Retention Mechanisms for Basic Drugs in the Submicellar and Micellar

Anal. Chem. 2008, 80, 9705–9713

Retention Mechanisms for Basic Drugs in the Submicellar and Micellar Reversed-Phase Liquid Chromatographic Modes ´ ngel,* J. R. Torres-Lapasio ´ lvarez-Coque M. J. Ruiz-A ´ , and M. C. Garcıá-A Departament de Quı´mica Analı´tica, Universitat de Vale`ncia, c/Dr. Moliner 50, 46100, Burjassot (Spain) S. Carda-Broch Departament de Quı´mica Fı´sica i Analı´tica, Universitat Jaume I, Cra. Borriol s/n, 12071, Castello´ (Spain) The reversed-phase liquid chromatographic (RPLC) behavior (retention, elution strength, selectivity, efficiency, and peak asymmetry) for a group of basic drugs (β-blockers), with mobile phases containing the anionic surfactant sodium dodecyl sulfate (SDS) and acetonitrile, revealed different separation environments, depending on the concentrations of both modifiers: hydro-organic, submicellar at low surfactant concentration and high concentration of organic solvent, micellar, and submicellar at high concentration of both surfactant and organic solvent. In the surfactant-mediated modes, the anionic surfactant layer adsorbed on the stationary phase interacts strongly with the positively charged basic drugs increasing the retention and masks the silanol groups that are the origin of the poor efficiencies and tailing peaks in hydro-organic RPLC with conventional columns. Also, the strong attraction between the cationic solutes and anionic SDS micelles or monomers in the mobile phase enhances the solubility and allows a direct transfer mechanism of the cationic solutes from micelles to the modified stationary phase, which has been extensively described for highly hydrophobic solutes. The addition of surfactants to the hydro-organic mobile phase in reversed-phase liquid chromatography (RPLC) gives rise to significant changes in the chromatographic behavior of solutes.1 Particularly interesting is the use of ionic surfactants in the analysis of compounds bearing an opposite charge, due to the formation of ion-pairs between solutes and surfactant molecules. When added at low concentration (below the critical micellar concentration, CMC), ionic surfactants progressively coat the stationary phase, keeping a small amount of free monomers in the mobile phase. Attraction to the modified stationary phase affects the distribution of solutes, increasing their retention. This is the basis of what is commonly known as ion-pair chromatography (IPC).2-4 * To whom correspondence should be addressed. (1) Ruiz-Ańgel,M. J.; Carda-Broch, S.; Torres-Lapasio´, J. R.; Garcıá-A´lvarezCoque, M. C. J. Chromatogr., A DOI 10.1016/j.chroma.2008.09.053. (2) Knox, J. H.; Laird, G. L. J. Chromatogr. 1976, 122, 17–34. (3) Kord, A. S.; Khaledi, M. G. Anal. Chem. 1992, 64, 1901–1907. 10.1021/ac801685p CCC: $40.75  2008 American Chemical Society Published on Web 11/19/2008

When ionic surfactants are added above the CMC, the column reaches saturation or small changes in surfactant coating are yielded at increasing concentration of surfactant in the mobile phase, depending on the column and surfactant nature.1,5,6 This situation is not so different from IPC. However, major changes take place in the mobile phase, where surfactant monomers aggregate to form micelles, as first demonstrated by Armstrong and Henry.7 This notably modifies the solubility and the transference of solutes between mobile and stationary phases, which has particular implications with regard to retention, selectivity, and efficiency. Owing to the presence of micelles, this chromatographic mode has been named micellar liquid chromatography (MLC). In this mode, solute separation is achieved on the basis of the differential partitioning between bulk solvent and micelles and between bulk solvent and surfactant-coated stationary phase. With conventional columns, solutions containing only surfactant are too weak as eluents and give rise to poor efficiencies. For this reason, a small amount of organic solvent is usually added to enhance the elution strength3 and efficiency.8 The addition of organic solvent to a micellar mobile phase decreases its polarity and affects the amount of surfactant adsorbed on the stationary phase. Also, micelle parameters, such as the CMC and surfactant aggregation number (i.e., number of surfactant monomers associated in a micelle) are altered.5,9 When a certain content of organic solvent is reached, micelles disaggregate. This makes ion-pair interactions between solute and surfactant monomers prevail again. The chromatographic performance under micelle breakdown conditions has been scarcely studied. Li and Fritz10 and Jandera et al.11 examined the effect of the addition of large amounts of organic solvent to mobile phases (4) Jandera, P.; Fischer, J. J. Chromatogr., A 1996, 728, 279–298. (5) Berthod, A.; Garcıá-A´lvarez-Coque, M. C. Micellar Liquid Chromatography; Marcel Dekker: New York, 2000. (6) Ruiz-Ańgel, M. J.; Garcıá-A´lvarez-Coque, M. C.; Berthod, A. Sep. Purif. Rev. 2009, 38, 1–32. (7) Armstrong, D. W.; Henry, S. J. J. Liq. Chromatogr. 1980, 3, 657–662. (8) Dorsey, J. G.; DeEchegaray, M. T.; Landy, J. S. Anal. Chem. 1983, 55, 924–928. (9) Lo´pez-Grıó, S.; Baeza-Baeza, J. J.; Garcıá-A´lvarez-Coque, M. C. Chromatographia 1998, 48, 655–663. (10) Li, X.; Fritz, J. S. J. Chromatogr., A 1996, 728, 235–247. (11) Jandera, J.; Fischer, J.; Effenberger, H. J. Chromatogr., A 1998, 807, 57– 70.

