Adsorption Mechanism in RPLC. Effect of the ... - ACS Publications

data for phenol are best modeled with the bi-Langmuir and the tri-Langmuir ... effect of the nature of the organic modifier in the mobile phase in pre...
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Anal. Chem. 2005, 77, 4257-4272

Adsorption Mechanism in RPLC. Effect of the Nature of the Organic Modifier Fabrice Gritti and Georges Guiochon*

Department of Chemistry, University of Tennessee, Knoxville, Tennessee 37996-1600, and Division of Chemical Sciences, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831-6120

The adsorption isotherms of phenol and caffeine were acquired by frontal analysis on two different adsorbents, Kromasil-C18 and Discovery-C18, with two different mobile phases, aqueous solutions of methanol (MeOH/H2O ) 40/60 and 30/70, v/v) and aqueous solutions of acetonitrile (MeCN/H2O ) 30/70 and 20/80, v/v). The adsorption isotherms are always strictly convex upward in methanol/water solutions. The calculations of the adsorption energy distribution confirm that the adsorption data for phenol are best modeled with the bi-Langmuir and the tri-Langmuir isotherm models for Kromasil-C18 and Discovery-C18, respectively. Because its molecule is larger and excluded from the deepest sites buried in the bonded layer, the adsorption data of caffeine follow biLangmuir isotherm model behavior on both adsorbents. In contrast, with acetonitrile/water solutions, the adsorption data of both phenol and caffeine deviate far less from linear behavior. They were best modeled by the sum of a Langmuir and a BET isotherm models. The Langmuir term represents the adsorption of the analyte on the highenergy sites located within the C18 layers and the BET term its adsorption on the low-energy sites and its accumulation in an adsorbed multilayer system of acetonitrile on the bonded alkyl chains. The formation of a complex adsorbed phase containing up to four layers of acetonitrile (with a thickness of 3.4 Å each) was confirmed by the excess adsorption isotherm data measured for acetonitrile on Discovery-C18. A simple interpretation of this change in the isotherm curvature at high concentrations when methanol is replaced with acetonitrile as the organic modifier is proposed, based on the structure of the interface between the C18 chains and the bulk mobile phase. This new model accounts for all the experimental observations. Most chemical analyses, impurity detections, separations, and sample preparations are now performed by using RPLC techniques. The number of experimental parameters the analyst can adjust in order to achieve his goal is important. The degree of hydrophocicity of the RPLC column (length of the alkyl bonded chains attached to the solid support, density of these ligands on * To whom correspondence should be addressed. Fax: 865-974-2667. e-mail: guiochonutk.edu. 10.1021/ac0580058 CCC: $30.25 Published on Web 05/24/2005

© 2005 American Chemical Society

the surface, nature of the solid support), the temperature, the pressure, the composition of the mobile phase, and the elution mode (isocratic or gradient) are all experimental parameters that can be easily modified. To predict the analytical elution times or the shape of overloaded band profiles for single-component and multicomponent systems, it is crucial to be able to determine the adsorption equilibria (single and competitive isotherms) of the compounds between the mobile and stationary phases.1 The effect of the experimental parameters on the nature and on the parameters of the adsorption isotherm has gained some attention. The effects of the temperature,2-4 the pressure,5-7 the ionic strength of the liquid phase,8-11 the presence of a buffer,12 the nature and concentration of the buffer,13,14 and the quantity of organic modifier present in aqueous mobile phases15-17 were quantified by measuring adsorption data by frontal analysis (FA), following a systematic protocol. These studies have provided new and often unsuspected information regarding the fundamental role played by those parameters on the adsorption process. Some experimental data and more precise conclusions on the effect of the nature of the organic modifier in the mobile phase in preparative chromatography, whether acetonitrile or methanol, the two most commonly used organic modifiers in RPLC, are missing. The considerable chromatographic literature has been devoted nearly exclusively to attempts at understanding the dependence of the retention in RPLC under linear conditions on the presence and the concentration of the organic modifier.18-21 (1) Guiochon, G.; Shirazi, S. G.; Katti, A. M. Fundamentals of Preparative and Nonlinear Chromatography; Academic Press: Boston, MA, 1994. (2) Szabelski, P.; Cavazzini, A.; Kaczmarski, K.; Liu, X.; Van Horn, J.; Guiochon, G.; J. Chromatogr., A 2002, 950, 41. (3) Kim, H.; Gritti, F.; Guiochon, G. J. Chromatogr., A 2004, 1049, 25. (4) Gritti, F.; Guiochon, G. J. Chromatogr., A 2004, 1043, 159. (5) Liu, X.; Zhou, D.; Szabelski, P.; Guiochon, G. Anal. Chem. 2003, 75, 3999. (6) Zhou, D.; Liu, X.; Kaczmarski, K.; Felinger, A.; Guiochon, G. Biotechnol. Prog. 2003, 19, 945. (7) Liu, X.; Zhou, D.; Szabelski, P.; Guiochon, G. J. Chromatogr., A 2003, 988, 205. (8) Gritti, F.; Guiochon, G. J. Chromatogr., A 2004, 1033, 43. (9) Gritti, F.; Guiochon, G. J. Chromatogr., A 2004, 1033, 57. (10) Gritti, F.; Guiochon, G. J. Chromatogr., A 2004, 1047, 33. (11) Gritti, F.; Guiochon, G. Anal. Chem. 2004, 76, 4779. (12) Gritti, F.; Guiochon, G. J. Chromatogr., A 2004, 1028, 197. (13) Gritti, F.; Guiochon, G. J. Chromatogr., A 2004, 1038, 53. (14) Gritti, F.; Guiochon, G. J. Chromatogr., A 2004, 1041, 63. (15) Gritti, F.; Guiochon, G. J. Chromatogr., A 2003, 995, 37. (16) Gritti, F.; Guiochon, G. J. Chromatogr., A 2003, 1010, 153. (17) Gritti, F.; Guiochon, G. J. Chromatogr., A 2003, 1017, 45. (18) Jaroniec, M.; Martire, D. E. J. Chromatogr. 1987, 387, 55. (19) Stalcup, A. M.; Martire, D. E.; Wise, S. A. J. Chromatogr. 1988, 442, 1.

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Practically all theoretical models proposed aim at fitting the results of systematic measurements of the logarithm of the retention factor, ln k′, as a function of the volume fraction of the organic modifier (methanol, acetonitrile, and tetrahydrofuran among the most studied). These models assume that the stationary phase is homogeneous, and their conclusions apply only in the lowconcentration range. It is well known in linear chromatography that acetonitrile has a higher eluent strength than methanol and can be used to elute large hydrophobic compounds whose retention is too large using methanol/water mobile phases. Because the organic modifier is often present in concentrations 1 or 2 orders of magnitude higher than that of the compounds (in preparative chromatography, the content of the compound rarely exceed 10% because of its limited solubility in the mobile phase), it interferes with the analytes for the adsorption on the stationary phase. It has been experimentally shown that the organic modifiers adsorb selectively on the RP-HPLC stationary phase.22-25 Acetonitrile is definitely more stronly adsorbed than methanol and may compete with the compound for adsorption on the stationary phase. This explains partly why the elution times are systematically reduced when acetonitrile is used as the organic modifier. The increase of solubility of the solute in acetonitrile may also contribute to the loss of retention. The goal of this work is to improve our understanding of the influence of the nature of the organic modifier on the retention of small analyte molecules on RPLC stationary phases and on the ways in which this modifier affects the adsorption of the eluites. Our study is not limited to the linear range, traditionally investigated by analytical chemists. We investigated the dependence of the equilibrium constant in as wide a concentration range as possible. The data afforded by this approach provide original, more general, and more profound information on the interactions that take place in the adsorption of solutes on RPLC packings since linear chromatography is merely the particular case when the concentration of the sample tends toward zero and the retention factors inform only on the initial slope of the isotherm. For this purpose, two different columns were tested, a Discovery-C18 and a Kromasil-C18 column. Two solutes were analyzed, phenol and caffeine, which have been shown to be good solute models for the characterization of RPLC columns.26 The measurement of adsorption data under a wide range of experimental conditions, their modeling using consistent isotherm models, the analysis of the breakthrough curves, and the agreement between simulated and experimental band profiles will be discussed according to the nature of the organic modifier. Excess adsorption measurements will be acquired and compared in order to propose a consistent adsorption mechanism of low molecular mass compounds in RPHPLC using organic modifiers of different natures.

