Journal of Chromatography A, 1169 (2007) 111–124
Effect of the surface coverage of endcapped C18-silica on the excess adsorption isotherms of commonly used organic solvents from water in reversed phase liquid chromatography Fabrice Gritti a,b , Y.V. Kazakevich c , 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 c Department of Chemistry, Seton Hall University, Seton Orange, NJ, USA Received 11 June 2007; received in revised form 27 August 2007; accepted 30 August 2007 Available online 4 September 2007
Abstract The excess adsorption isotherms of methanol, ethanol, 2-propanol, acetonitrile, and tetrahydrofuran from water were measured on five different silica-based packing materials by the minor disturbance method. These materials were prepared with the same lot of 5- m particles (average pore ˚ all endcapped with trimethylchlorosilane (TMS), and bonded to octadecyl chains with different surface coverages (0, 0.42, 1.01, 2.03, size 90 A), and 3.15 mol/m2 ). The relative adsorption of one eluent by respect to a second one informs on the heterogeneity of the material (alkyl-bonded and bare silica regions) and on the accessibility of the unreacted silanol groups to the mobile phase. It is shown that the total surface area of the adsorbent decreases with increasing degree of surface coverage with octadecyl chains and that the relative surface area of the regions occupied by accessible silanol groups to the regions occupied by alkyl-bonded groups decreases. For the five columns, an average of 10% of the adsorbent surface area is covered of bare silica accessible to the liquid phase, with a minimum of 5% with tetrahydrofuran and a maximum of 12% with ethanol or 2-propanol. Increasing the surface coverage by the C18 chains causes a significant increase of the attraction potential of the hydrophobic surface toward the organic solvent. This result is confirmed by the increase of the number of adsorbate monolayers with increasing bonding density of the octadecyl chains. This number is twice larger for the 315C18 column than for the C1 column. © 2007 Elsevier B.V. All rights reserved. Keywords: RPLC; Excess adsorption isotherm; Binary liquid mixtures; Organic solvents; Surface heterogeneity; C18 surface coverage; Accessible residual silanols; Adsorbent surface area; B.E.T. nitrogen adsorption (LTNA); Methanol; Ethanol; 2-propanol; Acetonitrile; Tetrahydrofuran
1. Introduction Retention in liquid–solid chromatography is controlled by the distribution of the analyte between the bulk mobile phase and the adsorbed phase. Although investigations on the actual retention mechanisms in RPLC began 30 years ago [1–9], this topic is still not settled [10,11]. It is commonly accepted that retention should be due to adsorption of the analytes onto the alkyl-silica surface, or to partitioning of the analytes between the layer of bonded alkyl chains and the mobile phase, or to a combination of these two limit mechanisms. While these models remain pertinent due to the complex organization of the alkyl-bonded
∗
Corresponding author at: Department of Chemistry, University of Tennessee, Knoxville, TN 37996-1600, USA. Tel.: +1 865 974 0733; fax: +1 865 974 2667. E-mail address:
[email protected] (G. Guiochon). 0021-9673/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2007.08.071
layer, with the simultaneous presence of local aggregates of alkyl chains and of cavities between these aggregates [12,13] and with the possibility for the mobile phase components to interact with residual, unreacted silanol groups, they tend to ignore that the mobile phase composition is different in the bulk mobile phase and immediately above the adsorbent surface. This difference is characterized by the excess (or default) adsorbed amount of the organic modifier. In RPLC, this eluent modifier plays the same role in the adsorption process as do the analytes. They are all part of the same multi-component solution and their excess adsorbed amounts are not independent of each other. In other words, to understand from a fundamental point of view the distribution of one analyte at infinite dilution between the adsorbed phase and a bulk binary solution, one must also understand the excess adsorption of the organic modifier as well. This explains why excess adsorption isotherms of the binary mobile phases commonly used in RPLC has been studied in the
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past [14–18]. On the basis of such data, Kazakevich et al. built the general analyte distribution function across the column [11]. In this model, the surface fraction occupied by the solid silica is ignored because silica is impermeable to the eluent. The distribution function accounts for the amount of analyte present in the bulk liquid phase (phase 1), the amount dissolved in the adsorbed boundary layer of the eluent (phase 2), and the excess amount adsorbed on the surface (phase 3). This model identifies three distinct phases, instead of the conventional two, the bulk liquid phase and the adsorbed liquid, because the analyte is necessarily a part of the solution with the mobile phase components and is not an independent entity. Admittedly, in linear chromatography, the analyte concentration is so small compared to that of the eluent that its presence does not affect the composition of the adsorbed mobile phase. The fundamental problem is that the formation of the adsorbed layer of eluent is a direct consequence of the electric field of the adsorbent surface that attracts the organic modifier. Considering the adsorbent surface and the adsorbed layer of eluent as two independent phases is fundamentally erroneous because the presence of the former determines the formation of the latter. For this reason, one should only consider two phases in the distribution function, the bulk liquid phase and the adsorbed liquid phase. However, it is difficult to delimitate the boundary between these two phases. In the case of an adsorbent with a homogeneous surface, this problem is easily solved by determining from the excess isotherm where the adsorption capacity is maximum [19]. For heterogeneous surfaces, the problem is complex and an arbitrary assumption must be made, regarding the total amount adsorbed [20]. The advantage of measuring the excess adsorption isotherms of binary mixtures from the bulk