Heterogeneity of the Adsorption Mechanism of Low Molecular Weight

Jul 8, 2006 - The retention mechanism in RPLC mode was investigated based on the acquisition of adsorption isotherm data by frontal analysis measureme...
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Anal. Chem. 2006, 78, 5823-5834

Heterogeneity of the Adsorption Mechanism of Low Molecular Weight Compounds in Reversed-Phase Liquid Chromatography 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 production process of conventional C18-bonded silica phases inevitably leads to adsorbents having heterogeneous surfaces.1,2 At the molecular level, the surface of the bare silica contains segregated elemental impurities (e.g., Fe2+, Al3+, B) that concentrate at the surface. The bond strains of the silicate tetrahedra located at the silica surface is another source of surface heterogeneity. The surface of silica gel is usually composed of different types of silicon atoms that are connected to none

(referred as the Q4 species in solid-state NMR notations), one (Q3), or two (Q2) hydroxyl groups. Furthermore, the surface of the porous silica particles used in RPLC is usually derivatized through reaction with octadecyldimethylchlorosilane, to bind C18 chains to it. This derivatization reaction cannot be complete, and it leaves a population of isolated silanol groups that are well known for interacting strongly with basic compounds, an interaction that has the potential of causing a nefarious peak tailing. To minimize the density of these residual silanol groups, the derivatized surface is reacted with trimethylchlorosilane, a reaction known as endcapping. Despite this end-capping reaction, only half of the 8 µmol/ m2 silanol groups initially present on the silica gel surface react and bind with either the C18 or the C1 alkyl chains, as determined by elemental analysis.3 This is due to the volume of the bulky methyl groups bonded to the silicon atom of the reagent. Steric hindrance limits to 50 the fraction of the initial silanol groups that are eliminated. As a result, the surface of derivatized silica adsorbents consists of C1 chains, C18 chains, free silanol groups accessible to the mobile phases, and isolated silanol groups that are trapped under the alkyl chains and are inaccessible to the mobile phase and, hence, to the analytes. Finally, the structure of the alkyl-bonded phase itself is also heterogeneous as suggested by results of molecular dynamics simulations.4,5 It was shown that the C18 chains present both trans and gauche conformations, an observation already made from solid-state NMR experiments.6 Note, however, that these calculations did not involve any liquid phase but assumed vacuum at the solid interface, leaving the alkyl chains aggregated onto the silica surface while they may be solvated over it in certain ranges of mobile-phase compositions. In reversedphase chromatography, a polar mobile phase is usually employed to transport the solutes. This mobile phase is often an aqueous mixture containing an organic solvent such as methanol, acetonitrile, or tetrahydrofuran. The presence of the organic modifier is essential to ensure that the mobile phase wets the hydrophobic surface and penetrates into the mesopore structure of the silica particles, offering a large surface area for the adsorption of the analytes. Otherwise, the mobile phase is expelled from the pores

* Corresponding author. Fax: 865-974-2667. E-mail: [email protected]. (1) Neue, U. D. HPLC Columns. Theory, Technology, and Practice; Wiley-VCH: New York, 1997. (2) Neue, U. D. Silica gel and its derivatization. In Encyclopedia of Analytical Chemistry; Meyers, R. A., Ed.; John Wiley & Sons: Chichester, 2000.

(3) Gritti, F.; Guiochon, G. J. Chromatogr., A 2006, 1115, 142. (4) Lippa, K. A.; Sander, L. C.; Mountain, R. D. Anal. Chem. 2005, 77, 7852. (5) Sander, L. C.; Lippa, K. A.; Wise, S. A. Anal. Bioanal. Chem. 2005, 382, 646. (6) Pursch, M.; Sander, L. C.; Egelhaaf, H.-J.; Raitza, M.; Wise, S. A.; Oelkrug, D.; Albert, K. J. Am. Chem. Soc. 1999, 121, 3201.

The retention mechanism in RPLC mode was investigated based on the acquisition of adsorption isotherm data by frontal analysis measurements and their modeling. This work is a review of the results of four years of adsorption data measurements. The data were acquired on a wide variety of brands of C18-silica columns (from Akzo Nobel, Bishoff, Hypersil, Merck, Phenomenex, Supelco, Vydac, and Waters) with several low molecular weight compounds such as phenol (94 g/mol), caffeine (194 g/mol), tryptophan (204 g/mol), sodium 2-naphthalenesulfonate (235 g/mol), and propranololium chloride (295 g/mol). The mobile phase was a mixture of methanol and water at variable composition. The adsorption isotherms were all convex upward (langmuirian), and the degree of heterogeneity of the adsorption system was determined from the calculation of the adsorption energy distribution using the expectation-maximization method. The adsorption isotherm parameters (number of types of adsorption sites, surface concentration of each type of site, and difference between the adsorption energies Ei - Ej on sites i and j), obtained from the mathematical fit of the adsorption data to the appropriate multi-Langmuir adsorption isotherm model, were analyzed and compared. The results allow the drawing of general conclusions regarding the relationships between the size of the analyte and the adsorption properties (saturation capacities, adsorption energies) characterizing the retention mechanism in RPLC mode for neutral, anionic, and cationic compounds.

