: column, MBondapak C-18 modified with adsorbed SDS; flow rate, 2.0 mL/min.
0.5
0 1
2
3
4
5
6
7
PH Flgure 8. Dependence of k’of bromocresol green on pH at various concentrations of SDS: (0)0.04, (A)0.06, (0)0.08, and (W) 0.10 M; column, 5 Fm ODS-bonded slllca modified with adsorbed SDS; flow rate, 1.0 mL/min; mobile phase, aqueous SDS in 0.01 M phosphate buffer. Solid lines were calculated by using eq 11. The parameter estimates fork:, k,‘, and pK, are (0)3.38, 0.05, and 5.7, (A)2.47, 0.05, and 6.0, (0)1.79, 0.05, and 6.2, and (m)1.55, 0.05, and 6.3.
of the isoeluting point, marking the change from increasing to decreasing k’ with micelle concentration (or vice versa), would be expected to be equal to the “apparent” acid dissociation constant, pKm. The pK, values (Table I) determined from the isoeluting points were 4.7 for benzoic acid, 6.3 for bromocresol green, 4.7 for phenyl acetic acid, 5.2 for bromophenol blue, and 5.3 for hydrocinnamic acid. Published work that does not take into account the pH of the mobile phase should be interpreted with caution (10, 11). Weak Organic Acids/Bases on ODS Columns. Micelle Bulk p H Effects. The data discussed so far has not addressed the validity of predictions using eq 11. By using a ODSbonded column where negatively charge surfactant monomers are adsorbed instead of the relatively unmodified cyano column, the effects of pH on capacity factor can be studied in more detail. The elution behavior of phenol and l-pyre-
nesulfonic acid, where hydrophobic effects would dominate, are very similar on ODS columns to that obtained using cyano columns. The less hydrophobic and negatively charged 2naphthalenesulfonate elutes very quickly (unretained) on ODS because of repulsion from both the micelle and the negatively charged modified stationary phase. Figures 8 and 9 show plots of k’vs. pH for bromocresol green and aniline that are typical of the behavior observed on ODS columns for the acids and bases studied. Inspection of the k’values in Figure 8 for a weak acid using an ODS column reveals that the largest k’ values occur in acidic solutions where the neutral form is present and is smallest in more basic solution where the anionic acid form is present (electrostatically repulsed by both the negative micelle and stationary phase). However, adsorption of anionic surfactant monomers on the surface of the ODS stationary phase causes protonated organic bases to be retained longer than the neutral free-base forms because of electrostatic attraction, and protonated bases elution behavior will mimic that of acids on ODs. Figure 9 shows the behavior of aniline on ODS columns, where k’is large a t low pH where the protonated species is present and attracted to the negatively charged stationary phase. On the other hand, inspection of the k’values for weak bases using cyano columns (Figure 10) show that the largest h’values occur in more basic solution where the neutral, free-base form is present and is smallest in acidic solution where the protonated,
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ANALYTICAL CHEMISTRY, VOL. 57, NO. 14, DECEMBER 1985
1.8 1.0 1.6 1.4 0.8 1.2
k’
k’l‘o
0.6
0.8 0.6 0.4
0.4 0.3
0.2
H;
0
0
1
Flgure 10. Dependence of k‘of aniline on pH at various concentratlons of SDS at (0)0.02, (A)0.05, (0)0.08,and (W) 0.10 M: column, 10 prn cyano-bonded silica; flow rate, 2.0 mL/mln; mobile phase, aqueous SDS in 0.01 M phosphate buffer. Solid lines were calculated by using eq 12. The parameter estimates for ki,k,’,and pK, are (0)1.75, 0.33, and 5.6, (A)1.48, 0.14, and 6.2, (0)1.16, 0.12, and 6.0, and (W) 1.09, 0.10, and 6.2.
positively charged form exists which has favorable electrostatic attraction to the negatively charged micelles. Therefore, the elution behavior vs. pH of protonated bases on ODS columns will be opposite to that observed on cyano-bonded columns. The variation in k’vs. pH resembles that of a conventional acid-base titration curve, and Figures 8 and 9 are mirror images of Figure 10. The pK, values were estimated from the isoeluting point in Figures 6 and 7 when using a cyano column. However, when no isoeluting point is observed as in Figures 8 and 10, it is assumed that pKamequals the pH where the change in k ’per unit change in pH is a maximum, and the pK, can be calculated by using a method similar to that described by Lingane (23). These calculated values are listed in Table I along with pK, values from the literature. The estimated limiting capacity factors of the neutral and anionic forms are also listed. Note that the pK, values obtained from the ODS column generally increase with increasing [SDS], and the average pKa, values obtained from four different SDS concentrations with the ODS column are in good agreement with those obtained from the isoeluting points using cyano columns (Table I). p K a Shifts. Shifts in pK, to larger values are induced by the anionic surfactant system. In anionic micellar solution, the pK, value shifts by approximately0.5 for benzoic acid and 3 for 4-dodecyloxylbenzoic acid (25). These shifts have been ascribed partly to the low dielectric constant at the micelle surface and partly to the surface potential (16). The electrical potential difference between the micelle surface and the bulk solvent has been described by eq 13 (26)
pKa = pK’ - $FJ2.3RT
(13)
where pKa is the apparent pK in the micellar system, pK’ is the intrinsic pK of the probe at the micellar surface, 11. is an interfacial potential, F is Faraday’s constant, R is the general gas constant, and T i s the temperature. The values in the SDS system are always negative with values of -135 and -108 mV reported (16). The 0.5 pKa shift observed for benzoic acid agrees well with that reported by Pelizzetti and Pramauro (25). Bromocresol green and bromophenol blue are large molecules that should have strong hydrophobic interactions with the micelle, and they are divalent which would increase their
+
6
7
Flgure 11. Salt effects on capacity factor of benzoic acid at various pH values: micellar mobile phase (0)0.05 M SDS in 0.01 M phosphate buffer, (0)0.08 M SDS in 0.01 M phosphate buffer (A) 0.05 M SDS in 0.01 M phosphate buffer with 0.10 M NaCI, (A)0.08 M SDS in 0.01 M phosphate buffer wlth 0.10 M NaCi; column, 10 pm cyano-bonded silica; flow rate, 1.0 mL/min.
