Report
THE NANOLITER NICHE The ideal analytical instrument rapidly provides a wealth of chemical and structural data from a minimal sample amount, but such a panacea seldom exists. Consequently, analytical scientists must evaluate the capabilities and limitations of techniques within practical constraints. NMR spectroscopy fulfills a critical role through its ability to produce unmatched structural information and provide data on intramolecular dynamics. For instance, NMR is the only technique available that C3T1 yield the three-dimensional structure of a protein in solution Furthermore NMR spectroscopy features an experimental versatility virtually unsurpassed in a nondestructive analytical method To accommodate the particular research nneds so fhe analvst NMR methods varv in comolexityfromonedimensional nroton snectra to multinuclear This Report examines the shortcomings of NMR that prevent its more extensive use for mass-limited samples. In addition to addressing probe hardware and performance specifications, we focus on nanoliter-volume NMR spectroscopy (which can provide increased mass sensitivity) and the combination of microseparations wiih NMR detection. (For information on NMR history, background, and theory, consult Refs. 1-7.) Despite continuous improvements in the homogeneity and field strength of superconducting magnets during the past 15 years, currently available magnetic fields yield NMR transitions of very low energy (~10~25 J/spin). Because the transition energy is small with respect to kT (Boltz-
NMR Detection for Trace Analysis and Capillary Separations
Microcoils significantly boost NMR mass sensitivity and provide new detection opportunities.
man constant x temperature) at room temperature, the population difference between the upper and lower energy states (as governed by the Boltzmann distribution) represents r T i n r ^ n t r a t i n n f ^ ^ n t a n d nKtain sper"tra
from lower concentrations of analytes Although the initial reports of coupling CE with NMR demonstrated its potential, original spectral LWs were larger than 7 Hz. Broad LWs make characterization of unknowns difficult, and the —100 mM LODs precluded most assays. The improvements in microcoil NMR probe design yield high-resolution spectra (typically, a LW ~1 Hz qualifiee sa sigh resolution) and improved LODs. With these advances, the combination of CE and NMR becomes practical in situations requiring separation and structural determination of mass-limited unknowns Though not yet performed for capillaries, the demonstrated nondestructive analytical capabilities of NMR include direct measurement of pH (26), selective detection of charged species (27), diffusion coef-
Figure 4. Field-amplified sample stacking in CE using NMR detection. A mixture of 15 nmol each of arginine (Arg) and triethylamine (TEA) was injected into the capillary behind a region of higher ionic strength, then stacked from 50 mM to 113 and 214 mM, respectively (middle spectrum). Arg signals appear at about 3.1 and 1.5 ppm; TEA, at 2.5 and 0.95 ppm.
ficients (28), mobilities (27,28), and flow imaging (29). In addition, electroosmotic flow and solvent composition can be monitored directly. For instance, preliminary data show an essentially linear relationship between LW and electroosmotic flow rate. In this fashion, LW can be used to measure the electroosmotic flow rate of a solvent and the electrophoretic migration rate of a charged species. By examining the LW, the coil residence time and flow rate of each analyte can be computed as it migrates through the detector. Such information is often dimcult to obtain with other detection methods without perturbing the separation. Itiis aspect of JNJV1K detection has not yet been fully exploited. A greater understanding of the fundamental separation mechanisms among the many CE modes of operation would aid in the development of improved or novel approaches. Expectations
Based on projected improvements in microcoil NMR figures of merit, we expect the flexibility and performance of nanolitervolume NMR probes to continue to advance. In the near future, deuterium lock channels, multinuclear probes, and higher field strengths undoubtedly will add to the capabilities of microcoil NMR. Other advances can also yield significant increases in performance. For example, cooling a copper detection coil to 77 K should yield a 2.5-fold enhancement of sensitivity
