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1H triple resonance experiments
Volume 192, number 4
CHEMICAL PHYSICS LETTERS
8 May 1992
The detection of weak heteronuclear coupling between spin 1 and spin l/2 nuclei in MAS NMR; 14N/’ 3C/ ‘H triple resonance experiments C.P. Grey and W.S. Veeman ’ NSR Centre for Molecular Design, Synthesis and Structure, Laboratory of Molecular Spectroscopy, University ofNijmegen. Toernooiveld, 6525 ED Nijmegen. The Netherlands
Received 3 1 December 199 1
Magic angle spinning and i4N irradiation have been employed to perturb the “‘N spin bath, in a new spin-echo triple resonance solid-state NMR experiment. Spinning introduces a time dependence in the 14Nquadrupolar interaction, permitting transitions between the three lZ, m)states, two or four times per rotor cycle; this alters the evolution of a i4N-coupled “C spin. The reduction in i3C echo intensity, on 14Nirradiation during the echo period, is greater for shorter C-N distances. Irradiation at a constant frequency is more effective than sweeping through the 14Nresonance. 14Ndouble-quantum spectra are obtained indirectly by irradiation of the double-quantum transition.
1. Introduction The observation of dipolar coupling between nuclei, in solid state NMR spectroscopy, can be exploited to obtain internuclear distances. Double resonance NMR methods have been developed to measure small dipolar couplings, which can often be obscured by other interactions. For instance, in SEDOR [ 1 ] (spin-echo double resonance) the dephasing and/or refocusing of the I nucleus in the spinecho experiment are modulated by applying x pulses to the coupled nucleus S, the strength of the dipolar coupling is then determined from the loss in refocused spin intensity I. REDOR [ 21 (rotational-echo double resonance ) , an extension of SEDOR for magic angle spinning (MAS) conditions, has enabled 13C15N distances of 4.0 8, to be measured with an accuracy of + 0.1 A in a crystalline peptide [ 31. SEDOR and REDOR experiments have been applied successfully to S= l/2 and I> l/2 coupled systems by monitoring the quadrupolar nucleus (I) [4]. These methods cannot be readily applied, however, ’ Present address: Physikalische Chemie, Fachbereich 6, Universitlt-GH-Duisburg, Lotharstrasse 1, W-4 100 Duisburg 1, Germany.
when the coupled S nucleus is quadrupolar, e.g. 14N. We have been interested in developing techniques for obtaining structural information from nitrogencontaining polymers. Isotopic substitution of i5N for the 99.6% abundant isotope 14N can often be prohibitively expensive and/or synthetically impossible. Furthermore, many studies with potential industrial applications require an investigation of polymeric material taken from large scale productions where isotopic substitution is clearly not feasible. The I= 1 nucleus, 14N, has quadrupole coupling constants, e*qQ/h, that range typically from 1 to 5 MHz [ 51. The first order quadrupole interaction shifts the 2Z+ 1 Zeeman levels of the 14N spin, resulting in resonances that are generally too broad to allow excitation and detection of the whole spectrum simultaneously. Hence, 14Nspectra of powdered materials have only been obtained indirectly [ 6 ] or from compounds with exceptionally small quadrupole coupling constants [ 71. The double quantum transition, Am = 2, for an I= 1 nucleus, is to first order, however, unaffected by the quadrupolar interaction; this has been employed to cross-polarize I= 1 nuclei [ 8 ] and to obtain 14N overtone spectra [ 91. We have explored two methods of exciting the 14N
0009-2614/92/S 05.00 0 1992 Elsevier Science Publishers B.V. All rights reserved.
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spins in polycrystalline solids: sweeping through the 14N resonance and continuous irradiation at one frequency. We chose, initially, to study a sample of glytine since the 13C spectrum of this material contains two discrete resonances arising from carbon atoms with different spatial proximity to the NH: group; a single-crystal 14N NMR study of this material gave values of 1.18 MHz and 0.54, for e2qQfh and the asymmetry parameter 9, respectively [ 10 1. The effect of 14N irradiation was monitored indirectly via the 13C spins, by observing changes in the 13C refocused intensity in a spin-echo experiment (fig. 1). The results described below suggest new methods for investigating 14N-13C coupling in rotating solids, and for the indirect detection of 14N double-quantum resonances.
2. Experimental methods The solid state NMR spectra were collected using a Bruker CXP300 at operating frequencies of 300.13, 74.54 and 21.69 MHz for ‘H, 13C and 14N, respectively. A home-built single coil, triple tuned probe was used which could be tuned to provide pulse lengths of 4.5 us for ‘H and 13C and 10 ps for the low y nucleus 14N. MAS was performed using a DOTY stator block and 5 mm zirconia rotors. Different 14N spectral offsets from a central carrier
k
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LETTERS
8 May 1992
frequency (chosen to be 21.686 MHz) were programmed into a home-built digital frequency synthesizer, designed to provide fast, phase-continuous frequency switching (described elsewhere [ 111) . This allowed either 14N frequency sweeps (from -200 to +200 kHz), or continuous irradiation at one frequency (where the offset could be changed automatically between experiments), to be performed. The output from this device was first fed into a Programmed Test Sources, Inc (PTS) 160 synthesizer, then into an EN1 3100L amplifier and tinally into a MSL300 broadband amplifier. The 14N pulse lengths were determined using a saturated solution of sodium nitrate, which gave a signal at approximately - 666 Hz from the carrier frequency of 2 1.686 MHz. Analytical-grade glycine and a sample of [2-13C] glycine, enriched to approximately 20% at C-2 (and prepared from 99.5% enriched [2-13C] glycine) were used.
