Trace Analysis by Low-Field NMR: Breaking the Sensitivity Limit

Aug 11, 2010 - Here, we report the feasibility of applying NH-PHIP in the low-field NMR. A trace ... A hitherto unknown potential opens up for trace a...
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Anal. Chem. 2010, 82, 7078–7082

Letters to Analytical Chemistry Trace Analysis by Low-Field NMR: Breaking the Sensitivity Limit Qingxia Gong, Ali Gordji-Nejad, Bernhard Blu¨mich,* and Stephan Appelt Institute of Technical and Macromolecular Chemistry, RWTH Aachen University, 52056 Aachen, Germany, and Central Institute for Electronics, Research Center Ju¨lich, 52425 Ju¨lich, Germany Sensitivity poses a persistent challenge to NMR spectroscopy and magnetic resonance imaging (MRI). Nonhydrogenative para-hydrogen induced polarization (NH-PHIP) has recently emerged as an efficient method to substantially increase the sensitivity of high-field NMR. Here, we report the feasibility of applying NH-PHIP in the low-field NMR. A trace amount of pyridine of just a few nanoliters (∼12 nmol) in a 0.4 mL NMR sample (a concentration of 31 µM or 1016/cm3) could be measured in a single scan by NH-PHIP. There is a striking difference in the signal-to-noise ratio (SNR) between thermal prepolarization and NH-PHIP: The SNR of the prepolarized 1H NMR signal decreases linearly with decreasing 1H concentration ([1H]) while the SNR in NH-PHIP experiments first increases with decreasing [1H], then remains constant over 2 orders of magnitude, and finally decreases linearly with decreasing [1H]. A hitherto unknown potential opens up for trace analysis by low-field NMR in the bio-, chemical, and material sciences. Low-field NMR without cryogens is inexpensive and can be made mobile. Its use promises new applications in well-logging, the analysis of objects of heritage culture, food quality control, and the chemical and material sciences which are prohibited by or difficult to perform with high-field machines. Compared to highfield NMR, the obvious disadvantage of low-field NMR is the inherently low sensitivity due to the small differences in the thermodynamic equilibrium populations of the spin states. Two general strategies known as thermal prepolarization and hyperpolarization have been demonstrated to overcome this disadvantage. Thermal prepolarization can be realized via establishing a new thermal equilibrium state of the sample in a strong but not necessarily homogeneous magnetic field before signal detection at low field.1 Hyperpolarization is achieved by transferring the high polarization of photons or the high spin order of a second group of spins to the target nuclei. Several hyperpolarization * To whom correspondence should be addressed. Fax: +49 241 802 2185. E-mail: [email protected]. (1) Appelt, S.; Ku ¨ hn, H.; Ha¨sing, F. W.; Blu ¨ mich, B. Nature Phys. 2006, 2, 105–109.