The experimental details of the present implementation of this method are given in the Experimental Section. The method of calculation of the amount adsorbed per unit volume of stationary phase was given in a previous publication.27 2. Models of Isotherm. The adsorption isotherm models best fitting the data acquired for the adsorption of phenol and caffeine on both C18-Discovery and C18-Kromasil from neat aqueous solutions of methanol or acetonitrile used as the liquid phase were either a multi-Langmuir isotherm model or the sum of the liquidsolid extended BET isotherm and the Langmuir isotherm. The multi-Langmuir isotherm can be written as the sum of N homogeneous Langmuir isotherms:

biC

i)N

q* )

∑q i)1

s,i

1 + biC

(1a)

where qs,i and bi are the monolayer saturation capacities and the low-concentration equilibrium constants on the sites of type i, respectively, and N is the number of distinct sites (degree of column heterogeneity) on the surface of the adsorbent. The equilibrium constants bi are associated with the adsorption energies a,i through the following equation:28

bi ) b0ea,i/RT

(2)

where a,i is the energy of adsorption, R is the universal ideal gas constant, T is the absolute temperature, and b0 is a preexponential factor that could be derived from the molecular partition functions in both the bulk and the adsorbed phases. b0 is often considered to be independent of the adsorption energy a,i.28 The derivation of the extended solid-liquid BET isotherm model is described elsewhere.15 The equation is

bSC q* ) qS (1 - bLC)(1 - bLC + bSC)

(1b)

where qS is the monolayer saturation capacity of the adsorbent, bS is the equilibrium constant for surface adsorption-desorption over the free surface of the adsorbent, and bL is the equilibrium constant for surface adsorption-desorption over a layer of adsorbate molecules. The adsorption energy distribution (AED) function of the multi-Langmuir isotherm is the sum of N Dirac functions: i)N

F() )

∑q

s,iδ(

- a,i)

(3)

i)1

THEORY 1. Determination of the Adsorption Isotherms. The adsorption data needed were acquired by the dynamic FA method. (20) Ying, P. T.; Dorsey, J. G.; Dill, K. A. Anal. Chem. 1989, 61, 2540. (21) Antia, F. D.; Horva´th, Cs. J. Chromatogr., A 550, 1991, 411. (22) Scott, R. P. W.; Kucera, P. J. J. Chromatogr. 1977, 142, 213. (23) Scott, R. P. W.; Kucera, P. J. J. Chromatogr. 1979, 175, 51. (24) Westerlund, D.; Theodorsen, A. J. Chromatogr. 1977, 144, 27. (25) McCormick, R. M.; Karger, B. L. Anal. Chem. 1980, 52, 2249. (26) Gritti, F.; Guiochon, G. Anal. Chem. 2003, 75, 5726.

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The energy distribution is N modal, and all the modes have a narrow width (in theory, 0 as a Dirac δ-function). 3. Calculation of the Adsorption Energy Distributions. The calculation of the AED was performed with the expectation(27) Gritti, F.; Go ¨tmar, G.; Stanley, B.; Guiochon, G. J. Chromatogr., A 2003, 988, 185. (28) Jaroniec, M.; Madey, R. Physical Adsorption on Heterogeneous Solids; Elsevier: Amsterdam, The Netherlands, 1988. (29) Gritti, F.; Piatkowsky, W.; Guiochon, G. J. Chromatogr., A 2002, 978, 45.

maximization method.30 The details of the algorithm applicable for any local isotherm (Langmuir, Jovanovic, Moreau, or BET) are given in a previous publication.11 4. Modeling of the Desorption Band Profiles in HPLC. The breakthrough curves of phenol and caffeine were calculated, using the equilibrium-dispersive model (ED) of chromatography.1,31,32 The ED model assumes instantaneous equilibrium between the mobile and the stationary phases and a finite column efficiency originating from an apparent axial dispersion coefficient, Da, that accounts for axial dispersion and for the mass-transfer resistances in the chromatographic column. This model has been successful to describe the overloaded band profiles of various low molecular mass compounds in RPLC,33,34 when the mass-transfer kinetics does not govern the shape of the band profiles but merely smooths the ideal band profiles predicted by pure thermodynamics. This is generally the case with small molecular size compounds. The ED model has the advantage of using little CPU time in comparison to what is required to calculate band profiles with more sophisticated models of chromatography, such as the lumped pore diffusion model or the general rate model.1 These latter models are usually preferred for detailed investigations involving slow mass-transfer kinetics. 5. Measurement of the Excess Isotherm of Acetonitrile. The excess adsorption isotherm of acetonitrile from its aqueous solutions was measured on C18-bonded Discovery-C18, using the minor disturbance method, also called the perturbation method, the step-and-pulse method, or the step-on-a-plateau method.35,36 This method consists of recording and measuring the reaction of a diphasic system under equilibrium to a small perturbation. The composition of the mobile phase is changed by increasing the volume fractions of acetonitrile stepwise from 0 to 1. A small perturbation of mobile-phase composition is injected on each successive plateau, and its retention time is measured. Detection of the perturbation is made with a UV detector, at 195 nm. According to Kazakevich and McNair,35 the thermodynamic void volume of the column, VM, is obtained by integrating the plot of the retention times of the perturbations obtained over the whole concentration range (from pure water to pure acetonitrile) and determining its average.