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and the retention of the compounds becomes negligibly small. To eliminate this problem, polar groups have been embedded in the bonded layer of the stationary phases (e.g., by binding such groups to some of the C18 chains), allowing the use of 100% water mobile phase.7 With classical end-capped C18-bonded phases, the strong hydrophobic character of the C18 chains leads to their folding on themselves, causing potential spaces to form between chains clusters, in which the mobile phase and analytes can penetrate. This is another source of surface heterogeneity for conventional C18-bonded silica phases. The heterogeneity of the solid-liquid interface in RPLC is beyond doubt based on the formulation of the packing material. Pemberton et al.8-12 studied the structure-function relationships on high-density octadecylsilane stationary phases by Raman spectroscopy, showed molecular pictures of the chromatographic interface, and displayed the structure of the alkyl chains and the interactions between them and low molecular weight aromatic compounds that are consistent with the heterogeneity of the hydrophobic interface. Unfortunately, the peak tailing observed in chromatography is still too often blamed only on strong interactions between isolated silanol groups and the molecules of analyte. While silanol groups do play a critical role in the retention of basic compounds and are at the origin of the tailing of their peaks, this is not so in many other cases, e.g., for neutral compounds or for anions for which the origin for skewed peak profiles must be found elsewhere. It has been demonstrated that the adsorption mechanism onto/into the hydrophobic layer is often heterogeneous and that the importance of this heterogeneity should not be neglected.13-15 The density of accessible isolated silanol groups on surfaces that have been end-capped is low. Access by analyte molecules to these groups is unlikely, due to their shielding by the bulky methyl groups bonded to the silicon atom holding the alkyl chains.3 If these silanol groups were inaccessible to the small molecules of the trimethylchlorosilane reagent in a good solvent (typically toluene), it is unlikely that larger solute molecules dissolved in a poor solvent of the alkyl groups could have close access to them. Admittedly, accessibilities for physisorption and for chemisorption are different. The success of the latter requires a closer distance of approach and a more specific orientation between the two reactive molecules than is required in the former case. Despite being inaccessible for reaction, some silanol groups can generate a sufficient attractive electrical field to adsorb sample molecules. Nevertheless, the interaction energy of phenol with the strongest sites found on the surface of RPLC materials seem to be too small to correspond to hydrogen-bonding interactions and cannot be associated with interactions with residual silanol groups because very similar adsorption isotherms were measured on (7) Layne, J. J. Chromatogr., A 2002, 957, 149. (8) Ho, M.; Pemberton, J. E. Anal. Chem. 1998, 70, 4915. (9) Pemberton, J. E.; M. Ho; Orendorff, C. J.; Ducey, M. W. J. Chromatogr., A 2001, 913, 243. (10) Ducey, M. W.; Orendorff, C. J.; Pemberton, J. E.; Sander, L. C. Anal. Chem. 2002, 74, 5576. (11) Ducey, M. W.; Orendorff, C. J.; Pemberton, J. E.; Sander, L. C. Anal. Chem. 2002, 74, 5585. (12) Orendorff, C. J.; Ducey, M. W.; Pemberton, J. E.; Sander, L. C. Anal. Chem. 2003, 75, 3369. (13) Gritti, F.; Guiochon, G. J. Chromatogr., A 2004, 1028, 75. (14) Gritti, F.; Guiochon, G. Anal. Chem. 2005, 77, 1020. (15) Gritti, F.; Guiochon, G. J. Chromatogr., A 2005, 1095, 27.