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techniques including spin exchange optical pumping,2,3 dynamic nuclear polarization (DNP),4-6 and spin polarization-induced nuclear Overhauser effect (SPINOE)7,8 have already been applied in low-field NMR. Para-hydrogen induced polarization (PHIP) as a chemical hyperpolarization method has also been proposed to improve the sensitivity of NMR.9-16 An apparent drawback of PHIP is that a chemical reaction of a substrate by hydrogenation is required. Recently, a new approach to generate PHIP sensitized materials without direct hydrogenation has emerged which is known as nonhydrogenativepara-hydrogeninducedpolarization(NH-PHIP).15,16 This technique involves a temporary association of a substrate and para-H2 (p-H2) via a transition metal based host, polarization transfer from p-H2 derived hydride ligand to the bound substrate via scalar coupling, and fast dissociation of the magnetically labeled substrate. A theoretical analysis of NHPHIP has indicated that the polarization transfer from p-H2 to the substrate is field- and time-dependent.17 (2) Happer, W. Rev. Mod. Phys. 1972, 44, 169–249. (3) Appelt, S.; Baranga, A. B.; Erickson, C. J.; Romalis, M. V.; Young, A. R.; Happer, W. Phys. Rev. A 1998, 58, 1412–1439. (4) Abragam, A.; Goldman, M. Rep. Prog. Phys. 1978, 41, 395–467. (5) Halse, M. E.; Callaghan, P. T. J. Magn. Reson. 2008, 195, 162–168. (6) Lingwood, M. D.; Ivanov, I. A.; Cote, A. R.; Han, S. J. Magn. Reson. 2010, 204, 56–63. (7) Navon, G.; Song, Y. Q.; Room, T.; Appelt, S.; Taylor, R. E.; Pines, A. Science 1996, 271, 1848–1851. (8) Appelt, S.; Ha¨sing, F. W.; Baer-Lang, S.; Shah, N. J.; Blu ¨ mich, B. Chem. Phys. Lett. 2001, 348, 263–269. (9) Bowers, C. R.; Weitekamp, D. P. Phys. Rev. Lett. 1986, 57, 2645–2648. (10) Natterer, J.; Bargon, J. Prog. Nucl. Magn. Reson. Spectrosc. 1997, 31, 293– 315. (11) Hu ¨ bler, P.; Giernoth, R.; Ku ¨ mmerle, G.; Bargon, J. J. Am. Chem. Soc. 1999, 121, 5311–5318. (12) Aime, S.; Canet, D.; Dastru, W.; Gobetto, R.; Reineri, F.; Viale, A. J. Phys. Chem. A 2001, 105, 6305–6310. (13) Bouchard, L. S.; Burt, S. R.; Anwar, M. S.; Kovtunov, K. V.; Koptyug, I. V.; Pines, A. Science 2008, 319, 442–445. (14) Kovtunov, K. V.; Beck, I. E.; Bukhtiyarov, V. I.; Koptyug, I. V. Angew. Chem. 2008, 120, 1514–1517. (15) Adams, R. W.; Aguilar, J. A.; Atkinson, K. D.; Cowley, M. J.; Elliott, P. I. P.; Duckett, S. B.; Green, G. G. R.; Khazal, I. G.; Lopez-Serrano, J.; Williamson, D. C. Science 2009, 323, 1708–1711. (16) Atkinson, K. D.; Cowley, M. J.; Elliott, P. I. P.; Duckett, S. B.; Green, G. G. R.; Lopez-Serrano, J.; Whitwood, A. C. J. Am. Chem. Soc. 2009, 131, 13362– 13368. (17) Adams, R. W.; Duckett, S. B.; Green, R. A.; Williamson, D. C.; Green, G. G. R. J. Chem. Phys. 2009, 131. 10.1021/ac101738f  2010 American Chemical Society Published on Web 08/11/2010

Figure 1. Schematic diagram of the low-field NMR spectrometer with three polarization devices: (1) 2 T Halbach magnet, (2) Rb-Xe gas-flow polarizer, and (3) gas-flow equipment to produce p-H2 enriched H2 gas.

Here, we report the feasibility of applying NH-PHIP to increase the sensitivity of low-field NMR. A continuous gas-flow apparatus is shown to utilize SPINOE and PHIP hyperpolarization techniques via easily switchable connections. We demonstrate how the NHPHIP technique differs from other hyperpolarization technologies in NMR experiments at the mT-regime. EXPERIMENTAL SECTION Sample Preparation. The NMR samples with pyridine were prepared by keeping [Ir(COD)(PCy3)(py)][PF6] (PCy3 ) triscyclohexylphosphine and py ) pyridine)/pyridine as 1:5 (wt: wt) in methanol-d4 which was adopted from the literature15,16 without further investigation. The ratio of pyridine to methanold4 was varied from 1:1 to 1:81 920. NMR Spectroscopy. In PHIP experiments, a 0.4 mL NMR sample was dispensed into a 10 mm glass NMR tube which was connected to the parahydrogen setup where 50% para-enriched H2 gas was produced by passing regular H2 through activated charcoal at 77 K. The pressure inside the NMR tube was set to 1.2, 3.5, or 7 bar via a pressure gauge. After the sample was shaken in a continuous flow of p-H2 gas at low field to ensure proper dissolution of fresh gas in the solution, the sample was inserted into the coil, and 1H NMR data was acquired after a 90° pulse excitation at B0 ) 3.9 mT (νH ) 166.3 kHz) in a single scan. Reference experiments before and after the PHIP NMR experiments were performed at 3.9 mT with samples that had been thermally prepolarized at 2 T. For SPINOE experiments,8 a 0.4 mL NMR sample in a 10 mm glass NMR tube which was connected to a Rb-Xe gas flow polarizer was immersed in a liquid nitrogen bath inside a 1 T Halbach magnet for 1 min to accumulate polarized Xe ice on top of the frozen sample. Then, the liquid nitrogen bath was removed, and the sample was heated to room temperature inside the magnet reaching an Ostwald equilibrium of Xe gas at 7 bar. Finally, the sample was placed into the low-field NMR spectrometer, and single-scan 1H NMR signals were recorded. The amplitude of