VM )

1 Cmax

∫

C)Cmax

C)0

VR(C) dC

(4)

this volume, VM, will be used for the calculation of the excess isotherm of acetonitrile in Results and Discussion, section 4. Knowing VM, it is possible to derive the excess amount of acetonitrile, Γ(C), that is adsorbed per unit surface area of the adsorbent (S). The definition of the excess amount, suggested by Gibbs,37 does not consider any specific parameter of the adsorption layer. Let Co and Ce (mol/L) be, respectively, the initial (30) Stanley, B. J.; Bialkowski, S. E.; Marshall, D. B. Anal. Chem. 1993, 65, 259. (31) Ruthven, D. M. Principles of Adsorption and Adsorption Processes; Wiley: New York, 1984. (32) Suzuki, M. Adsorption Engineering; Elsevier: Amsterdam, The Netherlands, 1990. (33) Gritti, F.; Guiochon, G. J. Chromatogr., A 2003, 1003, 43. (34) Gritti, F.; Guiochon, G. J. Chromatogr., A 2004, 1028, 105. (35) Kazakevich, Y. V.; McNair, H. M. J. Chromatogr. Sci. 1995, 33, 321. (36) Kazakevich, Y. V.; McNair, H. M. J. Chromatogr. Sci. 1993, 31, 317.

concentration of the analyte and its concentration after a volume V (L) of this solution has reached equilibrium with the amount of adsorbent having the surface area S (m2). Assuming that the partial molar volumes of all the solution components are the same in the two phases, the solution and the adsorbed monolayer, no changes in the volume of the solution take place upon the adsorption of the solute. Then, the excess amount of solute is defined by

Γ(C) ) (Co - Ce)V/S

(5)

The elution volume, VR(C), of the perturbation on the plateau at concentration C can be derived from the propagation velocity of this concentration C:36

VR(C) ) VM + S(dΓ/dC)C

(6)

After integration of eq 6, it is possible to calculate the experimental excess isotherm by

Γ(C) )

∫ (V (C) - V

1 S

C

0

R

M)

dC

(7)

Finally, the amount of preferentially adsorbed component nads per unit of surface area is obtained as follows:38

nads ) Cτ + Γ(C)

(8)

where τ is the thickness of the adsorbed layer and C is the concentration of the preferentially adsorbed component in the bulk phase. EXPERIMENTAL SECTION 1. Chemicals. The mobile phases used in this work were different mixtures of methanol or acetonitrile and water (40/60, 30/70, and 20/80, v/v), all HPLC grade and all purchased from Fisher Scientific (Fair Lawn, NJ). The solvents used to prepare the mobile phase were filtered before use on an SFCA filter membrane, 0.2-µm pore size (Suwannee, GA). Thiourea was chosen to measure the column holdup volume. Thiourea, phenol, and caffeine were all obtained from Aldrich (Milwaukee, WI). 2. Columns. The columns used in this work were a KromasilC18 (Eka Nobel, Bohus, Sweden) and a Discovery-C18 (Supelco Park, Bellefonte, PA) column supplied by the manufacturer. Their dimensions are 250 × 4.6 mm and 150 × 4.0 mm, respectively. The main characteristics of the bare porous silica and of the packing material used are summarized in Table 1. The holdup volumes of the columns were measured for each mobile-phase composition as the elution of a tracer compound (thiourea) that we assume to be unretained (See Table 1). This holdup volume was used to measure the adsorption data of phenol and caffeine on each column and for each organic modifier in Results and Discussion, sections 1-3. (37) Gibbs, J. W. On the equilibrium of heterogeneous substances; Collected Works. Longmans: New York, 1928. (38) Rustamov, I.; Farcas, T.; Ahmed, F.; Chan, F.; LoBrutto, R.; McNair, H. M.; Kazakevich, Y. V. J. Chromatogr., A 2001, 913, 49.

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Table 1. Physicochemical Properties of the Discovery-C18 and Kromasil-C18 Adsorbent Materials Packed in Stainless Steel Tubes (150 × 4.0 mm and 250 × 4.6 mm, Respectively)

particle shape particle size (µm) pore size (Å) specific surface (m2/g) (before derivatization) total carbon (%) surface coverage (µmol/m2) end capping void volume measurements (mL)

Discovery

Kromasil

spherical 5 180 200

spherical 5.98 112 314

12 3.0 yes 1.365a 1.340b

20 3.6 yes 2.515c 2.189d

a Elution of thiourea (MeOH/H O, 30/70, v/v). b Elution of thiourea 2 (ACN/H2O, 20/80, v/v). c Elution of thiourea (MeOH/H2O, 40/60, d v/v). Elution of thiourea (ACN/H2O, 30/70, v/v).

3. Apparatus. The isotherm data were acquired using a Hewlett-Packard (Palo Alto, CA) HP 1090 liquid chromatograph. This instrument includes a multisolvent delivery system (tank volumes, 1 L each), an autosampler with a 250-µL sample loop, a diode-array UV detector, a column thermostat, and a data station. Compressed nitrogen and helium bottles (National Welders, Charlotte, NC) are connected to the instrument to allow the continuous operations of the pump, the autosampler, and the solvent sparging. The extracolumn volumes are 0.035 and 0.29 mL as measured from the autosampler and from the pump system, respectively, to the column inlet. All the retention data were

corrected for these contributions. The flow rate accuracy was controlled by pumping the pure mobile phase at 22 °C and 1 mL/ min during 50 min, from each pump head, successively, into a volumetric glass of 50 mL. The relative error was less than 0.4%, so that we can estimate the long-term accuracy of the flow rate at 4 µL/min at flow rates around 1 mL/min. All measurements were carried out at a constant temperature of 22 °C, fixed by the laboratory air conditioner. The daily variation of the ambient temperature never exceeded (1 °C. 4. Measurements of the Adsorption Isotherms by FA. The maximum concentrations of phenol used in FA were fixed at 50 and 160 g/L with methanol as the organic modifier on KromasilC18 and on Discovery-C18, respectively, because of the important difference in the column saturation capacities of the two asorbents. The maximum concentrations of phenol with acetonitrile as the organic modifier were fixed at 40 and 30 g/L on the Kromasil-C18 and Discovery-C18 adsorbents because of the limited solubility of phenol in the corresponding acetonitrile/water mobile phases. The minimum concentration was chosen such that a symmetric breakthrough curve be obtained (e.g., the shape of the front and the rear parts of the curve are those of an erf function), attesting for the linearity of the isotherm at low concentrations. For each FA run, one pump of the HPLC instrument (pump A) delivers a stream of the pure mobile phase while the other pump (pump B) delivers a stream of one of the sample mother solutions. The concentration of the compound in the FA stream is determined by the concentration of the mother sample solution and the flow rate fractions delivered by the two pumps. The breakthrough curves were recorded at a flow rate of 1 mL min-1, with a

Figure 1. Adsorption isotherm data of phenol (A) on Kromasil-C18 with 40% methanol in water, v/v, corresponding AED (B), and a few breakthrough curves (C) recorded by UV at λ ) 291 nm. Flow rate, 1 mL/min, T ) 295 K. 4260 Analytical Chemistry, Vol. 77, No. 13, July 1, 2005

Figure 2. Comparison between experimental (dotted lines) and calculated (solid lines) band profiles of phenol using the best bi-Langmuir isotherm model. Same experimental conditions as in Figure 1. Column efficiency in the calculations, 2000 plates.

