Accelerated Articles Anal. Chem. 1994,66,2993-2999
I3C Dynamic Nuclear Polarization: A Detector for Continuous-Flow, On-Line Chromatography S. Stevenson and H. C. Dorn' Department of Chemistry, Virginia Pol'echnic
Institute and State University, Blacksburg, Virginia 2406 I
It is well recognized that NMR nuclides with low magnetogyric ratios (7) and/or low isotopic natural abundances suffer from sensitivitylimitations in many NMR experiments. Applications employing on-line, continuous NMR flow monitoring (e.g., chromatographicdetection) of weak NMR nuclides (e.g., I3C, lSN, and 2%i) are limited because of the corresponding poor signal strength. However, dynamic nuclear polarization (DNP) can alleviate this sensitivity constraint since the DNP signal enhancements are proportional to the electron-to-nuclear magnetogyric ratio ( ~ J T ~which ) , is on the order of 103-104 for most nuclides. In this paper, we report successful coupling of a continuous-flow,on-line l3C dynamic nuclear polarization detector with high-performance liquid chromatography (HPLCDNP). With this approach, scalar '3C DNP enhancements nearly 2 orders of magnitude larger than the thermal Boltzmann magnetization at 4.7 T have been obtained for favorable cases (HCCl3). The DNP enhancements of the chromatographic analytes are generated by solid-liquid intermolecular transfer (SLIT) from a silica phase immobilized nitroxide (SPIN) system at 0.33 T (0,/2?r = 9.3 GHz) and subsequent flow transfer to a high magnetic field for detection (4.7 T). In this first application, mixtures of halogenated compounds (e.g., HCClk CzQ, etc.) were separated with subsequent continuousflow, on-line DNP detection. The technique can be extended to other NMR nuclides and numerous applications requiring continuous-flow monitoring.
Historically,the interest in flow NMR monitoring has been piqued since Suryan's discovery' in 195 1 that the intensity of a 'H NMR signal could be enhanced for flowing liquids. In 1960, Forsen and Rupprecht2 investigated the relationship between flow rate and NMR signal intensity for 13Cnuclei. (1) Suryan, G. Proc. Indian Acad. Sci., Sect A. 1951, 33, 107-111. '(2) Forsen, S.;Rupprecht, A. J . Chem. Phys. 1960, 33, 1888-1889. (3) Grimaldi. J.; Baldo, J.; McMurray, C.; Sykes, B. D. J. Am. Chem. SOC. 1972, 94, 7641-7645.
0003-2700/94/0366-2993$04.50/0 0 1994 American Chemlcal Society
With further advances in the technique, flow NMR has been utilized to monitor kinetics and intermediate~,~-' I to measure flow rates,I2-l4 to modify nuclear relaxation rates ( l / T l ) , l J J 5 to probe flow dynamics,'"lg and to monitor biological systems ,2044 The early developments of L C - l H NMR occurred in 1978 when Watanabe25reported the on-line, stopped-flow NMR of an injected sample. From this point in time, other have contributed to the development of LC(4) Fyfe, C. A.; Cociverra, M.; Damji, S. W. H. Acc. Chem. Res. 1978, 11, 277-282. (5) KUhne, R. 0.;Schaffhauser, T.; Wokaun, A.; Ernst R. R. J . Magn. Reson. 