Deuterium Exchange

Jul 13, 2009 - ACS Journals. ACS eBooks; C&EN Global Enterprise .... 2. Hydrogen/Deuterium Exchange with Silanol Groups on the Surface of Fumed Silica...
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J. Phys. Chem. C 2009, 113, 13894–13898

Inverse Reaction Chromatography. 2. Hydrogen/Deuterium Exchange with Silanol Groups on the Surface of Fumed Silica V. A. Bakaev* and C. G. Pantano Materials Research Institute, The PennsylVania State UniVersity, UniVersity Park, PennsylVania ReceiVed: December 12, 2008; ReVised Manuscript ReceiVed: June 23, 2009

Hydrogen/deuterium (H/D) exchange between silanol groups on the surface of fumed silica, and deuterium oxide in the carrier gas, has been studied by a GC/MS chromatographic method using a residual gas analyzer as the mass sensitive detector (MSD) in GC/MS. The response of the MSD to the injection of deuterium oxide (DOD) at the inlet of the column packed with a mixture of fumed silica with inert silica particles showed water (HOH), HOD, and DOD. Each of their elution profiles were found to agree semiquantitatively with those obtained by computer simulation in the companion paper (Bakaev, V. A. J. Phys. Chem. C 2009). The concentration of silanol groups on the silica surface could be obtained from this experiment, and yielded a reasonable value. The rate constant of H/D exchange was also evaluated by comparison of computer simulation with experiment. It is concluded that this method can be applied to the study of other oxides surfaces, and possibly to various organic molecules, including biomolecules, adsorbed on solid surfaces. Introduction The hydrogen/deuterium exchange method is based on the fact that the rates of H/D exchange (hydrogen exchange (HX)1) can differ widely between the active sites of protein molecules. It also has been used for the determination of the silanol group concentration2 and the kinetics of H/D exchange3,4 on the surfaces of amorphous silica. At the present time, the main technique for H/D exchange is based on mass spectrometry. In this experimental study, we take a somewhat different approach using inverse reactive chromatography (IRC) combined with mass sensitive detection. According to ref 5, the first theoretical part of this work dealing with computer simulations, reactive chromatography is a chromatographic method accompanied by chemical reactions. The reactions occur in the mobile phase, while the stationary phase, which may contain a catalyst, remains intact. This new IRC method deals not with the mobile phase (as is the case for conventional reaction chromatography) but with transformation of the stationary phase as a result of its chemical interaction with the mobile phase. Of particular interest in this paper is the H/D exchange reaction between the deuterium oxide (DOD) and HOD in the carrier gas, and silanol groups on the silica surface. The chromatographic technique used here is basically GC/ MS. The peculiarity arises in using the split/splitless inlet of the GC. The computer simulation in the first part of this work5 is based on the concept of frontal chromatography whereby the solute concentration is a constant at the inlet. In contrast, the inlet of a conventional GC, such as we used in this work, is designed for creating narrow pulses of concentration. We found that, by maintaining the inlet at an unusually low (for analytic chromatography) temperature, a wide pulse of concentration is generated at the inlet which can be treated by computer simulation as in the case of frontal chromatography.5 Another issue in this work is the H/D exchange that occurs inside the mass selective detector (MSD) of the GC/MS which was partially overcome by introducing appropriate corrections. In the end, we obtained experimental elution profiles (EPs) which semiquantitatively agree with those obtained by the