sufficiently long time delay between each breakthrough curve to allow for the complete reequilibration of the column with the pure mobile phase. In practice, it was estimated that this reequilibration was achieved when the volume of mobile phase flushed through the column was equal to the sum of (1) the retention volume at infinite dilution of the compound studied, Vanalytical; (2) the extracolumn volume, Vext; (3) the volume of the plug of sample, Vp, injected to obtain the breakthrough curve; and (4) an arbitrary safety margin of 5 mL. This estimate of the reequilibration volume needed is justified by the reproducibility of the results that its use provided. The breakthrough curves were recorded at different wavelengths, as shown in the figures, to maximize the accuracy of the measurements. RESULTS AND DISCUSSION 1. Adsorption of Phenol on Kromasil-C18. The adsorption behavior of phenol on various C18-bonded silica packing materials, from aqueous solutions of methanol, has already been extensively investigated. The adsorption of phenol on these RPLC packing materials has revealed the heterogeneity of the surface of these adsorbents and that a bi-Langmuir isotherm is the best adsorption model that accounts for this behavior. Exceptionally, in the case of Chromolith-C18, a tri-Langmuir adsorption isotherm was found to be a better isotherm model, suggesting a higher degree of surface heterogeneity for this adsorbent. The influence of the

methanol content in the mobile phase and that of the temperature have been studied and the variations of the isotherm parameters with respect to these experimental parameters derived. These results are briefly summarized in the next section. The similar effect of another organic modifier, acetonitrile, on the adsorption of phenol on Kromasil-C18 is then investigated in more detail. (a) Methanol/Water (40/60, v/v) Mobile Phase. The adsorption data of phenol from methanol/water mobile phases are shown in Figure 1A for phenol concentrations between 0 and 50 g/L in the liquid phase. The isotherm was successfully fitted to a bi-Langmuir model, consistent with the results of the calculation of the AED (Figure 1B, 100 million iterations). This calculation shows a clearly bimodal energy distribution. The two saturation capacities were qs,1 ) 132.3 g/L and qs,2 ) 29.1 g/L and the equilibrium constants b1 ) 0.0169 L/g and b2 ) 0.107 L/g, respectively. A systematic series of selected breakthrough curves is shown in Figure 1C. These curves are fully consistent with the thermodynamical data and with the predictions of the ideal model,1 e.g., a front shock, followed, first, by a stable plateau (the slight fluctuations of this plateau around the feed concentrations are due to the imperfect behavior of the stream mixer) and, finally, by a diffuse rear desorption profile. An excellent agreement is found between the experimental and the calculated breakthrough curves, for all plateau concentrations injected between 0 and 100 g/L (Figure 2A-D). Analytical Chemistry, Vol. 77, No. 13, July 1, 2005

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Table 2. Best Isotherm Parameters Accounted for by the Adsorption of Phenol and Caffeine on Discovery- C18 and Kromasil-C18 versus the Mobile-Phase Compositiona phenol

caffeine

Kromasil-C18 mobile phase qs,1 (g/L) b1 (L/g) bL (L/g) qs,2 (g/L) b2 (L/g) qs,3 (g/L) b3 (L/g) a

MeOH/H2O 40/60 132.3 (124.5) 0.0169 (0.0149) 29.1 (39.6) 0.107 (0.0848)

MeCN/H2O 30/70 140.4 0.0188 0.0066 13.5 0.104

Discovery-C18 MeOH/H2O 30/70 1117 (1301) 0.00164 (0.00150) 78 (82.7) 0.0712 (0.0725) 5.6 (2.1) 0.305 (0.480)

MeCN/H2O 20/80 888.5 0.0020 0.0083 80.3 0.0850

Discovery-C18 MeOH/H2O 30/70 412.7 (634.0) 0.0077 (0.0058) 4.1 (4.2) 0.204 (0.187)

MeCN/H2O 20/80 137.4 0.01451 0.0040 3.9 0.116

The values in parentheses are the isotherm parameters derived from the calculated AED.

Figure 4. Comparison between two breakthrough curves of phenol with acetonitrile as the organic modifier, corresponding to the injection of a 40 g/L solution of phenol during 6 (solid line) and 8 min (dotted line), respectively. Note that the elution of the front of the hump on the equilibrium plateau shifts by 2 min, the time difference between the two injections. Same experimental conditions as in Figure 1.

Figure 3. Adsorption isotherm data of phenol (A) on Kromasil-C18 with 30% acetonitrile in water, v/v, and a few breakthrough curves (B) recorded by UV at λ ) 291 nm. Flow rate, 1 mL/min. T ) 295 K. Note the quasilinear behavior of the isotherm at high concentrations and the presence of a hump on the equilibrium plateau of the breakthrough curves.

(b) Acetonitrile/Water (30/70, v/v) Mobile Phase. The mobile-phase concentration of acetonitrile was adjusted in order to achieve about the same retention time of phenol (e.g., a similar Henry constant) as with methanol. The acetonitrile concentration was thus dropped to 30%, instead of 40% with methanol. The adsorption data obtained are reported in Figure 3A. A selection of the breakthrough curves recorded is shown in Figure 3B. The curvature of this isotherm is strikingly much less pronounced than that of the isotherm from the methanol solution. Accordingly, at 4262 Analytical Chemistry, Vol. 77, No. 13, July 1, 2005

high concentrations (C > 15 g/L) the concentration of phenol adsorbed on Kromasil-C18 from a methanol solution is lower than from an acetonitrile solution while at low concentrations (C < 15 g/L), the opposite is true. By contrast to the previous case (Figure 1), the isotherm from aqueous solutions of acetonitrile behaves almost linearly at high concentrations. The adsorption data could not be satisfactorily fit to a multi-Langmuir model but were successfully fitted to the sum of a BET and a Langmuir isotherm models. The isotherm parameters are reported in Table 2. It is noteworthy that the parameters of the Langmuirian term are of the same order as those of the term corresponding to the highenergy sites found when methanol is used as the organic modifier. These sites that control the retention of phenol at low concentrations are adsorption sites buried within the C18-bonded layer. Their number (qS,2) and their adsorption energy (b2) are slightly lower in the presence of acetonitrile than in that of methanol. On the other hand, the properties of the low-energy sites, those that control the retention behavior at high concentrations of phenol, are drastically changed. Low-energy sites fill more rapidly in the presence of methanol than in that of acetonitrile because only a monolayer forms in the first case while adsorption from acetonitrile leads to multilayers. Hence, the saturation capacity is smaller in the first case and the isotherm saturates at low concentration

Figure 5. Comparison between the experimental (dotted lines) and calculated (solid lines) band profiles of phenol using the best LangmuirBET isotherm model. Same experimental conditions as in Figure 3. Column efficiency in the calculations, 2000 plates. Note the important deviations at high concentrations because of the presence of the hump. Only the elution of the front matches the calculation results.

while in the second case more phenol molecules are adsorbed and the isotherm remains nearly linear from 15 to 40 g/L. Obviously, the adsorption mechanisms of phenol from acetonitrile and from methanol solutions on the low-energy sites (probably the sites at the top of the C18 bonded layer) are different. Phenol may form a multilayer adsorbed system in the presence of acetonitrile. A most surprising feature is the evolution of the shape of the breakthrough curves with increasing concentration of phenol. The higher the concentration of phenol, the larger the nick after the front shock. This second shock cannot be explained by any competitive adsorption of phenol and acetonitrile. As will be discussed later, the retention factor of acetonitrile in pure water is hardly 1.0 while that of phenol is ∼6.0. Figure 4 demonstrates that the concentration of phenol in the elution profile is higher after the second shock than that in the feed, but this profile is profoundly different from the one that would be observed if there were competitive adsorption of phenol and acetonitrile.1 The position of this shock depends on the duration, tp, of the injection (see the profiles with tp ) 6 and 8 min). A comparison of the experimental and the calculated breakthrough profiles (see Figure 5) shows obvious deviations, particularly at high phenol concentrations. After the expected front shock, a concentration plateau at Cfeed is eluted, followed by an unexpected raise of the phenol concentration that seems to be triggered when the stream of the pure mobile phase is resumed (Figure 4), at the end of the injection of the sample. Finally, the desorption of phenol begins