solutions [21] is that it is possible to extract from these data information on the heterogeneity of the adsorbent. For instance, it is important to understand the activity toward analytes of the unreacted silanol groups of RPLC stationary phases prepared by reacting surface silanols with alkylating reagents. This task has been attempted through frontal analysis measurements [10], which give the apparent concentration of analyte in the adsorbent volume as a function of the concentration of the compound in the bulk mobile phase. Analysis of the curvature of the isotherm reveals the degree of heterogeneity of the adsorbent surface and how complex are the interactions between analyte molecules and the adsorbed phase at high concentrations. Previous studies have been devoted to understand the competitive adsorption of water and methanol on RP-C18 surface [22,23]. In this work, we used the minor disturbance method [15] to measure the excess adsorption isotherms of several organic modifiers (methanol, ethanol, 2-propanol, acetonitrile, and tetrahydrofuran) from water solutions onto endcapped C18 bonded silica packing materials. These materials have different degrees of surface coverage in bonded C18 chains (0, 0.42, 1.01, 2.03, and 3.15 mol/m2 ). The effect of the surface coverage on the excess adsorption isotherm of these organic solvents from aqueous solutions water will be illustrated. The impact of the nature of the organic solvent on the apparent heterogeneity of the adsorbent surface will also be investigated. Finally, the excess
isotherm of a totally apolar solvent (hexane) dissolved in an apolar solvent (hexadecane) will also be measured on these stationary phases to demonstrate that the accessibility of the unreacted silanol groups depends strongly on the nature and composition of the mobile phase [21,25]. 2. Theory 2.1. The minor disturbance method From a simple column mass balance equation, Knox and Kaliszan [15] demonstrated that the elution volume, VR , of a pulse of the organic modifier in a column equilibrated with a binary eluent of volume fraction xA in eluent A is equal to: VR = VM
dyA dxA
(1)
where VM is the void volume defined as the sum of the volumes of all the eluent components in the column. Following the notation introduced by Knox [15], yA is the volume fraction of eluent component A inside the column bed at the equilibrium, and xA is the volume fraction of component A fed into the column. Note that Eq. (1) neglects the changes in partial molar volumes of the organic modifier upon its adsorption or its mixing to form the binary eluent. It also assumes that the binary solvent is not excluded from any part of the pore volume (no inaccessible micropores, no size exclusion effect). By definition of the excess adsorbed number of moles neA per column, neA =
VM y A VM xA − v∗A v∗A
and
yA = xA +
neA v∗A VM
(2)
where v∗A is the molar volume of liquid component A. Combining Eqs. (1) and (2) leads to: VR = VM + v∗A
dneA dne = VM + bA dxA dcA
(3)
b is the molar concentration of component A in the where cA eluent. According to Eq. (3), the excess number of moles of component A adsorbed is calculated from the following integral:
neA =
b cA
0
b b (VR (cA ) − VM ) dcA
(4)
The value of VM is calculated as follows: VM =
1 b,∗ cA
0
b,∗ cA
b b VR (cA ) dcA
(5)
b,∗ where cA is the molar concentration of component A in its pure liquid.
c
Measured from B.E.T. experiments. Measured from elemental analysis. %C1 is the carbon content measured after derivatization and before endcapping. %C2 is the carbon content measured after derivatization and after endcapping. ˚ 2. B.E.T. values corrected for a molecular surface area of nitrogen of 21 A a
4.81 4.81 4.81 4.81 4.81 4.81 150 × 4.6 150 × 4.6 150 × 4.6 150 × 4.6 150 × 4.6 150 × 4.6 Silica Endcapped C1 042C18 101C18 203C18 315C18
b
0.00 0.00 0.42 1.00 2.00 3.09 0 4.48 6.88 10.06 15.01 20.13
Particle size (m) Column dimension (mm × mm) Column
The minor disturbance measurements were all acquired using a Hewlett-Packard (Palo Alto, CA, USA) HP 1090 liquid chromatograph. This instrument includes a multi-solvent delivery system (tank volumes, 1 L each), an auto-sampler with a 25 L sample loop, a diode-array UV-detector, a RI-detector (HP1047), a column thermostat 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 0.041 mL from the auto-sampler needle seat to the UV cell and 0.211 mL from the auto-sampler needle seat to the RI cell. All retention data were corrected for these contributions. The flow-rate accuracy was controlled by pumping the pure mobile phase at 295 K and 1 mL/min during 50 min, from each pump head successively, into a 50 mL volumetric glass. The relative error was less than 0.25%, so we estimate the long-term accuracy of the flow-rate at less than 3 L/min at flow rates around 1 mL/min. All measurements were carried out at the constant temperature of 295 K, fixed by the laboratory air-conditioner. The variation of the ambi-
Table 1 Physico-chemical properties of the five different packing materials
3.3. Apparatus
Specific pore volume (cm3 /g)a
Average pore ˚ a radius (A)
Specific surface area (m2 /g)a
%C1 derivatization (%)b
Five chromatographic columns with different surface coverages were studied. They were all prototype columns, prepared and generously offered by their manufacturer (Waters, Milford, MA, USA). The column dimensions are 150 mm × 4.6 mm. The characteristics of these columns are listed in Table 1. They differ in the surface coverage of the C18 chains bonded to the silica based material. All the columns were prepared from the same batch of silica particles [24]. One column was endcapped with trimethylchlorosilane (TMS), the other four were first derivatized with octadecylsilane C18 at different surface coverages (0.42, 1.01, 2.03, and 3.15 mol/m2 ), then endcapped with TMS. The specific volumes of the packing materials were obtained from low temperature nitrogen adsorption (LTNA) measurements (Table 1). The density of the neat silica was measured by Helium pycnometry (ρSilica = 2.12 g/cm3 ). The interparticle volumes Vex of these columns were measured by inverse size exclusion chromatography (ISEC results are given in Table 2), using polystyrene standards excluded from the internal pore volume and of molecular weights MW = 90,000, 400,000, 575,000, and 900,000.
