Analysis of Multiple Samples Using Multiplex Sample NMR - American

Apr 24, 2001 - to excite and detect multiple samples in rapid succession. This method is advantageous for samples with relatively long longitudinal (T...
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Anal. Chem. 2001, 73, 2541-2546

Analysis of Multiple Samples Using Multiplex Sample NMR: Selective Excitation and Chemical Shift Imaging Approaches Ting Hou, Jay Smith, Ernesto MacNamara, Megan Macnaughtan, and Daniel Raftery*

H. C. Brown Laboratory, Department of Chemistry, Purdue University, West Lafayette, Indiana 47907-1393

Two improved approaches for the rapid analysis of multiple samples using multiplex sample NMR are described. In the first approach, frequency-selective 90° radio frequency pulses and large pulsed field gradients are applied to excite and detect multiple samples in rapid succession. This method is advantageous for samples with relatively long longitudinal (T1) relaxation times. In the second approach, chemical shift imaging is applied to acquire both the spectral and spatial information of multiple samples simultaneously. Chemical shift imaging is more time-consuming than selective excitation; however, it is advantageous for detecting samples with short T1’s and for signal averaging. Both approaches demonstrate the potential of multiplex sample NMR for carrying out highthroughput NMR detection. Due to their capabilities for providing efficient synthesis and product screening strategies, combinatorial chemistry methods have been rapidly and widely adopted with great impact in areas such as pharmaceutical research, organic synthesis, and catalyst discovery.1-4 As a result of the large numbers of samples that need to be analyzed in the combinatorial chemical process, highthroughput analysis methods are highly desired. In a number of analytical areas, efforts have been made to meet this requirement.5-8 For example, mass spectrometry methods have been developed to accommodate the high-throughput needs in analyzing thousands of compounds per day.7,8 Due to its high resolution and unique capabilities in structure determination and quantitative analysis, nuclear magnetic resonance (NMR) spectroscopy is extremely useful for a variety of chemical analyses. In particular, the need for high-throughput NMR approaches for drug discovery has been expressed and such * To whom correspondence should be addressed: (e-mail) [email protected]. (1) Czarnik, A. W.; Keene, J. D. Curr. Biol. 1998, 8, R705-R707. (2) Bolger, R. Drug Discovery Today 1999, 4, 251-253. (3) Lam, K.; Lebl, M.; Krchnak, V. Chem. Rev. 1997, 97, 411-448. (4) Ellman, J.; Stoddard, B.; Wells, J. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 2779-2782. (5) Fitch, W. L. Mol. Diversity 1998, 4, 39-45. (6) Boutin, J. A.; Hennig, P.; Lambert, P.; Bertin, S.; Petit, L.; Mahieu, J. P.; Serkiz, B.; Volland, J. P.; Fauchere, J. L. Anal. Biochem. 1996, 234, 126141. (7) Wang, T.; Zeng, L.; Strader, T.; Burton, L.; Kassel, D. B. Rapid Commun. Mass Spectrom. 1998, 12, 1123-1129. (8) Liu, H. H.; Felten, C.; Xue, Q. F.; Zhang, B. L.; Jedrzejewski, P.; Karger, B. L.; Foret, F. Anal. Chem. 2000, 72, 3303-3310. 10.1021/ac0100751 CCC: $20.00 Published on Web 04/24/2001