3. Results Fig. 2 shows the effect of 14N irradiation on the 13Cecho intensity in a MAS experiment. A 14N sweep results in an increasing loss in 13Cintensity, the longer the time taken for the sweep (experiment (i ), fig. 2a). The effect is the most pronounced for the meth-
Lkcouple
13c R__
l4N
14N
(1)
I
T
(II)
Fig. 1. The two-pulse sequences used. For the MAS experiments, both the “C 1c pulse (inserted to refocus the ‘% isotropic chemical shifts) and the start of the acquisition arc synchronised with the rotor echoes. The 14N spins are irradiated for one half of the echo period in scheme II, with I providing the control.
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I
(Without i4N irradiation;
(a)
;
(i)sweep
I
A
.”
Q
: ;;)co2
.
%
(ii) single frequency
II
(With “‘N irradiation)
Difference Spectrum
. (ii)
Fig. 2. (a) The loss in the “C intensity on “‘N irradiation. I is the carbon intensity with irradiation, and 1, without (schemes (II) and (I ) in fig. 1). In experiment (i ) a sweep of 400 kHz was performed in 100 steps through a central carrier frequency of 2 1.686 MHZ (each step lasting a time r/100). In experiment (ii) 14Nirradiation was applied at a constant frequency of 21.686 MHz. Spinning speeds of 3560 and 3610 Hz were used for (i) and (ii), respectively. The ‘% spectra that provide the data for the two ringed points in experiments (i), with r=2209 us, areshown in (b).
ylene carbon [ C( 2) ] of glycine which is directly bonded to the NH: group. This can be seen clearly in the spectra shown in fig. 2b. Reducing the number of steps in the sweep from 100 to 50 did not alter the results noticeably. A greater reduction in the echo can, however, be achieved by irradiating the 14Nspins at a constant frequency (experiment (ii), fig. 2a): after irradiation at 2 1.686 MHz for 2600 us (i.e. ten rotor periods) only 14% of the C (2) signal remains. This was initially surprising, in view of the large width of the 14N resonance (x 1.8 MHz). The experiments were then repeated without MAS to investigate the role of the sample spinning. Again,
both a r4N sweep and irradiation at a single frequency resulted in significant losses in signal intensity. Fig. 3a, however, demonstrates that the loss of r3C signal is extremely sensitive to the 14N irradiation frequency and at an offset of 75 kHz the effect has essentially disappeared. The experiment was repeated for smaller 14Noffsets with the sample of [ 2*‘Cl glycine. A significant dip in the 13Crefocused intensity was only observed over a 10 kHz range (fig. 3b), the minimum occurring at = -2 kHz. In contrast, a loss in signal occurs over a much larger range of r4N offsets in the spinning experiments (fig. 4). Furthermore, there is now a pronounced increase in 381
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7 (a)
/ 1I , nTpTTprTTrlTT
0 -600
-500
-400
-300
I,
-200
-100
0
100
“-1
500
’ 4N offset (k&Is) ’ 4N offset
(kHz) Fig. 4. The change in C( 2) intensity as a function of 14N offset for spinning speeds of (i) 2270 Hz and (ii) 4540 Hz. A constant value of T, equal to 88 1 ps, was used for both experiments (i.e. two and four rotor periods for (i) and (ii), respectively).
0.8 I/I0 0.6
C”2
-30
-20
-10 l4 N
0
10
20
30
offset (kHz)
Fig. 3. (a) The variation in the amount of refocused “C intensity, as a function of the 14N offset from the central carrier frequencyof21.686MHz (?= 1900 ps). (b) The ‘%Zintensity from the [ 2-“C] glycine sample for smaller “‘N offsets (r= 1000 ps). Both experiments are performed without MAS. Error bars in (a) take into account the fact that the signal-to-noise obtained in this experiment was significantly worse than in all the other reported experiments,
the amount of 13C refocused intensity at offsets around = -2 kHz. The loss in signal is extremely sensitive to the rate of sample spinning and is considerably reduced, for identical lengths of’s, on doubling the spinning speed (compare curves (i) and (ii), fig. 4). The width of the dip parallels the width of the tuning of the probe for 14N (which has a Qfactor of % 40). By performing a further series of measurements, where the 14N channel is retuned for each change in the 14N offset, a loss in signal intensity is still observed out to +700 kHz, which rep382
resents the band-width of the 14N channel. One explanation for the marked asymmetry of the observed dip about the central frequencies is the presence of a standing wave in the probe at approximately 20.5 MHz. This results in a reduction of the power levels that enter the coil, the closer the 14N frequency is to this standing wave.