0 0 3.39 7.64 13.90 19.61
%C2 derivatization & endcapping (%)b
3.2. Columns
354 314c 290c 287c 227c 172c
C18 surface coverage (mol/m2 )
The mobile phases used in this work were mixtures of water and methanol, ethanol, 2-propanol, acetonitrile, or tetrahydrofuran. Mixtures of hexane and hexadecane were also used in this study. All these solvents were HPLC grade, purchased from Fisher Scientific (Fair Lawn, NJ, USA). The polystyrene standards used to measure the external porosity of the column were purchased from Supelco (Bellefonte, PA, USA).
46.5 45.4 43.0 39.7 38.3 37.1
3.1. Chemicals
0.927 0.726 0.659 0.599 0.472 0.371
C1 surface coverage (mol/m2 )
3. Experimental
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0.00 3.87 3.33 2.65 1.53 0.92
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Table 2 Measurement of the interparticle volume (Vex ) and external porosities (e ) from ISEC Column
Vex (cm3 )
e
C1 042C18 101C18 203C18 315C18
0.994 0.955 0.943 0.927 0.931
0.399 0.383 0.378 0.372 0.373
ent temperature during the acquisition of the peak profiles never exceeded ±1 K. 3.4. Measurement of the perturbation with binary mixtures Each column was successively equilibrated with solutions of increasing volume fraction of the organic solvent in water (0, 0.5, 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 99, 99.5, and 100%). In order to observe the Gaussian signal corresponding to a linear perturbation of the equilibrium between the mobile and the stationary phases, 2 L of a solution having a concentration of organic modifier differing by less than 10% from the eluent composition at equilibrium was injected. The perturbations were detected with a RI detector at all mobile phase compositions. They were linear except in the extreme cases of 0 and 100% water solutions, for which an asymmetric peak shape (Langmuirian) was observed. Under these conditions, the perturbation volume was estimated from the extrapolation of the peak tail for zero concentration. The mobile phase velocity was set constant for all the measurements at 1.0 mL/min. The ambient temperature was 295 ± 1 K. 4. Results and discussion In the first part of this section, we discuss the dependence of the excess adsorption isotherm of the five organic modifiers dissolved in water (methanol, ethanol, 2-propanol, acetonitrile, and tetrahydrofuran) on the C18 surface coverage of the packing material. In the second part, we discuss how the nature of the organic modifier affects the excess adsorption amounts on these adsorbents. For instance, we compare the extent of the competition of these organic modifiers with water for adsorption onto the residual silanols. Finally, in the last part, we use a previously derived model of two-sites adsorption [20] to extract some qualitative information regarding the change in the heterogeneity of these five materials.
after bonding of the C18 and C1 groups onto the surface of the neat silica. According to [25], the void volume VM inside the chromatographic column is given by p
VM = VC
1 + (e /Vs )((fSilica /ρSilica ) + (fC1 /ρC1 ) + (fC18 /ρC18 )) p 1 + (1/Vs )((fSilica /ρSilica ) + (fC1 /ρC1 ) + (fC18 /ρC18 ))
(6)
where VC is the volume of the empty column tube (150 mm × 4.6 mm, VC = 2.493 cm3 ), e is the external porosp ity of the column (see Table 2), Vs is the specific pore volume of the packing material (cm3 /g, see Table 1), fSilica , fC18 , and fC1 are the mass fractions of silica, the C18 chains, and the C1 chains, respectively (see Table 3). ρC1 and ρC18 are the densities of the C1 and C18 chains, which are equal to 0.93 and 0.86 g/cm3 , the best estimates reported by Kazakevich by thermogravimetric measurements [25]. Finally, ρSilica is the density of silica and was estimated to be 2.12 g/cm3 as measured by Helium pycnometry. The mass of adsorbent mads packed in the chromatographic column is given by: mads =
VM − Vex p Vs
(7)
The total surface areas of silica present in each of the five columns studied in this work are SSilica = fSilica mads Sp
(8)
where Sp is the specific surface area of the neat silica (m2 /g). The mass of silica inside the five columns was estimated from the data published earlier [25]. The results of the calculations show that the surface areas of neat silica inside the column were 430, 435, 412, 407, and 391 m2 on the C1 , 042C18 , 101C18 , 203C18 , and 315C18 adsorbents, respectively. Thus, the relative variation of the silica surface area among all the columns does not exceed 4.3%. Since this value is within the range of experimental error, we can assume that all columns have the same silica surface area. The comparison of the experimental results expressed per column unit are the same as those calculated per m2 of neat silica surface. This is pertinent when we estimate the amount of residual silanols in each column, or compare the amounts of C18 and C1 chains. On the other hand, using the silica surface area is less judicious to compare the amounts of a compound adsorbed on the different columns because the effective surface area of the C18 -silica adsorbents differs significantly from one to another column (314, 290, 287, 227, and 172 m2 , respectively) (see Table 1). In any case, it is not straightforward to determine the true adsorption surface area occupied by a given adsorbate molecule on a given adsorbent.
