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Inverse Mixed-Mode Chromatography for the Evaluation of Multivalency and Cooperativity of Host-Guest Complexation in Porous Materials Qian-Hong Wan, Xiaohuan Wang, and Lei Chen Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.9b01764 • Publication Date (Web): 23 Jul 2019 Downloaded from pubs.acs.org on July 27, 2019

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Inverse Mixed-Mode Chromatography for the Evaluation of Multivalency and Cooperativity of Host-Guest Complexation in Porous Materials Qian-Hong Wan,* Xiaohuan Wang, and Lei Chen School of Pharmaceutical Science & Technology, Tianjin University, 92 Weijin Road, Tianjin 300072, China

ABSTRACT A new separation-based analytical method was developed to evaluate the multivalency and cooperativity of supramolecular host-guest complexation in porous materials. The method is based on inverse mixedmode chromatography in which a porous material with a multivalent functional group is packed into a column and bound with a complementary guest molecule to form a multivalent complex. The bound guest molecules are eluted in the mobile phase and detected by appropriate methods such as UV absorption. The retention factor of the guest molecule is determined and broken down into the contributions of non-covalent interactions between binding sites (e.g., hydrophobic and ionic components), thereby calculating the effective molarity and cooperativity factor of the complexation. Two model systems denoted as RP/SCX and RP/SAX were analyzed by the established method. On average, the RP/SCX system has an effective molarity (EM) of 0.14 M and a cooperativity factor () of 0.86, while the RP/SAX system has an EM value of 0.18 M and a  value of 2.3. Interestingly, experiments have shown that these values do not change with changes in the intrinsic binding strength of the constituent sites. In summary, the developed method allows for quantitative assessment of multivalency and cooperativity effects in porous materials, providing a valuable complement to the analytical toolbox for supramolecular chemists and materials scientists. Keywords: Inverse chromatography; Supramolecular chemistry; Host-guest interaction; Multivalency; Cooperativity

INTRODUCTION Multivalency and cooperativity have long been recognized as two fundamental principles in nature for regulating the behavior of complex molecular systems in chemistry and biology.1-5 Multivalency refers to the complexation between a multivalent receptor and a complementary ligand through multiple noncovalent interactions, such as van der Walls forces, hydrophobic effects, ion pairing, - stacking, hydrogen bonding and metal ion coordination. Consequently, the binding affinity that exists between host and guest is greatly enhanced by the combination of these forces compared with the single intermolecular interaction. Multivalent interactions play an important role in numerous biochemical and cellular processes, including protein formation, DNA replication, enzyme catalysis and cell adhesion. Cooperativity is a concept closely related to multivalency, which refers to an additional binding energy change due to interconnected binding sites. The formation of a multivalent complex can be seen as a stepwise process. The first step of the complexation is the intermolecular interaction, and the subsequent binding steps are the intramolecular interactions. The binding energy of the intramolecular interactions may be greater or less than the corresponding intermolecular interactions, which is considered to be a positive or negative cooperative