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H SPINOE signal at 7 bar is similar to that of the thermally prepolarized signal at 2 T. The SNR was determined from the FID according to SNR ) Vs/(2(2)1/2 × Vnoise) where Vs is the peak-to-peak voltage of the FID signal, and Vnoise is the rootmean-square voltage of noise. RESULTS AND DISCUSSION A low-field NMR spectrometer18 operating at frequencies from 5 to 250 kHz is used in this work. A schematic diagram of the experimental setup is shown in Figure 1. A 10 mm glass NMR tube containing a 0.4 mL sample is connected either to a Rb-Xe gas polarizer apparatus8 or to equipment that generates a continuous flow of p-H2 enriched hydrogen gas. The switch between different hyperpolairzation techniques can be easily performed. Reference experiments were executed with thermal prepolarization in a 2 T Halbach magnet.1,18 1 H free induction decay (FID) and corresponding spectra of NH-PHIP hyperpolarized pyridine at different concentrations (Figure 2, left) are compared to reference data acquired with thermally prepolarized pyridine (Figure 2, right). Pyridine (4.9 µL) can be detected in a single scan after prepolarization (Figure 2b, right). Surprisingly, a signal similar in amplitude can be obtained with as little as 4.9 nL of NH-PHIP polarized pyridine (Figure 2c, left). The limit of detection (LOD) of our equipment where signal-to-noise ratio SNR ) 1 is estimated to be about 1 µL with thermally prepolarized pyridine and about 1 nL with NHPHIP hyperpolarized pyridine. Therefore, the LOD of the NHPHIP method is about 1000 times smaller than that of the thermal prepolarization method using a 2 T Halbach magnet. Compared to the conventional high-field (10 T) NMR, our single-scan lowfield NMR spectroscopy presents comparable SNR. This is mainly because the loss of the SNR due to the Faraday induction term which is proportional to B0 is compensated by the more sensitive (18) Appelt, S.; Ha¨sing, F. W.; Sieling, U.; Gordji-Nejad, A.; Glo ¨ggler, S.; Blu ¨ mich, B. Phys. Rev. A 2010, 81, 023420.

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Figure 2. Comparison of NMR with NH-PHIP enhancement and with thermal prepolarization. Single-scan 1H FIDs and corresponding spectra (inset) of NH-PHIP polarized pyridine (left) and prepolarized pyridine (right) in 0.4 mL methanol-d4 solutions containing pyridine in amounts of (a) 0.2 mL, (b) 4.9 µL, (c, left) 4.9 nL, and (c, right) 156.2 nL. All spectra are plotted on the same vertical scale. The spectra in (b, right) and in (c) have been magnified 10 times.

receiver coil in the low-field NMR, the narrower bandwidth at low field, and the large polarization induced by NH-PHIP. The experimental results (Figure 2) at 3.9 mT show that the small 1 H chemical shift differences (e1 ppm) in pyridine are not resolved due to the inhomogeneity of the field (a few ppm/cm3), but the chemical-shift-resolved 1H NMR spectrum of acetic acid (∆δ ≈ 10 ppm) at this B0 field has been reported.19 For smaller chemical shift differences (methanol, ethanol), if a heteronuclear J-coupling exists in the sample, the chemical shift could also be measured.19 Further technological improvements considering the homogeneity and temporal stability of the B0 field will allow a spectral resolution in the subppm regime. (19) Appelt, S.; Glo ¨ggler, S.; Ha¨sing, F. W.; Sieling, U.; Nejad, A. G.; Blu ¨ mich, B. Chem. Phys. Lett. 2010, 485, 217–220.