4. Discussion Dipolar coupling to 14N will cause the 13C transverse magnetisation to evolve with three different frequencies w. Assuming the “high field approximation” [ 121 is valid for 14N then
w=oo-mmwd
(m=-l,O,
+l)
(1)
and od = D( 3 cos2tl- 1)) where D, the dipolar coupling constant, is 0.635f0.011 kHz for C(2)-N in glycine [ lo]. 8, the angle the C-N internuclear vector makes with the applied field, will be time dependent for MAS. (A breakdown of this high field assumption will lead to a mixing of the IZ, m) eigenfunctions and the observation of asymmetric doublets in the normal MAS spectra; this splitting has been observed for the methylene carbon in glytine at a field of 3.52 T [ 131 but is not observable at our higher field of 7.05 T.) The application of a rc pulse to the 13C spins in the static experiment, or
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CHEMICAL PHYSICS LETTERS
the action of MAS in the case of spinning, result in the refocussing of the 13Cspins, forming Hahn and rotational echoes, respectively. We believe that we are observing at least two different mechanisms that modulate the 13C-14N dipolar coupling during the period of 14Nirradiation; these prevent the 13Cspins from refocussing, resulting in the reduction in the 13C echo intensity. The magnitude of the dipolar coupling is strongly dependent on the C-N distance, which is consistent with the greater effects observed for the C ( 2 ) carbons. One mechanism is responsible for the sharp dip/peak around the central 14N frequencies (figs. 3 and 4) and the other is only operational under conditions of MAS. The second order quadrupolar interaction is expected to broaden and shift the double-quantum transition of 14N significantly. Following the approach of Tycko [ 141, this interaction is calculated to shift the resonance by + 3.25 kHz and produce a powder pattern 5.5 kHz wide with a maximum at + 6 kHz. Solid ammonium chloride was found to resonate at - 8.0 kHz from the 14Ncarrier frequency and the 14N resonance of glycine is expected to contain a similar contribution from the chemical shift. Thus the double-quantum resonance is predicted to occur at a similar range of 14N frequencies as the pronounced decrease in Z/Z,, observed in the static experiment (fig. 3). This suggests that double-quantum transitions (between the ( - 1) and I+ 1) levels) are occurring during 14Nirradiation. Doublequantum decoupling has been demonstrated for ‘H*H coupled systems by irradiation of the 2H doublequantum transition and the effectiveness of this decoupling mechanism was shown to decrease rapidly for small 2H resonance offsets [ 15 1. The 14N irradiation suppresses, at least partially, the dipolar evolution during the first half of the echo period by decoupling the I+ 1) and ( - 1) levels; the 10) level will not perturb a coupled 13C spin. In the second half of the echo period, there is no 14N irradiation and the 13Cspins will dephase according to eq. ( 1), leading to a loss in refocused intensity, as was observed experimentally. The quadrupolar splitting becomes time dependent on MAS. Assuming, for simplicity, an axial electric field gradient (EFG) tensor (i.e. q= 0), then the first order quadrupole splitting, 20,, is given by
8 May 1992
3 e2qQ = -47 (JZsin2/3cos(a+o,t) +sin2/3cos[2(cu+o,t)]},
(2)
where /3 and (Yare the polar angles defining the orientation of the z axis of the EFG tensor with respect to the spinning axis; wo=O either two or four times per rotor cycle ( 1/o,), depending on the value of @. If the 14N spins are irradiated at a the Larmor frequency, o,, a single-quantum transition can then, in theory, occur either two or four times per rotor cycle. This is demonstrated for 14N irradiation at a frequency w,-S in fig. 5, which shows a possible trajectory for a spin A originating in the I - 1) level. Considering a 13Cnucleus coupled to this 14Nspin: for the first part of the rotor period the spin will evolve under dipolar coupling to the ) - 1) state. Assuming the simplest case where the principle axis of the dipolar tensor is coincident with that of the electric field gradient (which is the case for an axial EFG aligned along the C-N bond and occurs in many -CN compounds) then @‘, the phase acquired at time t, is simply 0 L
l/or I tl
I 0
I tz
1
I
l/or
2/or I
I
wt
wt
2/w r
Fig. 5. The change in the 14Neigenstates with time for MAS. The energy levels are shown in the laboratory frame, for an axial EFG at 14N,and angles of 30” and 90”, for B and (Y,respectively (see eq. (2)). The possible transitions between levels for one 14Nspin, A, caused by 14Nirradiation at a frequency of w,- 6, are shown.
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@I= UWw){245sin28[sin(a+o,t,)-sina] +sin2/3{sin[2(a+w,f’)]-
sin2a)).
(3)
If a transition to (0) occurs at time t, then the evolution under the dipolar coupling is temporarily halted. At time f2, spin A then reverts back to the ( - 1) state and further evolution will occur. The total phase acquired at the end of a rotor period is then Qi,= (D/2w,)
+sin2P{sin[2(a+o,t’)] -sin[2((r+c0,t2)]j].
