A chromatographic estimate of the degree of surface heterogeneity of RPLC packing materials

Journal of Chromatography A, 1103 (2006) 69–82

A chromatographic estimate of the degree of surface heterogeneity of RPLC packing materials III. Endcapped amido-embedded reversed phase Fabrice Gritti a,b , Georges Guiochon a,b,∗ a

b

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

Received 15 August 2005; received in revised form 16 November 2005; accepted 21 November 2005 Available online 15 December 2005

Abstract The difference in adsorption behavior between a conventional monomeric endcapped C18 stationary phase (3.43 ␮mol/m2 ) and an endcapped polymeric RP-Amide phase (3.31 ␮mol/m2 ) was investigated. The adsorption isotherms of four compounds (phenol, caffeine, sodium 2-naphthalene sulfonate, and propranololium chloride) were measured by frontal analysis (FA) and the degree of heterogeneity of each phase for each solute was characterized by their adsorption energy distributions (AED), derived using the Expectation–Maximization method. The results show that only certain analytes (phenol and 2-naphthalene sulfonate) are sensitive to the presence of the polar embedded amide groups within the RP phase. Their binding constants on the amide-bonded phase are significantly higher than on conventional RPLC phases. Furthermore, an additional type of adsorption sites was observed for these two compounds. However, these sites having a low density, their presence does not affect much the retention factors of the two analytes. On the other hand, the adsorption behavior of the other two analytes (caffeine and propranololium chloride) is almost unaffected by the presence of the amide group in the bonded layer. Strong selective interactions may explain these observations. For example, hydrogen-bond interactions between an analyte (e.g., phenol or naphthalene sulfonate) and the carbonyl group (acceptor) or the nitrogen (donor) of the amido-embedded group may take place. No such interactions may take place with either caffeine or the cation propranololium chloride. This study confirms the hypothesis that analytes have ready access to locations deep inside the bonded layer, where the amide groups are present. © 2005 Elsevier B.V. All rights reserved. Keywords: Liquid chromatography; Column heterogeneity; Adsorption energy distribution; Chromatographic test; Adsorption isotherm; Frontal analysis; C18 -Bonded phase; Polar embedded RP phase; Multi-Langmuir isotherm; Phenol; Caffeine; Sodium naphthalene sulfonate; Propranololium chloride

1. Introduction The study of reversed-phase packing materials by methods initially developed to investigate nonlinear chromatography has recently brought new and interesting insights on the properties of the recent, high-performing RPLC columns [1] complementing previous understandings [2,3]. These results may have important consequences in many applied fields, including in pharmaceutical, biological, and food analysis, which strive for more rapid and more efficient sample analysis, separation, and/or purification by RPLC. The adsorption behavior of low molecular weight compounds (e.g., phenol and caffeine) that are highly soluble in the

∗

Corresponding author. Tel.: +1 865 974 0733; fax: +1 865 974 2667. E-mail address: [email protected] (G. Guiochon).

0021-9673/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2005.11.089

conventional mobile phases used in RPLC (aqueous solutions of methanol or acetonitrile), demonstrated the intrinsic heterogeneous character of chemically bonded alkyl-silicas [4]. The conventional thinking that the surface of RPLC packing materials is homogeneous seems a delusion. Treating them as such is not helpful to understand retention mechanisms in RPLC. The advantage of using methods of study that involve nonlinear chromatography arises from the fact that they provide information on the whole set of adsorption sites and allow, to some extent, if not the identification of most of their types, at least the measurement of their binding energies and their saturation capacities [1]. This is because the adsorption sites with the highest affinity for the solute fill first and, conversely, those with the lowest affinity fill last. In contrast, analytical, linear chromatography provides merely the retention factor at infinite dilution, which is the sum of the contributions of all the sites.

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Earlier results showed that the surfaces of RPLC packing materials are heterogeneous [1]. They are covered with sites belonging to different types. Low molecular weight compounds can recognize a maximum of four different types of adsorption sites [1,5]. In this work, we consider as small compounds having a molecular weight below 300 g/L and containing a maximum of three fused rings. The relative abundance of these sites varies considerably, their densities increasing typically by a factor 1 × 104 from the highest to the lowest energy types of sites. Thus, C18 -bonded silica adsorbents appear to have a fundamentally heterogeneous behavior regarding the adsorption of small analytes, probably because these compounds may penetrate deep inside the C18 -bonded layer and, accordingly, experience different micro-environments. This hypothesis was suggested a long time ago, based on results obtained in linear chromatography, regarding the retention behavior of series of homologous analytes with a wide range of alkyl chain length [6,7]. The most surprising conclusion of this work, however, is that the adsorption energy distribution is not continuous but consists of a series of discrete, well-resolved adsorption energy modes [8]. The differences between the adsorption energies measured is rather low (around 5 kJ/mol), however, excluding the involvement of ionic, hydrogen, or even strongly polar interactions. Nevertheless, they are sufficiently large to affect considerably the behavior of the columns under overloading conditions [9,10]. The fact that the molecules of analytes can penetrate inside the bonded layer of alkyl chains was previously demonstrated by studies of the adsorption energy distributions of four small compounds (phenol, caffeine, sodium naphthalene sulfonate, and propranololium chloride) on endcapped (GeminiC18 and Sunfire-C18 [11]) and non-endcapped (Prontosil-C30 [12] and Resolve C18 [13]) packing materials. Independently, this hypothesis was strongly suggested to account for the selective properties of alkyl bonded materials in linear chromatography [14]. There are more types of adsorption sites on a C30 bonded silica than on a C18 bonded phase, at least in part because the volume of the bonded phase is larger with C30 tethered chains and additional micro-environment may be compared to those in shorter C18 chains. For instance, the number of molecules of phenol adsorbed per alkyl bonded chain at saturation of the monolayer is about 5 with a C30 -bonded column and about 2 with regular C18 -bonded phases [12]. The new sites that are observed with the C30 column are not related to the absence of endcapping on this phase. As demonstrated in a previous work [13], the adsorption isotherm of phenol on endcapped and non-endcapped C18 columns is nearly identical. On the other hand, the absence of endcapping leads also to the appearance of new types of adsorption sites that are not necessarily related to ionic interactions between free silanols and the analytes. Probably, these new sites arise from the free space close to the surface where the analyte can be utterly embedded deep inside the bonded layer. This was observed with the neutral compound caffeine [13]. Obviously, with positively ionizable compounds like propranololium chloride, strong ion exchange interactions occur with negatively charged silanol groups. This was observed on the C30 phase [12]. This effect was even worse with non endcapped C18 where the