4.1. C18 -silica surface coverage and excess adsorbed amount of organic modifier from water solutions
Table 3 Repartition of the total mass of the adsorbent (1 g) between silica, C1 chains, and C18 chains for the five adsorbents used in this work
Before analyzing the excess adsorption data of the different organic modifiers onto the five different adsorbents, it is informative to estimate the amount of residual silanol groups on the surfaces of these adsorbents, based on the results of the BET experiments, the ISEC experiments, the Helium pycnometry experiments, and the carbon content analyses performed
First set of column
fSilica
fC18
fC1
C1 042C18 101C18 203C18 315C18
0.9092 0.8834 0.8487 0.7942 0.7328
0.0000 0.0406 0.0933 0.1745 0.2497
0.0908 0.0760 0.0581 0.0313 0.0175
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It is possible to estimate the amount of residual silanols nR SiOH in each column from the C1 and C18 surface coverages, dC1 and dC18 , assuming that the initial silanol surface concentrations dSiOH is the conventional 8 mol/m2 . Even though this value is most probably not accurate, the comparison of the values obtained for each column is meaningful because the five adsorbents were made with the same silica. Accordingly, nR SiOH = fSilica mads Sp dSiOH − dC1 − dC18
(9)
Accordingly, the numbers of residual silanols in the columns named C1 , 042C18 , 101C18 , 203C18 , and 315C18 are 1.59, 1.70, 1.73, 1.82, and 1.57 mmol, respectively. It is noteworthy that this number increases with increasing C18 surface coverage, except for the column 315C18 , which has the highest surface coverage in alkyl chains (3.09 mol/m2 ). Fig. 1A–D shows the excess adsorption amounts (mole per column) of methanol on the five columns studied. The calculations were based on Eq. (4). When the mobile phase is b rich in water or when CMeOH → 0, the adsorbent adsorbs
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preferentially methanol due to the hydrophobic nature of the surface. Despite that the C1 column has the largest surface area as measured by nitrogen adsorption and the smallest amount of residual silanols, it adsorbs the smallest excess amount of methanol. As the surface coverage increases, the excess amount of adsorbed methanol increases until the effect of the decreasing surface area becomes significant. This most likely explains why the excess number of moles of adsorbed methanol is smaller on the 315C18 column than on the 203C18 column. The amount of residual silanol groups is meaningless at high water concentrations because these groups are saturated with water. Indeed, at the other end of the concentration range, e.g. with methanol-rich mobile phases (>99%), the excess amount of adsorbed methanol is negative meaning that the excess amount of adsorbed water becomes positive. The residual silanol groups preferentially adsorb water rather than methanol. As seen in Fig. 1C, the variation of the maximum excess of adsorbed water is consistent with that of the amount of residual silanols inside the columns, as estimated above.
Fig. 1. Excess adsorption isotherms of methanol from water solutions onto five different endcapped (C1 ) RP-HPLC adsorbents vs. the concentration of methanol in the bulk mobile phase. The data were obtained at 295 K, using the minor disturbance method. The characteristics of each column are given in Table 1. (A) Full data. (B) Zoom of the excess methanol adsorbed from water-rich mobile phases. (C) Zoom of the excess water adsorbed from methanol-rich mobile phases. (D) Second zoom of the excess water adsorbed from methanol-rich mobile phases.