(4)
After two rotor periods @2r=20, and, hence, further dephasing will occur, the longer the 14N irradiation period. Considering now the whole polycrystalline sample, with the whole range of values for Q and 8, and crossings between all three states at different times: each 13C spin will acquire a different phase, leading to a loss of signal intensity at the rotor echo. Faster spinning will reduce the probability of transitions occurring between levels, increasing the intensity of the echo. Finally, sweeping the 14N frequency is unlikely to be any more effective at inducing single-quantum transitions than irradiation at one particular frequency. An alternate view of the experiment would be to consider the spinning as introducing an adiabatic passage from e.g. the 10) to the I - 1) states. Indeed, the adiabatic passage between spin-locked eigenstates due to MAS and continuous irradiation has been demonstrated I= 3/2 nuclei [ 161. When the 14Nirradiation frequency, w. - 6, occurs at the same frequency as the double-quantum transition, decoupling of the outer states occurs. Since this decoupling occurs for an integral number of rotor periods, this mechanism will not result in a decrease of the refocused 13Cintensity, and is responsible for the sharp spike in Z/Z0observed in fig. 4.
5. Conclusions The results presented above suggest a novel method for perturbing the entire 14N spin bath. Despite the 14N single-quantum spectrum of glycine spreading 384
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z 1.8 MHz, the combination of continuous 14N irradiation and sample spinning induces single-quantum transitions between all three 14N energy levels. This effect has been monitored indirectly via the dipolar coupling to “C nuclei, and is most noticeable for the C( 2) carbons which are adjacent to the nitrogen atom.s, but is also significant for the C ( 1) carbons, 2.49 A from the nitrogens. The 14N doublequantum transition can also be detected indirectly; a possible advantage of this method of recording the 14N double-quantum spectra, over those involving cross-polarization, is the less stringent requirement for the 14N power level. Further work is in progress to simulate the above results, with an aim of obtaining more precise distance information from the spectra. It is hoped that the sensitivity of the 13C nuclei to the proximity of the 14N spin in these experiments can be exploited and may provide a new technique for studying nitrogen containing materials, particularly in systems where “N enrichment is not a possibility.
Acknowledgement The authors wish to thank J.W.M. van OS for designing and building the triple-tuned probe; the help of J.W.M. van OS, W. Maas, E. van Eck and G. Nachtegaal with the NMR experiments is acknowledged gratefully. C.P.G. wishes to thank the Royal Society for a Levehulme William and Mary Research Fellowship.
References [ I] B. Herzog and E.L. Hahn, Phys. Rev. 103 ( 1956) 148; D.E. Kaplan and E.L. Hahn, J. Phys. Radium 19 ( 1958) 821. [2] T. Gullion and J. Schaeffer, J. Magn. Reson. 81 (1989) 196. [ 31 G.R. Marshall, D.D. Beusen, K. Kociolek, A.S. Redlinski, M.T. Leplawy, Y. Pan and J. Schaeffer, J. Am. Chem. Sot. 963 (1990) 112. [4] S. Shore, J.-P.H. Ansermet, C.P. Slichter and J.H. Sinfelt, Phys. Rev. Letters 58 (1987) 953; E. van Eck and W.S. Veeman, Solid State Nucl. Magn. Reson. 1 (1992) 1. [ 5 ] J.M. Lehn and J.P. K&zinger, in: Nitrogen NMR, eds. M. Witanowski and G.A. Webb (Plenum Press, New York, 1973) p. 80.
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[ 6 ] G. Schnur, R. Kimmich and F.J. Winter, J. Magn. Reson. 66 (1986) 295. (71 T.K. Pratum and M.P. Klein, J. Magn. Reson. 53 ( 1983) 473. [ 81 P. Bnmner, M. Reinhold and R.R. Ernst, J. Chem. Phys. 73 ( 1980) 1086; M. Reinhold, P. Brunner and R.R. Ernst, J. Chem. Phys. 74 (1981) 184; T.K. Pratum and M.P. Klein, J. Magn. Reson. 55 ( 1983) 421. [9] R. Tycko and S.J. Opella, J. Am. Chem. Sot. 108 (1986) 3531. [lo] R.A. Haberkom, R.E. Stark, H. van Willigen and R.G. Griffin, J. Am. Chem. Sot. 103 ( 1981) 2534.
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[ 111 J.W.M. van OS, G.J.W. Steeg and W.S. Veeman, Rev. Sci. Instr. 62 (1991) 1285. [ 121 D.L. VanderHart, H.S. Gutowsky and T.C. Farrar, J. Am. Chem. Sot. 89 (1967) 5056. [ 13 ] J.G. Hexem, M.H. Frey and S.J. Opella, J. Am. Chem. Sot. 103 (1980) 3531. [ 141 R. Tycko and S.J. Opella, J. Chem. Phys. 86 (1987) 1761. [ 151 R.C. Hewitt, S. Meiboom and L.C. Snyder, J. Chem. Phys. 58 (1973) 5089; L.C. Snyder and S. Meiboom, J. Chem. Phys. 58 ( 1973) 5096; A. Pines, S. Vega and M. Mehring, Phys. Rev. B 18 ( 1978) 112. [ 161 A.J. Vega, J. Magn. Reson., in press.