before this phenomenon is expected to take place (Figure 5), according to the calculations made with the ED model and based on the mass conservation of phenol. As the phenol concentration decreases, the difference between calculated and experimental profiles decreases but a disagreement between the two profiles persists until the end of the elution. A physically correct interpretation of this unusual phenomenon is not obvious. It is not simply related to the migration of the breakthrough curves of phenol. The sudden, rapid, abrupt drop of phenol concentration from Cphenol ) Cfeed to pure acetonitrile/water mobile phase (Cphenol ) 0) causes this phenomenon and the unusual desorption profiles. It is obvious that the desorption mechanism does not follow the mechanism expected on the basis of the ideal or the ED models and of the adsorption isotherm derived from the elution times of the front shocks of breakthrough curves. Once the solid adsorbent and the liquid phase are in equilibrium (see Figure 5A-D, the plateau concentration before the hump), a rapid return to the pure mobile phase causes the brutal desorption of a certain amount of phenol, so far held in the stationary phase, into the mobile phase. This result in the hump observed. A short, higher concentration plateau is generated. It propagates through the column and is detected after the elution of the first shock. Note that the size of this hump is independent of the duration of the rectangular injection (Figure 5). This phenomenon is independent of the flow rate and is not of kinetic origin. The exact same anomalous profiles are observed at flow rates of 1.0 and 0.1 mL/min (see later, Figure Analytical Chemistry, Vol. 77, No. 13, July 1, 2005

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Figure 6. Adsorption isotherm data of phenol (A) on Discovery-C18 with 30% methanol in water, v/v, corresponding AED (B), and a few breakthrough curves (C) recorded by UV at λ ) 289 nm. Flow rate, 1 mL/min. T ) 295 K. Note the larger saturation capacity of Discovery-C18 and the trimodal energy distribution, different from the bimodal one found with Kromasil-C18.

11), the only changes being an increase of the retention times of the different features of the profile (e.g., those of the two shocks) by a factor 10 and a slight increase of the dispersion on the diffuse boundaries. This observation shows that the second shock observed does not have a kinetic origin but that it mirrors some changes in the equilibrium between the stationary and the liquid phases. The breakthrough curves recorded are the result of the feed injection followed by a stepwise increase of the phenol concentration by a value corresponding to the excess mass of phenol retained. The difference between the apparent and the actual injection amounts resides in the fact that the mass corresponding to the second step comes from phenol already present in the column. There is an unusual redistribution of the mass of phenol inside the column when the stream of the pure mobile phase resumes. It seems that a cooperative adsorption of phenol and acetonitrile takes place and is at the origin of this phenomenon. At this point, we might suspect that, when acetonitrile is the organic modifier, the adsorption isotherm of phenol differs from its desorption isotherm. An isotherm hysteresis could account for this effect. It would be interesting to acquire adsorption and desorption data by using the staircase frontal analysis method with positive steps, from 0 to Cmax, followed by negative steps of the same amplitude, from Cmax to zero. This results of this experiment will be reported elsewhere. 2. Adsorption of Phenol on Discovery-C18. To determine whether the breakthrough profiles observed with Kromasil-C18 are specific to this packing material or apply to most C18-bonded 4264 Analytical Chemistry, Vol. 77, No. 13, July 1, 2005

silica materials, we repeated the measurements of the adsorption isotherms of phenol on another stationary phase, from aqueous solutions of methanol and acetonitrile. We found that the adsorption properties of Discovery-C18 were qualitatively similar to but quantitatively different from those of Kromasil-C18 and that some adjustments of the mobile-phase composition were required in order to achieve sufficiently large retention factors for the two compounds studied (phenol and caffeine), hence to measure accurate adsorption data by FA. So, the organic modifier concentrations were reduced by 10%. (a) Methanol/Water, 30/70, v/v. The adsorption data of phenol on Discovery-C18, from this aqueous solution of methanol, are shown in Figure 6A. As on Kromasil-C18, the isotherm is strictly convex upward and can be fitted to a multi-Langmuir adsorption model. Because the adsorbed concentration of phenol still increases rapidly at a mobile-phase concentration of 40 g/L (see Figure 6A), measurements were made with mobile-phase concentrations up to 160 g/L, in an attempt more closely to approach the saturation of the column and to improve the accuracy of the estimates of the isotherm parameters. The saturation capacity of the low-energy adsorption sites is obviously very high. The adsorption energy distribution calculated from the raw experimental data (Figure 6B) confirms a trimodal distribution, consistent with the best isotherm model being the tri-Langmuir model. The breakthrough curves recorded (see Figure 6C) are conventional. The best values obtained for the parameters are listed in Table 2. As expected from the carbon content being lower

Figure 7. Comparison between experimental (dotted lines) and calculated (solid lines) band profiles of phenol using the best tri-Langmuir isotherm model. Same experimental conditions as in Figure 6. Column efficiency in the calculations, 2000 plates.

for Discovery-C18 than for Kromasil-C18 (12 instead of 20%), and from the lower chain density (3.0 instead of 3.6 µmol/m2), the equilibrium constant on the low-energy adsorption sites (type 1 sites) is lower on Discovery-C18 than on Kromasil-C18 (Table 2). Surprisingly, however, despite the lower specific surface area of the bare silica used for Discovery-C18 (200 versus 314 m2/g), the saturation capacity of the type 1 sites on Discovery-C18 is nearly 8 times larger than that of the low-energy sites of Kromasil-C18. The smaller pore size and the higher carbon content of KromasilC18 may explain these differences. The volume occupied by the bonded layer in the latter material is a larger fraction of the mesopore volume; hence, the effective surface area of contact of this layer with the mobile phase is lower. In aqueous solutions of methanol, the total porosities of Kromasil-C18 and Discovery-C18 are 0.60534 and 0.7236, respectively. The adsorption capacity of Discovery-C18 is exceptionally high compared to that of other conventional RPLC materials, which oscillate around 200 g/L and is always lower than 300 g/L. Figure 7 illustrates the excellent agreement between calculated and experimental breakthrough curve profiles. In the whole range of concentrations investigated, the plateau concentration is always flat, as those recorded with Kromasil-C18. Using methanol as the organic modifier always leads to band profiles that are entirely predicted by the mere knowledge of the adsorption isotherm. No second shock is ever observed on these curves, and both the adsorption and the desorption profiles are consistent with the adsorption data measured by FA.

