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Solution State NMR Techniques Applied to Solid State Samples: Characterization of Benzoic Acid Confined in MCM-41 Thierry Azais,* Geoffrey Hartmeyer,† Sandrine Quignard, Guillaume Laurent, and Florence Babonneau UniVersite´ Pierre et Marie Curie-Paris6 and CNRS, UMR 7574, Laboratoire Chimie de la Matie`re Condense´e de Paris, Colle`ge de France, Paris, F-75005, France ReceiVed: NoVember 7, 2009; ReVised Manuscript ReceiVed: March 8, 2010
In this paper we present an NMR methodology to characterize small organic molecules confined in mesoporous materials. In particular, we demonstrate that NMR techniques issued from solution state NMR are well suited to characterize benzoic acid encapsulated in hexagonally ordered mesoporous silica MCM-41 possessing two different averaged pore sizes (30 and 100 Å). As evidenced by differential scanning calorimetry, entrapped benzoic acid molecules are highly mobile at room temperature due to confinement effect and possess a glass phase transition temperature around -55 °C. Thus, the 13C NMR characterization of encapsulated molecules has to be adapted to that particular behavior. In particular, the cross-polarization technique traditionally used in solid state NMR to record 13C magic angle spinning (MAS) spectra is of poor efficiency due to weak 1 H-13C dipolar interaction. Nevertheless, the presence of 1H-13C cross-relaxation phenomenon (nuclear Overhauser effect, NOE) allows us to record 13C spectra through power-gated techniques, routinely used in solution state NMR, in order to enhance the 13C signal through NOE. Furthermore, the long T2′(1H) values (up to 22 ms) are compatible with the setup of J-coupling-based experiments such as MAS refocused {1H}-13C INEPT experiments allowing us to characterize the sample through chemical bonds. These results combined with those of MAS 1H NOESY experiments lead us to distinguish unambiguously different benzoic acid populations within the large pore sample. Finally, we show that cooling down the samples at -35 °C diminishes the mobility and allows the reintroduction of the 1H-13C dipolar interaction. Thus, 2D MAS {1H}-13C HETCOR experiments can be performed at low temperature to explore spatial proximities. Introduction The case of confined molecules in porous matrices is unique in reason of the existence of confinement effect that implies a greater mobility of the trapped species when compared to the bulk. This typical behavior is a consequence of a depression of the thermodynamical parameters including the phase transition temperatures.1,2 Thus, the encapsulated molecules adopt a liquidlike behavior at room temperature, even if the pure substance is a solid under such conditions. This confinement effect, which is still not well understood, was essentially studied on simple liquids such as water,3,4 methanol,5,6 or benzene7-11 entrapped in model porous matrices that can be carbon or titania nanotubes or porous silica. In that latter case, the chosen materials are controlled porous glasses (CPGs)7,11 or mesoporous silica such as SBA-155,9,10 or MCM-41.5,6,10 These systems are characterized from ambient to low temperature by numerous techniques including differential scanning calorimetry (DSC),4,7,8,10 X-ray diffraction (XRD),5 quasi-elastic neutron scattering (QNS),6,12,13 and 1H or 2H NMR spectroscopy4,9-11 in order to study the physical behavior and to determine the phase transition temperatures of the encapsulated molecules. Recently, the characterization of confined organic species became essential in the field of pharmaceutical studies due to a growing interest in mesoporous silica-based systems used for the controlled release of hydrophobic drugs.14-21 Actually, from a pharmaceutical point of view, it is crucial to precisely * To whom correspondence should be addressed,
[email protected]. † Present address: IPCMS UMR7504 CNRS-ULP, Groupe des Mate´riaux Inorganiques, 23 rue du Loess, BP43 Strasbourg cedex 2, France.
