Anal. Chem. 1998, 70, 2623-2628
13
C Dynamic Nuclear Polarization: an Alternative Detector for Recycled-Flow NMR Experiments
S. Stevenson, T. Glass, and H. C. Dorn*
Department of Chemistry, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061
Static NMR experiments on insensitive nuclei (e.g., 13C) have been generally difficult because of low magnetogyric ratios and/or poor natural abundance, even at the highest magnetic fields available. One method of overcoming these sensitivity limitations is dynamic nuclear polarization (DNP), which can be utilized to enhance NMR signals relative to those obtained with static NMR detection. In this study, we report a recycled-flow DNP apparatus capable of monitoring 13C scalar-dominated enhancements 1-2 orders of magnitude greater than those obtainable using the conventional recycled-flow 13C NMR experiment. Specifically, a mixture of benzene and several chlorocarbons were continuously “recycled” through a 13C DNP spectrometer. Results indicate successful detection with the 13C DNP approach in scalar-dominated cases, where analytes could not be observed with conventional static or flow NMR detection in a reasonable length of time. In 1951, Suryan1 reported that increases in NMR signal strength could be achieved for flowing systems. From the 1950s to the 1970s, early flow NMR studies involved flow rate measurements, studies of flow dynamics, and measurements of spinlattice relaxation times (Tl’s).2-6 With the advent of pulsed FT techniques7 in the 1970s, it was possible to use flow NMR as a tool for investigating intermediates of chemical reactions.8-10 From the 1980s until the present, applications of flow NMR have rapidly spread, including the biological sciences.11-21 For example, in vivo 31P flow NMR studies14 of ATP metabolites in (1) Suryan, G. Proc. Indian Acad. Sci., Sect. A 1951, 337, 107-111. (2) Singer, J. R. Science 1959, 130, 1652. (3) Forsen, S.; Rupprecht, A. J. Chem. Phys. 1960, 33, 1888-1889. (4) Arnold, D. W.; Burichart, L. E. J. Appl. Phys. 1965, 36, 870. (5) Stejskal, E. O. J. Chem. Phys. 1965, 43, 3597. (6) McCormick, W. S.; Birkemeirer, W. P. Rev. Sci. Instrum. 1969, 40, 346. (7) Ernst, R. R.; Anderson, W. A. Rev. Sci. Instrum. 1966, 37, 93. (8) Grimaldi, J.; Baldo, J.; McMurray, C.; Sykes, B. D. J. Am. Chem. Soc. 1972, 94, 7641-7645. (9) Fyfe, C. A.; Ciciverra, M.; Damji, S. W. H. Acc. Chem. Res. 1978, 11, 277282. (10) Kuhne, R. O.; Schaffhauser, T.; Wokaun, A.; Ernst R. R. J. Magn. Reson. 1979, 35, 39-67. (11) Santos, H.; Turner, D. L. J. Magn. Reson. 1975, 68, 1201. (12) Ackerman, J. J. H.; Grove, T. H.; Wong, G. G.; Gadian, D. G.; Radda, G. R. Nature 1980, 283, 167. (13) Gonzalez-Mendez, R.; Wemmer, D.; Hahn, G.; Wade-Jardetsky, N.; Jardetzky O. Biochim. Biophys. Acta 1982, 720, 274-280. (14) Wyrwicz, A. M.; Schofield, J. C.; Burt, C. T. In Noninvasive Probes of Tissue Metabolism; Cohen, J. S., Ed.; Wiley: New York, NY, 1982; pp 149-171. (15) Albert, K.; Kruppa, G.; Zeller, K. P.; Bayer, E. Z. Naturforsch. 1984, 39c, 859. S0003-2700(97)01337-1 CCC: $15.00 Published on Web 05/09/1998