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The detection of weak heteronuclear coupling between spin 1 and spin l/2 nuclei in MAS NMR; 14N/’ 3C/ ‘H triple resonance experiments C.P. Grey and W.S. Veeman ’ NSR Centre for Molecular Design, Synthesis and Structure, Laboratory of Molecular Spectroscopy, University ofNijmegen. Toernooiveld, 6525 ED Nijmegen. The Netherlands
Received 3 1 December 199 1
Magic angle spinning and i4N irradiation have been employed to perturb the “‘N spin bath, in a new spin-echo triple resonance solid-state NMR experiment. Spinning introduces a time dependence in the 14Nquadrupolar interaction, permitting transitions between the three lZ, m)states, two or four times per rotor cycle; this alters the evolution of a i4N-coupled “C spin. The reduction in i3C echo intensity, on 14Nirradiation during the echo period, is greater for shorter C-N distances. Irradiation at a constant frequency is more effective than sweeping through the 14Nresonance. 14Ndouble-quantum spectra are obtained indirectly by irradiation of the double-quantum transition.
1. Introduction The observation of dipolar coupling between nuclei, in solid state NMR spectroscopy, can be exploited to obtain internuclear distances. Double resonance NMR methods have been developed to measure small dipolar couplings, which can often be obscured by other interactions. For instance, in SEDOR [ 1 ] (spin-echo double resonance) the dephasing and/or refocusing of the I nucleus in the spinecho experiment are modulated by applying x pulses to the coupled nucleus S, the strength of the dipolar coupling is then determined from the loss in refocused spin intensity I. REDOR [ 21 (rotational-echo double resonance ) , an extension of SEDOR for magic angle spinning (MAS) conditions, has enabled 13C15N distances of 4.0 8, to be measured with an accuracy of + 0.1 A in a crystalline peptide [ 31. SEDOR and REDOR experiments have been applied successfully to S= l/2 and I> l/2 coupled systems by monitoring the quadrupolar nucleus (I) [4]. These methods cannot be readily applied, however, ’ Present address: Physikalische Chemie, Fachbereich 6, Universitlt-GH-Duisburg, Lotharstrasse 1, W-4 100 Duisburg 1, Germany.
when the coupled S nucleus is quadrupolar, e.g. 14N. We have been interested in developing techniques for obtaining structural information from nitrogencontaining polymers. Isotopic substitution of i5N for the 99.6% abundant isotope 14N can often be prohibitively expensive and/or synthetically impossible. Furthermore, many studies with potential industrial applications require an investigation of polymeric material taken from large scale productions where isotopic substitution is clearly not feasible. The I= 1 nucleus, 14N, has quadrupole coupling constants, e*qQ/h, that range typically from 1 to 5 MHz [ 51. The first order quadrupole interaction shifts the 2Z+ 1 Zeeman levels of the 14N spin, resulting in resonances that are generally too broad to allow excitation and detection of the whole spectrum simultaneously. Hence, 14Nspectra of powdered materials have only been obtained indirectly [ 6 ] or from compounds with exceptionally small quadrupole coupling constants [ 71. The double quantum transition, Am = 2, for an I= 1 nucleus, is to first order, however, unaffected by the quadrupolar interaction; this has been employed to cross-polarize I= 1 nuclei [ 8 ] and to obtain 14N overtone spectra [ 91. We have explored two methods of exciting the 14N
0009-2614/92/S 05.00 0 1992 Elsevier Science Publishers B.V. All rights reserved.
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spins in polycrystalline solids: sweeping through the 14N resonance and continuous irradiation at one frequency. We chose, initially, to study a sample of glytine since the 13C spectrum of this material contains two discrete resonances arising from carbon atoms with different spatial proximity to the NH: group; a single-crystal 14N NMR study of this material gave values of 1.18 MHz and 0.54, for e2qQfh and the asymmetry parameter 9, respectively [ 10 1. The effect of 14N irradiation was monitored indirectly via the 13C spins, by observing changes in the 13C refocused intensity in a spin-echo experiment (fig. 1). The results described below suggest new methods for investigating 14N-13C coupling in rotating solids, and for the indirect detection of 14N double-quantum resonances.
2. Experimental methods The solid state NMR spectra were collected using a Bruker CXP300 at operating frequencies of 300.13, 74.54 and 21.69 MHz for ‘H, 13C and 14N, respectively. A home-built single coil, triple tuned probe was used which could be tuned to provide pulse lengths of 4.5 us for ‘H and 13C and 10 ps for the low y nucleus 14N. MAS was performed using a DOTY stator block and 5 mm zirconia rotors. Different 14N spectral offsets from a central carrier
k
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frequency (chosen to be 21.686 MHz) were programmed into a home-built digital frequency synthesizer, designed to provide fast, phase-continuous frequency switching (described elsewhere [ 111) . This allowed either 14N frequency sweeps (from -200 to +200 kHz), or continuous irradiation at one frequency (where the offset could be changed automatically between experiments), to be performed. The output from this device was first fed into a Programmed Test Sources, Inc (PTS) 160 synthesizer, then into an EN1 3100L amplifier and tinally into a MSL300 broadband amplifier. The 14N pulse lengths were determined using a saturated solution of sodium nitrate, which gave a signal at approximately - 666 Hz from the carrier frequency of 2 1.686 MHz. Analytical-grade glycine and a sample of [2-13C] glycine, enriched to approximately 20% at C-2 (and prepared from 99.5% enriched [2-13C] glycine) were used.