Figure 1. Schematic representation of benzoic acid with dimensions and labeling of C and H atoms.
characterize the physical state of a drug in the dosage form and C solid state NMR is a prefect tool for this purpose.22 Recent literature showed that drugs confined in porous matrices are submitted to a confinement effect that determines their physical state at room temperature23-26 and, consequently, influences strongly their release profile.27,28 In particular, we showed in a previous paper that such an effect must be taken into account for the NMR characterization of ibuprofen encapsulated in MCM-41.29 Indeed, in reason of the highly mobile behavior of the drug, the 13C NMR experiments on such solid samples are complicated by the partial averaging of heteronuclear 1H-13C dipolar interaction that prevents a high efficiency of the crosspolarization (CP) sequence, which is routinely used in solid state NMR. In this study we use benzoic acid (Figure 1) encapsulated in MCM-41 materials as a reference sample for the NMR characterization of small organic molecules confined in mesoporous silica materials. DSC and solid state NMR experiments 13
10.1021/jp910622m 2010 American Chemical Society Published on Web 04/21/2010
Characterization of Small Organic Molecules have evidenced a highly mobile behavior of the guest molecule at room temperature due to a confinement effect. We highlight in this paper that the sequences routinely used for solid state 13 C NMR characterization, such as cross-polarization-based techniques, are no longer suitable because of the poor efficiency of the CP process. We emphasize that this drawback can be circumvented by using NMR techniques derived from solution state NMR including NOE or J-coupling-based methods. We also point out that NOE enhancement of the 13C signal using magic angle spinning (MAS) power-gated techniques are particularly efficient in the sense that the signal-to-noise ratio can be doubled for some carbons. Furthermore, the long apparent transverse relaxation time T2′(1H) values allows performing MAS refocused {1H}-13C INEPT (insensitive nuclei enhanced by polarization transfer) experiments, where pumping and refocusing delays are rotor synchronized. Associated with 1H NOESY (nuclear Overhauser enhancement spectroscopy) experiments, we identify two benzoic acid populations in a large pore MCM-41 sample presenting two distinct pore sizes (45 and 110 Å). Finally, the reintroduction of the 1H-13C heteronuclear dipolar interaction is achieved by cooling down the samples to -35 °C. Interestingly, a 2D MAS {1H}-13C HETCOR experiment can be performed at a temperature above the solidification temperature determined by DSC measurements. More generally, we present an NMR approach that uses sequences derived from a solution state NMR adapted to solid state samples that are able to efficiently help in the characterization of organic molecules entrapped in porous materials and submitted to confinement effects.
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Figure 2. Schematic pulse sequences performed to record 13C MAS NMR spectra: (a) single pulse experiment, SPE; (b) single pulse experiment with power gate, SPE-PG; (c) cross-polarization, CP; (d) 13 C-{1H} MAS refocused INEPT.
Experimental Section MCM 41 Synthesis and Loading Procedure. MCM-41 with a mean pore diameter of 30 Å was obtained by mixing under stirring at room temperature H2O, NaOH, cetyltrimethylammonium bromide, and silica (Aerosil 200), in a given molar ratio of 20:0.25:0.1:1, respectively. MCM-41 with a mean pore diameter of 100 Å30 was prepared by mixing H2O, NaOH, cetyltrimethylammonium bromide, trimethylbenzene (swelling agent), and silica (Aerosil 200), with a molar ratio of 20:0.25: 0.1: 0.13:1, under stirring at room temperature. Then the mixture was heated at 120 °C for 24 h. The resulting white powders were filtered and plentifully washed with distilled water up to neutral pH, then dried at 70 °C for 48 h or 1 week, for the 30 and 100 Å material, respectively. The powders are then calcined at 600 °C for 6 h under air flux to remove the surfactant. Calcined MCM-41 powders are loaded with a solution of benzoic acid in ethanol (∼0.310 mol · L-1) through the “incipient wetness” procedure previously described.16 Four successive impregnations of 0.500 g of MCM-41 with a small amount of the solution are performed. The solvent is removed between two impregnations by heating at 70 °C overnight. Samples are quickly washed with ethanol to remove the excess of crystallized benzoic acid. The MCM-41 30 and 100 Å benzoic acid loaded samples will be referred in the forthcoming text as BA-30 and BA-100. Samples Characterization. Nitrogen adsorption/desorption isotherms were recorded at 77 K with a Micromeritics ASAP 2000 apparatus, after activation of the sample under vacuum (1 × 10-3 Torr) at 110 °C for the calcined samples or at room temperature during 15 h for the loaded samples. The specific surface area SBET is calculated according to the standard BET method,31 while the mean pore diameter is estimated by the BJH method at desorption.32