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effect. Otherwise, if there is no difference in binding energy between the two events, the binding system is said to be non-cooperative, which occurs independently. The chelation cooperativity associated with the formation of intramolecular interactions is quantified by effective molarity EM. 6-8 The term EM was first introduced in polymer chemistry and enzyme catalysis for evaluating the kinetically controlled cyclization rate. In this context, EM is known as kinetic effective molarity, defined as the ratio of the rate constants for intramolecular and intermolecular reactions. This concept was later transferred to supramolecular chemistry to describe the of thermodynamically controlled complex formation. It is defined as the ratio of the intramolecular equilibrium constant to the corresponding intermolecular equilibrium constant. EM has units of molar concentration, and high values indicate the preference of the intramolecular process to the intermolecular process. In the literature, the kinetic EM values as high as 1013 M are reported, but the thermodynamic EM values of most supramolecular complexes are much smaller, in the range of 1 mM to 10 M.9 The reason why the upper limit of the kinetic EM values is significantly larger than that of the thermodynamic EM values is not clear, but may depend on the nature of the two ring formation processes: permanent bonds are formed in covalent cyclization, rather than reversible supramolecular complexation, in which bond formation and cleavage occur continuously. Several experimental methods are available for measuring the thermodynamic or equilibrium EM values in solutions or at liquid-solid interfaces. The most common methods for solution systems include ultraviolet-visible, fluorescence, circular dichroism, nuclear magnetic resonance (NMR) spectroscopy, and isothermal titration calorimetry.10 Since many biomolecular interactions occur at the interface rather than in solution, it is necessary to extend EM measurements for the solid/liquid interface.5 As is typical in adsorption studies, the concentration of the species bound to the solid surface is expressed as the number of moles per surface area, rather than moles per volume for the solution species. Surface analytical techniques can be used to measure changes in the amount of adsorption during binding process, providing quantitative information about complexation on the surface. In this case, the most widely used techniques are surface plasmon resonance and quartz crystal microbalances.8 The analytical methods for porous materials are quite limited compared to the wide selection in solutions and interface systems. Recently, Xray and neutron diffraction, solid-state NMR and fluorescence spectroscopy, and computational models have been used to determine the structural and kinetic properties of host-guest interactions in metal-organic frameworks,11-13 however, quantitative estimation of multivalency and cooperativity effects in these materials remains a significant challenge. Porous materials have been increasingly used for gas storage and capture,14,15 separations,16-18 catalysis,19 drug delivery20-22 and sensing.23,24 The ability to characterize the supramolecular properties of these systems is critical to the development of high-performance products with advanced functions. As a powerful separation technique, mixed-mode chromatography has shown significant promise for characterizing porous materials. Conventional liquid chromatography uses a monofunctional stationary phase such as reversed phase or ion exchanger to separate the mixture, and the solute retention is primarily controlled by a single mode of action based on hydrophobic or ionic interactions. In contrast, mixed-mode chromatography uses a multifunctional stationary phase to achieve separation by multivalent complexation between a multivalent compound and a multivalent stationary phase.25,26 Recently, we reported studies of the retention behavior of divalent solutes (such as alkylanilines and alkylbenzoic acids) in mixed-mode chromatography and demonstrated that solute retention is highly dependent on the cooperative binding due to a combination of hydrophobic and ionic interactions.27,28 Liquid chromatography can be used not only as 2 ACS Paragon Plus Environment

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a separation method for a range of compounds, but also as a means of characterizing non-covalent interactions in porous materials. In the latter application, the porous material to be investigated is loaded into a chromatographic column and equilibrated with a mobile phase. A suitable molecular probe is then injected to reveal the binding properties of the material. Since the method of performing chromatographic analysis is contrary to the conventional method, this technique is generally known as inverse chromatography. In this paper, we describe an inverse mixed-mode chromatographic method for quantifying the effective molarity and cooperativity factor of host-guest complexation in porous materials. The method is based on the formation of transient complexes between the multifunctional host in the stationary phase and the complementary guest in the mobile phase. The retention factor of the guest molecules is measured and decomposed into components corresponding to different complex species present in the binding system. The values of effective molarity and cooperativity factor are calculated according to a binding model developed in this work. The proposed method was used to determine the effective molarities and cooperativity factors of the two model systems. APPROACH Two model systems were considered in this work to evaluate multivalency and cooperativity of supramolecular host-guest complexation in porous materials by inverse mixed-mode chromatography. As shown in Figure 1A, they are respectively composed of (1) benzenesulfonate bonded to porous silica as the host and a homogeneous series of alkylanilines as the guest, and (2) benzyltrimethylammonium bonded to porous silica as the host and a homogeneous series of alkylbenzoic acids as the guest. When the guest molecules are introduced into the chromatographic system, they are retained by the stationary phase due to the formation of transient complexes. As shown in Figure 1B, the

Figure 1. (A) Host-guest complexation systems studied in this work; (B) Formation of transient host-guest complexes during chromatography.