Analytical Chemistry, Vol. 80, No. 24, December 15, 2008

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containing different surfactants at high concentration (i.e., well above the CMC in aqueous solution), using a set of aromatic probe compounds. The authors observed that in these conditions, when compared to MLC, the retention behavior of solutes is different and the efficiencies higher. This chromatography was referred as a “bridge” between MLC and classical RPLC (i.e., with aqueous-organic mixtures).10 In this work, the behavior of cationic solutes (13 β-blockers) is explored using mobile phases containing the anionic surfactant sodium dodecyl sulfate (SDS) and acetonitrile. β-Blockers are basic drugs used in the treatment of neurological, neuropsychiatric and cardiovascular disorders,12 which are protonated at pH < 9 (the usual working pH range of most RPLC columns). The protonated basic solutes interact with free silanols on the stationary phase, which is generally accepted as the reason for peak tailing when these compounds are chromatographed with hydroorganic mobile phases.13-16 This interaction can be partially avoided by lowering the pH of the mobile phase to suppress silanol ionization. Another solution is the use of deactivated columns. The objective of this work is the comparison of the retention, peak shape, and selectivity of β-blockers in four regions of a twofactor space of concentrations of SDS and acetonitrile in RPLC (Figure 1a): hydro-organic, submicellar at low surfactant concentration and high concentration of organic solvent (IPC, low submicellar), micellar, and submicellar at high concentrations of both surfactant and organic solvent (high submicellar). The study revealed different retention mechanisms and changes in selectivity, depending on the coating of the stationary phase with surfactant and the existence of micelles or surfactant monomers in the mobile phase. EXPERIMENTAL SECTION Chemicals. The β-blockers were purchased from the following manufacturers: acebutolol (Italfarmaco, Alcobendas, Madrid, Spain), alprenolol, atenolol, nadolol, pindolol, propranolol, timolol (Sigma, St. Louis, MO), carteolol (Miquel-Otsuka, Barcelona, Spain), celiprolol (Rhoˆne-Poulenc Rorer, Alcorcoń, Madrid), esmolol (Du Pont-De Nemours, Le Grand Saconnex, Switzerland), labetalol (Glaxo, Tres Cantos, Madrid), and metoprolol and oxprenolol (Ciba-Geigy, Barcelona). Acebutolol, carteolol, celiprolol, labetalol, metoprolol, and oxprenolol were kindly donated by the pharmaceutical laboratories. The drugs were dissolved in a small amount of methanol and diluted with water. The concentration of the injected solutions was 40 µg/mL. The mobile phases were prepared with acetonitrile (Scharlab, Barcelona) and sodium dodecyl sulfate (SDS) (99% purity, Merck, Darmstadt, Germany). Acetonitrile-water mixtures in the absence of surfactant were also used. In all cases, mobile phases were buffered at pH 3 with 0.01 M citric acid monohydrate and sodium hydroxide (Panreac, Barcelona). The acidic SDS mobile phases were used during at least 1 month without observing any change (12) Cruickshank, J. M. β-Blockers in Clinical Practice; Churchill-Livingstone: New York, 1994. (13) Vervoort, R. J. M.; Maris, F. A.; Hindriks, H. J. Chromatogr. 1992, 623, 207–220. (14) Saarinen, M. T.; Sireń, H.; Riekkola, M. L. J. Chromatogr., B 1995, 664, 341–346. (15) Nawrocki, J. J. Chromatogr. A 1997, 779, 29–71. (16) Basci, N. E.; Temizer, A.; Bozkurt, A.; Isimer, A. J. Pharm. Biomed. Anal. 1998, 18, 745–750.

