Selective Sensitivity Enhancement in FT-NMR Thomas C. Farrar Department of Chemistry University of Wisconsin Madison, Wis. 53706 In this article the basic two-spin nuclear magnetic resonance ( N M R ) experiment and the new sensitiuity enhancement experiments are reuiewed. In port two of this two-part series o n oueruiew of two-dimensional NMR experiments will be presented. Part two will appear in the June 1 issue of ANALYTICAL CHEMISTRY.
From time to time experts in the area of NMR spectroscopy meet to discuss and to prognosticate future directions and developments in the field. Their conclusion is usually that NMR spectroscopy has reached a mature stage and that further developments in sensitivity, resolution, information content, new applications, and so on will be minimal. In fact, for the past three decades the field of NMR has advanced at a phenomenal rate of growth that shows no signs of abating. During the past decade enormous improvements in the sensitivity, speed, and reliability of modern analog and digital electronics and a concomitant decrease in cost have made it possible to design NMR spectrometers with capabilities undreamed of 5-10 years ago. These new analytical instruments are so powerful and so easy to operate that only the imagination of the spectroscopist limits the range of experiments that can be executed. Many of the substantial improvements in NMR are the result of the
spin gymnastics that can he orchestrated by the spectroscopist. That is, most of the selective sensitivity enhancement experiments and all of the twodimensional NMR experiments involve manipulation of the spins as they evolve or dephase for various periods of time, To explain these experiments we need to have a clear picture of how a simple time evolution experiment is carried out.
M, and the rf field, yB1, appear as stationary quantities. This rotating frame of reference greatly simplifies the vector pictures used to represent M,~ B I , Ha, and HB. When a 200-MHz (7r/2), pulse-a r/ 2 pulse applied about the x axis in the rotating frame of reference-is applied to the sample, the proton magnetization is rotated 90" ahout the I axis from an orientation along the magnetic field
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INS'TRLJMEN7A7lON One-dimensloMI NMR Let us consider the simple case of a Wenriched sample of chloroform. There are two spins each with spin quantum number I = 112. For a magnetic field of about 4.7 tesla (T), the proton NMR spectrum consists of a doublet centered a t about 200 MHz. The doublet separation is given by the carbon-hydrogen spin-coupling constant, JCH= 210 Hz. Similarly, the spectrum is a 210-Hz doublet centered at about 50 MHz. The proton and carbon spectra are shown in Figures l a and lh, respectively. The low-frequency line, Ha, in the proton spectrum arises from those molecules for which the proton is bonded to a L3C in the a-sDin state. The hiahfrequency proton line, HO, arises from those molecules for which the proton is bonded to a 13C in the &spin state. A similar situation holds for the 13C NMR spectral lines. If we represent the magnetization in a coordinate system that rotates at a frequency of we (200 MHz for protons), then the macroscopic magnetization, ~
Figure 1. H ' W h spectra at Bo = 4.7 T. (a) Proton spectrum. (b) W specbum.
Figure 2. Pulse ewects. (ad). A (*/2). pulse about t b x axis in the rotating hame rotates me proton
magnetization to a poalim a l q the yaxis, where it begins m &phase. (b) After a &phasing time r . the proton magnetizations have dephased because of chemical shin and spin-coupling interactions. A (n).pulse at Ihii point (b) rotatas bolh magnetization vectors about me xaxis imo the positions shown in b'. (c)~ n ean r additiona~ time 7 , the magnellzation vectors are refocused alow me -Y ais. 0003-2700/87/0359-679A/$01.50/0 0 1987 American Chemical Society
ANALYTICAL CHEMISTRY. VOL. 59, NO. 10. MAY 15, 1987
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Whenit comesto biotechnologyresearch I don't f i t yourway through theinformationmaze. Figure 5. Phase modulatbn of the NMR signal (seetext).
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ANALYTICAL CHEMISTRY, VOL. 59, NO.
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direction, the z direction in the rotating frame, to an orientation along the y axis in the xy plane (see Figure Za). For the Ha resonance line, which is very near the spectrometer reference frequency, o . , the magnetization will precess slowly in the rotating frame. For the H,3 resonance line, which is much further from o., the magnetization will precess much more rapidly in the rotating frame. Each of these magnetization vectors will generate an exponentially damped sine wave signal in the NMR receiver coil (Figures 3a and 3b). What one actually observes (Figure 3c) is the s u m of these two signals. The individual sine wave signals in Figures 3a and 3h are the result of the time evolution or the dephasing of the magnetization vectors Ha and HB. That is, as the H a and H,3 magnetizations get out of phase with one another and with the reference frequency wo, they generate the interference pattern shown in Figure 3c. This is referred to as phase modulation of the NMR signal. If at a time r after the initial (n/2),pulse we apply a pulse (Figures 2b and Zb'), the magnetization will hegin to refocus. At a time 2r after the initial (r/2), pulse, the magnetization will be refocused (Figure 2c). This experiment, sometimes called the spin-echo experiment, is the hasis for all the sensitivity enhancement and two-dimensional FT-NMR experiments that we will discuss later. Another situation that we encounter frequently is the effect of a r broadhand (i.e., nonselective) pulse on the magnetization of a given spin. If we apply a u pulse to the proton spins, we invert all of the proton spins. This inversion has the effect of interchanging the proton spin labels; the protons that were aligned with the magnetic field (proton a-spin) are now aligned against the field (proton &spin) as a result of
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the r rf pulse. Similarly, the @-spin states are converted to u-spin states hy the x pulse. We should probably mention that one cannot manipulate or measure the properties of individual nuclear spins; one can measure only the average property of a collection of spins. The effect of the (r/2), pulse then is to generate a transverse (i.e., in the xy plane) macroscopic magnetization, or magnetic coherence, for the Hu and the H@spin ensembles. A t this point the populations of all the energy levels are equal because there is no net macroscopic magnetization along the z axis.
Nuclear OvemauSar experlrnem Broad-band proton decoupling is normally employed in 'W NMR experiments for several r m n s . Consider methyl iodide, CHd, as an example. Proton decoupling collapses the 1% quartet (four lines with relative intensities of k331) to a single line with an intensity 24 timea greater than that of the outer linea in the quartet. One might expect the 1:331 quartet to collapse to a single line with an intensity eighttimeagreaterthanthatoftheouter lines. The additional threefold increase in sensitivity comes from the nuclear Overhauser enhancement (NOE) ( I ) . This is a cross-polarization phenomenon that is commonly used.
We will call the proton spin I and the carhon spin S. In the process of the Ispin decoupling, we transfer I-spin magnetization to the S-nucleus. The result is that we increase the maximum S-spin magnetization, Mz(S)mz,such that MJS),,
= 1 + Y&s
where y is the gyromagnetic ratio of the suhscript species. For the case I = proton and S = carbon (C),
M,(C),,
=
3.0
Thus in proton-decoupled 13C experiments we expect up to a threefold enhancement of the NMR signals. However, we realize this maximum enhancement only if the following conditions hold 1. The internuclear distance is short (about 1 A). 2. The relaxation is attributed to the nuclear dipole-dipole interaction. 3. The sample is in the extreme narT ~1). where wg is the rowing limit ( w ~