3. Results Fig. 2 shows the effect of 14N irradiation on the 13Cecho intensity in a MAS experiment. A 14N sweep results in an increasing loss in 13Cintensity, the longer the time taken for the sweep (experiment (i ), fig. 2a). The effect is the most pronounced for the meth-
Lkcouple
13c R__
l4N
14N
(1)
I
T
(II)
Fig. 1. The two-pulse sequences used. For the MAS experiments, both the “C 1c pulse (inserted to refocus the ‘% isotropic chemical shifts) and the start of the acquisition arc synchronised with the rotor echoes. The 14N spins are irradiated for one half of the echo period in scheme II, with I providing the control.
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I
(Without i4N irradiation;
(a)
;
(i)sweep
I
A
.”
Q
: ;;)co2
.
%
(ii) single frequency
II
(With “‘N irradiation)
Difference Spectrum
. (ii)
Fig. 2. (a) The loss in the “C intensity on “‘N irradiation. I is the carbon intensity with irradiation, and 1, without (schemes (II) and (I ) in fig. 1). In experiment (i ) a sweep of 400 kHz was performed in 100 steps through a central carrier frequency of 2 1.686 MHZ (each step lasting a time r/100). In experiment (ii) 14Nirradiation was applied at a constant frequency of 21.686 MHz. Spinning speeds of 3560 and 3610 Hz were used for (i) and (ii), respectively. The ‘% spectra that provide the data for the two ringed points in experiments (i), with r=2209 us, areshown in (b).
ylene carbon [ C( 2) ] of glycine which is directly bonded to the NH: group. This can be seen clearly in the spectra shown in fig. 2b. Reducing the number of steps in the sweep from 100 to 50 did not alter the results noticeably. A greater reduction in the echo can, however, be achieved by irradiating the 14Nspins at a constant frequency (experiment (ii), fig. 2a): after irradiation at 2 1.686 MHz for 2600 us (i.e. ten rotor periods) only 14% of the C (2) signal remains. This was initially surprising, in view of the large width of the 14N resonance (x 1.8 MHz). The experiments were then repeated without MAS to investigate the role of the sample spinning. Again,
both a r4N sweep and irradiation at a single frequency resulted in significant losses in signal intensity. Fig. 3a, however, demonstrates that the loss of r3C signal is extremely sensitive to the 14N irradiation frequency and at an offset of 75 kHz the effect has essentially disappeared. The experiment was repeated for smaller 14Noffsets with the sample of [ 2*‘Cl glycine. A significant dip in the 13Crefocused intensity was only observed over a 10 kHz range (fig. 3b), the minimum occurring at = -2 kHz. In contrast, a loss in signal occurs over a much larger range of r4N offsets in the spinning experiments (fig. 4). Furthermore, there is now a pronounced increase in 381
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7 (a)
/ 1I , nTpTTprTTrlTT
0 -600
-500
-400
-300
I,
-200
-100
0
100
“-1
500
’ 4N offset (k&Is) ’ 4N offset
(kHz) Fig. 4. The change in C( 2) intensity as a function of 14N offset for spinning speeds of (i) 2270 Hz and (ii) 4540 Hz. A constant value of T, equal to 88 1 ps, was used for both experiments (i.e. two and four rotor periods for (i) and (ii), respectively).
0.8 I/I0 0.6
C”2
-30
-20
-10 l4 N
0
10
20
30
offset (kHz)
Fig. 3. (a) The variation in the amount of refocused “C intensity, as a function of the 14N offset from the central carrier frequencyof21.686MHz (?= 1900 ps). (b) The ‘%Zintensity from the [ 2-“C] glycine sample for smaller “‘N offsets (r= 1000 ps). Both experiments are performed without MAS. Error bars in (a) take into account the fact that the signal-to-noise obtained in this experiment was significantly worse than in all the other reported experiments,
the amount of 13C refocused intensity at offsets around = -2 kHz. The loss in signal is extremely sensitive to the rate of sample spinning and is considerably reduced, for identical lengths of’s, on doubling the spinning speed (compare curves (i) and (ii), fig. 4). The width of the dip parallels the width of the tuning of the probe for 14N (which has a Qfactor of % 40). By performing a further series of measurements, where the 14N channel is retuned for each change in the 14N offset, a loss in signal intensity is still observed out to +700 kHz, which rep382
resents the band-width of the 14N channel. One explanation for the marked asymmetry of the observed dip about the central frequencies is the presence of a standing wave in the probe at approximately 20.5 MHz. This results in a reduction of the power levels that enter the coil, the closer the 14N frequency is to this standing wave.