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In Figure 2, the thermally prepolarized pyridine signal decreases with decreasing pyridine amount, whereas the NH-PHIP hyperpolarized pyridine in dense solution (containing 0.2 mL pyridine) produces smaller signal than that in dilute solution (containing 4.9 µL pyridine). In order to understand this intriguing behavior, we measured the dependence of the SNR on the pyridine concentration [py] over 5 orders of magnitude. Figure 3 compares the SNR in 1H NMR experiments with pyridine following three polarization methods: (1) thermal prepolarization at 2 T, (2) polarization by Rb-Xe SPINOE, and (3) polarization by NH-PHIP. The SNR is expected to depend linearly on the proton number density in the sample for 1H NMR experiments with polarization methods 1 and 2. This expectation is confirmed by the experimental results in Figure

Figure 3. Comparison of SNR in 1H NMR spectroscopy with pyridine following three polarization methods: (1) thermal prepolarization at 2 T (b), (2) Rb-Xe SPINOE (9), and (3) NH-PHIP (1, (, and O). Double logarithmic plot of the SNR versus the pyridine volume in a 0.4 mL methanol-d4 solution. The SNR data of the pyridine signal with methods 1 (b) and 2 (9) are fitted by a linear function of the pyridine volume. The NH-PHIP experiments were performed at three different p-H2 gas pressures: 7 bar (1), 3.5 bar ((), and 1.2 bar (O). The dashed lines separate the NH-PHIP experiments into three regions. In region I (4-40 nL), the SNR increases linearly with increasing [py]. In region II (900-36 000 nL), the SNR is constant independent of [py]. In region III (36 000-400 000 nL), the SNR decreases with increasing [py]. The arrows indicate where [py] ) [p-H2] at the given p-H2 gas pressures.

3, where the measured SNR values in methods 1 and 2 are well fitted by a linear function of the pyridine amount in the 0.4 mL methanol-d4 solution. The LOD of both methods can be extrapolated to be 1 µL. Interestingly, the SNR in NH-PHIP experiments depends on [py] (Figure 3) in a way completely different from the SNR following methods 1 or 2. We divide the concentration regime of pyridine into three regions (Figure 3): Region I is dominated by [py], region II is dominated by the concentration of p-H2 gas in the methanol-d4 solution ([p-H2]), and region III is dominated by [p-H2] and 1H T1-relaxation rate. The value of [p-H2] is calculated using Henry’s law constant of 620 MPa in methanol at room temperature.20 In our experiments at 7 bar p-H2 gas pressure, [p-H2] ) 1.7 × 1019/cm3. The three arrows in Figure 3 point to where [py] ) [p-H2] at the given p-H2 gas pressures above the solution. NH-PHIP is realized by transferring the polarization from p-H2 to pyridine when pyridine and p-H2 molecules are both temporarily bounded to a metal template which forms the p-H2 derived hydride ligand.16,17 Therefore, the enhancement in the NH-PHIP experiment is determined by the 1H T1-relaxation rate of pyridine and the polarization transfer rate Rp (the number of polarized pyridine per second in the solution). Rp is influenced by the formation rate of the p-H2 derived hydride ligand which depends on [py], [p-H2], and the metal template concentration [M]. Furthermore, simultaneous association of pyridine and p-H2 molecules on the metal template is required to form the hydride ligand. Because of the catalytic properties (20) Liu, Q. S.; Takemura, F.; Yabe, A. J. Chem. Eng. Data 1996, 41, 1141– 1143.