computer simulations in ref 5. The comparison of simulated EPs with experiment allows one to determine the concentration of silanol groups on the surface and the rate constant of H/D exchange, although the rate constant of H/D can be considered only as a rough estimate of the real value at this stage. Experimental Section A standard GC (Agilent 6890N) used earlier6 was equipped with a homemade MSD. The MSD consisted of a residual gas analyzer RGA100 (Standford Research Systems), turbomolecular vacuum station, and a restrictor capillary as an interface between the packed column and the MSD. The RGA100 is a relatively simple quadrupole mass spectrometer with electron ionization energy of 70 eV and electron multiplier gain established at 970. The RGA100 was connected to the vacuum station through a CF Tee flange of 35 mm inner diameter (i.d.). The RGA100 and the adapter flange connected to the restrictor capillary through a graphite ferrule are on the opposite sides of the Tee flange with the turbomolecular pump in the middle. The restrictor capillary is a fused silica capillary 25 µm i.d. and 16 cm length (Scientific Instrument Services cat. #062442). The restrictor capillary enters a wider capillary of 0.53 mm i.d. (Restek cat. #70081) connected to the output of the packed column. The wider capillary is inserted in copper tubing 1.6 mm i.d. (Restek cat. #21590) which together with the adapter flange was heated by a heater tape. The packed column6 is basically a glass tube 3.9 mm i.d., 23 cm long packed either with pure deactivated fused silica beads (inactive calibration column) or with a mixture of deactivated silica beads and 0.5 wt % of fumed silica (active column). The deactivated fused silica beads (Restek cat. #20791) are, in fact, not beads but irregularly shaped particles of fused silica 60/80 mesh size. Their average size can be estimated as 220 µm with surface area of 0.01 m2/g. Fumed silica (Sigma-Aldrich cat. #S5505) is a fine powder of silica particles 14 nm in size having surface area of 200 ( 25 m2/g. Since the packing of the active column contains only 0.5 wt % of fumed silica, its effective surface area is 1 m2/g, which is 100 times larger than the surface area of the calibration column. The total weight of both

10.1021/jp810984r CCC: $40.75  2009 American Chemical Society Published on Web 07/13/2009

Inverse Reaction Chromatography

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Figure 1. Elution profile after injection of water into calibration column at 100 °C.

the calibration and active column packing was about 3 g. Thus, the total surface area of silica in the active column was about 3 m 2. As in ref 6, the packed columns were connected to the split/ splitless injector through a guard column which was Silcosteel deactivated fused silica lined stainless steel tubing of 0.280 mm i.d., 6 m length (Restek old cat. #20572). The guard column was necessary because the packed columns had insufficient hydrodynamic resistance to the carrier gas, for the Agilent 6890N electronic pneumatic control (EPC) system, which is designed mainly for capillary columns. The split/splitless inlet had the splitless liner (Agilent #5062-3587) and worked in the split regime with a very low split ratio of 0.3. The total flow was 15.3 mL/min, the split flow was 3.6 mL/min, and the purge flow was 1.3 mL/min. The inlet temperature of 80 °C was below the oven (column) temperature of 100 °C, which is unusual for the conventional chromatography. The injection of 1 µL of water or deuterium oxide was performed with the Hamilton 7001 KH 1 µL syringe with a spacer so that the length of the needle entering the inlet was 37 cm. Results and Discussion Figure 1 shows the response of the MSD to the injection of 1 µL of HOH in the inlet of GC with the calibration column. This plot is already corrected for the background (see discussion of Figure 2). The symbols in this figure show the partial pressure of ionized water molecules corresponding to their molecular mass of 18 amu (here and below, the charges of ions are always unity and the ion masses are in amu). The partial pressure of OH, the main fragment of HOH, is considerablesca. 0.37 of that of HOHsbut is not shown in Figure 1. It should be emphasized that no signals for HOD and DOD were observed after the injection of HOH (in contrast with Figure 2). Due to the small split ratio, the amount of water injected into the calibration column was 0.74 µL. One can see from Figure 1 that the real time for removal of the injected water from the inlet is about 220 s (versus 14.5 s expected for the inlet temperature of 80 °C). This is the time required for the flow of carrier gas through the inlet to wash out the injected amount of water condensed on the walls of the liner. This means that the actual vapor pressure of water in the inlet was much lower than that corresponding to the inlet temperature. This is because the inlet temperature is, in fact, the temperature of its metal block maintained by the GC, while the actual temperature of the water droplets sitting on the liner in the inlet is much lower than that temperature. To explain the duration of the pulse shown in

Figure 2. Original elution profiles after injection of deuterium oxide into calibration column at 100 °C.

Figure 3. Corrected elution profiles after injection of deuterium oxide into calibration column at 100 °C.