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complexation between the host and guest can be seen as a stepwise process. The binding occurs first at one of the two binding sites to form monovalent complexes, followed by the formation of a divalent complex. Four types of guest molecules can be identified: unbound G, two monovalent 1:1 complexes HG1 and HG2 and a divalent 1:1 complex HG12. An expression for the concentration of bound G in this supramolecular system can be derived from the mass balance and equilibrium constant equations as shown in eq 1: [𝐺]bound = (𝐾1 + 𝐾2 + 𝐾1𝐾2EM)[G][H]

(1)

Here, K1 and K2 are the equilibrium constants of the monovalent binding interactions at sites 1 and 2, respectively. EM is an effective molarity that correlates with the probability of intramolecular interactions occurring. [G] and [H] are the concentrations of unbound guest and host molecules. As mentioned above, the bound species have the units of surface concentration expressed as number of moles per surface area. It is common in analytical chromatography that the concentration of the guest molecules in the sample injected into the column is relatively small compared to the host molecules bonded on the silica substrate. Therefore, under conditions of low site occupation, it can be safely assumed that [H] can be approximated as [H]0, the total ligand density of the host molecules immobilized on the solid substrate. Equation 1 can be written as [𝐺]bound = (𝐾1 + 𝐾2 + 𝐾1𝐾2EM)[G][H]0

(2)

Then, the distribution coefficient D of the guest between the stationary and the mobile phase is expressed as 𝐷 =

[G]bound [G]

= (𝐾1 + 𝐾2 + 𝐾1𝐾2EM)[H]0

(3)

It should be noted that if the surface bound species is expressed in the units of mole per surface area, then D will not be the dimensionless parameter as normally encountered in partition chromatography, but have a unit of length. The binding strength of a solute in chromatography is measured by a dimensionless retention factor k, which is defined as a mole ratio of the bound to unbound species, 29 given by eq 4: 𝑘 =  𝐷 =  (K1 + K2 + K1K2EM) [H]0 = k1 + k2 +

𝑘1𝑘2 EM [H]0

(4)

Here,  is the phase ratio defined by the surface area of the porous material packed in the column divided by the void volume of the mobile phase. Similarly, the phase ratio  is not a dimensionless parameter, but has a unit of length reciprocal. The product of  and [H]0 represents the molar concentration of the host in the packed column. Both  and [H]0 are characteristic properties of the porous material under study and can be determined by independent measurements. It can be seen from eq 4 that the retention factor k12 of the divalent cyclic complex HG12 is given by k12 =

k1k2 EM

(5)

 [H]0

According to eqs 4 and 5, the overall retention factor can be decomposed into hydrophobic, ionic and cooperative components k1, k2, and k12, corresponding to three bound species. If the values of k1 and k2 4 ACS Paragon Plus Environment

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are known, they can be subtracted from k to obtain the value of k12. Therefore, there is a need for a method of evaluating the values of k1 and k2 in order to quantify the cooperativity effects of a given binding system. In analogy to the double mutant cycle analysis, 30 the pairwise ionic and hydrophobic intramolecular interactions can be turned off stepwise to determine k1 and k2. For the specific examples shown in Figure 1, we assume that the divalent host has a hydrophobic binding site 1 and an ionic binding site 2. The ionic interaction at site 2 can be suppressed by using a buffer having a high salt concentration as the mobile phase. However, due to salting out effect,31 the hydrophobic interactions at site 1 will also be affected, which complicates the interpretation of the data. Therefore, the linear extrapolation to infinite salt concentration was used in this work to evaluate the hydrophobic component k1. The principle of this method can be illustrated by typical ion exchange equilibrium: (6)

HC + G ⇌ HG + C

Here, the charge signs for ionic species are omitted for clarity. C is a single-charged competitive ion. The ion exchange equilibrium constant Kix is given by 𝐾𝑖𝑥 =

[HG][C] [HC][G]

(7)

The distribution coefficient Dix of the guest is related to Kix by [HC]

(8)

𝐷𝑖𝑥 = 𝐾𝑖𝑥 [C]