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different C18-silica materials (end-capped, non-end-capped, hybrid silica) on the surface of which the densities of silanol groups are widely different.13-15 In conclusion, the mechanism of the heterogeneous adsorption on C18-bonded phases in RPLC is still not well understood. The origin of peak tailing in chromatography needs clarification that can be brought only by the acquisition of more relevant data, their comparison, and their proper interpretation. Linear chromatography measurements16-19 consist in determining the distribution constants of analytes between the stationary and the mobile phases in the linear domain of their respective equilibrium isotherms from the retention data of infinitely diluted sample pulses. It has been shown that the retention factor k′ measured or the free energy of transfer of a compound from the polar mobile phase to the hydrophobic stationary phase (∆G) can be expressed as the sum of elementary free energy contributions (∆G ) ∑∆Gi), each of which corresponds to a particular type of interaction (e.g., hydrophobic, steric, donor, and acceptor hydrogen bonding, ion exchange). A large variety of chromatographic tests allow to characterize HPLC packings regarding each of those types of intermolecular interactions.20 Analyzing the set of retention factors of a large number of compounds having different chemical properties, it is possible to characterize the polarity, the hydrophobicity, the hydrogen-bonding character, and other properties of a chromatographic system characterized by a packing material and a solvent. Such studies, however, ignore completely the heterogeneous character of the adsorption mechanism because the retention factors measured are the sums of all the different contributions of each type of adsorption site present on the surface. These elementary free energy contributions may originate from different sites that are present in various proportions and that cannot be separated. The log k′ plot cannot be directly resolved into the sum of individual free energy contributions.22 No information is obtained regarding the relative abundance of these sites or the adsorption energies of the compounds studied on each of them. The goal of this paper is to shed some light on the structural heterogeneity of the C18 phases that are extensively used in the clinical, pharmaceutical, biological, and environmental fields. A large number of commercialized C18-bonded columns were tested regarding the adsorption behavior of a variety of low molecular weight compounds in water-rich mobile phases. The degree of heterogeneity of these packing materials was determined based on the parameters of their adsorption isotherms, measured by frontal analysis (FA), and of their adsorption energy distributions. The fit of the adsorption data to a general multiLangmuir isotherm model allows quantitative estimates of the saturation capacities and the adsorption energies of each adsorption site. (16) Carr, P. W. Microchem. J. 1993, 48, 4. (17) Snyder, L. R.; Dolan, J. W.; Carr, P. W. J. Chromatogr., A 2004, 1060, 77. (18) Wang, A.; Carr, P. W. J. Chromatogr., A 2002, 965, 3. (19) Wilson, N. S.; Nelson, M. D.; Dolan, J. W.; Snyder, L. R.; Wolcott, R. G.; Carr, P. W. J. Chromatogr., A 2002, 961, 171. (20) Neue, U. D.; Tran, K. V.; Iraneta, P. S.; Alden, B. A. J. Sep. Sci. 2003, 26, 1. (21) Guiochon, G.; Felinger, A.; Katti, A. M.; Shirazi, D. Fundamentals of Preparative and Nonlinear Chromatography, 2nd ed.; Elsevier: Amsterdam, The Netherlands, 2006. (22) Gritti, F.; Guiochon, G. J. Chromatogr., A 2005, 1099, 1.

THEORY Frontal Analysis Method. All the conclusions of this work depend on the adsorption isotherms derived from modeling of the accurate and precise data acquired by frontal analysis on the systems studied. The different methods available to measure adsorption isotherm data have been reviewed.21 We chose the frontal analysis method because it is the most accurate and the most precise although it is also the one that requires the largest amounts of chemicals and is the most time-consuming. It is based on the successive and independent injection of concentration plugs of increasing concentrations of the probe compounds studied and on the application of the mass conservation law. More details on this method are given in a recent review.22 Calculation of the Adsorption Energy Distribution. Heterogeneous surfaces can be considered as a tessellation of a very large number of homogeneous surfaces on each of which the isotherm is given by a Langmuir or the Jovanovic isotherm model. The adsorption constant on each homogeneous surface is different, and there is an adsorption energy distribution on the heterogeneous surface. So, when the isotherm data show that there are no adsorbate-adsorbate interactions and that these data can be modeled by a convex upward isotherm (type I isotherm in the van der Waals classification), it is possible to decompose the isotherm into a sum of local convex upward isotherms, all following Langmuir or Jovanovic isotherm model behavior. A mathematical program has been elaborated by Stanley et al.,23 which allows the derivation of the adsorption constant distribution from the raw adsorption data, without any model assumption. This method is called the expectation-maximization method (EM). The iteration process converges toward a unique deconvolution, provided that the adsorption data are measured in a sufficiently wide range of concentrations. This range must include the linear domain of the isotherm, so the Henry constant is accurately measured. This means that the breakthrough profile recorded for the lowest concentration plateau is symmetrical with a front and a rear part that have similar slopes. Second, it was demonstrated24 that breakthrough profiles must be recorded for sufficiently high concentrations, so that the low-energy sites become filled up to ∼40% of their saturation capacity. The calculation of the distribution of the adsorption energy is critical in the determination of the most appropriate isotherm model that may best describe the experimental adsorption data. The mere fit of the adsorption data to different isotherm models might lead to suggest the choice of an adsorption isotherm model that is inconsistent with the adsorption energy distribution (AED). Specific details concerning the calculation of the AED from the raw adsorption data collected in this work are given in a recent review.22 Adsorption Isotherm Models. The adsorption data (or amounts adsorbed, q*, versus mobile-phase concentration C) acquired with the alkyl-bonded silica adsorbents used in HPLC are all described by a multi-Langmuir adsorption isotherm model

biC

i)N

q* )