© 2001 American Chemical Society

methods are being developed.9 For example, Fesik and co-workers described methods to investigate structure-activity relations (SAR) by NMR10,11 to determine protein-drug interactions and to aid in the synthesis of potential drug molecules. Twodimensional (2D) experiments were performed on 10-100 potential drug compounds at the same time at the rate of 1 every 10 min, allowing 1000-10 000 samples to be screened in a 24-h period using an automatic sample changer.11,12 Shapiro and co-workers developed a method based on ligand diffusion, which they called affinity NMR, to analyze small ligands binding to protein targets.13 A relatively large number of ligands can be analyzed simultaneously in the same NMR tube using this method. Another related technique called NOE pumping has also been reported by Shapiro and co-workers.14,15 To obtain improved structural information on compounds in complex mixtures, there are significant and increasing efforts to couple NMR with chromatographic separation methods such as LC, HPLC, and CE.16,17 The requirements for higher sensitivity and small working volumes imposed by chromatographic methods have led to the development, refinement, and application of a number of techniques, including microbore HPLC, flow-through probes, micro- or nanoliter volume probes, and solvent suppression pulse sequences.17-22 Besides the traditional automatic sample changer, flow probes17 have been adopted to achieve rapid NMR analysis of up to 500 samples/day.23 This technique has also been interfaced to standard 96-well microtiter plates used in the pharmaceutical industry. (9) Shapiro, M. J.; Gounarides, J. S. Prog. Nucl. Magn. Reson. Spectrosc. 1999, 35, 153-200. (10) Shuker, S. B.; Hajduk, P. J.; Meadows, R. P.; Fesik, S. W. Science 1996, 274, 1531-1534. (11) Hajduk, P. J.; Meadows, R. P.; Fesik, S. W. Science 1997, 278, 497-&. (12) Hajduk, P. J.; Gerfin, T.; Boehlen, J.-M.; Ha¨berli, M.; Marek, D.; Fesik, S. W. J. Med. Chem. 1999, 42, 2315-2317. (13) Lin, M.; Shapiro, M. J.; Wareing, J. R. J. Org. Chem. 1997, 62, 8930-8931. (14) Chen, A.; Shapiro, M. J. J. Am. Chem. Soc. 1998, 120, 10258-10259. (15) Chen, A.; Shapiro, M. J. J. Am. Chem. Soc. 2000, 122, 414-415. (16) Korhammer, S. A.; Bernreuther, A. Fresenius J. Anal. Chem. 1996, 354, 131-135. (17) Albert, K. J. Chromatogr., A 1995, 703, 123-147. (18) Wu, N.; Webb, A. G.; Peck, T. L.; Sweedler, J. V. Anal. Chem. 1995, 67, 3101-3107. (19) Olson, D. L.; Peck, T. L.; Webb, A. G.; Magin, R. L.; Sweedler, J. V. Science 1995, 270, 1967-1970. (20) Smallcombe, S.; Patt, S. L.; Keifer, P. J. Magn. Reson. Ser. A 1995, 117, 295-303. (21) Webb, A. G. Prog. Nucl. Magn. Reson. Spectrosc. 1997, 31, 1-42. (22) Lacey, M. E.; Subramanian, R.; Olson, D. L.; Webb, A. G.; Sweedler, J. V. Chem. Rev. 1999, 99, 3133-3152. (23) Keifer, P. A. Curr. Opin. Biotechnol. 1999, 10, 34-41.