Then, the retention factor kix of the guest is given by [H]0

[HC]

𝑘𝑖𝑥 = 𝐾𝑖𝑥 [C] = 𝐾𝑖𝑥 [C]

(9)

As mentioned above, under conditions of low site occupancy, [HC] can be approximately treated as [H]0. Therefore, the plot of the retention factor kix versus the reciprocal of the counterion concentration 1/[C] will produce a straight-line graph through the origin. The slope of the linear plot is proportional to the ion exchange equilibrium constant Kix and the molar concentration of the host [H]0 in the ion exchange system. The presence of hydrophobic interactions between the host and guest will result in an intercept on the k-axis, which corresponds to the hydrophobic retention factor. Both pure ionic and mixed interactions are eliminated at the infinite competing ion concentration (1/[C] = 0).32 Similarly, the ionic component k2 can be determined by extrapolating the overall retention factor k to the zero hydrophobic interaction. At a salt concentration no more than 100 mM (to mitigate the salting out effect), the retention of the guest molecules is controlled by multiple interactions as described in eq 4. For the homologous series sharing the same ionic head group, the retention is expected to increase with the length of the alkyl chain in the hydrophobic tail. Thus, the plot of the overall retention factor k versus the hydrophobic component k1 should be a straight line with both the intercept and slope proportional to the ionic component k2. Linear extrapolation of k to k1 = 0 will result in ionic component k2 due to the elimination of hydrophobic and cooperative interactions. The values of k1 and k2 are subtracted from k to obtain k12. Once the values of k1, k2 and k12 are known, effective molarity EM and cooperativity factor  can be evaluated using the following equations: EM =

𝑘12 𝑘1𝑘2

 [H]0

(10)


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β=

EM

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(11)

[H]0

Equation 11 is essentially identical to that defined by Ercolani and Schiaffino except that in their work the guest molecule is considered to be present in excess in the binding system.3

EXPERIMENTAL SECTION Chemicals and Reagents. Mercaptopropyltrimethoxysilane was purchased from Wuda Silicone Materials (Wuhan, China). Methanol, acetonitrile, and triethylamine were purchased from Concord Chemical Reagents (Tianjin, China). Sodium 4-vinylbenzenesulfonate, 4-vinylbenzyltrimethylammonium chloride and azobisisobutyronitrile were purchased from Guangfu Chemical Reagents (Tianjin, China). Porous silica microspheres, BaseLine Sil-10 (particle size 10 m, surface area 300 m2/g, pore size 10 nm), were supplied by BaseLine Chromtech Research Centre (Tianjin, China). Testing compounds including 4-alkylanilines, and 4-alkylbenzoic acids were of analytical grades and used as received. Synthesis and Characterization. The silica support was activated in the vapor phase with mercaptopropyltrimethoxysilane and then functionalized with sodium 4-vinylbenzenesulfonate or 4vinylbenzyltrimethylammonium chloride by a thiol-ene click reaction. In a typical synthesis, silica particles (4 g), mercaptopropyltrimethoxysilane (9.6 mmol) and triethylamine (7.2 mmol) were placed in a Teflonlined autoclave and heated at 150 ° C for 8 hours. The obtained particles were washed successively with methanol, 50% ethanol and methanol, and dried under vacuum at 80 ° C overnight. The thiol-bonded silica particles (4 g) were dispersed in 80 mL of methanol, followed by the addition of a mixture of sodium 4vinylbenzenesulfonate (0.8 mmol) and the catalyst azobisisobutyronitrile (0.24 mmol). The mixture was heated under reflux for 12 hours. The sulfonate-functionalized silica, designated RP/SCX, was filtered and washed successively with methanol, 50% ethanol and methanol. The ammonium functionalized silica, represented by RP/SAX, was prepared by essentially the same procedure, with an increased amount of 4vinylbenzyltrimethylammonium chloride (1.4 mmol) used to compensate for its lower reactivity. The carbon, sulfur, and nitrogen loadings of the synthesized phases were measured using a Vario Micro Cube elemental analyser from Elementar (Hanau, Germany). For the sulfonate and ammonium bonded particles, the surface coverages [H]0 was found to be 0.55 (0.50% S) and 0.26 (0.13% N) mol/m2, respectively. Chromatographic Measurement. Stainless steel columns (150 x 4.6 mm I.D.) were prepared in the laboratory by conventional slurry packing technique. An air driven liquid pump Haskel DSTV-115 (Burbank, CA) was used to drive the slurry through the column. The functionalized particles were dispersed in 30 mL of 1-hexanol/carbon tetrachloride (1:1, v/v), transferred to a high-pressure slurry reservoir and loaded into the column at 300 bar using n-hexane as the pushing solvent. The columns were flushed and equilibrated with the mobile phase prior to testing and use. Chromatography was performed on an Agilent 1100 series HPLC system (Palo Alto, CA, USA), equipped with a vacuum degasser, quaternary pump, autosampler, column oven, variable wavelength UV detector, and ChemStation version B 03.01. Conditions: column temperature, 30 oC; flow rate, 1.5 mL/min for RP/SCX column and 1.0 mL/min for RP/SAX column; detection wavelength, 254 nm; injection volume, 5 L. The mobile phases were prepared by dynamically mixing acetonitrile with an aqueous buffer with 6 ACS Paragon Plus Environment