4. Discussion Dipolar coupling to 14N will cause the 13C transverse magnetisation to evolve with three different frequencies w. Assuming the “high field approximation” [ 121 is valid for 14N then
w=oo-mmwd
(m=-l,O,
+l)
(1)
and od = D( 3 cos2tl- 1)) where D, the dipolar coupling constant, is 0.635f0.011 kHz for C(2)-N in glycine [ lo]. 8, the angle the C-N internuclear vector makes with the applied field, will be time dependent for MAS. (A breakdown of this high field assumption will lead to a mixing of the IZ, m) eigenfunctions and the observation of asymmetric doublets in the normal MAS spectra; this splitting has been observed for the methylene carbon in glytine at a field of 3.52 T [ 131 but is not observable at our higher field of 7.05 T.) The application of a rc pulse to the 13C spins in the static experiment, or
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the action of MAS in the case of spinning, result in the refocussing of the 13Cspins, forming Hahn and rotational echoes, respectively. We believe that we are observing at least two different mechanisms that modulate the 13C-14N dipolar coupling during the period of 14Nirradiation; these prevent the 13Cspins from refocussing, resulting in the reduction in the 13C echo intensity. The magnitude of the dipolar coupling is strongly dependent on the C-N distance, which is consistent with the greater effects observed for the C ( 2 ) carbons. One mechanism is responsible for the sharp dip/peak around the central 14N frequencies (figs. 3 and 4) and the other is only operational under conditions of MAS. The second order quadrupolar interaction is expected to broaden and shift the double-quantum transition of 14N significantly. Following the approach of Tycko [ 141, this interaction is calculated to shift the resonance by + 3.25 kHz and produce a powder pattern 5.5 kHz wide with a maximum at + 6 kHz. Solid ammonium chloride was found to resonate at - 8.0 kHz from the 14Ncarrier frequency and the 14N resonance of glycine is expected to contain a similar contribution from the chemical shift. Thus the double-quantum resonance is predicted to occur at a similar range of 14N frequencies as the pronounced decrease in Z/Z,, observed in the static experiment (fig. 3). This suggests that double-quantum transitions (between the ( - 1) and I+ 1) levels) are occurring during 14Nirradiation. Doublequantum decoupling has been demonstrated for ‘H*H coupled systems by irradiation of the 2H doublequantum transition and the effectiveness of this decoupling mechanism was shown to decrease rapidly for small 2H resonance offsets [ 15 1. The 14N irradiation suppresses, at least partially, the dipolar evolution during the first half of the echo period by decoupling the I+ 1) and ( - 1) levels; the 10) level will not perturb a coupled 13C spin. In the second half of the echo period, there is no 14N irradiation and the 13Cspins will dephase according to eq. ( 1), leading to a loss in refocused intensity, as was observed experimentally. The quadrupolar splitting becomes time dependent on MAS. Assuming, for simplicity, an axial electric field gradient (EFG) tensor (i.e. q= 0), then the first order quadrupole splitting, 20,, is given by
8 May 1992
3 e2qQ = -47 (JZsin2/3cos(a+o,t) +sin2/3cos[2(cu+o,t)]},
(2)
where /3 and (Yare the polar angles defining the orientation of the z axis of the EFG tensor with respect to the spinning axis; wo=O either two or four times per rotor cycle ( 1/o,), depending on the value of @. If the 14N spins are irradiated at a the Larmor frequency, o,, a single-quantum transition can then, in theory, occur either two or four times per rotor cycle. This is demonstrated for 14N irradiation at a frequency w,-S in fig. 5, which shows a possible trajectory for a spin A originating in the I - 1) level. Considering a 13Cnucleus coupled to this 14Nspin: for the first part of the rotor period the spin will evolve under dipolar coupling to the ) - 1) state. Assuming the simplest case where the principle axis of the dipolar tensor is coincident with that of the electric field gradient (which is the case for an axial EFG aligned along the C-N bond and occurs in many -CN compounds) then @‘, the phase acquired at time t, is simply 0 L
l/or I tl
I 0
I tz
1
I
l/or
2/or I
I
wt
wt
2/w r
Fig. 5. The change in the 14Neigenstates with time for MAS. The energy levels are shown in the laboratory frame, for an axial EFG at 14N,and angles of 30” and 90”, for B and (Y,respectively (see eq. (2)). The possible transitions between levels for one 14Nspin, A, caused by 14Nirradiation at a frequency of w,- 6, are shown.
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@I= UWw){245sin28[sin(a+o,t,)-sina] +sin2/3{sin[2(a+w,f’)]-
sin2a)).
(3)
If a transition to (0) occurs at time t, then the evolution under the dipolar coupling is temporarily halted. At time f2, spin A then reverts back to the ( - 1) state and further evolution will occur. The total phase acquired at the end of a rotor period is then Qi,= (D/2w,)
+sin2P{sin[2(a+o,t’)] -sin[2((r+c0,t2)]j].