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Figure 1. (a) Mobile phase compositions assayed to screen the properties of the different separation environments. (b) Weight of 50 drops of solutions containing SDS and acetonitrile (surfactant concentration: 0.05 M (3), 0.075 M (O), and 0.15 M (0)).

in retention times or peak shapes. Nanopure water (Barnstead, Sybron, Boston, MA) was used throughout. The drug solutions and mobile phases were filtered through 0.45 µm nylon membranes (Micron Separations, Westboro, MA). Experimental Design. In the hydro-organic mode, the experimental design consisted of four mobile phases with acetonitrile contents in the range 15-30% (v/v). In the other chromatographic modes, the mobile phases were prepared with SDS and acetonitrile, covering different concentration domains: 0.001-0.005 M SDS/30-50% acetonitrile, and 0.075-0.15 M SDS/5-50% acetonitrile (Figure 1a). The usual cautions required when working with micellar mobile phases were followed.17 Apparatus. A liquid chromatograph (Agilent, Waldbronn, Germany) equipped with an isocratic pump (Series 1200), an autosampler, and a UV-visible detector (series 1100) set at 225 nm was used. A Kromasil C18 column (125 mm × 4.6 mm i.d., 5 µm particle size, 320 m2/g surface area, and 110 Å pore diameter) was purchased from Ana´lisis Vıńicos (Ciudad Real, Spain) and (17) Ruiz-Ańgel, M. J.; Garcıá-A´lvarez-Coque, M. C. LC-GC Eur. 2008, 21, 420– 429.

connected to a similar 30 mm guard column (Scharlab). The flow rate was set at 1 mL/min. Duplicate injections were made using an injection volume of 20 µL. An analytical balance (±0.0001 g, Precisa, Dietikon, Switzerland) was also used. Data acquisition was carried out with an HPChemStation (Agilent, B.02.01), and mathematical treatment was performed in MATLAB 6.5 (The Mathworks, Natick, MA). RESULTS AND DISCUSSION Micelle Formation-Breakdown in Aqueous-Organic Solvent Mixtures. Among the large number of additives to aqueous surfactant systems, alcohols are by far the most frequent. Consequently, the reports about the formation and breakdown of micelles are mainly related to these additives.18 In contrast, only little information about aqueous surfactant-acetonitrile mixtures is available. In previous work, the CMC of SDS was measured in solutions containing several alcohols and acetonitrile.9 Acetonitrile was found to behave similarly to methanol: the CMC in water increased upon addition of both solvents, while it was reduced for longer alcohols, with an increasing rate depending on the chain length. Acetonitrile and methanol are more polar (the logarithm of octanol-water partition coefficient, log Po/w, is 0.18, 0.48, 2.2, 7.6, and 29 for methanol, ethanol, 1-propanol, 1-butanol, and 1-pentanol, respectively, and 0.46 for acetonitrile)19 and smaller in size than other alcohols, although acetonitrile is aprotic. Consequently, the available information on the effect of methanol and other short-chain alcohols on micelle formation-breakdown can be useful to understand the behavior of acetonitrile. The addition of alcohol to micellar solutions results in the partitioning of some or all of the alcohol into the micellar aggregates. Binding constants (mole fraction ratio of alcohol between bulk solvent and the micellar pseudophase) have been found to increase exponentially with the alcohol carbon number: 0.4, 1.1, 3.5, 6.3 and 15-21 (values per SDS molecule at 25 °C) for methanol, ethanol, 1-propanol, 1-butanol and 1-pentanol, respectively.20,21 Thus, the amount of methanol in the intermicellar solution is appreciably greater than for other alcohols. Methanol penetrates little, if at all, in micelles. If solubilized in the micelles, this alcohol would be located in the palisade layer, which contains the surfactant head groups, water, and counterions. Such intercalation of alcohol molecules leads to an increase in the distance between adjacent anionic sulfate headgroups in the SDS micelle. This decreases the surface charge density. Methanol and acetonitrile should be, however, considered more as cosolvents that act on the micellization process by modifying the properties of water and solvating the surfactant monomers. This is the reason for the need of a higher surfactant concentration in methanol-water and acetonitrile-water mixtures, in order to form micelles. The dielectric constant of water decreases upon alcohol addition.18 This is translated in a decreased surfactant aggregation number, even at high surfactant concentration. Some reports suggest that there is no sudden breakdown of micelles when the concentration of alcohol is increased but a progressive reduction (18) Zana, R. Adv. Colloid Interface Sci. 1995, 57, 1–64. (19) Leo, A.; Hansch, C.; Elkins, D. Chem. Rev. 1971, 71, 525–616. (20) Sepu´lveda, L.; Lissi, E.; Quina, F. H. Adv. Colloid Interface Sci. 1986, 25, 1–57. (21) De Lisi, R.; Milioto, S. J. Solution Chem. 1988, 17, 245–265.