1979, 35, 39-67. (6) Tan, L. K.; Cocivera, M. Can. J. Chem. 1982, 60, 778-786. (7) Chapman, B. E.; Kuchel, P. W.; Lovric, V. A.; Raftos, J. E.; Stewart, I. M. Br. J. Haematol. 1985, 61, 385-392. (8) Albert, K.; Dreher E.-L.; Straub, H.; Rieker, A. Magn. Reson. Chem. 1987, 25, 919-922. (9) OLeary, D. J.; Hawkes, S.P.; Wade, C. G. Magn. Reson. Med. 1987, 5, 572-577. (10) Davis, M. E.; Hathaway, P.; Morgan, D.; Glass, T.; Dorn, H. C. Stud. Surf Sci. Catal. 1987, 38, 263-27 1. (11) Trahanovsky, W.S.;Fischer,D.R.J.Am. Chem.Soc. 1990,112,49714972. (12) Singer, J. R. Science 1959, 130, 1652. (13) McCormick, W. S.;Birkemeirer, W. P. Reu. Sci. Instrum. 1969, 40, 346. (14) Morse, 0. C.; Singer, J. R. Science 1970, 170, 440-441. (15) Zhernovoi, A. I.; Latyshev, G. D. NMR in a Flowing Liquid; Consultants Bureau: New York, 1965. (16) Arnold, D. W.; Burichart, L. E. J. Appl. Phys. 1965, 36, 870. (17) Stejskal, E. 0. J . Chem. Phys. 1965, 43, 3597. (18) Grover, T.; Singer, J. R. J. Appl. Phys. 1971, 42, 938-940. (19) Hayward, R. J.; Packer, K. J.; Tomunson, D. J. Mol. Phys. 1972,23, 10831102. (20) Gonzalez-Mendez,R.; Wemmer, D.; Hahn,G.; Wade-Jardetsky,N.; Jardetzky, 0. Biochim. Biophys. Acta 1982, 720,274-280. (21) Wyrwicz, A. M.; Schofield, J. C.; Burt C. T. In Noninuasiue Probes in Tissue Metabolism; Cohen, J. S.,Ed.; Wiley: New York, 1982, pp 149-171. (22) Albert,K.; Kruppa,G.; Zeller, K.-P.;Bayer, E.; Hartmann, F. 2.Naturforsch. 1984, 39C, 859-862. (23) De Graaf, A. A.; Wittig, R. M.; Probst, U.; Strohhaecker, J.; Schoberth, S.M.; Sahm, H. J . Magn. Reson. 1992, 98,654459. (24) Chen, R.; Bailey, J. E. Eiotechnol. Eioeng. 1993, 42, 215. (25) Watanabe, N.; Niki, E. Proc. Jpn. Acad. Ser. B 1978, 54, 194-199. (26) Bayer, E.; Albert, K.; Nieder, M.; Grom, E.; Keller, T. J . Chromatogr. 1979, 186,497-507. (27) Haw, J. F.; Glass, T. E.; Hausler, D. W.; Motell, E.; Dorn, H. C. Anal. Chem. 1980, 52, 1135-1 140. (28) Haw, J. F.; Glass, T. E.; Dorn, H. C. Anal. Chem. 1980, 53, 2327-2332. (29) Haw, J. F.; Glass, T. E.; Dorn, H. C. Anal. Chem. 1981, 53, 2332-2336.
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'H NMR as a potential analytical tool. From 1985 until the present,3742further improvements in the signal-to-noise ratio have been achieved mainly by utilizing higher magnetic field instruments. Nevertheless, the LC-NMR technique is still limited by sensitivity constraints. The advantages of coupling the separation power of highperformance liquid chromatography (HPLC) to a structurerich 13CNMR detector are easily envisioned. The necessary increases in chromatographic column capacity and injection volume are readily achieved except for cases of limited sample quantities. On-line LC-NMR (or LC-DNP) detection is noninvasive and therefore permits the investigation of air- or UV-sensitive, labile compounds. In contrast with LC-IH NMR, the development of LCI3CNMR has not been clearly