© 1998 American Chemical Society
blood have been reported. Similar recycled-flow NMR experiments with other high-sensitivity nuclei (e.g., 2H, 19F, etc.) have also been performed.12,17 For flow experiments22-27 involving less sensitive nuclei (13C, 15N, and 113Cd) with low natural abundances and/or low magnetogyric ratios, reasonable S/N ratios are more difficult to achieve. For this reason, it has often been necessary to incorporate isotopically labeled samples to obtain an acceptable signal-to-noise ratio. For example, Albert et al.15 have studied the in vivo metabolism of 13C-enriched phenacitin in an isolated, perfused rat liver. Wilkins et al.23,24,28 have previously suggested utilizing the recycled-flow approach as a method for “reducing” 13C spin-lattice relaxation times for the “slower relaxing” carbonyl and quaternary carbons for large-volume samples in natural abundance. More recently, Sudmeier and co-workers29 have developed a model which critically assesses the relative merits of the flow and recycled-flow NMR and DNP experiments. This latter group concludes that the recycled-flow DNP has a significant S/N advantage per unit time in comparison with the conventional recycled-flow or static NMR experiment. Furthermore, the magnitude of the DNP enhancement is proportional to the electron-to-nuclear magnetogyric ratio (γe/γn), and large increases in the signal-to-noise ratio for low γ nuclei can be readily predicted. For example, a 13C flow DNP detector30 has recently been directly coupled to chromatography (HPLC-13C DNP) for continuous detection of a number of chlorocarbons. In the present study, recycled-flow 13C DNP detection is employed for both the liquid-liquid intermolecular transfer (LLIT) (16) Chiancone, E.; Drakenberg, T.; Teleman, O.; Forsen, S. J. Mol. Biol. 1985, 185, 201-207. (17) Eleff, S. M.; Schnall, M. D.; Ligetti, L.; Osbakken, M.; Subramanian, V. H.; Chance, B.; Leigh, J. S., Jr. Magn. Reson. Med. 1988, 7, 412-424. (18) Song, S. K.; Hotchkiss, R. S.; Karl, I. E.; Ackerman, J. H. Magn. Reson. Med. 1992, 25, 67-77. (19) Thompson, S. N.; Platzer, E. G.; Lee, R. W. K. Magn. Reson. Med. 1992, 28, 311-317. (20) De Graaf, A. A.; Wittig, R. M.; Probst, U.; Strohhaecker, J.; Schoberth, S. M.; Sahm, H. J. Magn. Reson. 1992, 98, 654-659. (21) Chen, R.; Bailey, J. E. Biotechnol. Bioeng. 1993, 42, 215. (22) Bayer, E.; Albert, K. J. Chromatogr. 1984, 312, 91. (23) Laude, D.; Lee, R. W.-K.; Wilkins, C. L. Anal. Chem. 1985, 57, 1281-1285. (24) Laude, D.; Lee, R. W.-K.; Wilkins, C. L. Anal. Chem. 1985, 57, 1286-1290. (25) Albert, K.; Nieder, M.; Bayer, E.; Spraul, M. J. Chromatogr. 1985, 346, 17. (26) Albert, K.; Sudmeier, J. L.; Sawkat, A. M.; Bachovchin, W. W. Magn. Reson. Med. 1989, 11, 309. (27) Zhang, Y.; Laude, D. A. J. Magn. Reson. 1990, 51, 527. (28) Laude, D. A.; Lee, R. W.-K.; Wilkins, C. L. J. Magn. Reson. 1984, 60, 464469. (29) Sudmeyer, J. L.; Gunther U. L.; Albert, K., Bachovchin, W. W. J. Magn. Reson. (A) 1996, 118, 145-156. (30) Stevenson, S. A.; Dorn, H. C. Anal. Chem. 1994, 66, 2993-2999.