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Despite these advances, however, NMR measurements are usually made on a single sample at a time, and throughput is therefore limited by this serial approach. To overcome this limitation, parallel NMR detection methods are highly desirable. For example, Oldfield reported the observation of three samples (one liquid and two solids) using separate probes tuned to observe different nuclei in the same high-field magnet.24 Recently, we reported a new approachsmultiplex sample NMRsfor improving NMR throughput.25-27 This methodology employs multiple coils connected in parallel that are tuned to the same resonant frequency to allow simultaneous detection of multiple samples. Two approaches have been adopted to achieve simultaneous detection. One approach is to construct separate resonance circuits with complete electrical and magnetic isolation in both the region of the detection coils and the rest of the rf circuitry.25 Using this method, it is possible to detect the signals emanating from all samples simultaneously without cross-talk. This approach has recently been optimized for small microcoil detection volumes using rf switches to couple the detection coils to the NMR receiver.28 A second approach with simplified hardware requirements is to construct parallel detection coils (four at present) in the same rf circuit.26,27 In this case, there is no ill effect of the coupling of different coils resonant at a common frequency. A field gradient is applied across the coils to differentiate the signals from each coil. Both 1D and 2D experiments have been carried out using this approach. Several spectral analysis methods can be used to identify the peaks in the spectrum, including peak picking, spectral subtraction, and multiplication methods. In this paper, we report further developments in the multiplex sample NMR methodology for rapid multiple sample detection. The exquisite control of rf pulses and pulsed field gradients (PFGs) currently available in modern NMR spectrometers allows a number of alternative approaches. Rapid sample selective excitation and chemical shift imaging (CSI) experiments are carried out with the application of large PFGs on the order of 10 G cm-1. In the selective excitation experiments, frequencyselective Gaussian-modulated rf pulses and large field gradients are applied to excite and detect multiple samples selectively and in rapid succession. This method is advantageous in analyzing samples with relatively long T1 relaxation times, such that one can collect the spectrum of one sample while the other samples relax to thermal equilibrium. When analyte T1 values are quite short, a promising alternative approach is to separate the spectral and spatial information into two dimensions using CSI methods.29,30 CSI techniques are well known in the magnetic resonance imaging literature and have been widely used to distinguish regions with different chemical compositions in human body images. Here, the CSI method gives both spectral and spatial information of multiple samples simultaneously, and it provides a means for fast detection (24) Oldfield, E. J. Magn. Reson. Ser. A 1994, 107, 255-257. (25) Fisher, G.; Petucci, C.; MacNamara, E.; Raftery, D. J. Magn. Reson. 1999, 138, 160-163. (26) MacNamara, E.; Hou, T.; Fisher, G.; Williams, S.; Raftery, D. Anal. Chim. Acta 1999, 397, 9-16. (27) Hou, T.; MacNamara, E.; Raftery, D. Anal. Chim. Acta 1999, 400, 297305. (28) Li, Y.; Wolters, A. M.; Malawey, P. V.; Sweedler, J. V.; Webb, A. G. Anal. Chem. 1999, 71, 4815-4820. (29) Brown, T. R.; Kincaid, B. M., Ugˇurbil, K. Proc. Natl. Acad. Sci. U.S.A. 1982, 79, 3523-3526. (30) Mansfield, P. Magn. Reson. Med. 1984, 1, 370-386.

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Figure 1. Schematic illustration of the multiplex sample NMR probe head with four sample coils located inside the inner pulsed field gradient coil. The outer gradient coil and the Teflon tubes connected to capillaries for flow sample introduction are omitted for clarity. See text for a detailed description.

of samples with short T1’s. The developments discussed here show the potential for the multiplex sample NMR methodology to impact areas that can benefit from high-resolution and high-throughput NMR detection. EXPERIMENTAL SECTION Chemicals. CDCl3 (99.8% D) and D2O (99.9% D) were purchased from Cambridge Isotope Laboratories (Andover, MA). 1-Propanol, ethanol, ethyl crotonate (96%), methyl ethyl ketone, Cr(acac)3, and 2,3-dibromopropionic acid (98%) were purchased from Aldrich (Milwaukee, WI). Pyridine and acetic acid were purchased from Mallinckrodt Laboratory Chemicals (Phillipsburg, NJ), and 2-propanol was purchased from Fisher Scientific (Pittsburgh, PA). A magnetic susceptibility matching fluid, Fluorinert FC-43, was obtained from Syn Quest Laboratories (Alachua, FL). All chemicals were used as received without further purification. NMR Probe. All spectral data were acquired on a home-built wide-bore (73-mm) probe. The probe head, which is illustrated in Figure 1, consists of four parallel sample coils that are surrounded by an actively shielded gradient coil pair. The construction of the four sample coils has been described previously,26 but with a few changes incorporated for the present experiments. Briefly, the solenoid geometry detection coils were fabricated by wrapping polyurethane-coated, high-purity (99.99%), 42-gauge (63.5-µm-diameter) copper wire (California Fine Wire Co., Grover Beach, CA) around glass capillaries (1.6-mm o.d., 0.8mm i.d.). The coils were attached to the capillary tubes using a cyanoacrylate adhesive (Krazy Glue, Borden Inc., Columbus, OH). Each coil consists of eight turns and has an inner diameter of 1.6 mm and a length of 0.6 mm. The sample tubes were mounted on to a rectangular support made of white Delrin, which holds the capillary tubes with an intercoil spacing (center to center) of 3.3 mm. An actively shielded gradient coil pair, which consists of oppositely wound inner and outer coils, was constructed to surround the four-sample coil array. The gradient coil design is based on Turner’s target field inversion technique.31,32 The gradient coils were formed by wrapping 21-gauge copper wire (31) Turner, R.; Bowley, R. M. J. Phys. E Sci. Instrum. 1986, 19, 876-879. (32) Turner, R. J. Phys. D Appl. Phys. 1986, 19, L147-L151.

