NMR spin-lattice relaxation studied by magnetization transfer

‘JOURNAL

OF

MAGNETIC

RESONANCE

39,169-174

(1980)

NMR Spin-Lattice Relaxation Studied by Magnetization Transfer Many proton NMR spectra contain groups of overlapping resonances which are difficult to resolve and assign by conventional methods; several steroids and carbohydrates provide examples of this problem. Recent two-dimensional Fourier transform methods (1-4) show promise for circumventing these difficulties by transferring proton magnetization to directly bonded carbon-13 nuclei, using the much higher dispersion of carbon shifts to separate the responses from different sites. The resulting spectrum runs in two orthogonal frequency dimensions (Fi and Fz) and correlates the proton shifts with those of the directly bound carbon nuclei, providing a “shift correlation map” (5, 6). In applications where it is necessary to measure proton spin-lattice relaxation times, extensive overlap of the interesting resonance lines often makes the problem intractable by direct methods. It is proposed that magnetization transfer to carbon13 could make these proton relaxation times accessible, albeit at the expense of a more complicated experiment and a significant loss of sensitivity. There may nevertheless be chemical applications where this additional effort is warranted. The relaxation rates determined in this way apply to protons bound to carbon-13 nuclei and therefore include a contribution from the CH dipolar interaction (7). For simplicity the present discussion disregards proton-proton cross-relaxation effects, and precautions are taken to suppress proton-carbon-13 cross-relaxation, giving exponential relaxation curves. The experiment follows the same course as chemical shift correlation by transfer of magnetization from protons to carbon-13 (1-6) except that the initial state of the proton spins is perturbed from Boltzmann equilibrium. The protons are first saturated by broadband noise irradiation and allowed to recover some longitudinal magnetization during a variable interval 7 prior to magnetization transfer to carbon-13; the method is thus analogous to the well-known saturation-recovery experiment. The carbon-13 intensities in the resulting shift correlation spectrum should then follow an exponential function of T: S, - S, = S, exp(-r/

Tin)

where T1n is the spin-lattice relaxation time of the directly bound proton. (Saturation-recovery is to be preferred over inversion-recovery for this application since it avoids the problem of inverted signals, allowing an absolute-value display to be used.) Since the measurement is applicable only to protons directly attached to carbon13 nuclei, and since the receiver detects carbon- 13 signals, there is a corresponding penalty in signal-to-noise ratio. This is somewhat alleviated by the fact that proton population differences are involved and it is the proton relaxation which determines the rate at which experiments can be repeated. 169

0022-2364/80/070169-06$02.00/O Copyright 0 1980 by Academic Press, Inc. All rights of reproduction in any form reserved. Printed in Great Britain

170

COMMUNICATIONS

FIG. 1. Pulse sequence used for determining T 1~ by observing the magnetization carbon-13 in a two-dimensional Fourier transform experiment.

transferred to

EXPERIMENTAL

The relaxation measurements were carried out on a Varian CFT-20 spectrometer modified for two-dimensional Fourier transformation (8). The pulse sequence is set out in Fig. 1. The initial preparation period T is used to achieve proton saturation by broadband irradiation and is followed by the vaFiable period r during which the proton magnetizations recover by spin-lattice relaxation. Throughout r a train of randomly spaced pulses is applied to carbon-13 to hold these spins saturated (9), thus eliminating carbon-proton cross-relaxation effects. In practice 90” pulses were used, timed by a pseudorandom sequence (10) with a mean interval of about 2.5 msec and a cycle time of about 5 sec. (During the period 7 this carbon-13 irradiation may cause the buildup of a small nuclear Overhauser enhancement of the proton resonance, thus determining the intensity of the asymptotic signal S,.) After this preparation and relaxation stage, the experiment follows the wellknown magnetization transfer pulse sequence (1-6). The first 90” pulse initiates free precession of transverse proton magnetization for the evolution period ti; then the second 90” pulse converts this back to longitudinal magnetization, the amplitude depending on the phase angle built up during tl. In this way proton shift and CH coupling information is coded into the tl dependence of the Z magnetization. The spin populations are thus modulated as a function of tl and this affects all connected carbon-13 transitions, The 90” carbon pulse converts this modulated Z magnetization into a detectable carbon-13 signal. A considerable simplification of the resulting two-dimensional spectrum is achieved by decoupling carbon from protons during both time intervals tl and tZ. This is accomplished during tl by setting a 180” carbon pulse at the midpoint of the evolution period to “refocus” the two components of the proton doublet (2,3), while straightforward proton noise irradiation is used during tz while the carbon-13 signal is being acquired. These decoupling operations would normally cause mutual cancellation of the antiphase components of the spin multiplets, but this is prevented (in both dimensions) by the fixed delays Al and AZ, calculated to allow 180” relative precession between the relevant magnetization vectors (2). This requires A = 1/(2JcH), where J oH is a direct coupling; long-range couplings are too weak to prevent such cancellation, so magnetization transfer is essentially restricted to directly bound CH groups.