affinity to bind with available protons over the proton binding affinity of singly charged species. These postulates are borne out by the remarkable shift in pKa of 1.4 found for bromocresol green and 1.0 for bromophenol blue, compared to average values of 0.5 for monovalent anions. The solid lines in Figures 8 and 10 were obtained by plotting eq 11 and 12 using parameter estimates shown in the figure captions. The good agreement between predicted behavior of k ’vs. pH and the experimental data points is a strong indication that these equations fit the experimental data and that the model is a reasonable representation of the chemical system. Salt Effects. Salts such as NaCl can directly influence chromatographic retention in several ways. The DebyeHuckel equation predicts a decrease in activity coefficient with increasing ionic strength, with a concomitantincrease in solute solubility (salting-in effect). In this context, the terms “salting-in” and “salting-out” apply to the solute becoming more or less soluble in the bulk aqueous phase, respectively, and does not refer to its behavior in the micelle. In micellar systems, added salt could cause a decrease in solvent activity as a result of strong ion-solvent binding, and as a consequence, the activity coefficient of a solute can increase (salting-out effect). The added salt would also be expected to affect the stationary phase, especially ODS columns. Because the solubility of organic species depends on both the ionic and the carbocyclic portion, the solubility behavior of ionic organic species as a function of salt concentration will be the result of a combination of electrostatic and hydrophobic effects (27). Added salt could cause a decrease in the interfacial potential at the micelle surface (16) which would allow stronger solute-micelle interaction. Therefore, the effect of added salt in the micellar mobile phase on the magnitude k ’gives some information on the magnitude of solute-micelle and solutestationary phase interactions. This is certainly part of the reason why the solute-micelle interaction of weak organic acids can change from so-called antibindingto binding behavior (11). The chromatographic behavior of benzoic acid and bromophenol blue plotted as k’vs. pH, both with and without added salt, are shown in Figures 11and 12. Large differences in k’occur at high pH, and the two curves tend to converge going toward low pH values, indicating that salt effects (electrostatic effects) should be large for anionic species and smaller for neutral species. Note that no isoeluting point occurs with salt added, and the slope of k’vs. SDS concentration would not change sign based on these data. Also, the
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PH Figure 12. Salt effects on capacity factors of bromophenol blue at various pH values: micellar mobile phase (0)0.05 M SDS in 0.01 M phosphate buffer, (0)0.08 M SDS in 0.01 M phosphate buffer, (A)0.05 M SDS in 0.01 M phosphate buffer with 0.10 M NaCI, and (A)0.08 M SDS in 0.01 M phosphate buffer with 0.10 M NaCI; column, 10-wm cyano-bonded silica; flow rate, 1.0 mL/mln.