∑q i)1

S,i

1 + biC

(1)

where bi is the equilibrium constant of the solute between the liquid and the fraction i of the surface of the solid phase and qS,i

is its saturation capacity. N is the number of homogeneous patches identified on the surface and is characterized by distinct adsorption energies and adsorption constants. It is an index of the degree of heterogeneity of the adsorbent. Each single term in eq 1 corresponds to the adsorption isotherm on the corresponding type of adsorption sites. The determination of the number N of distinct adsorption sites is done from the results of the calculation of the AED. The isotherm parameters discussed in this work were obtained by minimizing the sum of the squared relative residuals between the experimental data and the values predicted by the selected adsorption model. EXPERIMENTAL SECTION Chemicals. The mobile phases used were are all aqueous solutions of methanol or acetonitrile. Their compositions depend on the probe compound studied. They were chosen to achieve a suitable retention factor. All solvents were of HPLC grade, purchased from Fisher Scientific (Fair Lawn, NJ). The mobile phases were systematically filtered before use, on a surfactantfree cellulose acetate filter membrane, 0.2-µm pore size (Suwannee, GA). Some of the mobile phases contained some supporting salts (KCl, NaCl) or buffers (acetate at pH 4.75, phthalate at pH 2.75, phosphate at pH 2.75, succinate at pH 4.16, formate at pH 3.75, and citrate at pH 3.14). The hold-up tracer used was thiourea, which gives fair estimates of the total porosity of the adsorbent. All the compounds studied here (phenol, caffeine, tryptophan, sodium naphthalenesulfonate, propranololium chloride, hydrochloride nortriptyline) were purchased from Aldrich (Milwaukee, WI). The structure, molecular weight, and van der Waals volume of these compounds are summarized in Figure 1. Chromatographic Columns. The columns used in this work are from several manufacturers and represent a large section of the market of modern RPLC packing materials. Seven brands of manufacturer-packed columns and one commercial monolithic column were used to acquire the data reported and discussed in this study. All these stationary phases were C18- or C30-bonded materials. All columns were end-capped, except three: the Resolve-C18 column and two “polymeric” non-end-capped packed columns (Vydac-C18, Prontosil-C30), for which a trichlorooctadecylsilane was used in the bonding process instead of a monochlorooctadecylsimethylsilane. The silica-bonded materials used as stationary phases were Ascentis and Discovery (Supelco, Bellefontaine, PA), Chromolith (Merck, Darmstedt, Germany), HyPurity Elite (Hypersil, Runcorn, UK), Kromasil (Akzo Nobel, Bohus, Sweden), Luna and Gemini (Phenomenex, Torrance, CA), Resolve, Sunfire, Symmetry, and XTerra (Waters, Milford, MA), Prontosil (Bischoff, Leonberg, Germany), and Vydac 218TP (Vydac, Hesperia, CA). The main characteristics of the bare porous silica and of the materials used for the production of these brands of columns are summarized in Table 1. The best adsorption isotherm parameters were all normalized to the surface of the neat silica material and to the number of C18 (or C30) ligands present in the column. The parameters derived from frontal analysis are usually reported in moles of adsorbate per liter of stationary phase (saturation capacities) and in liters of stationary phase per mole of adsorbate (equilibrium constants). (23) Stanley, B. J.; Bialkowski, S. E.; Marshall, D. B. Anal. Chem. 1993, 65, 259. (24) Gritti, F.; Guiochon, G. J. Chromatogr., A 2005, 1097, 98.

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FBP )

1 %BP 1 - %BP + 2.15 0.795

(5)

In all these calculations, the contribution of the end-capping reagent (TMS) to the overall carbon content of the stationary phase was neglected. To report the amount of adsorbate at saturation for each type of adsorption site in number of adsorbed molecules per bonded alkyl chains, one needs an estimate of the concentration of these chains per unit surface area of silica gel. The corresponding bonded units are -Si(CH3)2-(CH2)17-CH3 (MW ) 311) on the end-capped C18 materials, -O-Si-(CH2)3-NHCO-(CH2)12-CH3 (MW 312) on the polymeric amido embedded stationary phase used, and -O-Si-(CH2)29-CH3 (MW ) 465) on the polymeric C30 phase. If SC is the mass fraction of the carbon atoms in the bonded chains and Sp the specific surface area of the neat silica solid support, the surface coverage CAlkyl or number of moles of alkyl bonded chains per squared meter of silica surface is equal to

Calkyl ) Figure 1. Chemical structures, molecular weight (MW), and van der Waals volume (vdW) of the four compounds, phenol, caffeine, sodium naphthalenesulfonate, and propranololium chloride.