demonstrated. The low natural abundance of the 13Cnuclide (1.1%) and low magnetogyric ratio (7)severely limit the signal-to-noise ratio (S/N) of the I3C NMR experiment-even for static measurements. In certain I3Cflow NMR monitoring experiments, the sensitivity can be improved by simply employing 13C-labeledsamples.43 However, this approach is not feasible for most applications. To successfully implement LC-I3C NMR, a significant increase in the 13C signal strength for natural abundance samples must be achieved in order to recover the sensitivity deficit relative to lH NMR. Specifically, the low natural abundance (1.108%) of the I3Cnuclide and low magnetogyric ratio ( y 5 / *dependence) yield natural abundance 13CNMR signals with S / N -2800 times lower than equivalent 'H NMR signals.44 One method of overcomingthis limitation is indirect detection techniques (via IH detection), where the 13Cspectral domain is observed (indirectly) by polarization transfer from a scalar coupled hydrogen.44However, this technique is limited by scalar coupling of an insensitive nuclide (e.g., I3C) to an abundant spin (lH). To improve the sensitivity of the 13C NMR experiment, dynamic nuclear polarization (DNP) can be utilized for enhancement of otherwiseweak I3CNMR signals. Theoretical and mathematical descriptions of DNP and flow DNP have been reported p r e v i ~ u s l y . ~ ~ - ~ ~ Haw, J . F.; Glass, T.E.; Dorn, H . C. Anal. Chem. 1983, 55, 22-29. Dorn, H. C. Anal. Chem. 1984, 56, 747A-758A. Bayer, E.; Albert, K.;Nieder, M.; Grom, E. J . Chromat. 1979,186,497-507. Bayer, E.; Albert, K.; Nieder, M.; Grom, E.; Wolff, G.; Rindlisbacher, M. Anal. Chem. 1982.54, 1747-1750. Buddruss, J.; Herzog, H . Org. Magn. Reson. 1980, 13, 153-155. Buddruss, J.; Herzog, H. Anal. Chem. 1983, 55, 1611-1614. Laude, D. A.; Wilkins, C. L. Anal. Chem. 1984, 56, 2471-2475. Laude, D.A.; Lee, R. W.-K.; Wilkins, C. L. Anal. Chem. 1985, 57, 14641469. Laude, D. A.; Wilkins, C. L. TrAC, Trends Anal. Chem. 1986,5,230-235. Albert, K.; Bayer, E. TrAC, Trends Anal. Chem. 1988, 7, 288-293. Albert, K.; Kunst, M.; Bayer, E.; Spraul, M.; Bermel, W. J . Chromat. 1989, 463, 355-363. Albert, K.; Kunst, M.; Bayer, E.; Hendrik, J. de J.; Genissei, P.; Spraul, M.; Bermel, W. Anal. Chem. 1989,61, 772-775. Grenier-Loustalot. M. F.; Grenier, P.; Bounoure, J.; Grall, M.; Panaras, R. Analusis 1990, 18, 2OC-207. Albert, K.; Kruppa, G.; Zeller, K. P.; Bayer, E. 2. Nufurforsch. 1984, 39C, 859-862. Ernst, R. R.; Bodenhausen, G.; Wokaun, Principles of Nuclear Magnetic Resonance in One and Two Dimensions: Oxford Press: New York, 1990. Bates, R. D. Magn. Reson. Rev. 1993, 16, 237-291. Gitti, R.; Wild, C.; Tsiao, C.; Zimmer, K.; Glass, T. E.; Dorn, H. C. J . Am. Chem. SOC.1988, 110, 2294-2296. Dorn,H. C.; Gitti, R.; Tsai, K. H.; Glass, T. E. Chem. Phys. Left. 1989, 155, 227-232. Tsai, K. H.; Dorn, H. C. Appl. Magn. Reson. 1990, I , 231-254. Tsai, K. H.; Dorn, H. C. J . Magn. Reson. 1990, 89, 362-366.