ionic compound is so strongly retained that it takes too long to elute it. Hence, the length of the alkyl chain as well as the degree of endcapping of the derivatized surface of silica plays a critical role on the degree of heterogeneity of the stationary phase because the liquid phase can access between the bonded chains and can help transport the analyte down to the silica surface. The goal of this work is to confirm the hypothesis of the penetration of solute molecules into the bonded alkyl layer by measuring the equilibrium isotherms of our four probe compounds on two different stationary phases and comparing the results. Our results permit also a generalization of former conclusions regarding the surface heterogeneity of actual chromatographic columns. Both adsorbents used were prepared with the same supporting silica. The first is a classical C18 -bonded RPLC material (Ascentis-C18 , Supelco), the other is a polar embedded phase containing bonded amido groups inside the bonded alkyl layer (Ascentis-RP-Amide). These polar embedded reversed phase have not been much studied yet in linear chromatography although they offer the possibility of using highly aqueous mobile phases [15–20]. The problems associated with the de-wetting of the surface by the liquid phase are avoided when a polar group is incorporated within the bonded chain or when the surface is endcapped with a polar group. The hypothesis motivating this work was that, if molecules can penetrate inside the bonded layer, some compounds should interact differently with layers made of C18 chains only and with those containing also bonded amido-alkyl chains. The four polar probes used were the same as those used in previous studies [11,12]. These differences in interactions should result in different adsorption isotherms and adsorption energy distributions. The adsorption data were acquired by frontal analysis (FA). 2. Theory 2.1. Determination of single-component isotherm data by frontal analysis The adsorption data of phenol, caffeine, sodium naphthalene sulfonate, and propra-nololium chloride were acquired by the frontal analysis method (FA), as explained previously [1]. The amount of compound adsorbed per unit volume of adsorbent is given by q∗ = Fv C

tF − t0 − text VC − F v t 0

(1)

where C is the equilibrium concentration in the bulk, tF is the elution time of the front shock, t0 the hold-up time of the column, text the extra-column time necessary for the mobile phase to percolate through the different connections between the mixer and the detector cell, VC the volume of the stainless steel tube, and Fv the flow rate applied during the FA runs. Because we have shown elsewhere [21] that thiourea is definitely retained on C18 bonded phases, we cannot consider that a measurement of the hold-up time derived from the elution time of thiourea gives a good estimate of the column hold-up volume.

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Table 1 Physico-chemical properties of the RP-C18 and RP-Amide Ascentis columns provided by the manufacturers (Supelco)

C18 -Ascentis (Supelco) RP-Amide Ascentis (Supelco) a b

Column dimension (mm × mm)

Particle size (␮m)

Mesopore ˚ size (A)

Specific surface (m2 /g)

Bonding process

Carbon content (%)

Surfacea coverage (␮mol/m2 )n.a.

Total porosityb

End-capping

150 × 4.6 150 × 4.6

5 5

100 100

450 450

Monomeric Polymeric

25 19.5

3.43 3.31

1.415 1.491

Yes Yes

n.a. = non available information from the manufacturer (but estimated from the specific surface and the carbon content). Estimated from pycnometric measurements (MeOH/CH2 Cl2 ).

The elution of thiourea always gives an overestimate of the true column hold-up volume, as shown below. A better estimate of the hold-up time was obtained from pycnometric measurements (see Table 1). The two solvents used (dichloromethane, ρCH2 Cl2 = 1.326 g/cm3 and methanol, ρMeOH = 0.791 g/cm3 ) were chosen for their significant difference in density. Both solvents spontaneously wet the surfaces of the Ascentis-C18 and RPAmide-C18 adsorbents. The values of the hold-up times of these two columns were 1.415 ± 0.001 min and 1.491 ± 0.001 min, respectively. Note that the estimate of the hold-up time from the elution time of the (slightly retained) marker thiourea is overestimated, at 1.458 min (+3%) and 1.595 min (+7%), respectively, due to the slight but non negligible retention of this compound.

ear regression analysis (MLRA), allows the selection of the best adsorption isotherm model. More detailed information are available in reference [1].

2.2. Calculation of the adsorption energy distributions (AEDs)

3.1. Chemicals

The AEDs of phenol, caffeine, sodium naphthalene sulfonate, and propranololium chloride were calculated using a program elaborated by Stanley et al. [22], based on the Expectation–Maximization method. This approach makes no assumption on the nature of the AED nor of the overall isotherm. The input of the program are: (1) the raw adsorption data, e.g., the N data points (q* versus C). (2) The minimum and the maximum values of the equilibrium constant, bmin and bmax , estimated from the highest and the lowest concentrations of the plateaus injected during the series of FA runs (Cmax and Cmin , respectively). In this study we systematically apply the following relationships: bmin =

1 10Cmax

(2)

and bmax =

1 Cmin

(3)

(3) the number of points M in the energy grid. The program converges after a certain number of iterations (typically around 1–10 million iterations, depending on the precision of the data and the highest concentration injected) and delivers the distribution of the equilibrium constant (q = f(ln b[εa ])). 2.3. From the isotherm data to the isotherm model The combination of the AED and of the results of the fitting of the adsorption data to isotherm models, using the multilin-

2.4. Determination of the molecular volumes of the analytes The molecular volumes of the different analytes used in this work were generated by the SYBYL7.0 molecular modeling package, as described previously [12]. The molecular volume of phenol, 2-naphthalene sulfonate sodium, caffeine, and pro˚ 3, pranololium chloride were 82.1, 128.8, 145.9, and 263.2 A respectively. 3. Experimental