The transformation from one to another unit is given by

qS (mol/m2) )

qS (mol/m3) FBP (kg/m3) × %silica × Sp (m2/kg)

(2)

where qS is the saturation capacity, FBP is the density of the packing material, Sp is the specific surface area of the underivatized matrix, and % silica is the mass percentage of silica in the packing material. Similarly, for the equilibrium constant

b (m2/mol) ) b (m3/mol)FBP (kg/m3) × %silica × Sp (m2/kg) (3) FBP was estimated from the mass content of BP in the stationary phase (%BP, hence %silica ) 1 - %BP), the density of neat silica (Fsilica ) 2.15 kg/m3), and the density of the alkyl bonded layer (Falkyl), which was assumed to be equal to the density of liquid octadecane, e.g. 0.795 kg/m3. The mass fraction of the bonded chains in the stationary phase is calculated from its carbon content (% C), knowing the number of carbon (NC), oxygen (NO), hydrogen (NH), nitrogen (NN), and silicon atoms (NSi) in the structure of the bonded moiety. Accordingly,

%BP ) (NCMC + NOMO + NHMH + NSiMSi + NNMN) × %C MW ) % C (4) NCMC NCMC where MC, MO, MH, MSi, and MN are the atomic weights of carbon, oxygen, hydrogen, silicon, and nitrogen, respectively, and 5826

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%C (mol/m2) (SC - % C) × MW × Sp

(6)

The surface concentrations given in Table 1 were derived from the overall carbon content (% C, including the amount brought by the end-capping), the molecular weight of the alkyl ligand, MW, the carbon content of the bonded phase, SC, and the specific surface area, Sp, of the underivatized matrix. These data were provided by the certificate of analysis supplied by the manufacturer and accompanying the chromatographic column. As a result, all the values given are slight overestimates of the true surface coverage. In addition, significant errors are made in cases in which the packing materials are hybrid organic silica materials (i.e., XTerra-C18 and Gemini-C18). The exact composition of their matrix is unknown. Finally, the results of the calculation made with eq 6 might be different from the values given by manufacturers because the latter use proprietary formulas. Since we compare a large series of RPLC columns in this work, we elected to provide consistent values of the surface coverages calculated with a unique equation (eq 6). The ratio of the number of moles of analyte occupying the lowest adsorption energy site i at saturation to the number of alkyl bonded chains is calculated as

N1 qS,1 ) NC18 Calkyl

(7)

Apparatus. The breakthrough curves and the overloaded band profiles of all the compounds studied were acquired using a Hewlett-Packard (Palo Alto, CA) HP 1090 liquid chromatograph. This instrument includes a multisolvent delivery system (three tank bottles, volume 1 L each), an autosampler with a 250-µL sample loop, a column thermostat, a diode-array UV detector, 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

Table 1. Physicochemical Properties of the Alkyl-Bonded Silica Columns column Waters C18-Resolve C18-Sunfire C18-Symmetry C18-Xterra Phenomenex C18-Luna C18-Gemini Supelco C18-Discovery C18-Ascentis Akzo Nobel C18-Kromasil Merck C18-Chromolith Vydac C18-Vydac Hypersil C18-Hypersil Bishoff C30-Prontosil c d

dimension (mm × mm)

particle size (µm)

mesopore size (Å)

specific surface (m2/g)

bonding process

%C

% silica

FBPa (g/cm3)

150 × 4.6 150 × 4.6 150 × 3.9 150 × 3.9

5 5 5 5

90 90 90 121

200 349 346 176

monomeric monomeric monomeric monomeric

10.2 17.5 19.5 15.2c

86.8 77.3 74.6 80.3

1.75 1.55 1.50 1.61

2.45 2.71 3.16 2.48

150 × 4.6 150 × 4.6

5 5

100 110

420 375

monomeric monomeric

18.2 14.0c

76.4 81.9

1.53 1.64

2.36 99%) of the compounds used. Two neutral compounds (phenol, caffeine), a zwitterionic compound (tryptophan), two cationic compounds (propranolol, nortriptyline), and one anionic compound (naphthalenesulfonate) were studied. The measurements were made under similar experimental conditions. The results presented here consolidate the adsorption data accumulated during the last four years in our laboratory and which were acquired to investigate specific issues, such as the effects of the temperature, the pressure, the mobile-phase composition (ionic strength, nature of buffer, pH, etc.), and the phase endcapping, on the adsorption behavior of these compounds and their retention mechanisms. They will now be brought together to demonstrate the heterogeneous character of the adsorption mechanism on RPLC packing materials and to quantify the degree of heterogeneity of each phase. Retention Mechanism of Phenol. Phenol was initially chosen for our systematic investigations because it has an exceptionally high solubility in methanol-water solutions. This solubility exceeds 200 g/L for solutions containing up to 70% water (v/v). Such high mobile-phase concentrations allow that FA measureAnalytical Chemistry, Vol. 78, No. 16, August 15, 2006

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Table 2. Adsorption Isotherm Parameters of Phenol on Various Brands of Alkyl-Bonded Stationary Phases column

ref

H2O (v/v %)

CH3OH (v/v,%)

N1/NC18

f1/2

C18-Symmetry

30 28 31 32 28 27 27 27 25 13 25 28 33, 34 29 35 28 35 13 28 26 28

70 70 70 70 70 70 55 40 60 75 70 70 85 70 70 70 70 70 70 70 70

30 30 30 30 30 30 45 60 40 25 30 30 15 30 30 30 30 30 30 30 30

1.6 1.2 1.1 1.1 1.1 1.2 1.0 1.1 1.1 2.7 14.0 1.6 2.1 1.0 1.6 1.2 1.2 1.9 1.6 4.5 0.9