Figure 1, the water temperature in the inlet is likely about 23 °C given the flow of the carrier gas through the inlet of about 14 mL/min. The processes in the split/splitless inlets of the GC after an injection of liquid are not well understood. It is assumed that the liquid injected in an inlet sits on the liner wall, its actual temperature being determined not by the temperature of the inlet metal block but by the balance between the cooling due to evaporation and heating due to the heat conductivity through the inlet walls.7 The solid line in Figure 1 is an approximation of the inlet partial pressure profile that was used in the computer simulations as reproduced in Figure 2 of the theoretical part of this work.5 The response of the MSD to the injection of DOD into the calibration column is shown in Figure 2. The main distinction of Figure 2 from Figure 1 are the elution profiles (EPs) of HOD (m ) 19) and HOH (m ) 18). These are not the result of the H/D exchange in the calibration column because the quantity of protons on the surface of that column is much smaller than the quantity of D atoms in the mobile phase. The H/D exchange in the calibration column manifests itself as sharp peaks of the m ) 18 and m ) 17 EPs at the shock position (87 s) in Figure 2 (cf. also HOH EP in Figure 3). However, the main part of these EPs resulted from H/D exchange inside the MSD. This is because the partial pressure of DOD is so low that the quantity of water (HOH) adsorbed on the MSD walls is much larger than the quantity of DOD molecules in the gas phase.

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Bakaev and Pantano

P(DOD) ) 1.46P0(DOD);

P(HOH) ) P0(HOH) 0.31P0(HOD)

P(HOD) ) 1.31P0(HOD) - 0.46P0(DOD)

Figure 4. Original elution profiles after injection of deuterium oxide into active column at 100 °C.

To make corrections for the H/D exchange in the MSD, it is necessary to reduce the EPs in Figure 2 to the background levels. The injection of DOD took place at t ) 60 s in Figure 2. During the first minute, only the background was recorded. The last 60 s of the EPs in Figure 2 were also used for determining the background. Thus, the background correction amounts to subtraction of the linearly interpolated background values from the EPs in Figure 2 and to shifting the time origin 60 s to the right. After the background correction, the HOH and HOD EPs in Figure 2 can be calibrated. The EP corresponding to m ) 18 in Figure 2 results not only from HOH but also from OD. The latter arise from the fragmentation of DOD molecules during ionization. From other experiments, it was determined that the partial pressure of OH (not shown in Figure 1) is 0.375 of the partial pressure of HOH (independent of time). We assume equal values for DOD and HOD. In the latter case, however, only half of the fragments are OD; the other half are OH. Thus, the EP of HOH can be obtained from that corresponding to m ) 18 in Figure 2 by subtracting 0.375 of the EP corresponding to m ) 20, and 0.1875 of the EP corresponding to m ) 19 in Figure 2. Accordingly, the EP corresponding to m ) 17 in Figure 2 should be eliminated by subtracting 0.375 of the EP of HOH (obtained after the above correction) and 0.1875 of that corresponding to m ) 19 in Figure 2. The corrected EPs of HOH, HOD, and DOD are shown in Figure 3. The partial vapor pressure of OH is also shown for comparison. It did not disappear as one would expect, but is slightly negative. This is likely due to overcorrection of the m ) 17 and m ) 18 EPs in Figure 2. Now, Figure 3 can be used to determine corrections for the H/D exchange in the MSD. These are the ratio of areas under the plot of HOD to that of DOD in Figure 3, which is 0.46, and that of HOH to HOD, which is 0.31. The MSD response to the injection of DOD into the active column is shown in Figure 4. Comparison with Figure 2 shows that the EP corresponding to m ) 18 (whose main source is HOH) is much larger in Figure 4 than in Figure 2. To obtain EPs of HOH, HOD, and DOD from those in Figure 4, three corrections were made. The first two corrections are the reduction to background and the extraction of fragments (as used for Figure 3). To correct these EPs for the H/D exchange in MSD, let the uncorrected EPs for the H/D exchange in the MSD be P0(HOH), P0(HOD), P0(DOD), whereas P(HOH), P(HOD), P(DOD)are the corrected partial pressures. Then, for all times

This essentially returns 46% of the amount transferred to the HOD EP (in the MSD) back to the DOD EP. The same is done to the HOD and HOH EPs. The EPs of Figure 4, corrected by the above methods, are compared in Figure 5 with the results of the computer simulations (taken from Figure 3 of the companion paper5). The correction procedure does not change the total area under all the EPs of HOH, HOD, and DOD. The total area of all EPs can be used to check the MSD constant. In the case of Figure 5, the constant is 6.6 × 108 nmol · Torr-1 s-1, which is the ratio of the injected amount of DOD to the total area under all EPs. The constant is slightly less than that determined with the calibration column from the experiments similar to those shown in Figures 1-3 (7 × 108 nmol · Torr-1 s-1). This is because the number of injected molecules which are still adsorbed in the column is larger in the active column than in the calibration column (at 100 °C). The area under the HOH EP × 2 plus the area under the HOD EP (in Figure 5) gives the total number of protons eluted from the column. The comparison with computer simulation (see below) shows that almost all OH-groups on the silica surface exchanged their protons to deuterium atoms. Thus, the total number of protons eluted from the column divided by the total area of the fumed silica in the column (3 m2) gives the concentration of silanol groups on the fumed silica surface, and corresponds to 7 silanols per nm2. The concentration of silanol groups on silica has also been determined by the static H/D exchange method.2 Basically, the method consists of adsorbing an excessive quantity of DOD on the silica surface, desorbing the H/D exchanged water, and measuring by a mass spectrometer the quantity of H2 (by decomposing water on the heated uranium foil) in the desorption products. It was shown that the concentration of silanol groups on various fully hydroxylated silica surfaces varies from 4.2 to 5.7 silanols per nm2 and was declared a physicochemical