Thermogravimetric analyses (TGA) were carried out on a TA equipment SDT 2960 and on a SETARAM TG-DTA Instruments under an air flow with a heating rate of 5 °C · min-1 up to 1000 °C. DSC measurements were carried out under a dynamic nitrogen atmosphere with a heating rate of 20 °C · min-1 on a TA Instruments DSC 2010 calorimeter equipped with a liquid nitrogen controlled cooling accessory from -120 to 150 °C. Room temperature 1H and 13C solid state NMR experiments were performed on a AV300 Bruker spectrometer operating at ν(1H) ) 300.13 MHz and ν(13C) ) 75.48 MHz. Zirconia rotors (4 mm) were spun at a MAS frequency νMAS of 14 kHz. The 13 C NMR spectra were recorded using different sequences schematically described in Figure 2: (a) 13C single pulse experiment, SPE, (b) 13C single pulse experiment with {1H} power gate decoupling, namely, waltz-1633 during recycle delay, SPE-PG, (c) {1H}-13C cross-polarization, CP, (d) {1H}-13C refocused INEPT. All 13C spectra were recorded with 1H TPPM decoupling34 during acquisition (typically νRF(1H) ) 60 kHz). For SPE-PG experiments, the low power {1H} radio frequency was set to 2.5 kHz. Refocused INEPT experiments were recorded at 14 kHz with rotor synchronized pumping (∆1) and refocusing (∆2) delays.35 The 1H NOESY experiment, schematically described in Figure 8 was recorded at 14 kHz with a rotor synchronized mixing time (∆mix). Recycle delay (RD) for 1H and 13C NMR experiments was set to 2 s. Low temperature experiments were performed using a BCU-Xtreme Bruker accessory to regulate temperature down to -35 °C. 1H MAS and 13C CP MAS spectra were recorded at νMAS ) 4740 Hz with RD ) 10 s, because of longer T1(1H) at low temperature. The contact time for the {1H}-13C HETCOR experiment was set to 1 ms. Temperature calibration was achieved using lead nitrate Pb(NO3)2.36 No temperature regulation was used for
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Figure 4. DSC measurement of BA-30.
Figure 3. (a) Nitrogen adsorption-desorption isotherms and (b) pore diameter distribution for both calcined (MCM-41 30 Å and MCM-41 100 Å) and loaded samples (BA-30 and BA-100).
experiments recorded at room temperature. The chemical shift reference (0 ppm) for 1H and 13C was tetramethylsilane (TMS). Results Porosity Characterization and TG Measurements. Nitrogen adsorption/desorption isotherms of calcined MCM-41 30 Å sample are presented in Figure 3. According to the IUPAC classification, this material exhibits a type IV isotherm and possesses a narrow distribution of uniform pores with a maximum of the repartition at 25 Å. The specific surface area (SBET), evaluated by BET calculations is 950 m2 · g-1. The mean pore diameter is 30 Å and the mesoporous volume (Vp) is 0.85 cm3 · g-1. Nitrogen isotherm of BA-30 is of type II, characteristic of nonporous materials that suggests the encapsulation of benzoic acid and the total pore filling. According to the IUPAC classification, the calcined MCM41 100 Å material exhibits a type IV isotherm (Figure 3). The specific surface area (SBET) is 750 m2 · g-1. The mean pore diameter is 100 Å, and a bimodal distribution of pore sizes is evidenced with ca. 80% of large pores having a maximum at 110 Å and ca. 20% of smaller pores having a maximum at 45 Å. The mesoporous volume (Vp) is 2.25 cm3 · g-1. Nitrogen isotherms of the loaded sample BA-100 keeps a type IV shape with a decrease of 46% in the specific surface area (400 m2 · g-1) and 37% in the mesoporous volume (1.40 cm3 · g-1) that also suggests the successful encapsulation of benzoic acid. We note that the porous distribution is still bimodal with maxima at 35 Å (10%) and 95 Å (90%). The amount of benzoic acid in BA-30 and BA-100 were determined by TG measurements. BA-30 and BA-100 contain 680 and 490 mg of benzoic acid per gram of silica, respectively.