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Analytical Chemistry, Vol. 66, No. 19, October 1, 1994
Unfortunately, a large number of molecules have not been examined by the 13C DNP technique. Most hydrocarbons examined to date (e.g., benzene) exhibit dipolar-dominated 13C DNP enhancement~.~5.~*-5* For example, in one recent study, the collisional dynamics of the fullerene c60, dissolved in deuterated benzene, was examined in the presence of the radical TEMPO.5o The ultimate 13C DNP enhancements (Aobs) for c 6 0 and the solvent (C6D6) were -250 f 20 and -200 f 20, respectively. In contrast, large scalar-dominated I3C DNP enhancements have usually been obtained for chlorinated hydrocarbons. For example, chloroform and deuterated chloroform exhibit liquid-liquid intermolecular transfer (LLIT) 13C DNP enhancements (with TEMPO) of +2200 and +2100, respectively, which are close to the scalar limit ( A , = +2600) for the 13C nuclide.48 In a different experiment, a solid-liquid intermolecular transfer (SLIT) was utilized in which a silica phase immobilized nitroxide (SPIN) radical transferred the polarization to flowingchloroform and deuterated chloroform samples. In the SLIT experiment, I3C DNP enhancements of +760 and +1100 were observed for chloroform and deuterated chloroform.51 Thus, in the present study halogenated compounds were examined in the LC-13C DNP approach because of potential environmental applications and the large DNP scalardominated enhancements expected for these compounds vide s ~ p r a . ~For ~ -favorable ~~ cases, an enhancement of 40-60 has been achieved in comparison with the I3C thermal Boltzmann magnetization at 4.7 T (50 MHz).48*49+51-53
EXPERIMENTAL SECTION A SSI (Model 200) HPLC pump was employed to deliver degassed (nitrogen) HPLC grade carbon tetrachloride (Aldrich) solvent to a Whatman (Partisil PAC 10 mm X 250 mm) column. The injection volumes for the halogenated samples ranged from 75 to 3000 FL (neat). The samples contained I3C in natural abundance (1.1%) and were utilized without purification (Aldrich, EM Science, Fisher, Mallinckrodt). Flow rates for LC experiments were selected at 2.33.5 mL/min, and a diagram of the on-line LC-13C DNP apparatus is presented in Figure 1. The separated analytes enter the low magnetic field region A (0.33 T). A silica phase immobilized nitroxide radicaWG53 (Figure 2a) was utilized as the unpaired electron source for the DNP experiment and was placed in the EPR flow ce1146,47,51-53 (Figure 2b). Microwave power was transferred to a microwave TE102 cavity from a klystron source (Bruker microwave bridge). The microwave source (9.3 GHz) was amplified to -5-15 W, which was sufficient for good saturation factors, s = 0.7-0.9. The volume in region A was 160 pL. N
(SO) Dorn, H. C.; Gu, J.; Bethune, D. S.;Johnson, R. D.; Yannoni, C. S.Chem. Phys. Left. 1993, 203, 549-554. (51) Dorn, H. C.; Glass, T. E.; Gitti, R.; Tsai, K. H. Appl. Magn. Reson. 1991, 2, 9-27. (52) Stevenson, S.Thesis, Virginia Tech, Blacksburg, VA, 1992. (53) Tsai, K. H. Dissertation, Virginia Tech, Blacksburg, VA, 1990. (54) Bates, R. D.; Wagner, B. E.; Poindexter, E. H. Chem. Phys. Left. 1972, 17, 328-331. (55) Hausser, K. H.; Stehlik, D. Ado. Magn. Reson. 1968, 3, 79-139. (56) Dwek, R. A.; Richards, R. E.; Taylor, D. Annu. Rev. N M R Spectrosc. 1969, 2, 293-344. (57) Potenza, J. A. Ado. Mol. Relaxation Processes 1972, 4, 229-354. (58) Mirller-Warmuth,W.; Meise-Gresch, K. Adv. Magn. Reson. 1983,11,1-45.
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The polarized flowing bolus was transferred with PEEK tubing (0.005 in. i.d., Upchurch) from the low-field to a high magnetic field (region C, JEOL FX-200,4.7 T) for 13CDNP detection. An approximate transfer volume of 80 pL can be estimated for region B. After exiting region B, the polarized analytes enter the 150 pL NMR glass flow cell in the high magnetic field (Figure 2c). A home-built flow NMR probe was employed with a Helmholtz detection coil tuned to resonance, wJ2a = 50.10 MHz.
BACKGROUND The well-known classical expression for the observed static Overhauser DNP enhancement, A , can be expressed as55-58 A = p.fs(Y,/Yn)
(1)
The so-called coupling factor, p , reflects the mode and time dependence of the nuclear-electron interaction. The maximum achievable value is +1/2 and -1 for dipolar- and scalardominated enhancements, respectively. For example, a maximum dipolar enhancement can be achieved when the condition we7,
.
l
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.
l
.
,
170
.