Analytical Chemistry, Vol. 70, No. 13, July 1, 1998 2623
Table 1. Composition of Mixture Used for Recycled-Flow Experiments compound chloroform trichloroethylene hexachloroethane hexachlorobenzene 1,3,5-trichlorobenzene tetrachloroethylene chlorobenzene benzene
13C
NMR δ (ppm)
77.5 116.7, 124.2 105.5 132.5 127.5, 135.8 120.6 126.6, 128.8, 129.9, 134.5 128.5
concn (M) 0.0447 0.0694 0.0714 0.0761 0.123 0.234 0.471 1.34
and solid-liquid intermolecular transfer (SLIT) techniques31-33 and critically compared with conventional recycled-flow NMR. Mixtures of chlorocarbons and hydrocarbons were dissolved in solvent (CCl4) and subsequently continuously recycled through a 13C DNP detector. The chlorocarbons were selected as analytes for this study because of potential environmental monitoring applications and the large scalar-dominated DNP enhancements usually observed30,31 for these compounds. With this technique, 13C NMR signal enhancements of 1-2 orders of magnitude have been established for the present recycled-flow DNP apparatus. Although 13C DNP observation was employed in the present study, applications of the recycled-flow DNP approach could also be extended to studies involving other nuclei (15N, 31P). EXPERIMENTAL SECTION Previously prepared31 silica-phase immobilized nitroxide (SPIN) radicals were employed for the solid-liquid intermolecular transfer (SLIT) experiments. For liquid-liquid intermolecular transfer (LLIT) studies,33 the radical TEMPO (Aldrich) was utilized as the unpaired electron source. HPLC grade carbon tetrachloride was used as the solvent and degassed with nitrogen gas to prevent dissolved oxygen from decreasing the DNP enhancements. The composition of the recycled-flow test mixture prepared for this study is presented in Table 1. All compounds and solvents contained 13C in natural abundance and were used directly from the manufacturers (Aldrich, EM Science, Fischer, Mallinckrodt) without further purification. The flow rates employed varied from 3.5 to 5.2 mL/min. The relatively large quantities of chlorobenzene and benzene were used to examine whether weak dipolar-dominated signals could be observed with the flow DNP technique. In contrast, chloroform, trichlorobenzene, trichloroethylene, hexachloroethane, hexachlorobenzene, and tetrachloroethylene were present in much smaller quantities because of the larger scalar-dominated enhancements expected in the recycled-flow DNP experiment. In addition, the latter three compounds are clearly examples where 1H NMR polarizationtransfer experiments are not readily feasible. A diagram of the recycled-flow 13C DNP apparatus is illustrated in Figure 1. To follow the flow DNP experiment, analytes from the test mixture enter the low magnetic field region A (0.33 T). A bed of SPIN radical sample was placed in an EPR flow cell and (31) Dorn, H. C.; Glass, T. E.; Gitti, R.; Tsai, K. H. Appl. Magn. Reson. 1991, 2, 9-27. (32) Tsai, K. H.; Dorn, H. C. Appl. Magn. Reson. 1990, 1, 231-254. (33) Dorn, H. C.; Gu, J.; Bethune, D. S.; Johnson, R. D.; Yannoni, C. S. Chem. Phys. Lett. 1993, 203, 549-554.
2624 Analytical Chemistry, Vol. 70, No. 13, July 1, 1998
Figure 1. Diagram of the
13C
DNP recycled-flow apparatus.