The chemicals used in this work were the same as those mentioned previously [12]. The mobile phase was a mixture of methanol and water (30:70, v/v) that contained also 25 mM sodium chloride for the study of the two ionizable compounds. Phenol, caffeine, sodium naphthalene sulfonate, and propranololium chloride were the four compounds tested. 3.2. Materials Two columns were used in this work. One is a classical monomeric, endcapped C18 -bonded silica, the other a polymeric, end-capped, C18 -bonded material with embedded amido groups. In this latter column, each bonded chain contains one peptide group, NH CO , located between the carbon atoms number 3 and 6 of a classical C18 -bonded chain, the peptide group replacing the carbon atoms number 4 and 5 (numbering starting from the silica surface). These two columns (both 150 mm × 4.6 mm) were the Ascentis-C18 and the Ascentis-RPAmide columns. They were gifts from Supelco (Bellefonte, PA, USA). Both materials were prepared with the same porous silica. The main characteristics of the bare porous silica and of the bonding material used are summarized in Table 1, according to the manufacturer. The structure of the bonded moiety given by the manufacturer is 0-Si (CH2 )3 NH CO (CH2 )12 CH3 , M = 312 g/mol. The bonding of the RP-Amide phase is achieved by reaction of a trifunctional silane, making the bonded phase polymeric. The other stationary phase studied is prepared by reaction with a monomeric silane fixing the group Si(CH3 )2 (CH2 )17 CH3 , MW = 311 g/mol, to the silica

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Fig. 1. Adsorption data of phenol, caffeine, 2-naphthalene sulfonate and pro-pranololium chloride on the Ascentis-C18 and Ascentis-RP-Amide columns measured by frontal analysis. The mobile phase is a mixture of methanol and water (30/70, v/v). 25 mM sodium chloride are added to elute the ionizable compounds (2-naphthalene sulfonate and propranololium chloride). T = 295 K. (A) 0–160 g/L range, (B) 0–5 g/L range, (C) 0–0.5 g/L range, (D) 0–0.05 g/L range.

surface. The surface coverages of the bonded chains were calculated according to the following relationship, with the surface coverage given in ␮mol of bonded moiety per m2 of neat silica: Surface coverage =

%C × 106 (SC − %C)MWSp

(4)

where %C is the carbon content of the stationary phase, SC is the mass fraction (%) of the carbon atoms in the bonded chain, and Sp the specific surface of the bare silica. SC = 61.5 and 77.1%, %C = 19.5 and 25.0%, and MW = 312 and 311 g/mol for the RPAmide and the RP-C18 phases, respectively. Sp is the same for both adsorbents, e.g. 450 m2 /g. The surface coverages of the RP-Amide and the RP-C18 packing materials are 3.31 and 3.43 mumol/m2 , respectively. Accordingly, it makes sense to compare directly the adsorption behavior of analytes on these two phases. The differences observed are essentially related to the presence of the embedded amido group that replaces two methylene groups.

3.3. Apparatus The breakthrough curves of the chemicals were acquired using a Hewlett-Packard (Palo Alto, CA, USA) HP 1090 liquid chromatograph. This instrument includes a multi-solvent delivery system (three tanks, volume 1 L each), an auto-sampler with a 250 ␮L sample loop, a column thermostat, a diode-array UVdetector, and a data station. Compressed nitrogen and helium bottles (National Welders, Charlotte, NC, USA) are connected to the instrument to allow the continuous operations of the pump, the auto-sampler, and the solvent sparging. The extra-column volumes are 0.044 and 0.840 mL, as measured from the autosampler and from the pump system, respectively, to the column inlet. All the retention data were corrected for this hold-up contribution. 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 ␮L/min at flow rates around 1 mL/min. All measurements were carried out at a constant temperature of 22 ◦ C, fixed by the laboratory

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Table 2 Best isotherm parameters accounted for by the adsorption of phenol, caffeine, naphthalene sulfonate and propranololium on the RP-C18 -Ascentis bonded HPLC column from a mixture of methanol and water (30/70, v/v) Probe solute

Phenol (neutral)

Caffeinea (neutral)

Naphthalene sulfonate (anionic −)

Propranolol (cationic +)

qS,1 (mol/L) b1 (L/mol) qS,2 (mol/L) b2 (L/mol)

1.40 (1.40) 1.03 (1.06) 0.62 (0.60) 10.1 (10.4)

0.85 (0.88) 2.16 (1.94) 0.07 (0.08) 17.3 (16.3)

0.60 (0.58) 2.31 (2.46) 0.019 (0.017) 68.3 (80.2)

0.83 (0.81) 13.3 (14.4) 0.021 (0.020) 913 (1580)

qS,2 qS,1 +qS,2

31% (30%)

7.6% (8.3%)

b2 b1

9.8 (9.8)

8.0 (8.4)

qS,3 (mmol/L) b3 (L/mmol) qS,4 (␮mol/L) b4 (L/␮mol)

– – – –

0.77a (0.77) 0.11 (0.12) – –

3.1% (2.8%) 29 (33) 6.6 (6.1) 0.80 (0.84) – –

2.5% (2.4%) 68 (110) 4.0 (1.2) 7.98 (19.1) – –

Twenty-five millimolars NaCl salt were added to measure the isotherms of the ionizable compounds. For the sake of comparison, the values in parenthesis are the isotherm parameters derived independently from the calculation of the AED. a Fit of the adsorption data of caffeine by maintaining constant q S,3 given by the AED.

air-conditioner. The daily variation of the ambient temperature never exceeded ±1 ◦ C. 3.4. Frontal analysis isotherm measurements The exact same procedure was followed as in a previous paper [11]. 4. Results and discussion The adsorption isotherms of the four compounds on the Ascentis-C18 and Ascentis-RP-Amide adsorbents are shown in Fig. 1A–D that encompass the whole concentration range studied. The best numerical estimates of the isotherm parameters are listed in Tables 2 and 3. The adsorption energy distributions, calculated from the raw adsorption data, are shown in Figs. 2–5 on both columns, for phenol (Fig. 2), caffeine (Fig. 3), 2-naphthalene sulfonate sodium (Fig. 4), and propranololium chloride (Fig. 5). The concentration range used is wider for phenol than for the other three compounds because it is more soluble. However, in all cases the concentration range exceeds 1 to 2 × 104 .