Figure 2. Schematic illustration of the selective excitation experiment (only two samples are illustrated here). Without the applied field gradient, the spectra for both samples are centered at a frequency f0 (a). Upon application of the pulsed field gradient G1, one of the samples is shifted to the specified spectral window centered at f0 + ∆f (b) and is excited by a selective 90° rf Gaussian pulse. Immediately after detection, a second gradient G2 is applied to shift the second sample to the specified spectral window (c). It is also excited and detected by a second 90° rf Gaussian pulse. The actual pulse sequence of the selective excitation experiments, including refocusing gradients, is shown in (d).

around the inner (50 mm high, 30-mm o.d.) and the outer (50 mm high, 38-mm o.d.) cylinder support, which are both made of white Delrin. A nonuniform winding pattern was adopted here to produce a linear field gradient inside the detection coils region. The winding direction of the wire is opposite for the inner and the outer coils in order to cancel magnetic field gradients far outside the gradient coil region that could otherwise produce large eddy currents in the aluminum probe shield and the magnet bore. The field gradient produced by these coils was along the superconducting magnetic field (z) direction. The position of each turn of the inner and outer coils was calculated using Mathcad software. Both the sample coil array and the gradient coil pair were housed in a removable PVC container that was filled with Fluorinert FC-43, a magnetic susceptibility matching fluid that has been shown to improve spectral line width.19 A single resonant circuit was constructed using the four parallel sample coils and two nonmagnetic tunable capacitors (Voltronics, Denville, NJ) to tune and match the circuit. The coil housing was mounted atop a home-built probe body and used a semirigid copper coaxial line to connect the resonant circuit at the top of the probe to a BNC connector at the base. To allow flow introduction of samples, Teflon tubes (Small Parts Inc., Miami Shores, FL) were connected

to the capillaries using polyolefin heat-shrink (Small Parts Inc.). Samples were loaded using a syringe. Spectra were collected on a Varian Inova NMR spectrometer operating at 300 MHz for 1H. Initial shimming of the four coils to a typical resolution of 1-3 Hz was carried out before any experiments were performed. NMR Spectroscopy: Selective Excitation. A schematic diagram of the selective excitation experiment is shown in Figure 2. In these experiments, the rf transmitter was offset from the frequency center of the normal spectral region (f0) by a frequency ∆f, chosen to be 6000 Hz (20 ppm) in our experiments. Next, a large field gradient G1 was applied to all samples, shifting the field center of the first sample volume into the chosen spectral region. During the application of this PFG, a Gaussian-shaped, frequencyselective 90° rf pulse33 excited the sample that had been shifted into the spectral window of the transmitter (centered at f0 + ∆f). After excitation, a smaller, reverse gradient G1′ was applied to refocus any partially dephased magnetization, and the free induction decay (FID) signal was then acquired for this first sample. Immediately after acquisition, and without changing the rf transmitter frequency, a new gradient value G2 was chosen and applied along with another 90° rf Gaussian pulse allowing a second (33) Bauer, C.; Freeman, R.; Frenkiel, T.; Keeler, J.; Shaka, A. J. J. Magn. Reson. 1984, 58, 442-457.