Figure 5. Comparison of corrected EPs (symbols) with those obtained by computer simulation (lines). Diamonds, HOH; triangles, DOD; squares, HOD.

Inverse Reaction Chromatography constant.2 Our value is 23% higher than the upper bound of this range. However, it is well-known that these surface concentration values vary depending on the detailed processing and history of the silica samples, so we believe it is a reasonable estimate which validates the reliability of our new method. The most probable source of any errors in our case is the H/D exchange inside MSD. In particular, Figure 5 suggested that the experimental HOD EP is overestimated, and lowering it would bring our silanol concentration in better agreement with ref 2. The experimental accuracy of this method can be considerably improved with the substitution of a modern commercial MSD such as, for example, the Agilent 5975C inert MSD. It allows one to raise the temperature of the MSD. A comparison of the experimental EPs in Figure 5 with those obtained by computer simulations in the accompanying paper5 shows semiquantitative agreement between them. The best agreement is between the EPs of HOH and DOD; the worst is with the EP of HOD. All three simulated EPs cross at one point. This is not the case for experimental EPs. The bell-shaped experimental HOD EP has a step at its left side which is absent in its simulated counterpart. The origin of this step is not clear. It might be a peculiarity of the H/D exchange in our MSD. The EPs in Figure 35 were obtained by fitting the shock (jump) of the simulated HOH profile (at the time of 27 s, which corresponds to the steep raise of the experimental EP) with values characteristic of water adsorption. This allows one to evaluate water adsorption at the silica surface at the temperature of this experiment. (The methods for measuring isotherms and heats of adsorption by inverse gas chromatography are described in ref 6.) The height of the plateau of the simulated HOH EP, which is connected with the MSD calibration constant, was fit to the maximum of the experimental HOH EP. In this way, the density of silanol groups on the silica surface can be determined as presented above. Similarly, the rate constant of the H/D exchange was selected to be k ) 60 M-1 s-1 (dm3 mol-1 s-1). The simulated EPs of HOH obtained with different values of k are shown in Figure 4 in the companion paper.5 It is clear from comparison of that figure with the experimental EPs in Figure 5 that the value of k ) 10 M-1 s-1 is definitely too low. The value of k ) 20 M-1 s-1 is probably also less than the real rate constant, but the values of k ) 40, 60, or 80 M-1 s-1 give the simulated EPs whose deviation from experimental ones are within the limits of experimental error. Moreover, it is seen from Figure 45 that, when values of k increase above 60 M-1 s-1, the simulated EPs converge. Thus, at this stage we can only establish that k is definitely larger than 20 M-1 s-1 and a reasonable value is k ) 60 M-1 s-1. The kinetics of the H/D exchange has also been studied on silica gel surfaces by the static method.3,4 It consists of bringing the silica gel in contact with the DOD vapor in a closed vial and measuring the change of the vapor composition with time. The measurement was achieved by periodical taking samples of vapor and measuring their composition with a mass spectrometer. The time scale of this exchange is on the order of hours, whereas our time scale is on the order of minutes. Especially slow kinetics (120 h equilibration time) was observed for a fine porous silica gel.4 This suggests that the kinetics of H/D exchange studied in refs 3, 4 was determined not by the H/D exchange per se but by diffusion of molecules in the stagnant gas, between grains of the sample, or inside the porous sample. In contrast to that static adsorption technique, our method of inverse chromatography is usually called a dynamic