elution behavior vs. micelle concentration at high pH with salt reverses compared to that without added salt, e.g., the bromophenol blue anion is less retained using 0.05 M SDS compared to 0.08 M SDS, but has the opposite behavior when NaCl is added, indicating anionic species in the presence of salt behave more like neutral species (i.e., sign of l / k ’ vs. [micelle] slope changes from negative to positive). This is likely the result of a decrease in interfacial potential on the micelle surface caused by the salt. The result that greater ionic strength fosters hydrophobic interactions likely results from intercalation of ions into the micellar head group and counterion structure which can influence electrostatic strength and acidity at the micelle-water interface. Bromophenol blue has a aqueous phase pK, that is very close to that of benzoic acid, but it has much more hydrophobic character which allows it to associate more strongly with both the micelle and stationary phase. Comparing Figures 11 and 12, the k’values and changes in k’between different micelle concentrations at high pH for bromophenol blue are appreciably larger than for benzoic acid, and no salt effect is observed for the more hydrophobic species at pH 3. This indicates that the greater the hydrophobicity of the solute, the greater are both the capacity factors and the solute-micelle equilibrium constant. Also, the larger the solute-micelle equilibrium constant, the larger the rate of decrease in k ’ with increasing micelle concentration. These observations are consistent with previous reports (13,14)and may be set as a rule in micellar chromatography. In terms of thermodynamics, the smallest partition coefficient possible is zero. Furthermore, if a solute retention is constant with increasing micelle concentration, this does not necessarily mean that the partition coefficient is zero. The isoeluting point (50% ionization point) in Figure 6 clearly demonstrates that the lack of change in k’in changing micelle concentrationin the mobile phase is the result of the balancing
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of opposing hydrophobic and electrostatic effects caused by change in the form of the species. This would not be observed often unless the pHs of the mobile phase happened to equal the pK,. Only one peak is observed in the chromatogram of a weak acid at pH = pK, because the prototropic equilibrium is much faster than the solute-micelle or solute-stationary phase dynamics. It is now possible to apply the present model to explain numerous types of unusual behavior of ionic and ionizable species reported in the literature ( 1 1 ) . For example, a rapid increase in the “coefficient” of bromophenol blue with increasing NaCl concentration compared to other ionizable species such as sodium 2-naphthalenesulfonate can be explained in the same manner given above for benzoic acid and bromophenol blue. Also, for species that have little hydrophobic character, such as ammonium thiocyanate, their behavior may be explained in terms of salting-in effects (into the bulk water) resulting in less micelle association and decreasing retention with increasing salt concentration.
ACKNOWLEDGMENT We thank L. S. Romsted for enlightening discussions on this work.
LITERATURE CITED Hlnze, W. L. I n “Solution Chemistry of Surfactants”; Mittal, K. L., Ed.; Plenum Press: New York, 1979; Vol. 1, pp 79-127. Albertson, P.-A. J . Chromatogr. 1978, 159,111-122. Femla, R. A,; Cline Love, L. J. Anal. Chem. 1984, 56,327-331. Cline Love, L. J.; Weinberger, R.; Yarmchuk, P. I n “Surfactants in Solution”; Mittal, K. L., Lindman, B., Eds.; Plenum Press: New York, 1984; VOI. 2, pp 1139-1 157. Woods, R.; Cline Love, L. J. SDectrochim. Acta, Part A 1984, 40A, 643-650. Granneman, G. R.; Sennello, L. T. J . Chromatogr. 1982, 229, 149-157. Pelizzettl, E.; Pramauro, E. J . f h y s . Chem. 1984, 88, 990-996. Dorsey, J. G.; DeEchegaray, M. T.; Landy, J. S. Anal. Chem. 1983, 55, 924-928. Yarmchuk, P.; Weinberger, R.; Hirsch, R. F.; Cline Love, L. J. J . Chromatogr. 1984, 283, 47-60. Armstrong, D. W.; Stine, 0 . Y. Anal. Chem. 1983, 55, 2317-2320. Armstrong, D. W.; Stine, G. Y. J . A m . Chem. SOC. 1983, 105, 6220-6223. Herries. E. G.: BishoD, W.; Richards, F. M. J . Phys. Chem. 1964, 68, 1842-1852. Armstrong, D. W.; Nome, F. Anal. Chem. 1981, 53, 1662-1666. Arunyanart, M.; Cline Love, L. J. Anal. Chem. 1984, 56, 1557-1561. Bunton, C. A.; Romsted, L. S.; Sepulveda, L. J . f h y s . Chem. 1980, 84, 2611-2618. Fernandez, M. S.; Fromherz, P. J . f h y s . Chem. 1977, 8 1 , 1755-1 761. Horvath, C.; Melander, W.; Molnar, I. Anal. Chem. 1977, 49, 142-1 54. Birdi, K. S.; Slngh, H. N.;Dalsager, S. U. J . f h y s . Chem. 1979, 83, 2733-2737. Knox, J. H.; Hartwick, R. A. J . Chromatogr. 1981, 204, 3. Sybilska, D.; Debowski, J.; Jurczak, J.; Zubowski, J. J . Chromatogr. 1984, 286, 163-170. Armstrong, D. W.; Stine, G. Y. J. A m . Chem. SOC. 1983, 105, 2962-2964. Kortum, G.; Vogei, W.; Andrussow, K. “Dissoclation Constants of Organic Acids in Aqueous Solutlon”: Butterworth: London, England, 1961. Lkgane, J. J. “Electroanalytical Chemistry”; 2nd ed.; Interscience: New York, 1958; p 93. Perrin, D. D. “Dissociation Constants of Organic Bases in Aqueous Solution”; Butterworth: London, England, 1965. Pelizzetti, E.; Prarnauro, E. Anal. Chim. Acta 1980, 717, 403-406. Mukerjee, P.; Banerjee, K. J . ’ f h y s . Chem. 1964, 88, 3567-3574. Long, R. A,; McDevitt, W. F. Cbem. Rev. 1952, 51, 119-169.
RECEIVED for review May 6, 1985. Accepted July 31, 1985. This work was supported in part by the National Science Foundation Grant No. CHE-8216878 to L.J.C.L.