3.1 2.6 3.3 3.3 2.2 2.1 4.0 7.0 4.5 2.9 14.3 2.6 2.2 2.3 2.0 1.6 2.7 3.1 2.0 5.1 2.0

C18-Kromasil

C18-Resolve C18-Discovery C18-Chromolith C18-Ascentis C18-Gemini C18-Luna C18-Sunfire C18-XTerra C18-Hypersil C30-Prontosil C18-Vydac

ments be carried out under such conditions that the adsorbed concentrations become close to the monolayer saturation capacity of the column. Then, the very good precision of the data permits precise estimates of the adsorption energy on the low adsorption energy sites that are the most abundant sites on the surface. The results are given in Table 2. This table gives the ratio of the numbers of molecules of phenol adsorbed at saturation of the adsorption sites of type 1 (lowest adsorption energy and most numerous sites in the column) to the number of alkyl-bonded chains, N1/NC18. It provides the ratio of the numbers of adsorption sites of types 1 and 2 (highest or intermediate adsorption energy), f1/2, and the ratio of the numbers of sites of types 2 and 3 (highest adsorption energy), f2/3. It also contains the difference between the adsorption energies on sites of types 2 and 1, E2 - E1, and the difference between the adsorption energies on sites of types 3 and 2, E3 - E2. It should be noted that N1/NC18 is between 1 and 2 on all the C18-bonded columns studied, except for the Discovery column, which has a much larger saturation capacity than all others.25 This result is surprising because this column has otherwise nearly the same physicochemical properties as the Hypersil column (See Table 1.). A possible explanation could be related to adsorbateadsorbate interactions beginning to take place at high solute concentrations and leading the program to give an erroneous value of the saturation capacity through an incorrect extrapolation of the FA data for phenol. However, the isotherm curvature remains convex upward in the whole range of concentrations studied, and phenol-phenol interactions take place in the adsorbed phase only at organic modifier concentrations that are much lower than the 30% methanol (v/v) used in the mobile phase. The C30-Prontosil column also has a larger value, but this might be explained by the longer alkyl chain, which provides more adsorption sites.26 Except for the Discovery column, all results are similar. Phenol is adsorbed on two distinct types of adsorption sites, 1 and 2, which (25) Gritti, F.; Guiochon, G. Anal. Chem. 2005, 77, 4257. (26) Gritti, F.; Guiochon, G. J. Chromatogr., A 2006, 1103, 43.

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f2/3

13.9 3.8

340

E2 - E1 (kJ/mol)

E 3 - E2 (kJ/mol)

6.9 6.0 4.1 4.6 5.3 5.7 3.9 7.0 4.5 6.5 9.3 5.5 6.5 5.7 5.0 5.6 5.0 7.7 5.9 7.1 4.7

3.6 2.7

8.7

are in the proportion of 2:3 to 1:1 in methanol-water mixtures (70/30, v/v). It is noteworthy that this ratio decreases with increasing concentration of the organic modifier in the mobile phase until quasi-homogeneous adsorption behavior is observed at high methanol concentrations.27 The physical origin of these different types of sites remains unknown. Based on chromatographic measurements alone, it is obvious that the high-energy sites (type 2 sites) involve neither free nor isolated silanol groups (through hydrogen-bonding or electrostatic interactions).13 The difference between the adsorption energies on the sites of types 2 and 1 is ∼5 kJ/mol, which is consistent with two adsorption sites involving dispersive interactions between phenol and the C18-bonded layer. Furthermore, free silanol groups are preferentially solvated by either water or methanol molecules that are present in a large excess, and most isolated silanol groups are unaccessible to phenol molecules because of steric hindrance.3 It is highly unlikely that phenol interacts with free silanol groups because the adsorption isotherms measured for this compound on end-capped and non-end-capped C18 are identical.13,14 It does not interact with isolated silanol groups either because the steric hindrance of these groups is too important. After the completion of the end-capping of the C18 bonded silica, the average distance between two dimethylalkylsilane groups is 5 Å and the size of these groups limits drastically the access of phenol molecules to the underlying silica surface, either for chemisorption or even for physisorption. Both types of adsorption sites certainly involve hydrophobic, dispersive interactions between the alkyl chains and the molecules of the analyte. The molecules of phenol are probably adsorbed in or on different regions of the hydrophobic layer. The low-energy sites could be located at the interface between the mobile phase and the alkyl layer, which provides the lowest surface area of contact of the analyte molecule and the surface, while the high adsorption energy sites could be located deeper inside the bonded alkyl layer, which would provide a larger surface area of contact with the analyte (27) Gritti, F.; Guiochon, G. J. Chromatogr., A 2003, 995, 37.