in the aggregation number.22 This has been estimated to be n ) 62 for SDS in water, whereas n ) 12, 8, 7. and 5 in 10, 20, 30, and 40% ethanol, respectively, and n ) 10, 9, 8, and 7 in 5, 10, 15, and 20% propanol, respectively.20 The aggregation number was found to be n ) 64 and 23 in 12 and 24% methanol, respectively.21 Unfortunately, similar data were not found for SDS/acetonitrile. Finally, at alcohol concentration high enough, no micelles are formed. This has been observed for 22% 1-propanol, 30% ethanol, and 40% methanol.18 According to Martıńez et al.,23 percentages of acetonitrile >50% cannot be used, since SDS micelles would be destroyed. However, this figure must be taken only as a rough estimation. The modification in the micelle structure can be monitored by following changes in the surface tension of SDS solutions. Orientation of surfactant molecules at the air-water interface reduces this property and, therefore, the solvent drop size, depending on the free surfactant monomers. An experimental study was performed by weighting 50 drops (delivered from a buret) of solutions containing SDS and acetonitrile, in order to monitor changes in the surface tension that could provide information about micelle existence.9 In that study, the CMC was estimated in the acetonitrile concentration range 1-20%. It was found that it increased slightly up to 15% acetonitrile, to further undergo a sudden increase to a larger rate. The CMC of SDS was ∼0.03 M at 20% acetonitrile, whereas it is 8.2 × 10-3 M in water. Following this previous study on micelle formation, we applied the drop weighting procedure to solutions containing three different concentrations of SDS (0.05, 0.075, and 0.15 M) and eight different levels of acetonitrile between 0 and 40%. The results are plotted against the acetonitrile content in Figure 1b. As observed, the drop weight remained approximately constant in the 5-15% acetonitrile range, followed by a gradual decrease in the 20-40% range. This reveals some micelle perturbation and a possible disaggregation. Hydro-Organic RPLC. The hydrophobic interaction of solutes with the alkyl-bonded layer of the stationary phase and the solubilization power of the aqueous-organic solvent mixture are the main reasons for the separation in conventional RPLC (Figure 2a). Additional mixed mechanisms involving ion-pair formation, salting-out effects, or ion-exchange interactions with free silanols on the packing may also take place with positively charged solutes, such as β-blockers. Some experiments were performed in this chromatographic mode to get a reference for exploring the interactions that take place in the presence of surfactant. The classical RPLC behavior (i.e., decreased retention at increasing organic solvent content) was observed (Figure 3a). The polarity range of β-blockers (1 < log Po/w < 3)24 limited the feasible acetonitrile content to 15-30%. This volume fraction range prevented large retention times for the most hydrophobic β-blockers and void volume elution for the most polar ones. Low Submicellar RPLC. In this chromatographic mode, the stationary phase is coated with SDS. The hydrophobic tail of (22) Rafati, A. A.; Gharibi, H.; Rezaie-Sameti, M. J. Mol. Liq. 2004, 111, 109– 116. (23) Martıńez, V.; Lo´pez, J. A.; Alonso, R. M.; Jimeńez, R. M. J. Chromatogr., A 1999, 836, 189–199. (24) Detroyer, A.; Vander Heyden, Y.; Carda-Broch, S.; Garcıá-A´lvarez-Coque, M. C.; Massart, D. L. J. Chromatogr., A 2001, 912, 211–221.


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Figure 2. Solute environment in an octadecylsilane chromatographic system for the four examined regions: (a) hydro-organic, (b) low submicellar, (c) micellar, and (d) high submicellar.

the surfactant associates with the alkyl chains bonded to the silica stationary phase, with the polar head groups oriented away from the surface (Figure 2b). This creates a negatively charged asymmetric bilayer, which reduces the pore size and affects the penetration depth of solutes into the bonded phases.25,26 Cationic solutes can interact hydrophobically with the uncovered alkyl-bonded layer or with the adsorbed surfactant monomers through electrostatic attraction. As long as the adsorbed amount of surfactant does not reach the maximal capacity of the column, surfactant coating on the stationary 9708


phase increases with its concentration in the mobile phase. This results in larger retention times. Under these conditions, the amount of free surfactant molecules in the mobile phase is low (below the CMC).5 This indicates that an ion-exchange retention mechanism is dominant and ion-pair formation with the surfactant in the mobile phase is practically inexistent. The addition of organic solvent increases the elution strength, as a result of the decreased mobile phase polarity, and the competition between organic solvent and surfactant molecules for adsorption sites, which