Characteristic porosity parameters of the samples and the amount of incorporated benzoic acid are summarized in Table 1. DSC Measurement. Figure 4 depicts the DSC measurement done on BA-30. We note a variation of the heat flow at -55 °C which is similar to a glass transition (Tg) in a polymer. This thermal event is not detected on unloaded MCM-41 materials while pure benzoic acid, which is a crystalline solid, shows a melting temperature at 125 °C. A similar result is obtained on BA-100 where the Tg is measured at -58 °C (Table 1). We associate these temperatures to a phase transition from an amorphous solid phase to a fluid phase, characteristic of the entrapped benzoic acid. This phenomenon is a consequence of the confinement effect that depresses phase transitions temperatures and is commonly observed for confined molecules in porous materials.1 Furthermore, we do not observe any exothermic peak around +125 °C for both BA-30 and BA-100, indicating that benzoic acid did not recrystallize in our materials, which is confirmed by X-ray diffraction data (not shown). Thus the totality of benzoic acid in BA-30 and BA-100 is confined in the pores and is under a fluid state at room temperature. Solid State NMR. In order to characterize in detail the guest molecule, solid state NMR spectroscopy experiments have been carried out. 13C MAS NMR spectra of BA-30 recorded at room temperature with various techniques described in the Experimental Section are shown in Figure 5 with peak assignments. The standard solid state CP MAS technique (tCP ) 3 ms and νMAS ) 5 kHz) is particularly inefficient to record 13C spectra with significant signal-to-noise ratio S/N because of a confinement effect that implies a weak heteronuclear 1H-13C dipolar interaction due to the high mobility of the molecules. This was already observed for benzoic acid blended with folded sheet mesoporous material.37 Even if the SPE sequence is more efficient, best results in terms of S/N are obtained with the SPEPG sequence that is routinely used in solution state NMR. For this latter sequence a continuous low power (2.5 kHz) proton decoupling is applied during the recycle delay allowing 1H-13C cross relaxation. Thus, using power gated conditions the 13C signal is enhanced by a heteronuclear 1H-13C nuclear Overhauser effect (NOE). High power proton decoupling (60 kHz)
TABLE 1: Porosity Characteristics (BET Surface, SBET, Porous Volume, VP, and Average Pore Diameter), Amount of Incorporated Benzoic Acid, Number of Molecules per nm2, and Glass Transition Temperature (Tg) for Unloaded (MCM-41 30 Å, MCM-41 100 Å) and Loaded Materials (BA-30, BA-100)
sample
SBET ((10 m2 · g-1)
VP ((0.05 cm3 · g-1)
MCM-41, 30 Å BA-30 MCM-41, 100 Å BA-100
950 160 750 400
0.85 0.15 2.25 1.40
average pore diameter [maxima of the porous distribution] ((2 Å)
amount of benzoic acid ((10 mg · g-1)
no. of benzoic acid per nm2
Tg ((1 °C)
680
3.53
-55
490
3.22
-58
30 [25] 100 [110;45] 100 [95;35]
Characterization of Small Organic Molecules
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η ) (S - S0)/S0
Figure 5. 13C MAS NMR spectra of BA-30 (with assignments) recorded using (a) SPE-PG, (b) SPE, and (c) CP (tCP ) 3 ms) sequences (number of scans NS ) 288 for each spectrum).