furnished the unpaired electron reservoir in region B. A klystron source (Bruker microwave bridge) was amplified by a Varian TWT amplifier, and the microwave power (5-15 W) was transferred to the microwave X-band TE102 cavity; saturation factors of 0.7-0.9 were easily obtained for the electron spin system. The total volume in region A was ∼160 µL. After polarization, the flowing bolus was quickly transferred with PEEK tubing (0.005 in. i.d., Upchurch) from region A to the high magnetic field region C (JEOL FX-200, 4.7 T) for subsequent 13C DNP detection. Typical transfer volumes for region B were ∼80 µL. The LLIT technique in particular requires short residence times in region B in order to avoid DNP transfer losses. Upon entering region C, the polarized bolus was detected in the NMR glass flow cell (150 µL). The repetition times for the pulses in the NMR or DNP experiment were 3.6 s, which is somewhat longer than average residence times in the flow cell (1.9-2.7 s). This was done in order to ensure complete flushing between scans for nonplug flow patterns in the detector cell (e.g., laminar flow), but at the expense of optimium S/N per unit time. The flow NMR probe was home-built and had a 13C resonant frequency, ωn/2π ) 50.10 MHz. In summary, a flowing bolus generates the dynamic nuclear polarization in region A and is quickly transferred (through region B) for high-field 13C DNP detection in region C to obtain optimal results. An SSI (model 200) pump was utilized for all recycled-flow DNP experiments. BACKGROUND Dynamic nuclear polarization (DNP) provides a method by which NMR signal intensities can be enhanced by saturating the electron spin system to which the nuclear spins (e.g., 13C) are coupled. The DNP nuclear-electron interaction can be either a time-dependent (Overhauser) or a time-independent interaction. Applications of DNP have encompassed the dynamics of motions in solution, solute/solvent interactions, and weak hydrogenbonding interactions.34 More recently, DNP has been utilized as (34) Bates, R. D. Magn. Reson. Rev. 1993, 16, 237-291. (35) Motchane, J. L.; Erb, E.; Uebersfeld, J. C. R. Acad. Sci. (Paris) 1958, 246, 1833. (36) Dwek, R. A.; Richards R. E.; Taylor, D. Annu. Rev. Nucl. Magn. Reson. Spectrosc. 1969, 2, 293. (37) Hausser, K. H.; Stehlik, D. Adv. Magn. Reson. 1968, 3, 79.
a new detector for liquid chromatography (HPLC-13C DNP).30 The time-dependent Overhauser DNP enhancement (A) for static experiments can easily be expressed and is discussed in further detail in several reviews.35-38
A ) Ffs(γe/γn)
(1)
The coupling, leakage, and saturation factors are represented by the symbols F, f, and s, respectively. Briefly, the mode and time dependence of nuclear-electron interactions are represented by the coupling factor, F. The fraction of the total relaxation due to the nuclear-electron interaction is represented by the leakage factor. A measure of the extent of saturation of the electronic transitions at the Larmor frequency, ωe, is represented by the saturation factor. The term (γe/γn) represents the magnetogyric ratios of the electron and nuclear spins, respectively. The maximum achievable values for F are +1/2 and -1 for the coupling factor and +1 for a dominant dipolar and scalar interaction, respectively. With this approach, ultimate scalar and dipolar Overhauser 13C DNP enhancements of -1300 and +2600 can be readily calculated (f,s ) 1).32 When the flow-transfer DNP experiment is used, a modification of eq 1 is necessary, since polarization buildup and NMR detection occur in spatially separated regions. Thus, a model has been employed32 in which the enhanced flow DNP signal, Aobs, is related to the static thermal Boltzmann magnetization by
Figure 2. SLIT recycled-flow spectra (3.5 mL/min): (a) recycledflow 13C DNP, 896 scans, 56 min of acquisition time; (b) recycledflow 13C DNP, 56 scans, only 3.5 min of acquisition time (the intense peak at 96.5 ppm is due the solvent (CCl4)); (c) recycled-flow 13C NMR spectrum (without DNP), 896 scans, 56 min, 5.2 mL/min; and (d) static 13C NMR spectrum (without DNP), 396 scans, 15 h of acquisition time.