(b) Acetonitrile/Water, 20/80, v/v. To increase the retention factor and improve the accuracy of the measurements, the acetonitrile content of the mobile phase was lowered from 30 to 20%. However, the solubility of phenol in this solution is now less than 40 g/L, and a maximum concentration of 30 g/L was used in the FA runs. The adsorption data measured by FA and the corresponding breakthrough curves are shown in Figure 8A and B, respectively. The isotherm is no longer convex upward, but it has an inflection point. The breakthrough curves are characteristic of an S-shaped isotherm, exhibiting a front shock at low concentrations and a diffuse front at high concentrations.1 Conversely, a rear shock is observed at high concentrations and a rear diffuse boundary at low concentrations. This result is similar to that observed with Kromasil-C18. The clear downward convexity of the isotherm suggests that, in this case, adsorbate-adsorbate interactions take place between the molecules of phenol. As for Kromasil-C18, the best isotherm model is the sum of the BET model (for the low-energy sites) and the Langmuir model (for the high-energy sites). The attempt of adding a third type of sites to the model, which would be consistent with the results obtained with methanol (Table 2), was unsuccessful, the calculation program failing to converge. The parameters obtained for the BET-Langmuir model are listed in Table 2. The values confirm the very large saturation capacity of Discovery-C18 (∼900 g/L), 6 times larger than that of Kromasil-C18 (∼140 g/L). It is striking that the properties of the type 2 sites are almost the same with methanol and acetonitrile. This confirms that these sites are Analytical Chemistry, Vol. 77, No. 13, July 1, 2005

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Figure 8. Adsorption isotherm data of phenol (A) on DiscoveryC18 with 20% acetonitrile in water, v/v, and a few breakthrough curves (B) recorded by UV at λ ) 289 nm. T ) 295 K. Note the nearly linear behavior of the isotherm, the slightly convex downward curvature at high concentrations, and the presence of a hump on the equilibrium plateau of the breakthrough curves when acetonitrile is used instead of methanol.

not exposed to the liquid phase but are located inside the C18bonded layer. Figure 9 shows the generally good agreement between the calculated and the experimental breakthrough curves, except for two discrepancies. First, the experimental diffuse boundary tails longer than the calculated one. Second, there is a second shock on the concentration plateau that is not accounted for in the ED model calculations. Figure 10A compares the experimental breakthrough curves recorded for injection having different durations (2, 4, and 6 min). It shows that the position of the second shock depends directly on the time at which the injection ends, suggesting that this shock is triggered by the replacement of the sample solution with the pure mobile phase, as is happening with Kromasil-C18. This phenomenon should be related to the presence of acetonitrile in the mobile phase and to its interactions with phenol. Figure 10B compares the breakthrough curves obtained upon the injection of a 4-mL sample at two different flow rates, 1 and 0.1 mL cm-3. The diffuse parts of the profiles (the front at high concentrations, the tail at low concentrations) are influenced by kinetics effects. In contrast, the elution volumes of the two shocks (3.92 and 6.34 mL) are identical in both cases and independent of the flow rate. The second shock is controlled by the equilibrium between the solid and the liquid 4266 Analytical Chemistry, Vol. 77, No. 13, July 1, 2005

phases. It cannot be the result of some unusual mass-transfer effects in the column. 3. Adsorption of Caffeine on Discovery-C18. Caffeine was chosen to complement phenol in this study. Its molecule is larger than that of phenol (Mw ) 194 versus 94 g/mol; it has two aromatic rings instead of one), it contains no acidic hydrogen atom that could take part in hydrogen bond interactions with methanol, but it has four mildly basic nitrogen atoms (caffeine is a hydrogen bond acceptor only). Accordingly, the solubility of caffeine in methanol/water solutions is lower than that of phenol (less than 40 g/L for caffeine, more than 160 g/L for phenol). However, phenol is more retained on RPLC columns26 due to its higher accessibility to the surface. The experiments reported in this section aim at finding whether the anomalous second shock on the breakthrough curves of phenol with Kromasil-C18 and DiscoveryC18 is a consequence specific to some property of this compound or is associated with the replacement of methanol with acetonitrile as the organic modifier. (a) Methanol/Water, 30/70, v/v. The adsorption isotherm data of caffeine are plotted in Figure 11A. The best isotherm model is the bi-Langmuir isotherm, a result confirmed by the AED, which is bimodal (Figure 11B). The best parameters of the bi-Langmuir isotherm are listed in Table 2. Obviously, caffeine does not have access to all the type 1 sites on which phenol can adsorb. The amount of caffeine adsorbed on the sites of this type is twice lower than the amount of phenol (meaning fewer molecules still). Similarly, the amount of caffeine that saturates the type 2 sites is 20 times less than that of phenol, and type 3 sites are not recognized by caffeine. They are inaccessible to this molecule. (b) Acetonitrile/Water, 20/80, v/v. The adsorption data of caffeine are plotted in Figure 12A. These data could be successfully fitted to the Langmuir-BET isotherm model. The best isotherm parameters are listed in Table 2. This result demonstrates that it is the use of acetonitrile as the organic modifier that leads to a change in the isotherm model accounting for the adsorption mechanism and in the sign of the curvature of this isotherm at high concentrations. The retention of caffeine at low concentrations is controlled by its adsorption on the high-energy sites of type 2, whether the mobile phase is a methanol/water or an acetonitrile/water solution. In the high-concentration domain, the adsorption mechanism is completely different in the two mobile phases. There is a finite saturation capacity in the presence of methanol. With acetonitrile, there seems to be none. The solid adsorbent keeps adsorbing more solute as its mobile-phase concentration increases up to saturation of the mobile phase. The behavior of the same adsorbent is profoundly different with methanol, the isotherm remains strictly convex upward, and the concentration in the adsorbed phase tends uniformly toward the finite saturation capacity. It is important to note, however, that the breakthrough curves of caffeine (Figure 12B) do not contain any anomaly, second shock, or hump similar to those recorded with phenol on both Kromasil-C18 and Discovery-C18 when acetonitrile is used as the organic modifier. So, the replacement of methanol with acetonitrile is not the essential cause for the occurrence of this phenomenon. Rather, specific molecular interactions between phenol and acetonitrile should be involved that do not take place with caffeine.

Figure 9. Comparison between experimental (dotted lines) and calculated (solid lines) band profiles of phenol using the best Langmuir-BET isotherm model. Same experimental conditions as in Figure 8. Column efficiency in the calculations, 2000.

4. Adsorption Mechanism on RPLC Adsorbents from Methanol and Acetonitrile Aqueous Solutions. Based on the measurement of the adsorption isotherms of these two compounds, the experimental results reported earlier in this work demonstrate that the adsorption mechanisms of phenol and caffeine on Kromasil-C18 and Discovery-C18, from aqueous solutions of methanol or acetonitrile, are quite different. In aqueous solutions of methanol, the adsorption isotherms are strictly convex upward and are well accounted for by a bi-Langmuir or a tri-Langmuir model, depending on the solute and the column. However, in aqueous solutions of acetonitrile, the isotherm, which is convex upward at low concentrations, becomes convex downward at high concentrations of solute and does not exhibit any saturation. The adsorbent has one or two types of sites, the behavior of which is not affected by the nature of the mobile phase, and a large number of low-energy adsorption sites, the behavior of which changes drastically when methanol is replaced with acetonitrile. It is important to understand what makes this change of behavior to take place, what causes the reversal of the isotherm curvature at high concentrations to happen. So much is known about the general behavior of isotherms. (a) An isotherm is convex upward if the surface has a finite number of adsorption sites and adsorbate-adsorbate interactions are weak or negligible. The number of adsorption sites available on the surface decreases progressively as the solute concentration in the mobile phase increases and q increases more slowly than C.