J. Phys. Chem. C, Vol. 113, No. 31, 2009 13897 adsorption technique. Its important advantage over the static method consists of diminishing the effects of diffusion in the kinetics of the H/D exchange. This is because the time scale of diffusion is proportional to R2/Deff where Deff is an effective diffusion coefficient and R is the characteristic length scale for diffusion. In the static adsorption techniques, R is on the order of the vial size for diffusion through the stagnant gas phase, whereas in the dynamic adsorption technique, R is the thickness of the stagnant gas film surrounding the grains of adsorbent (which is at least 3 orders of magnitude less than the typical vial size). The dynamic adsorption technique does not eliminate diffusion inside the porous nanostructure of an adsorbent, but the fumed silica used in this work is nonporous. The values of rate constant for H/D exchange for biomolecules in the gas phase is in the range 109-1012 M-1 s-1.8 These values are much higher than the value of the rate constant estimated above. On the other hand, the time scale of the H/D exchange for H in the main chain amide NH in liquid D2O is on the order of minutes or even hours,1 which suggests rate constants lower than ours. Thus, the rate constant of the H/D exchange in the adsorbed state estimated above should not be considered unrealistically low. Conclusion The main result of this work is summarized in Figure 5. It shows that the theoretical model of H/D exchange in a chromatographic column qualitatively agrees with experiment. Both the theoretical model and the experiment include certain approximations, and their qualitative agreement shows that those approximations grasp the essential features of the phenomenon under consideration. However, both the theoretical model and the experiment leave considerable room for improvement. It seems that the first candidate for improvement is to replace our MSD with a modern commercial one. It is probably worth commenting about where the inverse reaction chromatography can be applied. Clearly, it can be used for determination of the OH group concentration on the surfaces of various oxides as has already been done for the silanol concentration. It may turn out that our dynamic method is simply more convenient than the static methods which have been used earlier. In general, the comparison of experiment with computer simulation (performed here) would not be needed. On the other hand, such a comparison is essential when one tries to determine the H/D exchange rate constants for OH groups on the surfaces. One should expect different rate constants for different oxides because these constants differ by several orders of magnitude, e.g., for various biomolecules both in the gas phase and in solution. The rate constants also depend on the exchange reagents: one can use not only D2O as an exchange reagent but also, for example CD3OD, or other deuterium-exchanged molecules. Another possibility is to immobilize biomolecules on a surface of column packing and study the H/D exchange of these molecules in the adsorbed state. In addition, the H/D exchange is the simplest chemical reaction one can study by this inverse reaction chromatography. Other chemical reactions such as, for example, etherification of the silica surfaces by alcohols, in principle, can also be studied by this method. However, in this case, the theoretical model considered in the first part of our work would require considerable modification because the basic assumption of that model was the identical adsorption isotherms for all components in the mobile phase, which is adequate only for isotopes.

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Finally, it should be mentioned that this work is only the first, preliminary, step in the experimental application of the inverse reaction chromatography. The next step will include two experimental improvements. First, as mentioned above, we are going to switch from our homemade MSD to a commercial one. Second, we are going to substitute the injection into a relatively cold inlet used in this work by a frontal chromatography injection system which accurately maintains constant vapor concentration at the inlet of the column. Acknowledgment. This work was supported with funding from the National Science Foundation, through grant DMR0809657.

Bakaev and Pantano References and Notes (1) Englander, S. W. J. Am. Soc. Mass Spectrom. 2006, 17, 1481. (2) Zhuravlev, L. T. Langmuir 1987, 3, 316. (3) Gorelik, R. L.; Zhuravlev, L. T.; Kiselev, A. V. Kinet. Catal. 1971, 12, 450. (4) Gorelik, R. L.; Zhuravlev, L. T.; Kiselev, A. V.; Nikitin, Yu. S.; Oganesyan, E. B.; Shengelaya, K. Ya. Kolloid J USSR (Kolloidnii zhurnal, English translation) 1971, 33, 51. (5) Bakaev, V. A. J. Phys. Chem. C, 2009, DOI: 10.1021/jp810982w. (6) Bakaev, V. A.; Bakaeva, T. I.; Pantano, C. G. J. Phys. Chem. C 2007, 111, 7473. (7) Grob, K. Split and Splitless Injection for QuantitatiVe Gas Chromatography, 4th completely reVised edition; Wiley-VCH: Weinheim, 2001. (8) Campbell, S.; Rodgers, M. T.; Marzluff, E. M.; Bauchamp, J. L. J. Am. Chem. Soc. 1995, 117, 12840.

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