(2)
nuclear spin relaxation rates reduce the efficiency of the transfer of polarization to the high magnetic field detector (4.7 T).
where the terms E1a ) e-ta/T1a, E1b ) e-tb/T1b, and E1c ) e-tc/T1c represent the buildup (E1a) and decay (E1b, E1c) of the polarization in regions A, B, and C. The constant K is the ratio of high to low field strengths (14.4). Residence times for a polarized bolus are defined as ta, tb, and tc in regions A, B, and C, respectively, and corresponding nuclear spin-lattice relaxation times are defined by T1a, T1b, and T1c, respectively. In this study, flow 13C DNP signals have been generated by both SLIT31-33 and LLIT33 techniques. In the SLIT approach, SPIN samples are localized in a homemade EPR flow cell in region A. Since the radical is fixed in the low-field region throughout the SLIT DNP experiment, the observance of 13C scalar contact shifts and/or concomitant spectral line broadening due to the presence of the radical is avoided in the high magnetic field detector. Other advantages of the SLIT approach include (1) facile detection of scalar-dominated signals, (2) good transfer efficiencies in region B, and (3) a wide range of accessible flow rates. However, inherent disadvantages of the SLIT approach are possible: longer correlation times (τc), poor leakage factors, and possible threespin effects31 (via 1H interactions spins). In contrast, the LLIT approach contains a radical (e.g., TEMPO) as an integral part of the solvent system. The advantages of the LLIT experiment include favorable detection of dipolar-dominated 13C enhancements and suppression of threespin effects at high radical concentrations. However, with the LLIT approach, it is often necessary to maintain rapid bolus transfer times, since analyte-radical interactions and subsequent
RESULTS AND DISCUSSION SLIT 13C Recycled-Flow DNP Experiments. As previously noted, the SLIT 13C DNP experiment provides facile enhancement of carbon sites in a given molecule which are dominated by a scalar interactions; however, carbon sites with dipolar-dominated enhancements are not as easily observable in the SLIT approach. For the present study, a mixture (Table 1) of compounds which exhibit both scalar and dipolar enhancements was employed. As anticipated, large scalar-dominated enhancements (Figure 2a) were observed for carbon tetrachloride (δ, 96.5 ppm), tetrachloroethylene (δ, 120.6 ppm), hexachloroethane (δ, 105.5 ppm), and chloroform (δ, 77.5 ppm). The peak at 116.7 ppm which corresponds to the carbon directly attached to a hydrogen in trichloroethylene also exhibits a strong scalar-enhanced signal, in contrast with the nonobserved (weak dipolar or scalar enhancement) sp2-hybridized carbon containing two chlorine atoms. Consistent with these results, the aromatic carbons (C-2, C-4, C-6) containing one attached hydrogen in 1,3,5-trichlorobenzene also exhibit a strong scalar enhancement (δ, 127.5 ppm). For the case of chlorobenzene, a large scalar-dominated enhancement was observed for the C-2 carbon (δ, 128.8 ppm) as well as weaker, scalar-dominated signals of C-3 and C-4, 129.9 and 126.6 ppm, respectively, whereas the C-1 carbon (δ, 134.5 ppm) exhibited a small dipolar-dominated enhancement. The latter, small dipolardominated enhancement is observable in part because of the relatively high concentration of chlorobenzene in the mixture. However, the dipolar-dominated signals for benzene (δ, 128.5 pnm) and C-1, C-3, and C-5 carbon atoms of 1,3,5-trichlorobenzene (135.8 ppm) were not observable.
Aobs ) (A/K)(1 - E1a)(E1b)(E1c)
(38) Muller-Warmuth, W.; Meise-Gresch, K. Adv. Magn. Reson. 1983, 11, 1.