COMMUNICATIONS PROTON

RELAXATION

171 IN

SUCROSE

The proposed technique is illustrated by the example of sucrose, for which there is considerable overlap in the proton spectrum, all the resonances except one falling in a range of less than 1 ppm. The sample contained 30% of sucrose by weight in solution in D20. The carbon-13 assignment is that given by Pfeffer et al. (11). Note that G3 and G5 could not be resolved at this field strength; consequently the relaxation time of the composite peak is reported. Figure 2 shows the twodimensional shift correlation spectrum for the case where the initial proton populations correspond to Boltzmann equilibrium. Table 1 sets out the relaxation times of the protons (T& and the assignment of the carbon-13 resonance (II). STUDIES

OF

=CH

CROSS-RELAXATION

Investigation of cross-relaxation effects can yield intimate details of molecular motion and structure (12-15). A combination of experimental techniques is required

CH,OH /hqyl

G

5 iiHzOH

HO

mmI

“Ffj F6

-40

G4 GQ5

F2

Carbon shifts ,60

5

F,, Proton

0

shifts

FIG. 2. The two-dimensional Fourier transform spectrum of sucrose carbon-13 shifts (F2) and the shifts of directly bound protons (F1).

showing

the correlation

between

172

COMMUNICATIONS TABLE

1

SPIN-LATTICE RELAXATION TIMESOFPROTONSBOUNDTO CARBON-13 INSUCROSE Site" G6 Fl F6 G4 G2 G3, F4 F3 F.5 Gl

G5b

Tl (se4 0.17*0.01 0.15ztO.02 0.26ztO.06 0.37ztO.08 0.40* 0.09 0.30 * 0.03 0.33 *0.08 0.29i 0.06 0.29rtO.04 0.27 + 0.04

’ The assignments are indicated in Fig. 2. b These peaks are not resolved in the carbon-13 spectrum.

to extract all the pertinent relaxation transition probabilities, and several authors have emphasized the importance of monitoring the recovery of the difference magnetization between two components of a spin multiplet (16-18), since this can be shown to follow a pure exponential curve, in contrast to the biexponential recovery of the individual lines. Measurement of this “multiplet asymmetry relaxation time” Tld (18) normally involves a selective 180” pulse or an incomplete adiabatic fast passage in order to invert one component of the multiplet without perturbing the nearest neighbor. Since the magnetization transfer sequence prepares the carbon-13 spin multiplet components with antiphase 2 magnetizations, the time evolution of the difference magnetization is easily followed, giving TIA directly. This method has the advantage over selective pulse experiments in that it operates over a wide band of proton frequencies, so that a prior determination of the appropriate proton frequencies is not necessary. Only a very simple modification of the basic pulse sequence is required (Fig. 3), introducing a variable interval r between the second proton pulse and the carbon-13 “read” pulse (19). Consider a two-spin AX system consisting of a proton and a carbon-13 nucleus. By writing down the rate equations for spin populations, one can readily show that the difference magnetization (proton or carbon-13) decays with a pure exponential with rate constant

If the pulse sequence of Fig. 1 is used to measure the proton relaxation time TlH indirectly, and if T,, and the nuclear Overhauser enhancement factor are measured in the conventional way, then all four transition probabilities, Win, Wit, WZ, and

173

COMMUNICATIONS 90’

90”

-

‘H

G-t,/2 FIG.

parameter

3. Pulse sequence in cross-relaxation

used

At,/-jA,

to monitor studies.