4.1. Adsorption isotherm of phenol The adsorption isotherms of phenol on the C18 -bonded and on the RP-Amide stationary phases are drastically different (Fig. 1). Among the four compounds tested, phenol is the only one that shows a significantly much stronger adsorption on the RP-Amide phase that on the conventional C18 -bonded phase. This result is consistent with previous data that were used to suggest that this increase of retention is due to the hydrogen basicity of the polar group embedded in this stationary phase [16]. The detailed analysis of the isotherm parameters is given in Table 2. As observed on many other endcapped C18 -bonded phases [4,11], the adsorption energy distribution of phenol is again bimodal on the Ascentis-C18 column. The low-energy sites are about 2.5 times more abundant than the high-energy sites (saturation capacities, 1.40 versus 0.60 mol/L for the two types of sites). An energy difference of about 5 kJ/mol is measured between the adsorption energies on the two types of sites. These quantitative characteristics of the heterogeneity of the Ascentis-C18 adsorbent for phenol match exactly those found on other brands of monomeric endcapped C18 -bonded phases [4].

Table 3 Best isotherm parameters accounted for by the adsorption of phenol, caffeine, naphthalene sulfonate and propranololium on the RP-C18 -Amide bonded HPLC column from a mixture of methanol and water (30/70, v/v) Probe solute

Phenol (neutral)

Caffeine (neutral)

Naphthalene sulfonate (anionic −)

Propranolol (cationic +)

qS,1 (mol/L) b1 (L/mol) qS,2 (mol/L) b2 (L/mol)

2.21 (2.27) 2.45 (2.57) 0.46 (0.40) 18.4 (21.1)

0.90 (0.96) 2.44 (2.07) 0.046 (0.060) 18.8 (16.6)

0.66 (0.65) 2.14 (2.19) 0.014 (0.014) 172 (197)

1.07 (1.03) 8.16 (8.93) 0.022 (0.020) 822 (1270)

qS,2 qS,1 +qS,2

17% (15%

4.9% (5.9%)

2.1% (2.1%)

b2 b1

7.5 (8.2)

7.7 (8.0)

80 (90)

qS,3 (mmol/L) b3 (L/mmol) qS,4 (␮mol/L) b4 (L/␮mol)

0.31 (0.36) 2.3 (2.0) – –

– – – –

4.2 (3.4) 1.13 (1.30) 16.2 (9.2) 0.05 (0.09)

2.0% (1.9%) 101 (142) 3.3 (1.2) 7.66 (16.1) – –

Twenty-five millimolars NaCl salt were added to measure the isotherms of the ionizable compounds. For the sake of comparison, the values in parenthesis are the isotherm parameters derived independently from the calculation of the AED.

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Fig. 2. AEDs of phenol calculated by the EM method from the raw adsorption data given in Fig. 1. The number of iteration was fixed at one hundred millions, the number of energy grid points was 225 and the energy grid range was bmin = 1/10Cmax and bmax = 1/Cmin . Cmin and Cmax are the lowest and highest mobile phase concentrations used in FA, respectively. (A) Ascentis-C18 , (B) Ascentis-RP-Amide.

The adsorption behavior of phenol on the RP-Amide column is quite different. First, the degree of heterogeneity has changed: an additional type of adsorption site (sites of type 3) is seen on the AED, Fig. 2. This type of sites has a high adsorption energy, about 15 kJ/mol higher than the lowest adsorption energy. Secondly, the equilibrium constants on the two low adsorption energy sites, b1 and b2 , are about twice as large as those measured on the Ascentis-C18 adsorbent. The differences of adsorption energy between the corresponding types of sites in the two columns are 2.1 and 1.5 kJ/mol, respectively, for the type-1 and type-2 sites. Two different interpretations are likely to account for this increase in the binding energies: (1) in addition to the dispersive interactions with the alkyl chains in the bonded layer, phenol interacts selectively with the amido groups that are dispersed more or less homogeneously within the alkyl layer, that is can be involved in interactions with sites of types 1, 2, or 3; (2) phenol interacts only with the amido groups located on sites of type 3 and the larger binding constants observed on sites of

Fig. 3. Same as in Fig. 2, except the solute, caffeine.

types 1 and 2 are due to a different structure of the hydrophobic part of the RP-Amido phase. The absence of sites of type 3 on the surface of the Ascentis-C18 adsorbent seems to invalidate the first hypothesis, although it could be assumed that the alkyl chains somehow shields phenol from the amide groups on sites of types 1 and 2. The appearance of sites 3 with the RP-Amido phase, however, most likely shows that this site is directly related to the embedded amide groups. Accordingly, this would suggest that phenol molecules penetrate the bonded layer and reaches the inner part of the bonded layer, where the amide groups are located. Adsorption data need to be measured for other compounds to shed more light on the retention mechanism involved on the RP-Amide column and the role played by the amide group. 4.2. Adsorption isotherm of caffeine In contrast to phenol, the adsorption isotherms of caffeine are very close on both columns (Fig. 1A–D). A slightly higher isotherm is observed on the RP-Amido phase but only at mobile

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Fig. 5. Same as in Fig. 2, except the solute, propranololium chloride. Fig. 4. Same as in Fig. 2, except the solute, naphthalene sulfonate.

phase concentrations larger than 10 g/L. At lower concentrations, the two overall isotherms match each other perfectly and the two adsorbents cannot be distinguished. The two AED are different, however, as shown in Fig. 3. The distribution is trimodal on the conventional C18 -bonded phase and simply bimodal on the RP-Amide phase. It is noteworthy that the convergence of the AED on the RP-Amido phase is not complete despite the largest number of iteration applied, e.g., 1 Giga iterations. We have no explanations, except that the highest concentration at which the FA measurements were carried out, a concentration determined by the solubility of caffeine in the mobile phase, is not large enough ( 35 g/L) for a good estimate of the low-energy adsorption mode. In contrast to what was observed on other brands of endcapped C18 -bonded phases [4,11], caffeine adsorbs on AscentisC18 on three different types of adsorption sites, not on two types as usual on these packing materials. A similar behavior was already found on non-endcapped C18 -bonded phases, like the Resolve-C18 column [13]. The adsorption energy of