Analytical Chemistry, Vol. 70, No. 13, July 1, 1998
2625
The examples above are generally consistent with a model of transient bond formation between the SPIN surface and the more acidic carbon-hydrogen (C-H) bonding site in a given molecule. For example, the strong scalar-dominated transient bonding of chloroform and TEMPO is well documented in previous 13C DNP and scalar contact shift studies.30-32 For this favorable example, it is important to note an observable 13C DNP signal for chloroform (as well as other scalar-dominated signals) after a scan time of only 3.5 min (Figure 2b) at a concentration of 0.0447 M. The advantages of the recycled-flow 13C DNP experiment are clearly evident in comparisons with either the corresponding recycledflow NMR (Figure 2c) or the static 13C NMR experiment (Figure 2d). In these latter experiments, the only 13C signals observable were benzene in high concentration (δ ) 128.5 ppm) and the solvent (carbon tetrachloride, δ, 96.5 ppm)). In contrast, none of the chlorocarbon signals for the remaining components of the mixture were observed. However, it is important to note that the benzene signal was only weakly observed in the recycled-flow 13C DNP experiment because of a weak dipolar enhancement and strong three-spin interactions (via hydrogen), as anticipated. In situations represented by the case of benzene, the LLIT experiment (vide infra ) can be utilized to retrieve signals dominated by three-spin interactions. An alternative approach applicable for the SLIT experiment is to employ low-field (0.33 T), 15-MHz 1H spin saturation in the recycled-flow DNP experiment. LLIT 13C DNP Recycled-Flow Experiments. As noted in the previous section, dipolar-dominated signals with strong threespin interactions are difficult to observe in the SLIT experiment. In contrast, the LLIT experiment allows easy observation of both dipolar- and scalar-dominated 13C DNP signals, but the transfer efficiency of the experiment is degraded relative to that of the SLIT experiment. In addition, the LLIT experiment has the additional disadvantage of potential scalar contact 13C NMR shifts and broader spectral lines because of the presence of the radical (TEMPO) in the solution. Figure 3a illustrates the 13C LLIT DNP spectrum of the test mixture after 54 min of elapsed time. In contrast with the SLIT experiment (Figure 2), the chloroform signal is not observed. For these experimental conditions, the 13C spin-lattice relaxation time for the polarized CHCl bolus is 3 so short that it cannot be transferred in sufficient time to region C for detection (i.e., the generated polarization decays too rapidly). However, the dipolar-dominated hexachlorobenzene (negative) signal, which was not previously observed with the SLIT experiment, is now readily observed. Also, the LLIT dipolar-dominated signals from chlorobenzene and trichlorobenzene now exhibit improved signal-to-noise ratios. As expected, benzene now exhibits a large, negative 13C dipolar enhancement and is the dominant signal of all the analytes in the mixture. Of the compounds present in trace amounts, all of the expected 13C DNP signals could be observed for chlorobenzene, tetrachlorethylene, and hexachloroethane. Analogous with the SLIT results, the trichloroethylene signal corresponding to the carbon bonded to two chlorine atoms could not be observed. This is undoubtedly due to a nearly equal admixture of both scalar and dipolar contributions to the overall enhancemement at this carbon, since three-spin contributions are clearly not a contributing factor. However, the trichloroethylene carbon, which is bonded to a hydrogen, clearly exhibits a scalar-dominated signal, consistent 2626 Analytical Chemistry, Vol. 70, No. 13, July 1, 1998
Figure 3. LLIT recyled-flow 13C DNP NMR spectra, 5.2 mL/min, (TEMPO, 0.0179 M), with total experimental times of (a) 54 min (896 scans) and (b) only 11.5 min (192 scans). (c) Conventional static 13C NMR spectrum (without DNP), 196 scans, and 3.5 h of acquisition time.