Table 3. Adsorption Isotherm Parameters of Caffeine on Various Brands of Alkyl Bonded Stationary Phases column

ref

H2O (v/v %)

CH3OH (v/v %)

N1/NC18

f1/2

C18 -Symmetry

36 28 31 32 28 36 13, 14 25 28 33, 34 29 35 28 35 13 28 26 28

70 70 70 70 70 70 75 70 70 85 70 70 70 70 70 70 70 70

30 30 30 30 30 30 25 30 30 15 30 30 30 30 30 30 30 30

0.62 0.61 0.70 0.71 0.66 1.03 1.09 2.5 0.84 0.92 0.59 0.87 0.82 0.67 0.77 0.87 1.22 0.50

23.4 22.5 32.6 26.2 17.8 8.4 7.4 100 20.5 5.8 12.1 13.8 16.1 10.9 9.9 14.9 9.6 16.3

C18-Kromasil C18-Resolve C18-Discovery C18-Chromolith C18 -Ascentis C18-Gemini C18-Luna C18-Sunfire C18-XTerra C18-Hypersil C30 -Prontosil C18-Vydac

molecules. Other characterization techniques are needed to progress in the identification of the adsorption sites. Retention Mechanism of Caffeine. Caffeine was chosen as another probe because it has a larger molecular size than phenol, although it is less soluble in methanol-water mixtures ( 20 kJ/mol

(2) Application to Linear Chromatography. The relatively low density of the high-energy sites of types 2, 3, or 4 compared to the relatively high density of the low-energy sites of type 1 could suggest that their practical contribution to chromatographic results under analytical conditions is negligible. This would be incorrect. Each of these sites contributes to the retention factor. This contribution is characterized by the corresponding value of the Henry constant Hi ) qS,ibi (see eq 1, k′/F is the limit of q*/C when C tends toward 0, F being the phase ratio). Because low values of the saturation capacity of the high-energy sites are associated with high values of the equilibrium constant, the contributions to the Henry constant of the different types of sites tend to be of comparable size. If we assume that the preexponential factor b0 of the equilibrium constants is independent of the adsorption energy, Ei, the equilibrium constants bi and b1 of two different types of sites, i and 1, are related by

bi ) b1 exp

(

)

Ei - E1 RT

(8)

The corresponding Henry constants are related by

(

)

Hi qS,i Ei - E1 ) exp H1 qS,1 RT

(9)

From the experimental data measured by FA and their modeling, as discussed earlier, we can reasonably estimate that, on the average, qS,1 ≈ 3 µmol/m2, qS,2 ≈ 0.1 µmol/m2, qS,3 ≈ 0.01 µmol/m2, and qS,4 ≈ 0.1 nmol/m2 and E1 ≈ 3 kJ/mol, E2 ≈ 10 kJ/mol, E3 ≈ 15 kJ/mol, and E4 ≈ 25 kJ/mol. Let us arbitrarily normalize the contribution H1 to 1. Accordingly, H2/H1, H3/H1, and H4/H1 are equal to 0.56, 0.42, and 0.24, respectively. Clearly, the contributions to retention of the adsorption sites of types 1, 2, 3, and 4 (when the latter two exist) are comparable. Adsorption sites that have very low densities, densities that are several orders of magnitude lower than that of the low-energy sites of type 1, may affect the retention factor to nearly the same degree as the most abundant sites. This is why it is so important to understand the exact origin of these high-energy adsorption sites, so that the manufacturers of packing materials could learn to manipulate the experimental conditions of their preparation to achieve the production of more homogeneous surfaces. Finally, the occurrence of a strongly tailing profile for the peaks of very low sample sizes of certain compounds should not come as a surprise. Especially with highly efficient (N > 5000), modern