Figure 3. Dependence of the retention factor with the acetonitrile content for four representative β-blockers under several eluent conditions: (a) hydro-organic, (b) submicellar at 0.001 M SDS, and (c,d) micellar and submicellar at 0.075 M (c) and 0.15 M (d) SDS. Compounds: alprenolol (0), carteolol (3), oxprenolol (∆), and timolol (O). The plots obtained for the remaining nine β-blockers were similar to those depicted in this figure.

reduces the amount of surfactant adsorbed on the stationary phase. The β-blockers were eluted with six mobile phases at two SDS concentrations below the CMC (10-3 M and 5 × 10-3 M), each one at three acetonitrile levels (Figure 1a). Retention factors increased significantly when the concentration of SDS increased from 10-3 to 5 × 10-3 M as a result of the strong attraction of the cationic solutes by the adsorbed monomers of SDS. The addition of a large amount of acetonitrile, in the range 30-50%, was necessary to get practical retention times. The change in retention upon addition of acetonitrile is shown in Figure 3b for some representative β-blockers. Micellar Liquid Chromatography. Above the CMC, coating of octadecylsilane columns with SDS reaches saturation27 and the electrostatic interaction of cationic β-blockers with the anionic surfactant monomers on the stationary phase becomes the prevalent equilibrium. A secondary equilibrium is established with micelles in the mobile phase, as a result of electrostatic and hydrophobic interactions (Figure 2c). Because the column is saturated with surfactant, and further addition of surfactant increases the amount of micelles, the retention of β-blockers decreases with the concentration of SDS. Acetonitrile desorbs the surfactant coating similarly to the submicellar region, but the organic solvent also affects the distribution of solutes between bulk solvent and micellar aggregates. (25) Armstrong, D. W.; Ward, T. J.; Berthod, A. Anal. Chem. 1986, 58, 579– 582. (26) Lavine, B. K.; Hendayan, S.; Cooper, W. T.; He, Y. ACS Symp. Ser. 1999, 740, 290–313. (27) Berthod, A.; Girard, I.; Gonnet, C. Anal. Chem. 1986, 58, 1356–1358.

The chromatographic behavior in MLC for mobile phases covering a domain from 0.075 to 0.15 M SDS, and from 5 to 20-30% acetonitrile, can be observed in Figure 3c,d. The retention capability of the stationary phase increased with respect to conventional RPLC (Figure 3a), but it was smaller when compared with IPC at similar acetonitrile contents (Figure 3b). Since the surfactant coating is constant in MLC and retention decreases with addition of more surfactant, this behavior should be attributed to the presence of more micelles in the mobile phase, which enhances the solubilization capability. High Submicellar RPLC. An acetonitrile content of 15-20% is usually considered as the limiting concentration that preserves the integrity of micelles. The results derived from Figure 1b indicate that above 20% acetonitrile, the changes in micelle structure become significant. The dominant retention mechanism in this region depends on the amount of surfactant that has been swept off the alkyl-bonded phase by the organic solvent and the existence of micelles. As long as a certain amount of surfactant remains adsorbed, and micelles exist, the retention mechanism will be the typical of the micellar mode. When micelle disaggregation occurs, a submicellar situation is achieved, where ion-pair interactions with surfactant monomers in the bulk mobile phase will replace those with micelles (Figure 2d). In order to check the retention mechanism in this region, β-blockers were eluted with 12 mobile phases containing SDS and acetonitrile in the ranges 0.075-0.15 M and 20-50%, respectively (Figure 1a). The plot of log k versus acetonitrile Analytical Chemistry, Vol. 80, No. 24, December 15, 2008

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Figure 4. Box-and-whisker plots depicting the dependence of (a) efficiency (N) and (b) asymmetry factors (B/A) with mobile phase composition in the four separation environments, considering the 13 β-blockers.

content is shown in parts c and d of Figure 3 for 0.075 and 0.15 M SDS, respectively. As observed, retention factors were still larger than those in the classical RPLC mode (Figure 3a) for equivalent acetonitrile contents. This suggests that in the 20-50% range, a certain amount of SDS remains adsorbed on the column and ion-exchange with cationic β-blockers is taking place. The smaller retention at increasing acetonitrile content should be explained mainly by desorption of surfactant from the column and the decreased mobile phase polarity. As observed, the slopes of the plots (Figure 3c,d) in the range 30-50% acetonitrile are different from those in the range 5-20% acetonitrile (micellar region). This indicates a change 9710


in the chromatographic system. As micelle existence is unlikely in the 30-50% acetonitrile range, simultaneous dynamic ionexchange on the stationary phase and ion-pair formation of solutes with the free surfactant monomers in the bulk solvent should exist. A transition region may exist between 20-30% acetonitrile (Figure 1a), with a retention mechanism similar to that in MLC but with micelles starting to be significantly perturbed. Elution Strength. The elution strength in the hydro-organic mode was measured conventionally as the slope (a1) of a linear equation relating log k with the acetonitrile content (φ):

log k ) a0 + a1φ

(1)