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the concentration and the pH adjusted to desired values. Sample solutions were prepared by dissolving probe molecules in acetonitrile at about 5 g/mL. The void volume of the column was evaluated by multiplying the flow rate by the dead time t0 measured by an unretained marker such as urea or acetone. The retention factor k is calculated from k = (tR – t0)/t0, where tR is the retention time of the solute. The packing material in the column was unpacked at the end of the chromatographic experiment. After washing with ethanol and water, the material was dried in an oven and weighted. The void volume and the mass of the packing in the columns were determined to be 1.5 mL and 1.5 g, respectively, which gives a phase ratio  = 0.3 x 106 m2/L. The host concentrations in RP/SCX and RP/SAX column are [H]0 = 0.17 M and 0.078 M, respectively.

RESULTS AND DISCUSSION Separation of simple amphiphilic molecules is problematic for conventional chromatography on reversed phase or ion exchange columns because they retain quite weak in the former, while resolution is generally poor in the latter. In contrast, mixed-mode chromatography provides superior performance due to multivalent interactions. Figure 2 shows respective separation of alkylanilines on benzenesulfonate bonded silica (RP/SCX) and alkylbenzoic acids on benzyltrimethylammonium bonded silica (RP/SAX). All probe molecules are baseline resolved with symmetrical peaks and eluted in increasing order of alkyl chain length. These probe molecules are expected to bind to the complementary host through ionic, hydrophobic, and mixed interactions because they have an ionic head and a hydrophobic tail.

Figure 2. (A) Representative separation of alkylanilines on RP/SCX column. Chromatographic conditions: column, 150 x 4.6 mm; temperature, 30 oC; mobile phase, acetonitrile (ACN)/60 mM potassium phosphate, pH 2.5 (45:55, v/v); flow rate, 1.5 mL/min, detection, UV 254 nm. Solute: 1, 4-toluidine; 2, 4-ethylaniline; 3, 4-propylaniline; 4, 4-butylaniline; 5, 4-pentylaniline. (B) Representative separation of alkylbenzoic acids on RP/SAX column. Chromatographic conditions: same as for (A) except for mobile phase, which was ACN/50 mM ammonium formate, pH 6.3 (35:65, v/v); and flow rate, which was 1.0 mL/min. Solute: 1, 4methylbenzoic acid; 2, 4-ethylbenzoic acid; 3, 4-propylbenzoic acid; 4, 4-butylbenzoic acid; 5, 4pentylbenzoic acid.