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Figure 3. Pulse sequence for the CSI experiment. See text for a detailed description.

sample to be excited and detected. The procedure was repeated sequentially for all four samples. An alternative method to excite the samples selectively is to use the same gradient value and instead vary the transmitter offset. In this case, however, it is necessary to change the spectral observation window, which makes referencing more difficult. Signal averaging was achieved by looping over the samples multiple times, with careful tracking of the assignments of the samples and their corresponding FIDs in the data array. The actual pulse sequence used in these experiments is shown in Figure 2d. The pulse width of the rf Gaussian pulses was 2 ms, and 16 transients were acquired for each sample. The total acquisition time for four samples was ∼3.5 min. The optimal pulsed field gradient applied to each sample was determined in the following way: The duration of the first gradient G was kept constant (2010 µs), and its amplitude was arrayed from -30 to 30 G cm-1 in 21 steps with an increment of 3 G cm-1. In this arrayed experiment, the duration of the refocusing field gradient (G′) was set to 2 ms, and its amplitude was chosen to be half that of the first gradient. 1D spectra using such gradient arrays were obtained. The approximate amplitude of the first pulsed field gradient G for each coil was chosen such that only the sample in that coil was detected and with the best S/N. Then for each coil, the gradient G was further optimized for the best S/N and selectivity. Optimal values for the first gradient G for the four coils were -22, -8.5, 5.2, and 17.5 G cm-1. The amplitude of the refocusing gradients G′ was chosen to be half of that of the sample selective gradient G. Further optimization of the duration of the refocusing gradient G′ was carried out to obtain the best S/N for each coil. Thus, the following G′ duration values were obtained: 2.0, 2.1, 1.3, and 2.1 ms for the four coils, respectively. Chemical Shift Imaging. As described in the introduction, CSI methods can be utilized to obtain both spectral and spatial information of multiple samples simultaneously. In the multiplex sample NMR probe described above, the four sample coils are located in different positions along the z-direction, so only a single field gradient along the z-direction is needed to incorporate or encode the spatial information into the data set. The pulse sequence of the CSI experiment is shown in Figure 3. After the 90° excitation pulse (pulse width 8.5 µs), a PFG along the z-direction was applied during the t1 period for 600 µs during which the magnetization of the different samples evolved under both the chemical shift interaction and the applied field gradient. After the gradient was turned off, a small delay was added to allow eddy currents induced by the PFGs to dissipate. Then a 180° pulse (18 µs) was applied to refocus the chemical shift evolution. A second 90° (projection) pulse was applied after the echo to eliminate the phase modulation invoked by the applied gradient. In this manner, pure amplitude-modulated cosine and sine signals could be 2544