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As the surface coverage increases, the maximum of the excess adsorption isotherm barely shifts toward lower methanol concentrations. On the average, over the five columns, the maximum excess is of the order of 1 mmol per column. This corresponds to about two excess molecules of methanol for three bonded alkyl chains in the adsorbed phase (either C18 or C1 chains, the total number of these chains being nearly constant at 1.5 mmol per column) by respect to the bulk concentration. This excess is maximum for the 101C18 and 203C18 columns (1.1 mmol for these intermediate bonding densities) and minimum for the C1 and 315C18 columns (0.8 mmol for the lowest and highest bonding densities). Fig. 2A–D shows the same experimental results as in Fig. 1A–D, with ethanol as the organic solvent. Again, with mobile phases rich in water, the excess amount of organic solvent adsorbed increases with increasing C18 surface coverage, but it begins to decrease for the 315C18 column. Residual silanols are saturated with water and play a minor role in the preferential adsorption of the ethanol on the hydrophobic alkyl chains. Again, the maximum excess amount of ethanol adsorbed is of the order of 1 mmol per column, with a maximum of 1.2 mmol for the 101C18 and 203C18 columns. In contrast, the situation is quite different when the adsorbent is nearly saturated with ethanol. The preferential adsorption of water is more important
in the presence of ethanol than of methanol. The maximum is nearly twice as large (0.15–0.30 mmol of water per column) and it takes place for a smaller volume fraction of the organic solvent. Both observations are consistent. The preferential adsorption of water onto the silanol groups increases with increasing surface coverage in C18 chains. We will return to this observation later. Fig. 3A–D presents the results of the measurements made with 2-propanol/water mixtures. These results are similar to those obtained with the previous two organic solvents. The excess amounts of 2-propanol adsorbed increase with increasing C18 surface coverage for water-rich eluents, except for the column 315C18 as previously reported for ethanol. Note that the maximum amount adsorbed is larger for 2-propanol that for methanol or ethanol. On the average, the excess number of moles of 2-propanol adsorbed is 1.4 mmol, meaning that there is at most about one molecule of propanol more adsorbed per alkyl-bonded chain than corresponds to the bulk mobile phase concentration. Fig. 4A–D shows the excess adsorption isotherms of acetonitrile. In water-rich mobile phases, the excess amount of acetonitrile adsorbed increases with increasing surface coverage in alkyl chains. This excess is maximum for the 101C18 and 203C18 columns, as it is with the other organic solvents. Note that the average maximum is of the order of 2.5 mmol of acetoni-
Fig. 2. (A–D) Same as Fig. 1, for ethanol.
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Fig. 3. (A–D) Same as in Fig. 2, for 2-propanol.
trile adsorbed per column, corresponding to about two molecules of acetonitrile more adsorbed per bonded alkyl ligand than there are in the bulk phase. It is important to note that, as the surface coverage increases, the maximum excess adsorption of water decreases. This would be consistent with a reduced access of the mobile phase to the bare silica surface when the thickness of the bonded layer increases. The excess adsorption isotherms of tetrahydrofuran from water on the same adsorbents are shown in Fig. 5A–D. These results are similar to those obtained with acetonitrile. The density of the bonded alkyl chains has the same effect on the adsorption excess amounts of tetrahydrofuran. In summary, the systematic effect of the surface coverage in C18 chains on the excess amounts of organic solvents adsorbed from aqueous solutions is two-fold: • It contributes to increase the excess amount of the organic modifier adsorbed in the column in water-rich eluents, despite the fact that the available surface area of the adsorbent in the column decreases. Increasing the bonding density of C18 chains in endcapped columns amounts to strengthening the attractive potential between the organic solvent and the adsorbent surface. Because the pore volume is finite, the excess amount of organic modifier adsorbed eventually decreases
when the volume occupied by the bonded layer becomes high enough (like it is with the 315C18 column). • For non alcoholic organic modifier (acetonitrile and THF), increasing the C18 bonding density tends to decrease the maximum excess amount of water adsorbed from the bulk eluent. The C18 chains may behave like a shield and contribute to make the adsorbent surface more homogeneous. On the other hand, the density of bonded C18 chains is not the only characteristics that controls the excess adsorption behavior of organic solvents from aqueous solutions. In the next section, we discuss the effect of the nature of the organic modifier on their excess amount adsorbed onto these stationary phases. 4.2. Effect of the nature of the organic modifier on the excess adsorption onto RPLC adsorbents We now compare the excess amounts of the five organic solvents adsorbed from water on a given column. In this comparison, we consider two different regions of the excess adsorption isotherm: (1) the concentration range in which the largest excess amount of organic modifier is adsorbed (typically between 20 and 40% of organic solvent, v/v) and (2) the concentration range
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Fig. 4. (A–D) Same as in Fig. 3, for acetonitrile.
in which the alkyl layer is nearly saturated with the organic solvent (about 96–99% of organic solvent, v/v), which allows the measurement of the maximum excess amount of water adsorbed onto the silanol groups. It is striking to see that the relative variation of these two maximum excesses of adsorption are nearly independent of the surface coverage of C18 chains. All the curves in Figs. 6–10 are similar. For water-rich solutions, they show that the excess of adsorption of the alcohols increases with increasing number of carbon atoms, from 1 to 3. This was expected in RPLC. The figures show also large differences between the maximum adsorption excesses of these alcohols and of acetonitrile and tetrahydrofuran. On the average, the maximum adsorption excesses of acetonitrile and tetrahydrofuran are about twice as large as those of the alcohols. This observation is consistent with the fact that the persistence distance over which the adsorption forces are effective on organic molecules in the bulk solution is larger with acetonitrile and tetrahydrofuran than with the alcohols. For solutions rich in organic solvent, the excess adsorption of water increases from methanol to ethanol and to 2-propanol. The larger and the more hydrophobic the alcohol molecules,
the less they can compete with water for adsorption onto the silanol groups. Tetrahydrofuran behaves like acetonitrile with respect to adsorption onto the hydrophobic regions of the adsorbent surface but this has no equivalent regarding their respective adsorption onto the silanol groups. Acetonitrile is the organic solvent that competes the less with water for adsorption on these sites. Tetrahydrofuran behaves more like ethanol and methanol at low and at high C18 surface coverages. The excess adsorbed amount of water is always maximum with acetonitrile. The effects of the residual silanols that are observed with polar mobile phases are not surprising. The question arises of whether the silanol groups are active in the presence of apolar solvents. To investigate this point, we measured the excess adsorption isotherm of the pair hexadecane/hexane, on the same adsorbents. The results are shown in Fig. 11. The excess adsorption isotherms of hexane are nearly symmetrical, showing that the adsorbed and bulk phases are nearly ideal. With respect to hexadecane and hexane, the surfaces of our adsorbents appear to be almost homogeneous. This result demonstrates that the silanol groups do not affect much the adsorption behavior of hexane or hexadecane. Interestingly, and for entropic reasons [26], the excess adsorption of hexane
F. Gritti et al. / J. Chromatogr. A 1169 (2007) 111–124
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Fig. 5. (A–D) Same as in Fig. 4, for tetrahydrofuran.