(4)
After two rotor periods @2r=20, and, hence, further dephasing will occur, the longer the 14N irradiation period. Considering now the whole polycrystalline sample, with the whole range of values for Q and 8, and crossings between all three states at different times: each 13C spin will acquire a different phase, leading to a loss of signal intensity at the rotor echo. Faster spinning will reduce the probability of transitions occurring between levels, increasing the intensity of the echo. Finally, sweeping the 14N frequency is unlikely to be any more effective at inducing single-quantum transitions than irradiation at one particular frequency. An alternate view of the experiment would be to consider the spinning as introducing an adiabatic passage from e.g. the 10) to the I - 1) states. Indeed, the adiabatic passage between spin-locked eigenstates due to MAS and continuous irradiation has been demonstrated I= 3/2 nuclei [ 161. When the 14Nirradiation frequency, w. - 6, occurs at the same frequency as the double-quantum transition, decoupling of the outer states occurs. Since this decoupling occurs for an integral number of rotor periods, this mechanism will not result in a decrease of the refocused 13Cintensity, and is responsible for the sharp spike in Z/Z0observed in fig. 4.
5. Conclusions The results presented above suggest a novel method for perturbing the entire 14N spin bath. Despite the 14N single-quantum spectrum of glycine spreading 384
LETTERS
8 May 1992
z 1.8 MHz, the combination of continuous 14N irradiation and sample spinning induces single-quantum transitions between all three 14N energy levels. This effect has been monitored indirectly via the dipolar coupling to “C nuclei, and is most noticeable for the C( 2) carbons which are adjacent to the nitrogen atom.s, but is also significant for the C ( 1) carbons, 2.49 A from the nitrogens. The 14N doublequantum transition can also be detected indirectly; a possible advantage of this method of recording the 14N double-quantum spectra, over those involving cross-polarization, is the less stringent requirement for the 14N power level. Further work is in progress to simulate the above results, with an aim of obtaining more precise distance information from the spectra. It is hoped that the sensitivity of the 13C nuclei to the proximity of the 14N spin in these experiments can be exploited and may provide a new technique for studying nitrogen containing materials, particularly in systems where “N enrichment is not a possibility.
Acknowledgement The authors wish to thank J.W.M. van OS for designing and building the triple-tuned probe; the help of J.W.M. van OS, W. Maas, E. van Eck and G. Nachtegaal with the NMR experiments is acknowledged gratefully. C.P.G. wishes to thank the Royal Society for a Levehulme William and Mary Research Fellowship.
References [ I] B. Herzog and E.L. Hahn, Phys. Rev. 103 ( 1956) 148; D.E. Kaplan and E.L. Hahn, J. Phys. Radium 19 ( 1958) 821. [2] T. Gullion and J. Schaeffer, J. Magn. Reson. 81 (1989) 196. [ 31 G.R. Marshall, D.D. Beusen, K. Kociolek, A.S. Redlinski, M.T. Leplawy, Y. Pan and J. Schaeffer, J. Am. Chem. Sot. 963 (1990) 112. [4] S. Shore, J.-P.H. Ansermet, C.P. Slichter and J.H. Sinfelt, Phys. Rev. Letters 58 (1987) 953; E. van Eck and W.S. Veeman, Solid State Nucl. Magn. Reson. 1 (1992) 1. [ 5 ] J.M. Lehn and J.P. K&zinger, in: Nitrogen NMR, eds. M. Witanowski and G.A. Webb (Plenum Press, New York, 1973) p. 80.
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[ 6 ] G. Schnur, R. Kimmich and F.J. Winter, J. Magn. Reson. 66 (1986) 295. (71 T.K. Pratum and M.P. Klein, J. Magn. Reson. 53 ( 1983) 473. [ 81 P. Bnmner, M. Reinhold and R.R. Ernst, J. Chem. Phys. 73 ( 1980) 1086; M. Reinhold, P. Brunner and R.R. Ernst, J. Chem. Phys. 74 (1981) 184; T.K. Pratum and M.P. Klein, J. Magn. Reson. 55 ( 1983) 421. [9] R. Tycko and S.J. Opella, J. Am. Chem. Sot. 108 (1986) 3531. [lo] R.A. Haberkom, R.E. Stark, H. van Willigen and R.G. Griffin, J. Am. Chem. Sot. 103 ( 1981) 2534.
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[ 111 J.W.M. van OS, G.J.W. Steeg and W.S. Veeman, Rev. Sci. Instr. 62 (1991) 1285. [ 121 D.L. VanderHart, H.S. Gutowsky and T.C. Farrar, J. Am. Chem. Sot. 89 (1967) 5056. [ 13 ] J.G. Hexem, M.H. Frey and S.J. Opella, J. Am. Chem. Sot. 103 (1980) 3531. [ 141 R. Tycko and S.J. Opella, J. Chem. Phys. 86 (1987) 1761. [ 151 R.C. Hewitt, S. Meiboom and L.C. Snyder, J. Chem. Phys. 58 (1973) 5089; L.C. Snyder and S. Meiboom, J. Chem. Phys. 58 ( 1973) 5096; A. Pines, S. Vega and M. Mehring, Phys. Rev. B 18 ( 1978) 112. [ 161 A.J. Vega, J. Magn. Reson., in press.
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