(b) An isotherm can have a linear behavior for one of three possible reasons. (1) The analyte concentration is very low and the limited accuracy of the measurements does not allow the detection of the curvature of the isotherm within the range of concentrations used. All that can be measured is the Henry constant. (2) The number of adsorption sites available is practically infinite, and the two phases keep having an ideal behavior (no solute-solute or adsorbate-adsorbate interactions). This is a high-concentration version of case 1. (3) There is a compensation between the effects of a finite number of adsorption sites (causing a negative curvature) and a real phase behavior (with strong adsorbate-adsorbate interactions causing a positive curvature). This compensation tends to be accidental. It gives an inflection point at a certain concentration, and the linear behavior of the isotherm does not persist when the mobile-phase concentration is increased and adsorbate-adsorbate interactions become stronger. (c) An isotherm is convex downward when the compound accumulates in the adsorbed phase faster than in the liquid phase (q increases faster than C). This takes place when there are adsorbate-adsorbate interactions that are stronger than the solute-solute interactions. According to these considerations, no or very weak adsorbateadsorbate interaction take place with either compound studied when methanol is used as the organic modifier. The surface of the adsorbent is progressively filled with a monolayer of the analyte until its complete saturation (which is never observed Analytical Chemistry, Vol. 77, No. 13, July 1, 2005

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Figure 10. (A) Comparison between three breakthrough curves of phenol (A) with acetonitrile as the organic modifier, corresponding to the consecutive injection of a 40 g/L solution of phenol during 2 (solid line), 4 (dashed line), and 6 min (dotted line). As with Kromasil, the elution of the front of the hump on the equilibrium plateau shifts by 2 min, the time difference between two consecutive injections. Same experimental conditions as in Figure 9. (B) Comparison between the breakthrough curves of phenol recorded at two different flow rates, 1.0 and 0.1 mL/min. Note that the elution volume of the hump is the same while the profiles of diffuse boundaries at high concentrations depend on the flow rate.

experimentally because the solubility of the analyte in the mobile phase is limited). On the other hand, the quasilinear adsorption isotherm (with a weak upward curvature) of phenol and caffeine at high concentrations in aqueous solutions of acetonitrile suggests that there are only two possibilities: (1) Significant phenol-phenol and caffeine-caffeine interactions can take place in the adsorbed phase when the mobile phase is acetonitrile (but not if it is methanol). (2) The number of adsorption sites available increases considerably when acetonitrile is substituted to methanol. The assumption of phenol-phenol interactions taking place in an aqueous solution of acetonitrile is implausible. No such interactions take place in aqueous solutions of methanol even at the high concentration of 300 g/L. Rather, the increase of the number of the apparent adsorption sites may explain its unusual adsorption isotherm in the presence of acetonitrile, but then what could cause it? Many papers by different authors39 attempt to quantify the adsorption of organic modifiers on RPLC adsorbents and to improve our understanding of the retention mechanisms in RPLC. Because of their hydrophobic expulsion from aqueous 4268

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mobile phases, organic solvents are expected to distribute between mobile and stationary phases in RPLC. McCormick and Karger25 compared the distribution isotherms of methanol and acetonitrile on octyl bonded Hypersil and showed that a maximum of 20 mg of methanol and 100 mg of acetonitrile were adsorbed per gram of adsorbent. It is surprising to observe that only 625 µmol of methanol but 2440 µmol of acetonitrile (∼4 times as many molecules) can adsorb on the surface of 1 g of this adsorbent. Given the specific surface area of the bare Hypersil (180 m2/g) and assuming monolayer adsorption for both solvents, methanol and acetonitrile would occupy average surface areas of 48 and 12 Å2/molecule, respectively. Based on the average van der Waals radii of these compounds, their average molecular areas are 28 and 25 Å2, for methanol and acetonitrile, respectively. So, this is equivalent to saying that methanol forms a relatively dense monolayer and acetonitrile forms two such layers on a C8 bonded phase. More recently, Kazakevich et al.40 measured the excess isotherms of methanol and acetonitrile on several C18 bonded phases, using the minor disturbance method. They concluded that the former modifier forms an adsorbed monolayer while the latter one gives an adsorbed multilayer system, containing about four layers. To generalize these conclusions and extend to our packing materials, we measured the excess isotherm of acetonitrile on Discovery-C18, from a series of aqueous mobile phases containing from no to 100% acetonitrile. An example of the chromatograms recorded using the perturbation method is shown in Figure 13 with either a positive or a negative perturbation of acetonitrile, for a concentration of acetonitrile of 15% (v/v). The perturbation signal is then related to the distribution of acetonitrile between the liquid and the stationary phases. The evolution of the elution time of the perturbation of the acetonitrile plateau concentration and the corresponding excess isotherm calculated from eq 7 (with S ) 160 m2; see adsorbent properties) are reported in Figure 14. On the basis of the work of Everett,41 we can assess the average area occupied by the organic modifier molecules by using the model of an adsorption layer with a finite thickness. The concentration of the adsorbate in the finite thickness layer could be found by extrapolating the slope of the excess adsorption isotherm in the linear region to intercept the y-axis (Figure 14B). We found Cads ) 26 µmol/m2, meaning that one molecule of acetonitrile would occupy an average surface area of 6.4 Å2, a value that is well below the average molecular area of a molecule of acetonitrile, 25 Å2. Accordingly, acetonitrile forms up to four adsorbed layers on Discovery-C18. The molar volume of acetonitrile taken in the liquid state is v ) MCH3CN/dCH3CN ) 52.2 mL/mol. Accordingly, the maximum volume, Vads, occupied by the adsorbed layers of acetonitrile are given by

Vads ) SCadsv ) Sτ

(9)

with S ) 160 m2. The volume of adsorbed acetonitrile is then 0.217 mL and its average thickness is τ ) 13.6 Å. Equation 8 gives the total amount of acetonitrile adsorbed as a function of its concentra(39) Alvarez-Zepeda, A.; Martire, D. E. J. Chromatogr., A 1991, 550, 285. (40) Kazakevich, Y. V.; LoBrutto, R.; Chan, F.; Patel, T. J. Chromatogr., A 2001, 913, 49. (41) Everett, D. H. Pure Appl. Chem. 1986, 58, 967.

Figure 11. Adsorption isotherm data of caffeine (A) on Discovery-C18 with 30% methanol in water, v/v, AED (B), and a few breakthrough curves (C) recorded by UV at λ ) 308 nm. Flow rate, 1 mL/min. T ) 295 K. Note the bimodal energy distribution (the convergence is incomplete because the solubility of caffeine in the liquid phase is only ∼35 g/L) and the presence of a little hump on the equilibrium plateau of the breakthrough curves, previously absent with phenol (Figures 1 and 7).