with the SLIT results (vide supra ). To further demonstrate the potential S/N advantage of the recycled-flow DNP experiment, it should be emphasized that the recycled-flow 13C LLIT DNP spectrum presented in Figure 3b has been generated with only 11.5 min of instrumental time. In contrast, Figure 3c represents the static 13C NMR spectrum obtained with conventional conditions, but it took 3.5 h. However, it is important to emphasize that a detector volume of only 150 µL was used for the static NMR measurements (Figures 2d and 3c), whereas a total volume (pump reservoir, regions A, B, and C, etc.) of ∼3 mL was required in the recycled-flow NMR and DNP experiments. Nevertheless, even these qualitative S/N comparisons dramatically illustrate the advantages of the SLIT and LLIT recycled-flow DNP approach. It should be noted that the concentration of radical can have a significant influence on both scalar- and dipolar-dominated signals in the corresponding LLIT DNP spectrum. At low radical concentrations, a partial dipolar-dominated DNP enhancement can occur, but the overall signal intensity can still be positive or very small. At higher radical concentrations, an increasing negative dipolar DNP enhancement will be observed. Thus, there exists a specific radical concentration where a “null” can be achieved from the transition of a positive to a negative signal. To illustrate this phenomenon, the DNP enhancements at various free radical concentrations are summarized in Figure 4. For example, the benzene signal is positive in spectrum b, is a “null” in spectrum in c, and achieves its maximum, negative, dipolar-dominated enhancement in spectrum d. In addition, dipolar-dominated signals from chlorobenzene and trichlorobenzene also approach their maximum, negative intensity in spectrum d. The trend established clearly shows that larger enhancements are possible at higher radical concentrations for dipolar-dominated signals. For scalar-dominated interactions, the signals are always positive, but generally shorter relaxation times are observed. For example, the
Table 2. Sensitivity Comparisons of Static NMR and 13C Recycled-Flow DNP for Equivalent Sample Volumesa HClCdCCl2 HCCl3 13C
DNPb
SLIT recycled-flow (polarized at 0.33 and 4.7 T, 50-MHz detection) static 13C NMR, 50 MHz, 4.7 Tc static 13C NMR, 100 MHz, 9.4 Td
C(1)
C(2)
C2C16
23 (10)
nd
31 (13)
45 (19)
nd 0.9 (5)
nd 3.4 (19)
nd 1.3 (7.5)
2.3 (6) 10 (56)
a See text for sample volume discussion; S/N (rms) with the same observation time in each case (56 min); static 13C NMR measurements assuming an average T1 of 30 s, T ) 4T1; nd, not detected in this observation time. b S/N values with volume correction factor of 2.2 applied are given in parentheses, (3 mL/0.6 mL)0.5 for the 13C recycledflow DNP experiment experiment. c See text, corrected with S/N commercial instrumentation factor 2.6 for Bruker WP 200 (preamplifier, filling factor etc.); value without any correction is given in parentheses. d See text, corrected with S/N commercial instrumentation factor 6.1 for Varian VX-400 (filling factor preamplifier, etc.), but not corrected for differences in magnetic field strength; values without any correction are given in parentheses.
Figure 4. LLIT recycled-flow 13C DNP spectra obtained with various concentrations of the TEMPO added to the test mixture, 5.2 mL/min, and 896 scans for each spectrum.
tetrachloroethylene signals maximize in spectrum c; however, the trichloroethylene and hexachloroethane signals maximize in spectrum d. It should also be noted that increasing radical concentrations too can often lead to poor transfer efficiencies (E1b, E1c ≈ 0, see Experimental Section and Background), and poor DNP enhancements will occur. Comparisons of Static NMR and 13C Recycled-Flow DNP. Although the 13C recycled-flow DNP experiment invites quantitative sensitivity comparisons with commerical static 13C NMR instruments, these comparison are complicated by several factors. As already noted above, the DNP experiment yields a wide range of enhancement factors, dependent on whether a scalar or dipolar interaction dominates in a given molecular system (e.g., chloroform versus benzene). Furthermore, the 13C DNP enhancements were generated at 0.33 T but monitored at 4.7 T (50 MHz). Thus, another factor to consider in the transfer DNP experiment is whether to make comparisons (with the NMR experiment) relative to the monitored (4.7 T) or polarization field strength (0.33 T). From a purist viewpoint, these should be compared at the same polarizing field strength (NMR and DNP), but this is experimentally impractical on both accounts. Also, most static NMR measurements would be conducted on samples without the presence of a free radical (e.g., TEMPO). Thus, the SLIT DNP experiment is more suitable than the LLIT DNP for these comparisons. It should be duly noted that most commerical high-resolution instruments are optimized for static measurements with sample volumes of ∼0.6 mL (samples in 5-mm tubes). In contrast, the 13C recycled-flow DNP experiment is optimized for smaller detection volumes (160 µL), but a larger total volume is required (∼3 mL), although the latter volume can be minimized to as little as ∼1 mL for recycled-flow DNP experiments. Finally, compari-