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Monovalent Hydrophobic Retention. To assess the contribution of hydrophobic interaction to the retention, two series of experiments were carried out: (1) the retention factors of the alkylanilines were measured on RP/SCX column as a function of ionic strength in the mobile phase over a range of 30 to 70 mM and (2) the retention factors of the alkylbenzoic acids were measured on RP/SAX column as a function of ionic strength in the mobile phase over a range of 20 to 60 mM. The values of k obtained in (1) and (2) were plotted against the reciprocal of the competing ion concentration, 1/[C]. As shown in Figure 3, the retention factor increases with increasing value of 1/[C] and increasing length of the alkyl chain in the hydrophobic tail. A linear relation exists between k and 1/[C] for all probe molecules, with positive intercept and slope values for each plot (Table 1). As described above, the positive intercept reflects the contribution of the hydrophobic interaction between the hydrophobic tail of the amphiphilic probe and the spacer connecting the ionic head group to the silica, since at this point the charge on the host has been completely screened. Therefore, these intercepts can be used as a hydrophobic component k1, which increases with the hydrophobicity of the probe molecule. A positive slope indicates the contribution of ionic interaction between the ionic head of the probe molecule and the complementary charged group in the host. The results of the regression analyses of the graphs shown in Table 1 indicate that both hydrophobic and ionic interactions contribute to the solute retention in this mode of chromatography.

Figure 3. Plots of k versus 1/[C] for (A) a homologous series of alkylanilines on RP/SCX column: (■) 4Toluidine; (●) 4-Ethylaniline; (▲) 4-Propylaniline; (▼) 4-Butylaniline; (◀) 4-Pentylaniline; and (B) a homologous series of alkylbenzoic acids on RP/SAX column: ( ■ ) 4-Methylbenzoic acid; ( ● ) 4Ethylbenzoic acid; (▲) 4-Propylbenzoic acid; (▼) 4-Butylbenzoic acid; (◀)4-Pentylbenzoic acid.

To further confirm the hydrophobic nature of the intercept, we constructed the relationship between the log k1 of two homologs and the number of carbon atoms nC in the side chain alkyl groups. As shown in Figure S1, a linear plot with a positive slope was observed in both cases. This behavior is well documented in reversed phase chromatography and is attributed to the cumulative contribution of the methylene groups in the alkyl chain to the hydrophobic interaction between the analyte and the stationary phase.33 As shown in Table 1, the slope of the k versus 1/[C] curve also increases with the length of the alkyl chain. From the standpoint of independent ion and hydrophobic interactions, this result seems puzzling because, according to eq 9, the slope of the curve is independent of the hydrophobicity of the 8 ACS Paragon Plus Environment

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solute. However, if we consider that these two interactions work together to create a cyclic complex, the puzzle is easily resolved. It can be seen from eqs 4 and 9 that the slope is not only related to the ion exchange equilibrium constant and the host molar concentration, but is also proportional to the hydrophobic component k1 due to cooperative binding. The slope of the plot on the RP/SCX column is 4-6 times higher than that of the RP/SAX column, in part because of its higher ion exchange capacity. However, this difference can only be explained in detail after measuring the relative magnitudes of k2 on the two columns.

Table 1. Intercepts and Slopes of k versus 1/[C] Plots for Homologous Series Column

Solute

Intercept

Slope (mM)

RP/SCX

4-Toluidine

0.48

281

4-Ethylaniline

0.74

327

4-Propylaniline

1.07

379

4-Butylaniline

1.44

444

4-Pentylaniline

1.84

515

4-Methylbenzoic acid 4-Ethylbenzoic acid

0.53

42

0.79

53

4-Propylbenzoic acid 1.15

68

4-Butylbenzoic acid

1.68

90

4-Pentylbenzoic acid

2.35

119

RP/SAX

Monovalent Ionic Retention. To assess the ionic component k2, we further examined the overall retention factor k of the probe as a linear function of the hydrophobic component k1, and extrapolated to k1 = 0 to eliminate the hydrophobic contribution. Figure 4 shows that two homologs exhibit a good linear relationship between the k and k1 values, consistent with the expectations of eq 4. Positive intercept and slope values corresponding to contributions of ionic and cooperative interactions were obtained in both cases (Table 2). As the salt concentration increases, the intercept and slope values decrease because the ionic contribution is reduced at higher salt concentrations. The intercept and slope values observed on the RP/SCX column are greater than on the RP/SAX column, probably because the former retains more ion exchange capacity, expressed as [H]0. The k2 value obtained represents a hypothetical amine or acid retention factor with no contribution of hydrophobic interaction under the conditions tested. This is confirmed by a linear plot of k2 versus 1/[C], which passes through the origin (Figure S2) because all interactions are now cancelled.