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obtained. As such, the signal was acquired during the t2 period in a hypercomplex format in which signals in the real and imaginary parts of the FID were accumulated independently and stored into two separate data sets.34 Thus, pure absorption 2D spectra were obtained upon Fourier transformation. Phase cycling with eight steps for both the real and imaginary data sets was used to eliminate artifacts caused by rf field inhomogeneities during the 180° pulses. A delay of 3 s between each scan was used, and four dummy scans were also applied before the entire experiment. The total acquisition time for experiments with 12 gradient increments and 8 transients for each gradient increment was ∼13 min. The 2D data set obtained in the CSI experiment was transferred from the spectrometer to a personal computer (PC) and analyzed using Mathcad software. Typically, 12 gradient increments were used, and the total accumulated phase evolution for the different samples due to the PFGs was between π and 4π. Linear prediction35 was applied in the t1 dimension prior to 2D Fourier transformation to extend the data set and limit truncation artifacts. RESULTS AND DISCUSSION Selective Excitation. Two examples of the results obtained using the selective excitation experiment are shown in Figure 4. Figure 4a-e shows the 1D spectra of 1-propanol, ethanol, acetic acid, and 2-propanol (all 0.5 M) in D2O obtained using the rapid selective excitation method. Figure 4f-j is the selective excitation result for a second set of samples: 0.5 M pyridine, ethyl crotonate, 2,3-dibromopropionic acid, and methyl ethyl ketone in CDCl3. The spectral line width of the four coils in the selective excitation experiments ranges from 2 to 4 Hz, slightly larger than that observed in the Bloch decay experiments (1-3 Hz). This small broadening effect is due to residual eddy currents caused by application of the gradients. Nevertheless, the results here show clearly that the selective excitation method can give clean and highly resolved spectra for each sample in the multiple coils. As discussed in the introduction, high throughput can be obtained even though we excite only one sample at a time. After exciting and detecting one of the four samples, it is not necessary to wait for a time of 3T1 for the excited sample to relax back to equilibrium. Instead, one can excite and detect other samples, while the excited sample relaxes back to equilibrium.28 The selective excitation approach is especially advantageous when the T1’s of the analytes are 1-2 s or longer. In fact, as long as 3T1 is larger than the number of coils times the acquisition time per coil, the selective excitation method will be highly efficient. In the selective excitation experiments performed here, the T1’s for the samples used are in the range of 3-5 s. Using conventional 1D NMR methods, the detection of these four samples acquired with 16 transients each would require 10-16 min, plus additional time for changing samples. In the experiments described here, the delay time was set to 2 s (limited by the long T1’s of the samples) between each scan, the acquisition time was 0.6 s, and the total time to acquire the spectra of four samples with 16 transients each was only 3.5 min. It is clearly shown here that (34) States, D. J.; Haberkorn, R. A.; Ruben, D. J. J. Magn. Reson. 1982, 48, 286292. (35) Barkhuijsen, H.; Debeer, R.; Bovee, W.; VanOrmondt, D. J. Magn. Reson. 1985, 61, 465-481.

Figure 5. 2D contour plot and spectra of 2,3-dibromopropionic acid, ethyl crotonate, methyl ethyl ketone, and pyridine (0.1 M in CDCl3) generated using the CSI pulse sequence shown in Figure 3. A total of 96 transients were acquired in hypercomplex mode. See text for data transformation parameters.

Figure 4. Isolated 1H spectra of two sets of samples obtained using the selective excitation methodology. Normal 1H Bloch decay spectrum of the first set of four samples (a) and the resulting subspectra of 0.5 M (b) 1-propanol, (c) 2-propanol, (d) acetic acid, and (e) ethanol in D2O. 1H Bloch decay spectrum of the second set of four samples (f) and the resulting subspectra of 0.5 M (g) pyridine, (h) ethyl crotonate, (i) 2,3-dibromopropionic acid, and (j) methyl ethyl ketone.

the selective excitation method provides a considerable gain in throughput. Less signal averaging would reduce this time considerably. There are some improvements that can be made at the present time. Currently, the selective excitation spectra show a small, 1020% loss in S/N compared to a single-pulse 1H Bloch decay experiment. Some of this loss is due to imperfect refocusing of the magnetization and the presence of residual eddy currents, resulting in slightly larger peak widths, and/or incomplete excitation using the selective rf Gaussian pulses. Also, as shown in Figure 4, the isolation between samples and the phase characteristics of the spectra are not yet perfected. Currently, the observed cross contamination between spectra is roughly 2-7%. These imperfections are caused by the somewhat broadband π/2 Gaussian pulse, which is known to have inferior spectral phase characteristics.36 To improve the selectivity and to reduce the phase distortion, 3π/2 Gaussian pulse37 or EBURP-1 pulses36 could be used, and these approaches are currently under investigation in our laboratory. Chemical Shift Imaging. Figure 5 shows the experimental results for 0.1 M ethyl crotonate, 2,3-dibromopropionic acid, (36) Geen, H.; Freeman, R. J. Magn. Reson. 1990, 87, 415-421. (37) Emsley, L.; Bodenhausen, G. J. Magn. Reson. 1989, 82, 211-221.