In the surfactant-mediated modes, there are significant interactions between surfactant and acetonitrile. These interactions were evaluated by performing a screening study. Since the real variations are complex, the effects were assayed at two extreme levels by fitting the experimental data to the following polynomial using 22 experimental designs: log k ) a0 + a1φ + a2[M] + a12[M]φ

(2)

where [M] is the concentration of surfactant. This means that there are no degrees of freedom left, and the polynomial will only allow a rough estimation of the factor effects and their interaction. The mean parameters in eqs 1 and 2 for the group of β-blockers in different concentration windows are given below. The surfactant levels were 10-3 and 5 × 10-3 M for the low submicellar mode and 0.075 and 0.15 M for both micellar and high submicellar modes (the acetonitrile content is given in parentheses). For comparison purposes, molar concentrations of both surfactant and organic solvent were considered in eq 2 (10% acetonitrile corresponds to ∼1.91 M): Hydro-organic (15-30%): a0 ) 1.8 ( 0.9,

a1 ) -0.30 ( 0.09

Low submicellar (40-50%): a0 ) 2.7 ( 0.6, a1 ) -0.26 ( 0.04,

a2 ) +147 ( 21,

a12 ) -7.2 ( 2.1

Micellar (5-15%): a0 ) 2.4 ( 0.3, a1 ) -0.033 ( 0.014,

a2 ) -4.29 ( 0.25,

a12 ) -0.32 ( 0.14

Micellar/high submicellar (15-30%): a0 ) 2.37 ( 0.22, a1 ) -0.04 ( 0.03,

a2 ) -4.50 ( 0.20,

a12 ) -0.24 ( 0.14

Micellar/high submicellar (20-50%): a0 ) 2.9 ( 0.4, a1 ) -0.195 ( 0.014,

a2 ) -7.9 ( 0.8,

a12 ) +0.58 ( 0.12

High submicellar (30-50%): a0 ) 3.5 ( 0.7, a1 ) -0.25 ( 0.04,

a2 ) -10.7 ( 2.0,

a12 ) +0.87 ( 0.23

The effect of acetonitrile on retention in both submicellar chromatographic modes (a1 ) -0.26 and -0.25) is not far from that in the hydro-organic mode (-0.30), which denotes that the change in the nature of the mobile phase is nonsignificant with regard to the hydro-organic medium. The small difference could be explained by the facts that the relationship between log k and φ is not strictly linear and the acetonitrile ranges are different between the hydro-organic and submicellar modes. Also, because of the stronger interaction of solutes with the modified stationary phase, the origin of retention is shifted to higher values for the submicellar modes. It should be noted that the organic solvent strength is appreciably smaller in the micellar mode. The highly positive value obtained for a2 at low SDS concentration (i.e., a strong increase in retention with surfactant concentration) evidences the dominance of an ion-exchange retention mechanism. In the micellar and high submicellar modes, the effect of the surfactant is opposite (a2 is negative), as a consequence of