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Figure 4. Plots of k versus k1 for (A) a homologous series of alkylanilines on RP/SCX phase at various salt concentrations (mM): (■) 30; (●) 40; (▲) 50; (▼) 60; (◀) 70; and (B) a homologous series of alkylbenzoic acids on RP/SAX phase at various salt concentrations (mM): (■) 20; (●) 30; (▲) 40; (▼) 50; (◀) 60.

Table 2. Intercepts and Slopes of k versus k1 Plots for Homologous Series Column

[C] (mM)

Intercept

Slope

RP/SCX

30

6.64

6.67

40

5.00

5.26

50

3.99

4.41

60

3.32

3.84

70

2.85

3.43

20

0.93

3.14

30

0.63

2.42

40

0.48

2.07

50

0.37

1.86

60

0.31

1.72

RP/SAX

Divalent Hydrophobic/Ionic Retention. The divalent retention component k12 was evaluated by subtracting the hydrophobic and ionic components (k1 and k2) from the total retention k. The obtained values of k12 together with those of k, k1, k2 are summarized in Tables S1 and S2 in the Supporting Information, respectively, for the RP/SCX and RP/SAX systems. As expected, k12 increases with the length of the alkyl chain and decreases with the competing ion concentration as a result of increased hydrophobic and reduced ionic contributions, respectively. Effective Molarities and Cooperativity Factors. The effective molarities and cooperativity factors were calculated from the data of k1, k2, k12 and [H]0 according to eqs 10 and 11, and summarized in Tables S1 and S2 for the RP/SCX and RP/SAX systems, respectively. On average, the RP/SCX system 10 ACS Paragon Plus Environment

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has an EM value of 0.14 M and a  value of 0.86, while the RP/SAX system has an EM value of 0.18 M and a  value of 2.3. As mentioned above, the EM value reflects the likelihood that an unbound portion of a partially bound monovalent HG complex will find a complementary site. This can also be explained as the host concentration experienced by the guest. An EM value higher (or lower) than the actual host concentration indicates positive (or negative) cooperativity, thus providing a way to estimate whether the sequential binding steps in the formation of multivalent complexes behave in an independent or cooperative manner.3,8,9 The non-unity  values seem to confirm that the multivalent interactions in both host-guest complexation systems studied are regulated by cooperativity effects. As the length of the alkyl group of the probes increases and the salt concentration increases, the EM and  values for both systems remain unchanged. In studying the relationship between chemical structure and effective molarity for intramolecular hydrogen bonding, Hunter and coworkers point out that the value of EM does not vary with the intrinsic binding strength, but rather depends on supramolecular architecture and geometric complementarity.34,35 The invariant EM and  values of the two systems observed in this work support their view that the cooperativity in multivalent complexes is a structural property essentially determined by geometric and charge complementarity. The positive cooperativity shown by the RP/SAX system indicates that the geometric structure between the host and the guest has a good match to form a ring complex. In contrast, the negative cooperativity observed in the RP/SCX system suggests that the cyclic complex formed is strained. This may be because the - stacking interaction interferes with the ionic interaction between the host and the guest due to shorter spacing between the benzene ring and the acidic moiety in the host. In addition, negative cooperativity can also result from preferential solvation of the host and guest molecules in the binary solvent mixture, which causes conformational changes and leads to the formation of strained rings.36 The solvent effect on effective molarity and cooperativity factor of host-guest complexation is currently being investigated in our laboratory.