from hexadecane is positive. The average maximum excess of hexane adsorbed is about 0.2 mmol per column, at least five times less than the excess adsorbed of the organic solvents from water.
Fig. 6. Plot of the maximum excess adsorbed amounts of methanol, ethanol, 2-propanol, acetonitrile, and tetrahydrofuran measured on the C1 -silica column.
4.3. Silanol activity of RPLC adsorbents and number of molecular layers in the adsorbed phase The measurements of excess adsorption isotherms of binary solutions clearly demonstrated that an assessment of the hetero-
Fig. 7. Same as in Fig. 6, for the 042C18 -silica column.
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Fig. 8. Same as in Fig. 7, for the 101C18 -silica column.
geneity of the surface of C18 adsorbents depends on the choice made for the composition of the bulk liquid phase. The use of aqueous solutions of organic solvents reveal the presence of surface silanol groups, whether the solvent used is polar or not. A question remains, which type of silanol groups are actually seen. The surface of RPLC adsorbents is made of unreacted silanol groups, some directly accessible to the mobile phase, others trapped between or under alkyl bonded chains. This takes place with the C18 alkyl-bonded chains in contact with the mobile phases studied in Section 4.1. Recent NMR investigations [27] confirmed the conclusion of Kazakevich et al. [28] concerning the absence of swelling of the bonded phase layer upon solvation of the C18 chains by these mobile phases. The alkyl chains are and stay collapsed, so it is likely that only a fraction of the residual silanols are accessible to the mobile phase. The use of aqueous solutions of methanol, ethanol, 2-propanol, acetonitrile, and tetrahydrofuran may reveal the existence of only the silanols that are not trapped between or under the collapsed C18 chains. In a previous paper [20], it was demonstrated that the excess amount of component 1 adsorbed at equilibrium from the solu-
Fig. 9. Same as in Fig. 8, for the 203C18 -silica column.
Fig. 10. Same as in Fig. 9, for the 315C18 -silica column.
tion of components 1 and 2 is ne1 =
j=N
Aj t(K1/2,j − 1)x1l (1 − x1l ) K1/2,j a1∗ x1l + a2∗ (1 − x1l ) j=1
(10)
where a1∗ and a2∗ are the molar surface areas of these components. The molar surface areas of water, methanol, ethanol, 2-propanol, acetonitrile, and tetrahydrofuran were reported [19]. Aj is the surface area occupied by the sites of type j and K1/2,j is the selectivity of component 1 with respect to component 2 upon adsorption on sites j: K1/2,j =
a xl x1,j 2 a x1l x2,j
(11)
Fig. 11. Excess adsorption isotherms of hexane from hexadecane solutions onto five different endcapped (C1 ) RP-HPLC adsorbents vs. the concentration of hexane in the bulk mobile phase. The data were obtained at 295 K using the minor disturbance method. Note that the excess amount of hexane adsorbed is positive over the whole range of concentrations in contrast with Figs. 1A–5 A. The adsorbent surface is apparently homogeneous with respect to the adsorption of hexadecane/hexane mixtures.
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Fig. 12. Illustration of the convention chosen (dna1 = 0, number of adsorbate monolayers t ” ) based on the profile of the total amount adsorbed na vs. the mole 1
fraction in the bulk x1l .