caffeine on this third type of sites was 10.5 kJ/mol higher than on type 1 adsorption sites. The same energy difference is found in this work with the endcapped Ascentis-C18 adsorbent (εa,3 − εa,1 = 9.9 kJ/mol) which suggests that the sites of type 3 might be physically similar on these two C18 phases. The main difference observed is the saturation capacities of the sites of this type: it was found to be about 12 mmol of caffeine per liter of stationary phase on the non-endcapped Resolve-C18 adsorbent but it is less than 1 mmol/L on the endcapped Ascentis-C18 column. The difference is probably explained by the removal of most of these sites during the endcapping of the silica. Nevertheless, it seems that all these sites were not completely eliminated on the endcapped Ascentis-C18 column and that FA can still detect those remaining. On the other hand, it is not the case with the endcapped Ascentis-RP-Amide column. No such sites of type 3 are measured and the bi-Langmuir model accounts well for the FA data on this adsorbent. Surprisingly, the binding constants of caffeine on the sites of types 1 and 2 are practically the same on both Ascentis columns

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(b1  2.2 L/mol and b2  17 L/mol). Because a significant difference was observed for the adsorption constants of phenol (b1 increased from 1.0 to 2.5 L/mol and b2 from 10 to 20 L/mol), it seems probable that the embedded amide groups are present in the sites of types 1 and 2. Otherwise, the binding constants of phenol on the sites of types 1 and 2 would be the same. The first hypothesis made in the previous section seems to be validated. Accordingly, the adsorption behavior of caffeine is not affected by the presence of the amide groups in the bonded layer. It is likely that the selective interactions with the CO NH groups on the surface of the Ascentis-RP-Amide phase involves hydrogen bond donor interactions like those in which phenol can be involved. Caffeine having no exchangeable proton, there are almost no differences between the adsorption behavior of caffeine on Ascentis-C18 and Ascentis-RP-Amide.

Although the presence of the amide group within the alkyl bonded layer does not change much the overall saturation capacity of the packing material for the negatively charged analyte, the detailed mechanism of adsorption becomes fundamentally different and the band profiles are drastically affected. The presence of a fourth type of adsorption sites with an extremely high binding constant and the larger values of the intermediate equilibrium constants on sites of types 2 and 3 lead to a significant increase of the retention factor at infinite dilution (as it does for phenol too) and to a strong peak asymmetry with a long tailing at sample size that are conventionally considered as within the analytical standard range of sizes. The resulting closeness of the overall adsorption isotherm is explained by the very low saturation capacity of type 4 sites which makes theses sites inoperative at significant concentrations. 4.4. Adsorption isotherm of propranololium chloride

4.3. Adsorption isotherm of sodium 2-naphthalene sulfonate The overall adsorption isotherms of sodium 2-naphthalene sulfonate on the two adsorbents are close. At low concentrations, the high-energy adsorption sites fill up with increasing concentration and the anion seems slightly more retained on the RP-Amide phase than on the RPLC phase (Fig. 1D). Surprisingly, however, the AEDs on the two columns are different (Fig. 4). There are three distinct adsorption sites on the conventional Ascentis-C18 column, their saturation capacities and binding constants being close to those found for these three sites with other brands of endcapped C18 -bonded columns (e.g., Gemini-C18 and Sunfire-C18 [11]). These three sites correspond to three distinct hydrophobic micro-environments that can accommodate molecules of 2-naphthalene sulfonate within the C18 -bonded layer. The differences between the adsorption energies on sites of types 2 and 1 and between those on sites of types 3 and 1 are 8 and 14 kJ/mol, respectively, for the AscentisC18 phase, and 9.5 and 14.5 kJ/mol [11] for both GeminiC18 and Sunfire-C18 , suggesting a great similarities between the behavior of these different layers of bonded octadecyl chains. In contrast, there are four distinct modes in the adsorption energy distributions of naphthalene sulfonate sodium on Ascentis-RP-Amide. The differences between the two adsorbents are due to (1) a new, type 4, of ultra-high energy adsorption sites and (2) higher values of the equilibrium constants on the sites of type 2 and 3 (about 180 versus 75 L/mol on type 2 sites and 1200 L/mol versus 800 L/mol, on type 3 sites). Despite the relatively poor accuracy of the isotherm parameters of the type 4 adsorption sites (qS,4 is of the order of 10 ␮mol/L and b4  70 000 L/mol), it seems highly probable that this fourth type of adsorption sites on the RP-Amide phase arises from some hydrogen-bond interactions between the S O bonds of the sulfonate ion and the N H bonds of the embedded amide group. No ion-exchange were possible in the range of pH investigated in this work because the pK␣ value of the amido nitrogen is less than 0. The bonded chain remains definitely neutral.

The adsorption isotherms of propranololium chloride on the two columns are shown in Fig. 1A–D. The two isotherms cross each other. In the low concentration range (from 0 to about 20 g/L), the adsorption of propranololium chloride is stronger on the C18 than on the RP-Amide phase and the reverse is observed at higher concentrations. As expected, the AEDs (see Fig. 5) exhibit only three modes, with binding constants in the same range of values as those for the corresponding modes of phenol and caffeine. No ion-exchange interaction can take place between two ionic species having the same electrical charge. However, it is observed that the equilibrium constants are higher on the C18 -bonded phase than on the amide phase, showing that propranololium chloride interacts less strongly with the amide phase than with the regular C18 one. Note that the two isotherms cross each other at a relatively high concentration, because the saturation capacity of the low-energy type 1 sites is larger on the RPAmide than on the C18 phase (1.07 mol/L versus 0.83 mol/L). These results are qualitatively and quantitatively in excellent agreement with the parameters of the adsorption isotherms measured previously on Gemini-C18 and Sunfire-C18 for the same compounds, under the same experimental conditions [11]. In conclusion, the adsorption mechanisms of caffeine and propranololium chloride on a conventional endcapped monomeric C18 adsorbent and on an endcapped, monomeric C18 , amide-embedded adsorbent made with the same silica are very similar. In contrast, the adsorption behaviors of phenol and sodium naphthalene sulfonate on these same two phases are markedly different. Yet, all the analyte molecules have access to the same embedded amide groups. However, all may not interact selectively with them. Some give merely dispersive van der Waals interactions, while others give strongly polar, hydrogendonor, or ion-exchange interactions, explaining, in these cases, the presence of an additional type of high-energy adsorption sites. Interestingly, the binding constants measured on the lowor moderate-energy sites (sites of types 1 and 2 for phenol, of types 2 and 3 for naphthalene sulfonate) are larger on the Ascentis-RP-Amide phase than on the Ascentis-C18 phase. This

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Fig. 6. Contribution of each type of adsorption sites (qS,i bi ) to the overall retention of the compounds in linear conditions on the Ascentis-C18 column. For each compound, the comparison between the Henry constants found from the best fitting parameters (left rectangles) of the adsorption data and the calculated AED (right rectangles) is given.