of the metal template and the transient nature of the temporary association, Rp is determined by the smaller value of [py] and [p-H2]. A standard NMR sample usually has a substrate/solvent volume ratio from 1/10 to 1/1000. Samples in this concentration range were measured at low field, and the SNR results are shown in region II in Figure 3. In region II, [py] is about 1.8 × 1019-6.6 × 1020/cm3 and [py] > [p-H2]. The SNR is constant and does not depend on [py]. This is only possible when the 1H T1-relaxation rate of pyridine and Rp are constant. In region II (and also in region I), the pyridine solution is so dilute that intramolecular relaxation mechanisms dominate, resulting in a constant 1H T1-relaxation rate of pyridine. Because [py] > [p-H2], Rp is proportional only to [p-H2]. This is confirmed by experimental data in Figure 3 which show that at 7 bar p-H2 gas pressure, the SNR is 2 times larger than that at 3.5 bar and 6 times larger than that at 1.2 bar. At the point where [py] ) [p-H2] (see the arrows in Figure 3), region II is left and region I is entered through a transition regime. As in region I [py] < [p-H2], Rp should be proportional to [py]. Because pyridine has a constant 1H T1-relaxation rate, the SNR is expected to be proportional to [py], which is experimentally confirmed in Figure 3. The similar SNR of NHPHIP experiments at three different p-H2 gas pressures with pyridine less than 20 nL in the solution further confirm the independence of [p-H2]. From region I, we can extrapolate the LOD of the NH-PHIP with pyridine to be 1 nL (1.8 × 1016/ cm3). In region III where [py] > [p-H2], the SNR decreases with increasing [py]. Although Rp is assumed constant at a given [p-H2], the 1H T1-relaxation rate of pyridine is not constant anymore and increases with [py] and [M]. This is due to the strong intermolecular interactions and the increased viscosity of the solution at high [py] and [M]. The linear dependence of the SNR on [p-H2] is observed at three different p-H2 gas pressures due to the fact that Rp ∝ [p-H2]. The NH-PHIP method favors the detection of pyridine at concentrations over almost 6 orders of magnitude. This large gain in sensitivity represents a large potential for trace analysis. On the basis of the reported works15,21 and our own experiences, analytes containing a nitrogen, oxygen, or sulfur atom in the aromatic ring, such as caffeine, nicotine, pyrrol, thiophene, furan, and their derivatives, could be NH-PHIP polarized. This selectivity of the NH-PHIP method for a certain class of substrates out of a complex mixture opens up the potential for biological and chemical applications, such as the blood analysis and drug detection. The selectivity of the NH-PHIP method could be extended in the future by the development of other specific catalytic systems. CONCLUSIONS A continuous gas flow apparatus has been described to utilize SPINOE or PHIP hyperpolarization technique to significantly (21) Atkinson, K. D.; Cowley, M. J.; Duckett, S. B.; Elliott, P. I. P.; Green, G. G. R.; Lopez-Serrano, J.; Khazal, I. G.; Whitwood, A. C. Inorg. Chem. 2009, 48, 663–670. (22) Greenberg, Y. S. Rev. Mod. Phys. 1998, 70, 175–222. (23) Savukov, I. M.; Romalis, M. V. Phys. Rev. Lett. 2005, 94, 123001. (24) Savukov, I. M.; Lee, S. K.; Romalis, M. V. Nature 2006, 442, 1021–1024.

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enhance the 1H low-field NMR signals. A trace amount of pyridine (∼1 nL or 12 nmol) in a 0.4 mL NMR sample (a concentration of 31 µM or ∼1 × 1016/cm3), which could not be detected via either SPINOE method or thermal prepolarization method at 2 T, could be measured in a single scan via the NHPHIP method. Motivated by its unprecedented sensitivity, the NH-PHIP method can be combined with low-field NMR spectroscopy and find application for trace analysis in the bio-, chemical, and material sciences. Time-resolved low-field NMR measurements of rare spins such as 13C and 15N can also be foreseen. It is reasonable to expect that the SNR of NH-PHIP enhanced low-field NMR spectroscopy can be improved further

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and the principle of NH-PHIP be extended to a broad range of other systems. p-H2 gas (100%) could be adopted to improve the NH-PHIP polarization and, thus, reduce the LOD to picoliters. The use of more sensitive detection methods, such as atomic magnetometers, SQUIDs, and optical detection methods22-24 could further lower the LOD to the femtoliter regime.

Received for review July 1, 2010. Accepted August 4, 2010. AC101738F