a ) is In Eq. (11), the molar fraction in the adsorbed phase (x1,j calculated for a number t of adsorbed monolayers, t being measured according to the convention chosen for the delimitation between the adsorbed and the bulk liquid phases. This convention was explained in detail in [20]. Fig. 12 describes on which basis we chose arbitrarily this convention. The plot of the total number of adsorbed molecules, na , versus the organic modifier concentration must have an horizontal inflection point. This corresponds to the lowest acceptable number of adsorbed layers if we assume that na is an increasing function of the concentration of organic modifier in the mobile phase. The number of adsorbed monolayers was calculated as follows [20]:
1 t= − A
dne1 dx1l
Fig. 13. Plot of the minimum number of adsorbate monolayers vs. the C18 surface coverage of the columns used in this work, according to the convention described in Fig. 12. Table 4 Best fitting parameters (A, CH3 , KCH3 , KOH ) of Eq. (13) to the experimental excess amount of methanol Methanol
A
CH3
KCH3
KOH
C1 042C18 101C18 203C18 315C18
380 301 284 213 154
0.913 0.914 0.926 0.919 0.893
4.55 6.17 7.32 9.05 11.37
0.0307 0.0168 0.0090 0.0060 0.0083
l l (x1,I a1∗ +[1 − x1,I ]a2∗ ) + (a2∗ I
− a1∗ )[ne1 ]I (12)
where I is the inflection point of the excess adsorption isotherm l , [ne ] , and [dne /dxl ] observed in its decreasing branch, x1,I 1 I 1 I 1 are the molar composition, the excess adsorbed amount, and the derivative of the excess adsorbed amount with respect to the molar fraction of component 1 at the inflection point I, respectively. The results are shown in Fig. 13 according to the experimental data given in Figs. 1A–5A. As the surface coverage in C18 chains increases, the number of adsorbed monomolecular layers t increases. From the C1 column to the 315C18 column,
(1) The number of types of adsorption sites is only 2. The first type is related to the preferential adsorption of the organic component (KCH3 ) on the alkyl-bonded chains of surface area (ACH3 ). The second type is due to the preferential adsorption of water (KOH ) on the accessible free silanol groups (surface area A − ACH3 ). (2) For the convenience of the fit, we assumed that the adsorption constants KCH3 and KOH are independent of the bulk composition, x1l .
Due to these simplifying assumptions, the results of the fit are only qualitative. The fitted parameters are CH3 = ACH3 /A, A, KCH3 , and KOH . Eq. (10) writes: CH3 (KCH3 − 1)x1l (1 − x1l ) (1 − CH3 )(KOH − 1)x1l (1 − x1l ) e n1 = At + (13) KCH3 a1∗ x1l + a2∗ (1 − x1l ) KOH a1∗ x1l + a2∗ (1 − x1l )
t is almost doubled. As discussed before, increasing the density of the C18 chains amounts to enhancing the attractive potential of the surface of the adsorbent. If the maximum adsorption excess observed for the 315C18 is actually smaller than for the 203C18 column, this is because the specific surface area of the former material is much lower than that of the latter material [24]. Knowing the value of t makes possible a fit of the experimental excess adsorption data to Eq. (10). We made two assumptions:
The numerical results of the fit are shown in Tables 4–8 for methanol, ethanol, 2-propanol, acetonitrile, and tetrahydrofuran, respectively. The statistical errors varied from 5 to 15%. The fit of the data was made by minimizing the sum of the squared relative deviations between experimental and theoretical data, which explains why the fit is excellent for the small excess amounts (e.g. for the water-rich and the organic modifierrich mobile phases). The agreement is only approximate for the
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high excess adsorbed amounts. Examples of the agreement are shown for the five organic solvents on the 315C18 column in Fig. 14A–E. There is an excellent agreement between the best values of the overall surface areas of the different adsorbents, A, given by the fit and those derived from the analysis of the experimental BET
data given in Table 1. Much general, qualitative information can be derived from the results of these fits. The fraction of the silica surface covered with bonded alkyl chains increases just barely with increasing bonding density of these chains. This means that the density of the residual silanol groups and the access to them of the organic modifier molecules is nearly independent of the
Fig. 14. Comparison between the experimental excess adsorbed amounts and the best theoretical prediction based on Eq. (13). The best parameters are given in Tables 4–8 for methanol (A), ethanol (B), 2-propanol (C), acetonitrile (D), and tetrahydrofuran (E), for the 315C {18}-silica column.
F. Gritti et al. / J. Chromatogr. A 1169 (2007) 111–124 Table 5 Best fitting parameters (A, CH3 , KCH3 , KOH ) of Eq. (13) to the experimental excess amount of ethanol Ethanol
A
CH3
KCH3
KOH
C1 042C18 101C18 203C18 315C18
414 383 310 242 179
0.853 0.869 0.889 0.896 0.878
4.08 5.75 6.95 8.44 9.05
0.0177 0.0161 0.0096 0.0083 0.0066
Table 6 Best fitting parameters (A, CH3 , KCH3 , KOH ) of Eq. (13) to the experimental excess amount of 2-propanol 2-Propanol
A
CH3
KCH3
KOH
C1 042C18 101C18 203C18 315C18
287 249 231 187 141
0.835 0.860 0.908 0.911 0.895
7.23 11.91 15.82 20.97 22.11
0.0068 0.0090 0.0057 0.0050 0.0037
Table 7 Best fitting parameters (A, CH3 , KCH3 , KOH ) of Eq. (13) to the experimental excess amount of acetonitrile Acetonitrile
A
CH3
KCH3
KOH
C1 042C18 101C18 203C18 315C18
401 389 318 226 177
0.884 0.903 0.917 0.925 0.920
2.858 2.834 3.407 4.20 4.167
0.0147 0.0149 0.0126 0.0087 0.0078