77

ity of the Ascentis-C18 adsorbent on the retention of the analytes studied is similar to those of the Gemini-C18 and Sunfire-C18 adsorbents. Nearly the same plots are obtained for all three columns. Note that the retention factor of phenol is almost double on the Ascentis-RP-Amide column than on the Ascentis-C18 one, essentially because the adsorption parameters of the type 1 adsorption sites is larger on the former. The sites of type 3 play a rather minor role and contribute little to explaining the difference of retention between the two Ascentis columns. The same observation applies to the retention of naphthalene sulfonate, although in this case, it is the change in the contribution of the sites of type 2 which is the most important. Again, the additional type 4 of adsorption sites has a rather weak impact on the overall retention factor. These two Figures also confirm that the retention mechanisms of caffeine and propranololium chloride are quite similar on the two adsorbents. None of the

suggests that the amide groups are spread rather homogeneously across the whole bonded layer. 4.5. Surface heterogeneity of the stationary phases Figs. 6 and 7 show the contributions of the different types of adsorption sites, i, to the overall retention factors, k = FH (with F, phase ratio, and H, Henry constant) of the four analytes studied, on the two Ascentis columns. We have H=

n
1

Hi =

n


qS,i bi

(5)

1

The great interest of the measurements of the adsorption isotherms is now obvious. The detailed analysis of the different isotherms and the use of their parameters give accurate new insights on the retention mechanisms of each compound by allowing the quantification of the roles played by the different adsorption sites at infinite dilution. Linear chromatography gives a single number, k ot H, and leaves clueless regarding the different underlying mechanisms. The effect of the heterogene-

Fig. 7. Same as in Fig. 6, except the column Ascentis-RP-Amide.

Fig. 8. Relationship between the thermodynamic parameters found for sites 1, qS,1 (A) and ln b1 (B), and the molecular volume of the analyte. Note the different trends, the equilibrium constant increases, typical of hydrophobic interactions.

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Fig. 10. Same as in Fig. 8, except the type of sites 3.

Fig. 9. Same as in Fig. 8, except the type of sites 2.

different contributions significantly varies from one column to the other. Figs. 8–10 illustrate the evolution of the isotherm parameters (qS,i and bi ) with the molecular size of the analyte. Obviously, this size is not the only descriptor governing solute retention in RPLC (the overall polarity of the solute is another critical factor too) but the saturation capacity and the equilibrium constants are expected to increase and decrease, respectively, with increasing molecular size of analytes. The saturation capacities should decrease because of solute exclusion and the binding constants should increase because of the larger area of contact. These trends hold approximately for the sites of types 1 (Fig. 8) and 2 (Fig. 9). For the sites of type 3, the results are not conclusive. Figs. 11 and 12 compare the differences between the adsorption energies of the four solutes on the different types of adsorption sites. A minimum value of 5 kJ/mol is measured between any pair of successive adsorption sites. The difference between the energies of adsorption on the strongest type of adsorption

Fig. 11. Difference between the adsorption energies on sites 4, 3, 2, and sites 1 for the different compounds studied on the Ascentis-C18 column.

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Energy differences between the lowest and the highest adsorption energy types of sites larger than 25 kJ/mol have never been measured obtained in the calculations of AEDs. This value corresponds to an energy of approximately 10 times RT = 2.45 kJ/mol. This can be explained by the following considerations. The probability, Pi , for an adsorbed molecule of analyte to desorb i.e. to escape from the surface by gaining the adsorption energy Ei from its surrounding is given by the Boltzman equation:   Ei b (6) Pi = exp − = RT b0

Fig. 12. Same as in Fig. 11, except with the column Ascentis-RP-Amide.

sites and on the sites of type 1 is at most 25 kJ/mol. Even though the actual physical meaning of these different types of adsorption sites has not yet been established, and probably cannot be based merely on the modeling of the FA data and on the results of the AED calculations, the accumulation of data obtained on a number of packing materials of different natures [11–13] shows that: • Adsorption on the first two types of sites (types 1 and 2) involve dispersive interactions between the alkyl chains and the solute molecules. Sites 1 are located at the interface of the mobile phase and the bonded layer, sites 2 are located deep inside this bonded layer. The adsorption energy on the sites of type 1 is of the order of a few kJ/mol [23] (the weakest interaction energy between the analyte molecules and the surface). The adsorption energy on the sites of type 2 is usually slightly less than 10 kJ/mol. • The third type of adsorption sites is usually found with large molecular size compounds (e.g. naphthalene sulfonate, propranololium chloride), which fit inside large cavities located in the bonded layer. For instance, these sites are observed on non-endcapped stationary phases. The adsorption energy on these sites is of the order of 15–20 kJ/mol. • The fourth type of adsorption sites is rarely seen in RPLC. It is usually related to highly selective interactions like ionexchange or specific hydrogen bonding interactions. Ionexchange interactions or hydrogen bonding with residual, isolated silanols rarely happens on endcapped reversed phase because the access to them is sterically hindered by the voluminous CH3 Si CH3 group bonded to the silica surface. Although, there might be a very low density of silanol groups accessible to the solute in solution. The adsorption energy on these sites may vary from 20 to 30 kJ/mol and the amount is of the order of 1 nmol/m2 or less. Table 4 summarizes the properties of the four different adsorption sites that may be found on RPLC packing materials with most conventional low molecular weight compounds.