,
,
l
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'
l
'
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PPM Flgure 4. (a) LC-I3C DNP profile: 500 pL of HCClsinjected, 3.5 mL/ min, 10 scandfile, 31 s/file, and CCI, mobile phase. (b) LC-lSC NMR profile (withoutDNP): 500 pL of %CI3 injected, 3.5 mL/min, 10scans/ file, 31 slflle, and CCl, mobile phase.
injected HCC& (77.5 ppm) elutes from 6.2-8.8 minutes and also exhibits a large scalar-dominated enhancement. However, the enhancement for HCC13 is somewhat larger and has beenpreviously characterized in terms of a strong hydrogen-
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160
120
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PPM Flgure 5. LC-'% DNP profile: 1250 pL of tetrachloroethylene, 1250 pL of trichloroethylene, 2.3 mL/mln, 5 scanslfile, 20 s/flle, and CCI,, mobile phase; alternate flies shown.
bonding interaction with the immobilized nitroxide radical.51-53 Based on this data, one can easily estimate that an injection volume of -50 pL would still yield a detectable 13C DNP signal. For comparison, a second analogous experiment was performed, but without DNP enhancement under equivalent conditions except for the absence of the microwave source. Although the experimental conditions were not optimal (e.g., no high magnetic field preequilibration volume) for this "classical" LC-13C NMR approach, nevertheless, the absence of observable signals (Figure 4b) clearly demonstrates the advantages of the LC-13C DNP approach. To further illustrate the approach, Figure 5 is the LC-13C DNP profile for a two-componentmixture which is not resolved in the chromatographic dimension. Specifically, tetrachloroethylene elutes slightly later than trichloroethylene. However, these two compounds are easily resolved in the '3C chemical shift dimension with strong scalar-dominated signals observed at 120.6 and 116.7 ppm, respectively. It is important to note that a strong scalar-dominated 13C DNP signal is easily observed for C2Cl4 which is difficult to observe in the usual 13C NMR spectrum because of a long spin-lattice relaxation time ( Tlno)and the absence of a nuclear Overhauser effect. This example also illustrates the advantage of the I3C DNP approach for samples not containing hydrogen, where lH polarization transfer (or indirect detection) experiments cannot be employed.44 On the other hand, it is important to note a significant disadvantage of the present SLIT I3C DNP approach. Specifically, a signal was not observed for one of the nonequivalent carbon signals (124.2 ppm) in C2HC13 because of a weak scalar (or dipolar) enhancement. However, in a fashion similar to chloroform, a strong scalar-dominated signal (1 16.7 ppm) was observed for the carbon containing a directly attached hydrogen. In order to explore the sensitivity limits of the present experimental LC-13C DNP apparatus, an experiment similar to the one above was performed in which reduced quantities of C2C14, C2HC13, and HCCL were injected. Once again, resolution in the chromatographic dimension was not observed, but characteristic 13CDNP signals for these three compounds were clearly discernible in the 13C DNP dimension (Figure 6). This spectrum represents a 35 s window of the LC-13C DNP profile centered at an 11.0 min elution time. In this example, a total volume of only 250,85, and 75 pL of C2C14, CzHC13, and HCCl3, respectively, were injected into the
I20
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PPM Figure 6. LC-l% DNP [window (35 s) centered at 11.0 min elution time]: 75 pL of chloroform, 85 pL of trichloroethylene, 250 pL of tetrachloroethylene,2.8 mL/mln, 10 scans/fiie, and CCl, mobile phase.
chromatographic column. Although the present 13C DNP detection limits are not impressive in comparison with high magnetic field LC-*H NMR approaches, they are adequate for semipreparative to preparative chromatographic separations. Furthermore, additional S / N improvements by at least 1 order of magnitude are clearly feasible vide infra. To illustrate the separation of a more complex mixture, Figure 7 represents the LC-13C DNP profile for the injection of chloroform, tetrachloroethylene, hexafluorobenzene, benzene, and 1,1,1-trichlorotrifluoroethane. Although a largescale (preparative) injection volume was utilized, the components are fairly well resolved in the chromatographic dimension. The corresponding elution order is HCCl3, tetrachloroethylene, hexafluorobenzene, 1,1,1-trichlorotrifluoroethane, and benzene. The DNP enhancements for compounds in this mixture exhibit both scalar-dominated (absorption) and dipolar-dominated (emission) enhancements. In this mixture, intermolecular interactions of chloroform and tetrachloroethylene with the SPIN radical system are scalardominated. The corresponding I3CDNP signals exhibit large increases in intensity because of the strong scalar interaction. However, molecular interactions of benzene and hexafluorobenzene with the SPIN surface radicals are primarily dipolar-dominated, and the corresponding 13CDNP signals are significantly reduced in intensity. For example, hexafluorobenzene exhibits a negative dipolar 13CDNP signal which was observablein theSLITexperiment. Thiscan becompared to previous 19FDNP low-field studies for C6F6 in which weak dipolar-dominated 19F enhancements were observed for nitroxide radical^.^^ As reported in a previous study,5' the SLIT 13C DNP dipolar-dominated signal for benzene is significantly reduced in intensity because of three spin effects (via hydrogen) and a reduced dipolar enhancement. Nevertheless, the benzene 13C DNP signal was still observed (Figure 7, 12-14 min) in the present experiments. It should also be noted that the solvent peak is decreased in the 10-13 min region, presumably due to the large quantities of benzene and hexafluorobenzene injected.