surface coverage by C18 chains. This result is consistent with the concentrations of the residual silanols increasing only from 4.0 (for C1 ) to 4.1 mol/m2 (for 315C18 ). Although the surface heterogeneity is minimum with tetrahydrofuran (CH3 0.96) and maximum with ethanol and 2-propanol (CH3 0.88), the experimental results show that, on the average, 10% of the adsorbent surface is covered with silanol adsorption sites, despite the fact that the alkyl chains are collapsed against the surface. The selectivity KCH3 between the organic solvent and water increases with increasing C18 density. This is due to the increase of the adsorption potential when increasing density of the C18 chains bonded to the surface. The largest selectivity is observed for 2-propanol, the lowest for acetonitrile. The large excess of acetonitrile adsorbed is explained by the large number of adsorbed monolayers formed. On the other hand, the preferential adsorption of water with respect to the organic solvent (1/KOH ) Table 8 Best fitting parameters (A, CH3 , KCH3 , KOH ) of Eq. (13) to the experimental excess amount of tetrahydrofuran Tetrahydrofuran
A
CH3
KCH3
KOH
C1 042C18 101C18 203C18 315C18
427 336 285 206 153
0.945 0.952 0.966 0.969 0.967
3.06 4.20 5.77 9.48 12.3
0.0125 0.0117 0.0090 0.0070 0.0065
123
onto the free silanol groups is much stronger than the preferential adsorption of the organic solvent onto the alkyl-bonded chains. Also, 1/K0H increases with increasing surface coverage in alkyl chains. For instance, it increases from 33 to 120, 57 to 151, 147 to 270, 68 to 128, and 80 to 154 with methanol, ethanol, 2propanol, acetonitrile, and tetrahydrofuran, respectively, while KCH3 remains between 3 and 22. 5. Conclusion Increasing the degree of surface coverage of silica with bonded C18 chains does not reduce significantly the surface heterogeneity of the adsorbent. Its surface is covered in part with alkyl-bonded chains, in part with residual silanol groups that are freely accessible to the organic modifier molecules. Simple calculations show that the amount of unreacted silanol groups is nearly the same for all five columns, at 1.6 mmol/column. While the overall surface area of the adsorbent in contact with the mobile phase decreases with increasing C18 chain density, the ratio of accessible surface silanols to surface bonded chains remains constant. This ratio varies with the nature of the organic modifier, from 5 to 15%. It is minimum for tetrahydrofuran (5%) and maximum for 2-propanol (15%). The accessible silanol groups adsorb water selectively about 10 times more strongly than the alkyl chains adsorb selectively the organic solvents. While increasing the C18 bonding density does not reduce the proportion of the surface area covered with silanol groups, it increases markedly the adsorption strength of the organic solvent onto the alkyl chain surface and the number of the adsorbed monomolecular layers of these solvents adsorbed against the surface. The excess amount adsorbed decreases at high-bonding density (3.1 mol/m2 ) but only because so does the surface area of the adsorbent. Previous frontal analysis measurements had already demonstrated evidence of this result [24]. When the pore size is small (e.g., for the 315C18 column, the pore ˚ and acetonitrile or tetrahydrofuran are used as the size is 35 A) organic modifier, the entire pore volume is filled with adsorbed molecules of these solvents, forming at least five to six monolayers. Our results demonstrate that it is impossible to avoid the nefarious effects of residual silanols in RPLC, even with endcapped alkyl-silica adsorbents and aqueous organic mobile phases. The negative excess amounts of the commonly used organic solvents clearly show why. These results raise a few important questions. Could the method used see all the silanol groups? What kind of silanols are we seeing, free silanols? isolated silanols? Would these silanol groups be seen with different mobile phase mixtures? For instance, would the same degree of heterogeneity be observed with mixtures of hexadecane and ethanol or 2-propanol, instead [21]. Hexadecane could be expected to solvate the tethered C18 chains better than the organic modifiers used and certainly better than water. With such a strong solvent of the ligands, the silanols which are shielded from contact with aqueous organic mobile phases under conventional RPLC conditions could become accessible to the mobile phase components. They could make a third type of adsorption
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sites or behave like the silanols accessible in our experiments, merely increasing their number. Finally, surface excess isotherms of organic modifiers should be measured on other types of columns to check the validity of the conclusions presented here. Particularly interesting would be data acquired on a C18 -Xterra column, which has few residual silanols on its surface and a C18 -Resolve column that is not endcapped. This work is in progress. Acknowledgments This work was supported in part by grant CHE-06-08659 of the National Science Foundation, by Grant DE-FG05-88-ER13869 of the US Department of Energy, and by the cooperative agreement between the University of Tennessee and the Oak Ridge National Laboratory. We thank Mariana Kele and Uwe Dieter Neue (Waters, Mildford, MA, USA) for the gift of the prototype columns used in this study. References [1] H. Colin, G. Guiochon, J. Chromatogr. 141 (1977) 289. [2] W.R. Melander, Cs. Horv´ath, in: Cs. Horv´ath (Ed.), High Performance Liquid Chromatography, vol. 2, Academic, New York, 1980, p. 113. [3] Cs. Horv´ath, W.R. Melander, I. Molnar, J. Chromatogr. 125 (1976) 129. [4] L.R. Snyder, J.W. Dolan, P.W. Carr, J. Chromatogr. A 1060 (2004) 77.
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