L-2.+

the decay

rate

Noise

-

‘*,Lt,+

of the difference

magnetization

(Ti,),

a key

IV0 are readily obtained (18). Note, however, that all these measurements are made by detecting carbon-13 signals only; hence there is less likelihood of introducing systematic errors by changing spectrometers. This technique has been employed to evaluate the relaxation parameters of the three CH sites in bromobenzene (20). SENSITIVITY

IMPROVEMENT

The poor sensitivity of the methods described above is partly attributable to the long times required to gather the data for the two-dimensional Fourier transformation. In situations where the separation of the proton shifts is not essential to the investigation, then a simplified experiment would provide improved sensitivity and a one-dimensional spectrum. This is the “INEPT” technique (21), where magnetization transfer from protons to carbon-13 is accomplished by setting tl = 1/(2Jcn) in order to achieve a maximum transfer of polarization. An additional 180 refocusing pulse applied to the protons at it1 makes the result independent of the proton shifts. After the initial saturation recovery sequence on the proton spins (see Fig. 1) the pulse sequence would be Protons: 90’(X) . . . it1 . . . 180”(X) . . . iti . . .90”(* Y) . . .A2 . . . Noise Carbon:

. . . itI . . . 180”(X).

. . it1 . . .90’(X).

. . A*. . . Acquisition

Note that a 90” phase shift is required for the last proton pulse, and that this is alternated in order to cancel carbon-13 magnetization not transferred from the protons (21). Only one-dimensional Fourier transformations are carried out (as a function of t2) giving one-dimensional carbon-13 spectra decoupled from protons, but with intensities .following Eq. [l]. A similar modification can be made to the cross-relaxation experiment (Fig. 3). ACKNOWLEDGMENTS This work was made possible by an equipment grant from the Science Research Council. The authors are pleased to acknowledge several discussions with Dr. Gareth Morris and Dr. Geoffrey Bodenhausen.

174

COMMUNICATIONS REFERENCES

1. A. A.MAUDSLEYANDR.R.ERNST, Chem.Phys.Lett. 50,368 (1977). 2. A. A.MAUDSLEY,L.M~LLER, AND R.R.ERNsT, J. Magn.Reson.28,463 (1977). 3. G.BODENHAUSENANDR.FREEMAN,J. Magn.Reson.28,471 (1977). 4. G.BODENHAUSENANDR.FREEMAN,J. Am. Chem.Soc. 100,320(1978). 5. R. FREEMANAND G. A. MORRIS,J. Chem. Sot. Chem. Commun. 684 (1978). 6. R. FREEMANAND G. A.MORRIS, Bull. Magn.Reson. 1,5 (1979). 7. H. OZAWA,~. ARATA, AND S.FUJIWARA,J. Chem.Phys. 57,1613 (1972). 8. G.BODENHAUSEN,R.FREEMAN,R.NIEDERMEYER,ANDD.L.TuRNER, J.Magn.Reson.26, 133 (1977).

J.L.MARKLEY, W.J.HORSLEY,ANDM.P. KLEIN,J. Chem.Phys.55,3604 (1971). 10. R. R. ERNST,J. Chem. Phys. 45,384s (1966). 11. P.E.PFEFFER,K.M.VALENTINE,ANDF.W.PARRISH, J. Am. Chem.Soc. 101,126s 12. I.SOLOMONANDN.BLOEMBERGEN,J. Chem.Phys.25,261 (1956). 13. J. H. NOGGLE,J. Chem. Phys. 43,3304 (1965). 14. R.FREEMAN,S.WI?TEKOEK,ANDR. R. ERNST,J.Chem.Phys.52,1529 (1970). 15. T.D. ALGER,~. W.COLLINS,ANDD.M.GRANT, J. Chem.Phys.54,2820 (1971). 16. H.SHIMIZUAND S.FUJIWARA,J. Chem.Phys.34,1501(1961). 1% E.L.MACKORANDC.MACLEAN, J. Chem.Phys.42,4254(1965). 18. C.L.MAYNE,D. W.ALDERMAN,ANDD. M. GRANT,J. Chem.Phys.63,2514(1975). 19. G. BODENHAUSEN,~II~~~~~S~~~ results. 20. A. G. AVENT,Part11Thesis,Universityof Oxford. 21. G. A.MORRISAND R.FREEMAN,J. Am. Chem. Sot. 101,760 (1979). 9.

(1979).

ANTHONY G. AVENT" RAY FREEMAN Physical Chemistry Laboratory Oxford University South Parks Road Oxford, England Received December 26, 1979 * Presentaddress:Department of Chemistry,Universityof Southampton.