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(12) Barbara,T. M.J. Magn. Reson., Ser. A 1994,109,265-69. (13) Keifer, P. A; Baltusis, L; Rce, D. M.; Tymiak, A A; Shoolery, J. N./. Magn. Reson., Ser. A 1196,119, 65-75. (14) Webb, A G. Pro,. Nucl. Magn. Reson. Spectrosc. .997,31,1-42. (15) Callaghan, P. T.J. Magn. Reson. 1990, 87, 304-18. (16) Wu, N.; Peck, T. L.; Webb, A G.; Magin, R. L.; Sweedler, J. Y.J. Am. Chem. Soc. 1994,116,7929-30. (17) Peck, T. L.; Magin, R L; Lauterburr P. C. /. Magn. Reson., Ser. B 1995,108,114-24. (18) Albert, K. et al. Anall Chem. 1994, 666 3042-46. (19) Wu, N.; Webb, A; Peck, T. L; Sweedler, J. V. Anal. Chem. 1995, 67,3101-07. (20) Dorn, H. C. Anall Chem. 1984,56,747 A-58A (21) Korhammer, S. A; Bernreuther, A Freseniusj. Anal. Chem. 1196,354,131-35. Recent efforts have included coupling (22) Behnke, B. el al. Anal. Chem. 1996,68, standard HPLC/NMR to MS (32). We pre1110-15. (23) Schlotterbeck, G.; Tseng, L-H.; Handell H.; dict that this combination will be demonBraumann, U.; Albert, K. Anall Chem. strated soon on the nanoliter scale. The 1997,69,1421-25. separation and characterization of compo(24) Wu, N.; Peck, T. L; Webb, A G.; Magin, nents of complex, mass-limited mixtures R L ; Sweedler, J. V. Anal. Chem. 1994, 66,3849-57. may prove to be the main driving force be(25) Huang, X.; Coleman, W. F.; Zare, R N. hind the advancement of microcoil NMR / Chromatogr. 1989,480,95-110. and its coupling to other methods. (26) Lawrence, B. A; Poise, J.; DePina, A; Allen, M. M.; Kolodny, N. H. Curr. Microbiol. .997,34,280-83. We gratefully acknowledgefinancialsupport (27) Heil, S. R; Holz, M.Angew. .hem., Int. Ed. from the Nattonal lnstitutes of Health (PHS1 Engl. 1996,35,1717-20. R01 GM53030) and the David and Lucile Pack(28) Hinton, D. P.; Johnson, C. S.J. .olloid Inard Foundation. An NSF graduate fellowship to terface eci. 1995,173, 3,4-717 MEL is appreciated as well as the assistance of (29) Wu, D.; Chen, A; Johnson, C. S.J. Magn. the Varian Oxford Instruments Center for ExcelReson.. Ser. A A195,115,123-26. lence in NMR (VOICE Lab) in the School of (30) Navon, G.; Song, Y-Q.. Room, T; Appelt, ,.; Chemical Sciences at the University of Illinois. Taylor, R E.; Pines, A Science e196,271, Cover photo courtesy of MRM Corp., Savoy, ,L. 1848-51. (31) Rogers, J. A; Jackman, R. J.; Whitesides, References G. M.; Olson, D. L; Sweedler, J. V. Appl. Phys. Lett. 1197, 70,2464-66. (1) Ernst, R R; Bodenhausen, G.; Wokaun, A Principles of Nuclear Magnetic Resonance (32) Shockcor, J. P.; Unger, S. E.. Wilson, ,I D.; Foxall, P.J.D.; Nicholson, J. K; Lindon, ,J C. in One and Two Dimensions; Oxford UniAnal. Chem. 1196,68,4431-35. versity Press: Oxford, 1991. (2) Derome, A E. Modern NMR Techniques for Chemistry Research; Pergamon Press Ltd.: Dean L. Olson is a postdoctoral associate New York, 1987. whose research focuses on enzyme kinetics, (3) Shoolery, J. N. Anal. Chem. 1193, 65, bioanalytical methods, and developing new 731A-41A applications of microcoii NMR. Michael E. (4) Waugh, J. S.Anal. Chem. 1193, 65, 725A-29A. Lacey is a graduate student whose research (5) Freeman, R.Anal. Chem. 1193, 65, focuses on applying microcoil NMR to biologi743A-53 A cal systems through hapillary preconcentration (6) Noble, D. Anall Chem. 1994, 66, 658 Aand separation. Jonathan V. Sweedler, associ61A ate professor, focuses his research on develop(7) Komoroski.RA Ana/. Chem. .994,66, 1024A-33A. ing new analytical techniques to assay (8) Olson, D. L.; Lacey, M. E.; Sweedler, ,. V. nanoliter-volume samples and identifying Anal. Chem. 1198, 70, 645-5-5 new signaling molecules and understand(9) Olson, D. L.; Peck, T. L; Webb, A. G.; Maing theirfunction in invertebrate model systems. gin, R. L.. Sweedler, J. V. Science 1995, Correspondence ebout this article cac be ad270,1967-70. (10) Freeman, R A Handbook ofNMR; Longdressed to Sweedler at Debt of Chemistry and man Scientific & Technical: New York, Beckman Institute University ofIllinois at 1988; p 220. Urbana-Champaisn Urbana IL 61801 (11) Hoult, D. I.; Richards, R. E.J. Magn. Reson. (sweedler@bozo scs vivv edue 1976,24, 71-85. (11), and using a superconducting coil material could provide even greater improvement. In some applications, using hyperpolarization transfer or an optically pumped system may result in significant enhancements (30). Coils fabricated by microcontact printing may offer superior spectral line shapes without shimming or the use of magnetic susceptibility-matching fluid but require the conductance of the coil material to be increased (31). In addition, microfabricated coils including planar coils offer even smaller sample volume requirements in the picoliter range possibly higher mass sensitivities and may find application for routine chemical assays (14)
Analytical Chemistry News & Features, April 1, 1998