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AnalyticalChemlstry, Vol. 66, No. 19, October 1, 1994
2997
TIME
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PPM Flgure 7. LC-% DNP profile: 1000 pL of chloroform, 1500 pL of tetrachloroethylene, 2500 pL of hexafluorobenzene, 1500 pL of l , l , l trichlorotrifluoroethane,3000 pL of benzene mixture, 2.5 mL/min, 5 scans/file, 19 s/file, and CC, mobile phase. (lgF)decouplingwas not employed. I3CNMR signals for 1,1,l-trlchlorotrlfluoroethane(*& quartet under heavy apodization,line broadening 1 1 Hz) and hexafluorobenzene(lJCFdoublet) are observed.
For the two nonequivalent carbon atoms for the l , l , l trichlorotrifluoroethane molecule, one carbon (CF3) was not observed while the other carbon atom (CCl3) exhibits a fairly strong scalar-dominated interaction with the SPIN surface radicals. As expected, thecarbon bonded to thechlorine (CC13 group) appears as a quartet (see expanded inset, Figure 7) with spin-spin coupling 2 J c ~ These . results are consistent with previous s t ~ d i e swhich ~ ~ , indicate ~~ a general dominance of the scalar mechanism for the more chlorinated carbon in chlorofluorocarbons. However, a dipolar-dominated 19FDNP enhancement for CF3CC13 has been reported in low-field DNP studies.45 It should also be noted that {I9F)decoupling could also be employed64 to further improve the S / N for these fluorocarbon samples. (62) Chandrakumar, N.; Narashimhan, P. T. J. Chem. Phys. 1982, 77, 26972699. (63) Bates, R.D. J . Chem. Phys. 1977, 66, 1759-1760. (64) Allen, L. A,; Spratt, M. P.; Glass, T. E.; Dorn, H. C. Anal. Chem. 1988,60,
675-679.
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CONCLUSIONS The results of the present study clearly demonstrate that the flow 13C DNP detector can provide significant enhancements (101-102) in favorable cases which allow detection in semipreparative to preparative chromatographic applications of 13C natural abundance samples. The technique is not limitedto chromatographic applications and is applicable to numerous flow NMR monitoring experiments. In addition, the flow transfer DNP experiment is not limited to the 13C nuclide, and numerous other NMR nuclides could easily be monitored (e.g., 19F,29Si,31P,and 15N). The advantages of the DNP approach can be significant for nuclides with low magnetogyric ratios and/or molecules not containing abundant spins (lH, 19F). In addition, further S / N improvements in the transfer 13C DNP detector by at least another order of magnitude are clearly feasible. For example, it should be emphasized that the DNP polarization in the present study was generated at
-
only 0.33 T. The maximum achievable enhancements in the present study are governed by the A/K ratio, which is 180 for a scalar-dominated enhancement. Since the maximum experimental I3C DNP SLIT enhancements observed were significantly lower (-60), further improvements are feasible. For example, a significant reduction in the nuclear-electron correlation time ( T J is clearly possible with the corresponding LLIT experiment, with elevated temperatures, or by employing supercritical fluids. A reduced correlation time is significant for helping fulfill the condition W ~