methyl ethyl ketone, and pyridine in CDCl3, which were loaded into the four-coil probe and acquired simultaneously using the CSI method. The normal T1 relaxation times are 4-6 s for these four samples, so a relaxation reagent, 1 mM Cr(acac)3, was added to shorten the experimental time. In the 2D contour plot shown in Figure 5, the four analytes in different coils are clearly spatially separated. By taking traces along the chemical shift dimension at the corresponding coil positions, clean and well-separated spectra of all four samples were obtained. The resolution obtained for the CSI experiment ranged from 2 to 5 Hz. Since all samples were excited and detected at the same time in the CSI experiments shown here, it was necessary to wait for a time of at least 3T1 to let all the samples relax back to equilibrium between scans. Also the imperfection of the 180° pulses due to the sizable rf field inhomogeneity of the short solenoid geometry coils required phase cycling. These considerations make the total acquisition time relatively long compared to the selective excitation method. The CSI methodology will therefore be advantageous in terms of high throughput for samples with short T1’s (less than 1 s). We note parenthetically that the T1 times decrease with molecular size and that T1 values of e1 s are observed for molecules with molecular weight greater than 500. In our experiments, Cr(acac)3 was added to shorten the T1’s of the samples to ∼1 s. As a result, the delay between each scan could be reduced to 3 s. A distinct advantage of the CSI method is that signals from all samples contribute to each transient, such that good S/N is obtained for lower concentrations. The S/N ratio of the CSI experiment is ∼70% of the standard single pulse (Bloch decay) spectrum, assuming equal concentrations and signal acquisitions. The observed cross contamination between spectra is in the range of 1-5%. One important point to consider is whether the S/N of the multiplex sample NMR probe is degraded compared to a singlecoil probe. In a parallel, two-coil circuit consisting of two identical coils, the inductance and resistance of the circuit are both reduced by a factor of 2; however, their ratio, L/R, is the same as for a single-coil circuit. Assuming that the noise is determined by the coil resistance, the S/N is proportional to Q1/2, where the quality factor Q ) ωL/R. One would therefore expect that the S/N would be independent of the number of coils. In line with this reasoning, we found that the parallel coil configuration in our multiplex Analytical Chemistry, Vol. 73, No. 11, June 1, 2001

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sample NMR probe does not adversely affect the S/N. 1H Bloch decay experiments to compare the S/N of the one- and four-coil probes show that a four-coil probe has almost exactly the same S/N as the single-coil probe with the same coil dimensions (spectra not shown). CONCLUSIONS We have described two improved approaches for achieving rapid NMR analysis of multiple samples using parallel detection coils. One approach employs selective sample excitation using bandwidth-limited 90° rf Gaussian pulses and large pulsed field gradients on the order of 10 G cm-1. Separate 1D spectra of samples in each coil can be obtained in rapid succession. The second approach uses a chemical shift imaging methodology, which gives both spectral and spatial information of multiple samples in a 2D spectrum. This method is somewhat more timeconsuming but can still achieve rapid sample detection on samples

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with short T1’s. The two approaches discussed here show the potential of multiplex sample NMR to increase the throughput of NMR analysis for a variety of high-throughput applications. ACKNOWLEDGMENT We express our appreciation to Dr. Tom Barbara at Varian Inc. for his aid in the gradient coil design, and Randall Replogle for his precision machine work. We also thank the reviewers for insightful comments. M. M. thanks the DoD for a graduate fellowship. This project has been funded in part by the NSF (CHE 95-31693), the Purdue Research Foundation TRASK Fund, and the Alfred P. Sloan Foundation.

Received for review January 17, 2001. Accepted March 20, 2001. AC0100751