TABLE 2: 1H and 13C Isotropic Chemical Shifts (δiso) and 1 H Apparent Transverse Relaxation Times T2′(1H) for BA-30 and BA-100 and 13C NOE Enhancement Factor η for BA-30, BA-100, and Benzoic Acid in CDCl3 1
1
δiso( H) T2′(1H) in ms
BA-30 BA-100 BA-30 BA-100
H Site H3/H7
H4/H6
H5
7.6 8.0/7.6 22.7 16.7/20.2
6.9 7.3/6.8 18.5 14.7/16.2
7.0 7.4/7.0 19.7 16.0/15.8
13
C Site
δiso(13C) BA-30 BA-100 η BA-30 BA-100 benzoic acid in CDCl3
C1
C2
173.3 173.4 0.39 0.21 1.09
130.5 130.6 0.28 0.16 0.4
C3/C7 C4/C6 131.5 131.5 0.88 0.76 1.85
129.8 129.6 0.89 0.84 1.81
C5 135.1 134.9 0.69 0.65 1.94
is applied during acquisition to remove residual 1H-13C dipolar interaction and to make sure that acquisition conditions are identical for safe comparison between SPE and SPE-PG spectra. The NOE enhancement factor η for a nuclear spin S (following saturation of the I spin reservoir sufficiently long to allow the S spins to reach the steady state) is defined considering the relative values of the transition probabilities38
η ) (γI /γS)(W2 - W0)/(2W1 + W2 + W0) where γI and γS are the gyromagnetic ratios of I and S nucleis respectively, and W2, W1, and W0 are the second, first, and zero quantum transitions rates, respectively. The NOE between coupled I and S spins is maximized for molecular motions with a fast correlation time (τC < 10-10 s). In this extreme narrowing motional regime, η reaches a theoretical maximum value of 1.99 for 1H-13C NOE. Usually this phenomenon is not observed in the solid state as the molecular reorientation time is too long or inexistent (η ) 0). In Table 2 we report η for BA-30, BA-100, and a CDCl3 solution of benzoic acid according to the definition
where S refers to the 13C intensity following proton saturation, i.e., 13C intensity corresponding to SPE-PG experiment, while S0 is the 13C intensity without proton irradiation prior to excitation, i.e., 13C intensity corresponding to the SPE experiment. The η values found for each carbon site are significant for confined benzoic acid and are ranging from 0.28 to 0.89 and from 0.12 to 0.89 for BA-30 and BA-100, respectively (Table 2). For bulk benzoic acid, the NOE enhancement is not observed (η ) 0), as expected. Finally, highest η values are found for BA in CDCl3 solution with a minimum value of 0.4 for the ipso carbon (C2) which is not protonated and a maximum value of 1.94, close to the theoretical value in the extreme narrowing motional regime, for the carbon in the para position (C5). The intermediate η values observed for BA-30 and BA10 correspond to moderate correlation times of the confined molecules and are characteristic of the viscous-like behavior at room temperature. Such 1H-13C cross-relaxation phenomenon was observed in the solid state for polymers39 or mobile organic compounds40 and similar intermediate NOE enhancement factors were determined. Room temperature 1H MAS spectrum of BA-30 is presented in Figure 6a,b. The extremely sharp lines observed confirm a highly mobile behavior of the molecules within the pores and can be safely assigned to protons in ortho (δ(C3/C7) ) 7.6 ppm), meta (δ(C4/C6) ) 6.9 ppm), and para positions (δ(C5) ) 7.0 ppm). Furthermore, a shoulder is observed at 7.9 ppm for BA30 arising from the silanols SiOH and is easily evidenced by 2D1H-29Si CP MAS experiments (see Supporting Information). It is worth noting that the proton signal coming from the COOH group is not visible implying that this proton is in chemical exchange at room temperature as already observed for fatty acids confined in MCM-41.41 For comparison purposes, the 1H MAS spectrum of pure benzoic acid is given in Figure 6c where the carboxylic proton signal is visible at 13 ppm as a broad resonance. This spectrum is characteristic of a solid organic sample as it is broadened by the presence of a strong homonuclear 1H-1H dipolar coupling42 which is absent in the case