the additional ion-pair interactions with micelles or free surfactant monomers in the mobile phase. Note that the sign of the interaction term (a12) on retention is opposite in the high submicellar mode. In the micellar and high submicellar modes, the surfactant was stronger than acetonitrile, especially in the former mode. The reason for this behavior is the electrostatic association of β-blockers with the anionic micelles or surfactant monomers, which are stronger than hydrophobic forces with organic solvent molecules. We fitted the retention data to diverse acetonitrile concentration windows with an upper limit at 50%, for the same SDS window (between 0.075 and 0.15 M), and observed that the parameters associated with the organic solvent (a1) and SDS (a2) gradually changed when the acetonitrile window was shifted to larger values (see coefficients above). Also, it should be noted that all parameters in eq 2 are remarkably similar in the 5-15% and 15-30% ranges, which suggests that micelles exist at least up to this acetonitrile content. Peak Shape. Chromatographic efficiencies, expressed as theoretical plates (N), were estimated at 10% peak height, according to the equation of Foley and Dorsey.28 Peak asymmetries were measured as the tailing-to-fronting half-width ratio (B/A), also at 10% peak height. The corresponding box-andwhisker plots for N and B/A are depicted in parts a and b of Figure 4, respectively. The smallest values for N were obtained for the hydro-organic mode (N ) 800-1700). Efficiencies improved in the micellar mode (N ) 1000-3300). In previous work, we observed a larger enhancement in efficiencies between the hydroorganic and micellar modes, using a Spherisorb column (100-1600 versus 2000-4000).29 However, the most outstanding enhancements were observed in the submicellar modes, with N values frequently in the 4000-9000 range. The enhancement was already remarkable in the transition region (between 15 and 30% acetonitrile at high SDS concentration), where micelles are getting smaller and eventually disaggregate. The undesirable interaction of β-blockers with ionized silanols on conventional silica-based stationary phases, using hydro-organic eluents, is a slow process, which results in poor peak shape.15 Under submicellar conditions at low surfactant concentration, adsorbed SDS monomers mask silanols, while β-blockers interact with the SDS layer through an ion-exchange mechanism, which seems to be a fast process,29,30 giving rise to a large increase in column efficiency. However, the efficiency significantly worsens at increasing acetonitrile content, revealing surfactant desorption, which favors solute penetration and interaction with the buried silanols. In the high submicellar mode, the magnitude of the efficiencies is similar to that in the low submicellar and appreciably larger than in the micellar mode. Note that in the submicellar mode, the change in SDS at low concentration (from 10-3 to 5 × 10-3 M) gives rise to a slight improvement in efficiency, but at high SDS concentration (from 0.075 to 0.15 M SDS), there is a certain trend to worsen. The smaller efficiencies in the micellar mode can be explained by the thicker SDS layer on the stationary phase. This produces (28) Foley, J. P.; Dorsey, J. G. Anal. Chem. 1983, 57, 730–737. (29) Ruiz-Ańgel, M. J.; Carda-Broch, S.; Torres-Lapasio´, J. R.; Simo´-Alfonso, E. F.; Garcıá-A´lvarez-Coque, M. C. Anal. Chim. Acta 2002, 454, 109–123. (30) Ruiz-Ańgel, M. J.; Torres-Lapasio´, J. R.; Carda-Broch, S.; Garcıá-A´lvarezCoque, M. C. J. Chromatogr. Sci. 2003, 41, 350–358.


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Figure 5. Experimental optimal chromatograms obtained for the four separation environments. Mobile phase composition: (a) 15.0% acetonitrile, (b) 0.0047 M SDS/42.1% acetonitrile, (c) 0.107 M SDS/5.0% acetonitrile, and (d) 0.100 M SDS/36.4% acetonitrile. Compounds: (1) Carteolol, (2) pindolol, (3) timolol, (4) acebutolol, (5) metoprolol, (6) esmolol, and (7) celiprolol.

a significant decrease in the pore volume, dramatically reducing the active surface area.31 Also, a significant difference between the submicellar and micellar modes is the existence of micelles to which the cationic β-blockers are strongly bound. The coefficients in eq 2 for the micellar mode indicate the main role of the surfactant in sweeping the solutes off the column. This means that a direct transfer mechanism of these solutes between micelles and stationary phase is likely, which has been reported previously for highly hydrophobic solutes.32 With conventional porous RPLC stationary phases, the micelles are largely excluded from the pores by steric constraints and, therefore, do not have access to the analytes, except when they have diffused out of the pores into the interstitial region.33 Also, when ionic surfactants are used, the resulting charge build-up on the stationary phase within the pores gives rise to a Donnan-like potential that tends to repel like charged species from the pores, especially large structures (such as micelles, whose dimensions are commensurate with pore diameters of typical octadecylsilane (31) Borgerding, M. F.; Hinze, W. L.; Stafford, L. D.; Fulp, G. W.; Hamlin, W. C. Anal. Chem. 1989, 61, 1353–1358. (32) Borgerding, M. F.; Quina, F. H.; Hinze, W. L.; Bowermaster, J.; McNair, H. M. Anal. Chem. 1988, 60, 2520–2527. (33) McCormick, T. J.; Foley, J. P.; Riley, C. M.; Lloyd, D. K. Anal. Chem. 2000, 72, 294–301.