sons should ideally be made with the same detector coil, preamplifier, and magnetic field homogeniety for both the NMR and the recycled-flow DNP experiments. This is also impractical since the DNP experiments were monitored with a home-built flow probe with older instrumentation (e.g., preamplifier). Despite the reservations above, Table 2 compares the 13C recycled-flow DNP experiment and static NMR measurements. The latter were performed with commerical 200- and 400-MHz (4.7 and 9.4 T) NMR instruments. For the same sample (see Table 1), static 13C NMR signal-to-noise (S/N) measurements for the solvent CCl4 yielded relative values of 12.5, 72, and 506 for the recycled-flow NMR (DNP) and 200-, and 400-MHz NMR instruments, respectively. Even correcting for differences in sample volumes, (150 µL for “DNP” detector/600 µL for static NMR)0.5, as suggested by Sudmeier et al.,29 still leads to a factor of 2.9 improvement in S/N for the commerical 200-MHz instrument in comparison with the DNP apparatus. In similar fashion, the 400-MHz instrument exhibts a better static S/N by a factor of 6.1, even after correcting for the volume change and the difference in magnetic field strength (i.e., Bo7/4). These results are in general agreement with previous measurements in our laboratory and reflect the poorer filling factor, preamplifier, etc. employed in the NMR detector for the recycled-flow DNP apparatus. In agreement with the calculations presented by Sudmeier and co-workers,29 the results (Table 2) obtained for the three analytes with scalar-dominated enhancements at lowest concentration in the mixture generally exhibit a 1-2 orders of magnitude S/N advantage for the recycled-flow SLIT 13C DNP experiment in comparison witth conventional static NMR experiments. The only exception is the dipolar-dominated enhancement for the C(1) carbon of trichloroethylene, as previously discussed. In the most favorable case, HCCl3, the DNP enhancement exceeds the S/N of the 400-MHz instrument by a factor of nearly 5, even with no corrections for the magnetic field strength and instrumentation factors. However, it is important to emphasize the caveat that this S/N advantage is not reflected for molecular systems with dipolar-dominated enhancements (e.g., see C2HCl3 and benzene). Analytical Chemistry, Vol. 70, No. 13, July 1, 1998
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CONCLUSIONS In summary, a recycled-flow apparatus has been described that utilizes dynamic nuclear polarization as a technique to significantly enhance scalar-dominated 13C DNP signals. Several chlorocarbons that were present in low concentrations could not be detected with either conventional static or recycled-flow NMR detection alone. In contrast, these species were generally readily observed through the recycled DNP experiment. Although this study represents a contrived mixture, the recycled-flow 13C DNP spectrometer could easily be adapted for monitoring other nuclei (e.g., 15N, 31P) with scalar-dominated enhancements whose NMR spectra would be otherwise difficult, if not impossible, to obtain. Possible applications of flow DNP are readily envisioned. Flow DNP could be utilized to conveniently monitor environmentally significant samples from industrial waste. In addition, the technique could also be used in flowing biological systems similar to those discussed vide supra. The ability to observe the 13C
2628 Analytical Chemistry, Vol. 70, No. 13, July 1, 1998
spectra for isotopically unenriched species makes the DNP approach an interesting alternative to conventional static or recycled-flow NMR detection approaches. ACKNOWLEDGMENT The authors gratefully acknowledge partial financial support for this study from ACS-PRF, NSF, and the Environmental Protection Agency Office of Exploratory Research. The authors also acknowledge Juan Gu for technical assistance with the DNP experiment. In addition, Rossi Gitti kindly prepared the SPIN sample for the SLIT experiments.
Received for review December 8, 1997. February 12, 1998. AC971337V
Accepted