of confined molecules where the high mobility leads to the average of this interaction. Measurements of apparent transverse relaxation times T2′(1H) were done at νMAS ) 14 kHz using a solid echo sequence: π/2-τ-π-τ acquisition with τ corresponding to an entire number of rotor cycles (Table 2). The long values obtained (from 18.5 to 22.7 ms for BA-30 and from 15.8 to 20.2 ms for BA100) are unusual for a solid state sample and are once again characteristic of the highly mobile behavior of the entrapped molecules. For comparison, the T2′(1H) values of bulk benzoic acid are around 150 µs whereas in solution (CDCl3) these values are comprised between 600 and 700 ms. The T2′(1H) values of the confined benzoic acid that are intermediate between those observed in solution and in the solid state are consistent with the previously reported η values which indicates that MCM-41 trapped benzoic acid acts more as a viscous fluid than a pure liquid. In order to realize a spectral assignment of the encapsulated molecules, 2D 1H-13C NMR experiments are usually required. Although two-dimensional dipolar {1H}-13C based experiments such as HETCOR are prevented due to the mobility, the long T2′(1H) values allow running NMR experiments derived from solution state NMR that require long coherence lifetimes such as 13C-{1H} refocused INEPT. While the CP process is a through space magnetization transfer, the INEPT experiment
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Figure 6. 1H MAS spectrum of (a) bulk benzoic acid, (b) BA-30, and (c) zoom on the 6-9 ppm region (with assignments). Asterisks (*) denote residual ethoxy groups originating from the silica precursor.
is based on through bonds magnetization transfer using J couplings. This experiment needs coherence lifetimes related to 1/(4 × 1JCH) that are comprised between 1.6 ms for aromatic and 2 ms for aliphatic carbons (corresponding to 1JCH ) 155 and 125 Hz for aromatic and aliphatic carbons, respectively). These long magnetization evolutions are usually unreachable in solid state NMR because of too short transverse relaxation times. Figure 7a displays the 2D 13C-{1H} MAS refocused INEPT spectrum of BA-30 recorded at νMAS ) 14 kHz where pumping (∆1) and refocusing delays (∆2) were rotor synchronized to maximize S/N and to avoid reintroduction of dipolar interactions (∆1 ) ∆2 ) 1.57 ms). Each carbon C3/C7, C4/C6, or C5 correlates with a single proton resonance which corresponds to H3/H7, H4/H6, or H5, respectively, whereas the carboxylic carbon C1 and the ipso carbon C2 are not visible due to the absence of directly bonded protons. The 2D 13C-{1H} MAS refocused INEPT characterization of BA-100 is depicted in Figure 7b. Interestingly, we observe that each 13C signal is correlated to two distinct 1H peaks. Knowing that ∆1 and ∆2 are chosen to match with the 1JCH values, i.e., the through-bond magnetization transfer to 13C is coming exclusively from directly bonded protons, this result shows unambiguously the presence of two distinct types of guest molecules corresponding to two different populations that we named A and B. The relative percentages of these two populations estimated by deconvolution of the 1H MAS spectrum (not shown) are 57% and 43% for populations A and B, respectively. To explore the spatial proximities between the two types of benzoic acid in BA-100, we used 2D 1H NOESY experiment. This sequence is widely used in the field of solution state NMR and is based on nuclei magnetization exchange through homonuclear NOE. In our case, the mobility which is intermediate
Figure 7. Two-dimensional 13C-{1H} MAS refocused INEPT spectrum of (a) BA-30 and (b) BA-100 (with ∆1 ) ∆2 ) 1.57 ms, νMAS ) 14 kHz, NS ) 128, and TD(F1) ) 240).
between the liquid and the solid state, as previously mentioned, implies that this experiment is probably driven by NOE at short mixing time (