where b is the equilibrium constant and b0 the preexponential factor. The probability of desorption is obviously 1 when the adsorption energy Ei is zero. It drops to 0.13, 0.017, 0.00029, and 0.000005 when the adsorption energy increases to 5, 10, 20, and 30 kJ/mol, respectively. This means that if a molecule is adsorbed with an adsorption energy of 30 kJ/mol, on the average one molecule out of 2 × 105 desorbs at any one time (i.e., one molecule is found in the mobile phase for every 200,000 found adsorbed at equilibrium). The retention factor, k , can be calculated according to [24]: k =

1 − Pi Pi

(7)

The retention factor of compounds with adsorption energies of 5, 10, 20, and 30 kJ/mol on an homogeneous phase would be 6.7, 58, 3447, and 200,000, respectively. This means that adsorbents having a specific surface area of a few hundreds m2 /g with homogeneous surfaces covered with sites of types 3 or 4 could never be used in chromatography but with far stronger mobile phases than used in this work. The retention times observed on conventional chromatographic columns packed with such adsorbents (void volume V0 = 1 mL) would be about 60 h and 140 days, respectively. We can observe these high-energy sites because their density is very small with respect to the number of adsorption sites of types 1 and 2 (see relative abundances in Table 4), which is tantamount to adsorbents having correspondingly low specific surface areas. As a result, the contributions of the four types of sites to the retention factor in linear chromatography are of the same order of magnitude, as shown in Figs. 6 and 7. 4.6. Band tailing of “analytical” peaks The presence of high adsorption-energy sites on reversedphase stationary phases causes a large loss of sensitivity because peak tailing becomes important with increasing sample size. Depending on the concentration of the analyte injected and on the initial curvature of the isotherm, some bands may experience an intense strong tailing while others may remain symmetrical. For instance, we showed earlier in this study that sodium naphthalene sulfonate may adsorb on four different types of sites on the RP amido-embedded stationary phase while caffeine and phenol are adsorbed on only two and three types of sites, respectively. The highest energy type of adsorption sites of caffeine,

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Table 4 Properties (order of magnitude) of the four types of adsorption sites encountered on conventional RP-HPLC columns Type of site

Abundance (␮mol/m2 )

Adsorption energy (kJ/mol)

Interactions

Retention factora k

Sites 1 Sites 2 Sites 3 Sites 4

2.0–4.0 0.05–1.25 0.003–0.03 0.00002–0.0004

0–5 6–13 14–20 21–30

Dispersive Dispersive Dispersive and specific Specific

2.5 50 1000 35000

a

Assuming an homogeneous stationary phase made of one type of adsorption site.

phenol, and sodium naphthalene sulfonate are 90% filled when the mobile phase concentration of these compounds are 0.9/bmax , i.e., 9.3, 0.037, and 0.0042 g/L, respectively (see Table 3). Thus, nonlinear behavior can be observed for one, two, or possibly the three compounds when their mixture is injected, depending on their concentrations in the sample and on its size. Thus, for a mixture of equal concentrations, we expect that when the sample size is increased, the peak of sodium naphthalene sulfonate will tail first, followed by those of phenol and of caffeine. Fig. 13 shows four “analytical chromatograms” obtained upon the injection of 10 ␮L of solutions of equal concentrations, 0.0135, 0.09, 0.6, and 4.0 g/L. The sample did not contain propranololium chloride because its retention is too high compared to those of the other three compounds. For a sample concentration less than 0.0135 g/L, the three peaks are gaussian. Because bands dilute and broaden during

elution, the peak of naphthalene sulfonate remains gaussian at the outlet of the column. Typically, the ratio of the injected to the eluted concentration for a 15 cm long packed column is of the order of 10 [28], which explains why, despite an injected concentration larger than 0.0042 g/L, the peak of sodium naphthalene sulfonate appears symmetrical. For a sample concentration of 0.09 g/L, the tailing becomes obvious for this compound only, due to the saturation of the adsorption sites of type 4. For a concentration of 0.6 g/L, the peak of phenol remains symmetrical because the injected band broadens and dilutes rapidly. For a sample concentration of 4.0 g/L, nonlinear adsorption behavior of phenol is observed, due to the saturation of the adsorption sites of type 3. The peak of caffeine remains nearly gaussian, with only a slight asymmetry at 4.0 g/L of caffeine, over the whole range of concentration investigated. This is due to the adsorption behavior of the sites of type 2 being still linear. If we

Fig. 13. Injection of variable amounts of a solution of phenol, caffeine, and propranololium chloride (equal concentrations) on the Ascentis-RP-Amide column. Same experimental conditions as in Fig. 1. The three solute behave differently revealing the difference in the saturation capacities of their high energy sites.

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Fig. 14. Same as in Fig. 13, except with the column Ascentis-RP-C18 .

assume a dilution factor due to axial dispersion of a factor 10, the solution of caffeine injected would fill only 4% of the sites of type 2 at the column outlet. The same results were obtained under the same experimental conditions with the Ascentis-C18 column (Fig. 14). As expected from Fig. 6 and the values of the Henry’s constants, phenol and sodium naphthalene sulfonate almost coelute under linear conditions, although the latter is actually eluted first. The sites of highest adsorption energy of phenol and sodium naphthalene sulfonate are filled up to 90% when their mobile phase concentrations are 8.4 and 0.26 g/L, respectively. Accordingly, the bands of phenol and caffeine exhibit a similar profile when a 4.0 g/L solution is injected. As shown before, sodium naphthalene sulfonate tails more significantly on the amido-embedded than on the alkyl-bonded one because, if the former phase exhibit only three types of adsorption sites instead of four, the adsorption energy on the highest energy sites is high and these sites are active enough (b3  800 L/mol) to generate a strong band tailing with samples of a 0.6 g/L solution. Figs. 13 and 14 are consistent with the Henry’s constant shown in Tables 2 and 3. All the advantage of measuring adsorption isotherm become obvious when we want to understand why the peaks of certain analytes exhibit a deleterious peak tailing when others follow linear behavior. This tailing is not of kinetic origin as demonstrated in this work. It is a direct consequence of the presence of a low density of high adsorption-energy sites.