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phases).34 This effect has been corroborated using ultrawide pore columns, where the access of micelles to pores is easier.33 In conventional aqueous micelles (i.e., with no additives present), solute migration can occur either via the exit of the solute from the micelle, followed by its diffusion in the bulk solvent and subsequent association to another micelle, or by direct transfer through collisions and transient merging of intact micelles. This latter path is, in principle, applicable only to nonionic micellar systems, since ionic micelles strongly repel one another due to their high electrostatic surface charge density. However, it has been reported that for alcohol-modified surfactant solutions, which contain micelles and a variety of micellar fragments and surfactantstabilized alcohol clusters, this additional migration mechanism can become operative. Thus, the mixed micelle can lose a fragment or incorporate a fragment, eventually mediating the transfer of a solute between different micelles or clusters in the mobile phase or between a micelle in the mobile phase and the surfactant-coated (micellar-like) stationary phase.35 Coating of the stationary phase with the surfactant (and masking of silanols) is also revealed by the peak shapes of (34) Thomas, D. P.; Foley, J. P. J. Chromatogr., A 2004, 1061, 195–203. (35) Lo´pez-Grıó, S.; Garcıá-A´lvarez-Coque, M. C.; Hinze, W. L.; Quina, F. H.; Berthod, A. Anal. Chem. 2000, 72, 4826–4835.

β-blockers (Figure 4b). Thus, in the hydro-organic mode, poor peak shapes were obtained (B/A ) 1.5-3.0). In the low submicellar mode, they improved (B/A ) 1.0-2.6) but deteriorated at increasing acetonitrile in the mobile phase. In contrast, chromatographic peaks were almost symmetrical in both micellar and high submicellar modes (B/A ) 1.0-1.3), changing scarcely with mobile phase composition, except by the observation of a significant deterioration in the high submicellar mode in the presence of 50% acetonitrile. At this organic solvent content, the behavior in both submicellar regions were similar (see also the efficiencies plot in Figure 4a). This can be interpreted as the loss of a significant portion of the surfactant layer on the stationary phase, reducing thus the masking effect on the silanols. Selectivity. Figure 5 shows optimal chromatograms obtained for each of the four systems (hydro-organic, low submicellar, micellar, and high submicellar). The change in the nature of both stationary and mobile phases was translated into significant changes in selectivity. Not only is the elution strength modified but also the elution order and resolution performance. The chromatograms show the suppression of peak tailing observed in hydro-organic RPLC and an improvement in the efficiencies for the three surfactant-mediated systems, especially for the high submicellar mode. CONCLUSIONS Addition of SDS to a conventional hydro-organic RPLC system gives rise to different environments and behaviors for cationic solutes (such as β-blockers), depending on the concentration of surfactant and organic solvent. This results in significant changes in retention, peak shape, and selectivity. The surfactant layer adsorbed on the stationary phase interacts strongly with the basic drugs (increasing their retention) and masks the silanol groups that are the origin of the poor efficiencies and tailing peaks observed for these compounds in hydro-organic RPLC with (36) Knox, J. H.; Hartwick, R. A. J. Chromatogr. 1981, 204, 3–21. (37) Berthod, A.; Borgerding, M. F.; Hinze, W. L. J. Chromatogr. 1991, 556, 263–275.

conventional columns. Also, solubility in the mobile phase is enhanced by the presence of micelles or SDS monomers, to which the cationic solutes are strongly attracted. This implies a direct transfer mechanism from micelles to the stationary phase. The observation of the changes in the chromatographic behavior of basic drugs, upon addition of surfactant, evidences the processes that take place inside the RPLC system. The retention mechanism in the high submicellar mode (whose features have been scarcely investigated) has been inferred by comparison with the hydro-organic mode (without surfactant), low submicellar mode (with surfactant adsorbed on the stationary phase), and micellar mode (with micelles and adsorbed surfactant). Li and Fritz10 carried out several experiments to verify if some surfactant (such as SDS) remained adsorbed on the stationary phase and concluded that the surfactant was completely removed from the column. Our studies suggest that gradual changes in micelle structure happen with a likely breakdown at ∼30% acetonitrile (in the 0.075-0.15 M SDS range). Also, some anionic surfactant should remain adsorbed on the column in the high submicellar mode, at least up to 50% acetonitrile. This agrees with previous observation on the irreversible surfactant adsorption, due to a tight insertion of the surfactant alkyl-chains in the alkyl moieties of the bonded layer of the densely grafted phases.36,37 Confirmation of this hypothesis would require a detailed study, where surfactant adsorption isotherms at high surfactant concentration would be measured at varying volume fraction of organic modifier. ACKNOWLEDGMENT This work was supported by Project CTQ2007-61828 (Ministerio de Educacioń y Ciencia, MEC, of Spain) and FEDER funds. M.J.R.-A. thanks the MEC for a Ramoń y Cajal research contract.

Received for review August 11, 2008. Accepted October 20, 2008. AC801685P


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Retention Mechanisms for Basic Drugs in the Submicellar and Micellar

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