columns and for low molecular weight compounds, it is highly unlikely that the explanation for this tailing be found in the slow adsorption-desorption kinetics of the compound. In contrast, these nefarious tailings are well explained by the heterogeneous adsorption mechanism that affects certain compounds on RPLC packing materials. The compounds for which there is a type of adsorption site with a high adsorption energy and a low saturation capacity will exhibit significant tailing at low concentrations. Under conditions of fast mass-transfer kinetics, thermodynamics account for significant tailing as soon as the sample size reaches the range of a few percent of the saturation capacity of the column. We have in this work shown how the same stationary phases may offer for different compounds two, three, or four different types of adsorption sites. CONCLUSION Our accumulation of systematic measurements of accurate adsorption isotherm data by frontal analysis during these last four years allows the drawing of important conclusions regarding the adsorption mechanism of low molecular weight compounds in RPLC. We definitively established that the adsorption mechanism of most compounds on the surface of conventional C18-bonded silica materials is heterogeneous. This effect results from the bonded layer structure, the silica surface heterogeneity, and the randomness of the distribution of the alkyl C18 chains and of the end-capping groups. It is consistent with the results of independent NMR investigations,6 although it is not yet certain that the two techniques examine the same chemical environment. NMR observes the dynamic motion of the bonded alkyl chains while chromatography assesses the interaction energy between analyte molecules and alkyl chains. It is often considered that the equilibrium constant measured in linear chromatography expresses the distribution of the analyte between two immiscible phases, the liquid solvent and the solid adsorbent. This vision of a solid-liquid equilibrium system does not apply to RPLC systems. The analyte actually distributes between two liquid phases, the mobile phase and the bonded alkyl layer in equilibrium with the mobile phase. The composition of the mobile phase is more or less strongly different at the interface with the bonded layer and in the bulk, as shown in the case of an acetonitrile-water mobile phase.25 Finally, the organic modifier penetrates into the bonded layer. All these effects arise from the bonded alkyl chains not forming a solid layer. By its very nature, the bonded layer is heterogeneous. Our results show that an analyte can interact with the layer in several different ways, up to four in this work, but more could certainly be possible in other cases. This complexity of the adsorption mechanism is due to the structure of the bonded layer, a structure that is experienced differently by different analytes. It is not possible to relate the sites behaving in a certain way toward different compounds. The heterogeneity of RPLC adsorbents is not unique, and it does not remain the same for all compounds. The heterogeneous character of the adsorption mechanism results from the structure of the bonded layer, but its experimental consequences depend on the nature of the sample compound. For example, in the system made of the non-endcapped C18-Resolve and an aqueous solution of methanol having the same composition, the AED of phenol is bimodal, that of caffeine tetramodal. The size, the charge, and even the presence Analytical Chemistry, Vol. 78, No. 16, August 15, 2006

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of specific chemical groups of the analyte molecule affect the degree of heterogeneity of the adsorption. Conversely, embedding a polar group among the C18 chains changes the heterogeneity of the system for certain solutes.29 The only general conclusions at this time are that the adsorption mechanism is heterogeneous and that different analytes experience this heterogeneity differently. Yet, it is important to observe that the higher the adsorption energy on a certain type of adsorption sites, the lower the density of these sites. The exact physical nature of these different adsorption sites remains unknown, and they might very well be impossible to describe otherwise than in statistical terms. It is remarkable, however, that these different sites are definitely distinct. The distribution of the adsorption energy on the surface is not a continuous function. It is composed of several narrow modes separated by wide energy ranges in which no adsorption site is found. Only certain, specific values of the adsorption energy are allowed. Finally, the experimental results gathered in this work should encourage those trying to understand retention mechanisms in RPLC to see beyond the classical extrathermondynamic relationship.16-20,47-51 For a given column, one might encounter several types of adsorption sites with distinct adsorption energies. The extrathermodynamic LSER interpretation of retention data ignores (47) Croes, K.; Steffens, A.; Marchand, D. H.; Snyder, L. R. J. Chromatogr., A 2005, 1098, 123. (48) Marchand, D. H.; Croes, K.; Dolan, J. W.; Snyder, L. R.; Henry, R. A.; Kallury, K. M. R.; Waite, S.; Carr, P. W. J. Chromatogr., A 2005, 1062, 65. (49) Dolan, J. W.; Snyder, L. R.; Jupille, T. H.; Wilson, N. S. J. Chromatogr., A 2002, 960, 51. (50) Wilson, N. S.; Dolan, J. W.; Snyder, L. R.; Carr, P. W.; Sander, L. C. J. Chromatogr., A 2002, 961, 217. (51) Neue, U. D.; Tran, K. V.; Me´ndez, A.; Carr, P. W. J. Chromatogr., A 2005, 1063, 35.

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the fact that a fraction of the adsorption sites are stronger than the others and that distinct equilibrium constants Ki have to be defined instead. Interestingly, extrathermodynamics relationships coupled with the adsorption energy distribution function of the chromatographic system would give more realistic information. To avoid that the number of unknown parameters becomes exceedingly large, it could be reduced by assuming that the different adsorption sites differ in only one extrathermodynamical term (such as the hydrophobic term between sites 3, 2, and 1 or the hydrogen-bond term between sites 4 and the others). ACKNOWLEDGMENT This work was supported in part by grant CHE-02-44693 of the National Science Foundation, by Grant DE-FG05-88-ER-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 H. Liliedahl and L. Torstensson (Eka Nobel, Bohus, Sweden) for the generous gift of the Kromasil-C18 columns, U. D. Neue and M. Kele (Waters, Milford, MA) for the gift of the Symmetry-C18, XTerra-C18, SunfireC18, and Resolve-C18 columns, T. Farkas (Phenomenex, Torrance, CA) for the gift of the Luna-C18 and Gemini-C18 columns, K. Sinz, K. Cabrera, and D. Lubda (Merck KGaA, Darmstadt, Germany) for the gift of the Chromolith-C18 columns, and M. Sarker (Supelco, Bellefonte, CA) for the gift of the Discovery-C18 and Ascentis-C18 columns used in this work.

Received for review March 2, 2006. Accepted June 7, 2006. AC060392D