Their saturation capacity being low, peak tailing is observed even when very dilute samples are injected. 5. Conclusion This work demonstrates that the measurement of adsorption isotherm data for different low-molecular-weight compounds, the modeling of these data, and the calculation of the adsorption energy distribution provide accurate, detailed information on the complex retention mechanisms of chemically bonded adsorbents. The selectivity of the RP-Amide phase for certain analytes (e.g., phenol) can be explained by comparing the values of the isotherm parameters derived for this phase and a conventional C18 -bonded packing material. The methodology employed in this work allows the identification of the sites responsible for this selective recognition of certain polar compounds and affords precise quantitative details on these interactions. The origin and nature of the differences in the degree of heterogeneity of the two columns studied and of others to which the method would be applied becomes clear. It is consistent with the differences known in the composition of the adsorbents. The most serious inconvenient of this method is that it is time consuming since the acquisition of the adsorption data needed to obtain an accurate isotherm and a correct AED takes more time than the collection of the retention times of small pulses performed in linear chromatography. On the other hand, this

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approach is powerful. It allows a deep understanding of the behavior of packing materials and it is applicable to any mode of chromatography. Its use requires, first, the selection of a few (ca. 4–6) chemicals (neutral, positive and negative ions, small and large size) that all follow convex upward isotherm behavior (i.e., strictly langmuirian). These compounds must be highly soluble in the eluent selected and must give high response factors because accurate results require that adsorption data be acquired in a concentration range exceeding 1 to 1 × 104 . The interpretation of the adsorption data will inform on the overall saturation capacity of the column, the possible presence of polar or ionexchange groups in the bonded layer or at the interface. Note that certain compounds will “see” certain types of adsorption sites which are inert toward others. As shown earlier, caffeine exhibits the same behavior toward conventional C18 -bonded adsorbents and the RP-Amide column. Because we have not yet identified the physical nature of the sites observed, we cannot follow their behavior with different compounds. This study completes our demonstration of the heterogeneous character of the C18 -bonded silica adsorbents used in RPLC. This result was suspected on the basis of linear chromatography results but no quantitative description could be derived with this method. This shows that the properties of RPLC stationary phases cannot be fully elucidated from collections of retention times of small pulse injections. This shows also that retention models based on the Linear Solvation Strength Model (LSSM), which attempts to relate retention factors and volume fraction of an organic modifier in the main eluent, is a purely empirical construction that is not based on any actual physical property of RPLC adsorbents. The linear behavior observed is often accidental. It results from the combination of the independent behaviors of the different retention terms [25–27]. Because each one of these behaviors is usually given by a logarithmic dependence, the overall effect can be nothing short of complex. Similarly, a deeper look on the thermodynamic quantities, H0 and S0 , derived from plots of the logarithm of the retention factor versus the reciprocal temperature (Van’t hoff plots) is required. Most results found in the literature are spurious because the columns being heterogeneous, each type of sites has its own values of H0 and S0 . Thus, the distribution of analyte molecules between liquid phases and alkyl-bonded adsorbents appears far more complex than we thought.

Acknowledgments This work was supported in part by grant CHE-02-44693 of the National Science Foundation and by the cooperative agreement between the University of Tennessee and the Oak Ridge National Laboratory. We thank Motilal Sarker (Supelco, Bellefonte, CA) for the gift of the Ascentis-C18 and AscentisRP-Amide columns and for fruitful discussions. References [1] F. Gritti, G. Guiochon, J. Chromatogr. A 1099 (2005) 1. [2] W.R. Melander, Cs. Horvath, in: Cs. Horvath (Ed.), High Performance Liquid Chromatography, vol. 2, Academic, New York, 1980, p. 113. [3] J.G. Dorsey, K.A. Dill, Chem. Rev. 89 (1989) 331. [4] F. Gritti, G. Guiochon, Anal. Chem. 75 (2003) 5726. [5] F. Gritti, G. Guiochon, Anal. Chem. 77 (2005) 1020. [6] A. Tchapla, H. Colin, G. Guiochon, Anal. Chem. 56 (1984) 621. [7] A. Tchapla, S. Heron, H. Colin, G. Guiochon, Anal. Chem. 60 (1988) 1443. [8] F. Gritti, G. Gotmar, B.J. Stanley, G. Guiochon, J. Chromatogr. A 988 (2003) 185. [9] F. Gritti, G. Guiochon, J. Chromatogr. A 1090 (2005) 39. [10] F. Gritti, G. Guiochon, Anal. Chem. 77 (2005) 4272. [11] F. Gritti, G. Guiochon, J. Chromatogr. A 1103 (2006) 43. [12] F. Gritti, G. Guiochon, J. Chromatogr. A 1103 (2006) 57. [13] F. Gritti, G. Guiochon, J. Chromatogr. A 1028 (2004) 75. [14] L.C. Sander, S.A. Wise, Anal. Chem. 56 (1984) 504. [15] M.R. Euerby, P. Petersson, J. Chromatogr. A 1088 (2005) 1. [16] N.S. Wilson, J. Gilroy, J.W. Dolan, L.R. Snyder, J. Chromatogr. A 1026 (2004) 91. [17] J. Layne, J. Chromatogr. A 957 (2002) 149. [18] J. O’Gara, D. Walsh, C. Phoebe, B. Alden, E. Bouvier, P. Iraneta, M. Capparella, T. Walter, LC-GC 19 (2001) 632. [19] D. McCalley, J. Chromatogr. A 844 (1999) 23. [20] M.R. Euerby, P. Petersson, LC-GC Eur. 13 (2000) 665. [21] F. Gritti, G. Guiochon, J. Chromatogr. A 1075 (2005) 117. [22] B.J. Stanley, S.E. Bialkowski, D.B. Marshall, Anal. Chem. 65 (1993) 259. [23] F. Gritti, G. Guiochon, J. Chromatogr. A 1043 (2004) 159. [24] J.C. Giddings, Unified Separation Science, John Wiley & Sons Inc., New York, 1991. [25] F. Gritti, G. Guiochon, J. Chromatogr. A 995 (2003) 37. [26] F. Gritti, G. Guiochon, J. Chromatogr. A 1010 (2003) 153. [27] F. Gritti, G. Guiochon, J. Chromatogr. A 1017 (2003) 45. [28] F. Gritti, A. Felinger, G. Guiochon, J. Chromatogr. A, in preparation.