CONCLUSIONS Inverse mixed-mode chromatography has been successfully applied to the evaluation of multivalency and cooperativity of supramolecular systems in porous materials. Decomposing the observed retention factors into hydrophobic, ionic and cooperative components provides some invaluable insight into the driving forces of host-guest complexation and allows estimation of supramolecular binding parameters. Both negative and positive cooperativities were observed for the two systems consisting of porous silica bonded with benzenesulfonic acid and benzyltrimethylammonium as the host and the homologous series of alkylanilines and alkylbenzoic acids as the guest. Interestingly, the values of effective molarity and cooperativity factors are essentially independent of the intrinsic binding strength of the constituent sites, suggesting that they are structural properties of the binding system. Inverse-chromatography-based methods offer some unique advantages over conventional titration methods. First, the retention of the probe, rather than the signal intensity, is used to measure the strength of the interaction and the distribution of the different species. Thus, highly purified probe molecules are not required, as long as the impurities can be separated from the peak of interest. Secondly, weak binding interactions can be studied conveniently by varying the mobile phase composition, typically with a stability 11 ACS Paragon Plus Environment

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constant in the range of 10 to103 M-1. Most importantly, the method can be used to study supramolecular phenomena in porous materials and provide quantitative information about the effects of multivalency and cooperativity on molecular recognition, which is of fundamental importance for the development of nextgeneration materials for chromatography, catalysis, and drug delivery.

ASSOCIATED CONTENT

Supporting Information The supporting Information is available free of charge on the ACS Publications website at DOI: Linear plots of k1 vs. nc and k2 vs. 1/[C]; tables of retention factors, effective molarities and cooperativity factors obtained on two supramolecular binding systems. AUTHOR INFORMATION Corresponding Author E-mail address: [email protected]

Notes The authors declare no competing financial interest.

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Figure 1. (A) Host-guest complexation systems studied in this work; (B) Formation of transient host-guest complexes during chromatography.

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Figure 2. (A) Representative separation of alkylanilines on RP/SCX column. Chromatographic conditions: column, 150 x 4.6 mm; temperature, 30 oC; mobile phase, acetonitrile (ACN)/60 mM potassium phosphate, pH 2.5 (45:55, v/v); flow rate, 1.5 mL/min, detection, UV 254 nm. Solute: 1, 4-toluidine; 2, 4-ethylaniline; 3, 4-propylaniline; 4, 4-butylaniline; 5, 4-pentylaniline. (B) Representative separation of alkylbenzoic acids on RP/SAX column. Chromatographic conditions: same as for (A) except for mobile phase, which was ACN/50 mM ammonium formate, pH 6.3 (35:65, v/v); and flow rate, which was 1.0 mL/min. Solute: 1, 4methylbenzoic acid; 2, 4-ethylbenzoic acid; 3, 4-propylbenzoic acid; 4, 4-butylbenzoic acid; 5, 4pentylbenzoic acid.


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Figure 3. Plots of k versus 1/[C] for (A) a homologous series of alkylanilines on RP/SCX phase: (■) 4Toluidine; (●) 4-Ethylaniline; (▲) 4-Propylaniline; (▼) 4-Butylaniline; (◀) 4-Pentylaniline; and (B) a homologous series of alkylbenzoic acids on RP/SAX phase: (■) 4-Methylbenzoic acid; (●) 4-Ethylbenzoic acid; (▲) 4-Propylbenzoic acid; (▼) 4-Butylbenzoic acid; (◀)4-Pentylbenzoic acid. 335x133mm (96 x 96 DPI)


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Figure 4. Plots of k versus k1 for (A) a homologous series of alkylanilines on RP/SCX phase at various salt concentrations (mM): (■) 30; (●) 40; (▲) 50; (▼) 60; (◀) 70; and (B) a homologous series of alkylbenzoic acids on RP/SAX phase at various salt concentrations (mM): (■) 20; (●) 30; (▲) 40; (▼) 50; (◀) 60. 196x82mm (96 x 96 DPI)


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