tion in the mobile phase. The results are shown in Figure 15. When the acetonitrile content is between 20 and 30% v/v (see earlier), the amount adsorbed on Discovery-C18 is ∼15 µmol/m2, meaning that more than two layers of acetonitrile are covering the bonded C18 alkyl chains. These results show that the change in the isotherm behavior when methanol is replaced with acetonitrile as the organic modifier is due to the formation of a relatively thick, multimolecular adsorbed layer of acetonitrile, forming an intermediate liquid phase between the bulk mobile phase and the adsorbent. This liquid layer includes the C18 chains, which are dissolved in it but extends above them. This structure was proposed by Kazakevich,40 who suggested a retention model that accounts well for many retention data measured at infinite dilution. This model assumes two simultaneous equilibria, one between two liquid phases (the bulk mobile phase and an acetonitrile-rich adsorbed phase) and the other between the C18 bonded layer and the acetonitrile-rich adsorbed phase. The thick adsorbed layer of acetonitrile (τ ) 13.6 Å) can dissolve far more analyte molecules from the bulk than the bonded layer alone. The analyte molecules become encapsulated in the multilayer system of acetonitrile and concentrate in this region of high saturation capacity. It is expected that the concentration of the compound in this phase is higher than in the bulk mobile phase because the analyte solubility is higher in pure acetonitrile than in the water-rich mobile phase. This model explains also why the isotherms of phenol and caffeine follow BET behavior once the high-energy sites of type 2, located within the C18 bonded layer, are saturated. The adsorbateadsorbate interactions in the adsorbed layer and their interactions

with the adsorbed acetonitrile molecules allow the adsorption of a large amount of material and the buildup of a thick multilayer system. This model explains the anti-Langmuirian behavior of the isotherms that is systematically observed at high concentrations. A comparison between the adsorption mechanisms of phenol or caffeine on the RPLC materials from aqueous solutions of methanol and acetonitrile is illustrated in Figure 16. The profiles of the analyte concentration along the direction perpendicular to the surface of the adsorbent are schematized in Figure 17. CONCLUSION Our results demonstrate that the isotherm behavior of the adsorption of low-molecular-mass polar compounds depends strongly on the nature of the organic solvent used in RPLC. Typically, strictly convex upward isotherms (i.e., multi-Langmuir isotherms due to the surface heterogeneity of the adsorbent) are often found with methanol and S-shaped isotherms (convex upward at low concentrations, downward at high concentrations) with acetonitrile in the aqueous mobile phase. We propose an interpretation of these results that brings new insight to the adsorption mechanism in RPLC. This model is consistent with former measurements of excess adsorption isotherms of methanol and acetonitrile on RP-HPLC adsorbents,40 with methanol, a simple adsorbed monolayer of methanol dissolving the bonded alkyl chains and the solutes’ injected form on the surface of the packing material, and with acetonitrile, two successive layers form. The adsorbed layer of acetonitrile dissolving the bonded alkyl chains and the solutes is covered with Analytical Chemistry, Vol. 77, No. 13, July 1, 2005

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Figure 13. Positive (+) and negative (-) perturbations of the equilibrium between C18-bonded Discovery-C18 and the mobile phase (acetonitrile/water, 15/85, v/v) detected by UV (λ ) 195 nm) after injection of 2-µL perturbations of pure acetonitrile and pure water, respectively.

Figure 12. Adsorption isotherm data of caffeine (A) on DiscoveryC18 with 20% acetonitrile in water, v/v, and a few breakthrough curves (B) recorded by UV at λ ) 225 nm. T ) 295 K. Note the quasilinear behavior of the isotherm, the slight convex downward curvature at high concentrations, and the absence of hump on the equilibrium plateau of the breakthrough curves, previously observed with phenol (Figures 3 and 8).

an adsorbed layer enriched in acetonitrile, located between the mobile phase and the former layer. The characteristics of this second layer depend on the acetonitrile concentration of the mobile phase and on the local concentration of the injected solutes. The formation of this multilayer adsorbed system explains the differences observed in the adsorption isotherms of the same compounds in the presence of these two organic modifiers. The complexity of the structure of the adsorbed phase explains also the unusual breakthrough curves of phenol in the presence of acetonitrile and particularly the elution of a stream with a concentration higher than that of the feed. This last phenomenon illustrates the difficulty in delimiting the structure of the liquidsolid interface in RPLC. It may have considerable importance in preparative chromatography because the high concentrations of feed compounds injected may perturb the structure of the interface between the pure bulk mobile phase and the hydrophobic C18 bonded layer and, hence, affect the distribution of the feed components between the two phases. As long as very small samples are used, the adsorption of the organic modifier does not introduce complications in linear chromatography. The structure of the interface is not perturbed by the presence of the analytes. Because the surface of conventional RPLC adsorbents 4270 Analytical Chemistry, Vol. 77, No. 13, July 1, 2005

Figure 14. Dependence of the retention time of minor disturbance peaks in the acetonitrile/water system on Discovery-C18 (A) and corresponding excess adsorption isotherm of acetonitrile (B) calculated from eq 7. The dashed line extrapolates the linear part of the isotherm and gives the intercept with the y-axis to determine the surface concentration of acetonitrile in the adsorbed layer of finite thickness τ.

is heterogeneous, the retention behavior of analytes “at infinite dilution” is essentially governed by the distribution of these analytes between the adsorption sites located within the C18 layer

Figure 15. Adsorption isotherm of acetonitrile from water on Discovery-C18 according to eq 8. In the calculation,. the finite layer thickness was τ )13.6 Å.

Figure 17. Concentration profiles of phenol at the solid-liquid interface of Discovery-C18, based on the adsorption data acquired with methanol (A, tri-Langmuir isotherm model) and acetonitrile (B, Langmuir-BET isotherm) as organic modifiers.

Figure 16. Schematic comparison of the adsorption mechanisms of a solute from aqueous solutions of methanol (A) and acetonitrile (B) onto a RPLC material. The different shadings represent the three different “phases” involved in the chromatographic system. From top to bottom, the bulk mobile phase (a water-rich solution), the adsorbed mono- or multilayer of organic modifier molecules (a phase rich in adsorbed organic modifier), and the C18-bonded phase. The analyte (phenol or caffeine) is represented by small ovals.

(the highest energy sites) and either the bulk mobile phase (methanol/water) or the pure organic modifier (e.g., acetonitrile) in the multilayer adsorbed system. This model explains why acetonitrile is always reputed to be a stronger eluent than

methanol, despite the often lower solubility of analytes in acetonitrile/water mixtures (e.g., 160 g/L in methanol/water, 30/70, v/v). However, when the concentrations are high and the low-energy sites located at the interface of the mobile phase and the C18 chains begin to fill, the adsorbed amount of analyte becomes larger with acetonitrile/water than with methanol/water mixtures. Because their curvatures are so different, the two isotherms cross. So, the statement that acetonitrile is a stronger eluent than methanol does not make sense at high concentrations. (See the adsorption isotherms of phenol and caffeine on Discovery.) Still little is known of the adsorption mechanisms in RPLC. We are of the opinion that the results of mere measurement of retention factors under linear conditions obfuscate the issues, mask the physical reality, and obscure important details of the chromatographic system because this lumps the consequences of different effects in one meaningless parameter. The solid silica surface is heterogeneous. The distribution of the bonded chains on the silica surface is heterogeneous. The coverage is neither dense nor complete. The structure of the hydrophobic bonded layer is heterogeneous. The structure of the liquid Analytical Chemistry, Vol. 77, No. 13, July 1, 2005

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interface between the layer of bonded chains and the mobile phase is heterogeneous. Because the amount adsorbed on these different features of the RPLC material varies with the mobilephase concentration in different ways in different concentration ranges, the characteristics of the equilibrium isotherms can be used to shed new light on these different aspects of the adsorption mechanism and on the properties of the actual commercial columns. ACKNOWLEDGMENT This work was supported in part by Grant CHE-02-44693 of the National Science Foundation, by Grant DE-F.G.05-88-ER-

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13869 of the U.S. Department of Energy, and by the cooperative agreement between the University of Tennessee and the Oak Ridge National Laboratory. We thank Hans Liliedahl and Lars Torstensson (Eka Nobel, Bohus, Sweden) for the generous gift of the Kromasil-C18 column used in this work.

Received for review February 8, 2005. Accepted April 13, 2005.

AC0580058