2iero017 8-1 Relaxation in NMR Spectroscopy 8.1 Relaxation in NMR Spectroscopy © Copyright Hans J. Reich 2016 All Rights Reserved University of Wisconsin ‘An understanding of relaxation processes is important for the proper measurement and interpretation of NMR spectra. There are three important considerations. 1. The very small energy difference between a and B states of a nuclear spin orientation in a magnetic field results ina very small excess population of nuclei in the ground vs the excited states (typically less than 1 out of about 10,000 molecules). For many nuclei relaxation (i.e., return from excited to ground state) is a very slow process, with half-lives on the order of 0.1 to 100 seconds for a spin Ya nucleus (compare this with micro-, pico- and femtoseconds for relaxation of electronic and vibrational transitions). It is thus very easy to saturate an NMR transition (equalize populations of excited and ground state), with the resultant loss in signal quality, and failure to obtain correct peak areas. 2. NMR lines are extraordinarily sharp, and extraordinarily close together (In energetic terms) compared to higher energy spectroscopic methods. Sa much so that Heisenberg uncertainty broadening (which is a function of lifetime of a given energy state, and hence relaxation rates) is 2 dominant feature of many NMR spectra, and can limit our ability to measure and interpret spectra. When relaxation is very fast, NMR lines are broad, J-coupling may not be resolved or the signal may even be difficult or impossible to detect. 3. The success of many multipulse expe proper consideration of relaxation times. lents, especially 2D and 3D spectra, depends crucially on T, and Tz Relaxation. We distinguish two types of relaxation, Spin-Lattice (Tj, also known as longitudinal relaxation, or relaxation in the z-direction) and Spin-Spin (Tz, also known as transverse relaxation, or relaxation in the x-y plane). T; relaxation corresponds to the process of establishing (or re-establishing) the normal Gaussian population distribution of a and B spin states in the magnetic field, Ta is loss of phase coherence among nuclei. To is less than or equal to T; (R = relaxation rate, Rp = 1/T, Ro = Ri), since return of magnetization to the z-direction inherently causes loss of magnetization in the x-y plane. The line width of an NMR signal is determined by T - short Tz means broader lines (v42 = 1/nTo, v1/2 = width at half height). The maximum repetition rate during acquisition of an NMR signal is governed by T; - short T; means the magnetization recovers more rapidly, and a spectrum can be acquired in less time Sources of Line Broadening in NMR 1. Instrumental problems - tuning, mis-shapen NMR tube, etc 2. Sample problems: Sample inhomogeneity (poor mixing. solid particles) Temperature gradients across sample Paramagnetic impurities 3. Ty Relaxation (Spin-Lattice Relaxation): gain and loss of magnetization in the z-direction. NMR lines are at Jeast as wide as specified by the Heisenberg Uncertainty Principle broadening due to inherent lifetime of spin states (the actual width is governed by T2). For most spin 1/2 nuclei Tz is between 0,2 and 50 seconds. hitpswww' chem wise-edulareasitelehinmv/08-tech-01-0laxhim ane2iero017 8-1 Relaxation in NMR Spectroscopy AE-At = hie hbv-bt = nie vy2 >= 112zT; (Half-width at half height) Ny >= AAT, Vy >= WT] (Width at half height) Broadening due to paramagnetic impurities is a special case of T, broadening Place unmagnetized sample in magnetic field: establishment of the normal Boltzmann equilibrium between the a and B spin states has a first order rate constant of k = 1/T}: Mz = Moo (1 - eT) Or apply a 90° pulse, and watch the magnetization return to the z-direction: Mz = Me (1 - e"/T4) 7 — kh y vy The rate of spontaneous relaxation of nuclear spin orientations is almost zero. T; relaxation is caused by transient magnetic fields (usually due to molecular motion) at the Larmour precession frequency. In most situations T; relaxation is optimal if the average rate of molecular reorientation in space is at the Larmour precession frequency. In mobile liquids near room temperature the average rates of molecular rotation are several orders of magnitude higher than vo, so only a very small fraction of the motions are at the proper frequency, leading to very inefficient relaxation (long 71) For larger molecules and more viscous solutions molecular motions become slower and and more efficient relaxation results (T, shortens). However, at some point the average molecular motions become slower than Vg, and T; becomes longer again (Figure 8.1-1) hitpswww' chem wise-edulareasitelehinmv/08-tech-01-0laxhim ana2iero017 8-1 Relaxation in NMR Spectroscopy to L small Higher Lower \ Molecules FRI” = Fela ah Ty p % 107 + Ty, Th 10? + Large molecules Polymers, proteins yok 10 t 10% + ' We Solids 10%; tv, = 10% at 100 MHZ. a 107 40°" 10° 40° 10° 107 10° 10° 10° Te (correlation time) —— Increasing viscosity or molecular size —~ Figure 8.1-1. Behavior of T; and T2 as a function of correlation time. T = Molecular correlation time: the time it takes the average molecule to rotate one radian (adapted from Bloembergen, E.M. Purcell, R.V, Pound "Relaxation Effects in Nuclear Magnetic Resonance Absorption” Physical Review 1948, 73, 679-746) 4. Tz Relaxation (Spin-Spin Relaxation): Heisenberg Uncertainty Principle broadening due to lifetime of spin coherence - gain and loss of magnetization in the x,y-direction. Vy = nT Tasty For protons, 72 is usually between 1 and 10 seconds Tz = 18e¢, v% = tik = O3HZ Tz = 10sec, v% = 10-2 = 0.03Hz k- y “ ¥ y y Tp relaxation is caused by transient magnetic fields (usually due to molecular motion) at any frequency. Thus T keeps getting shorter as molecular reorientation rates slow down (Figure 8.1-1) Tp relaxation is also caused by swapping of chemical shifts or coupling constants (chemical exchange). Line broadening due to chemical exchange provides an Important tool for measurement of the rates of a variety of molecular processes (see Sect 8-TECH-3) hitpswww' chem wise-edulareasitelehinmv/08-tech-01-0laxhim a22ier2017 Spin ‘4 nuclei in magnetic field B, 8.1 Relaxation in NMR Spectroscopy Place samplein magnetic field z very short time 3 time h TT, ¥ Top: +p Bottom - Net Magnetization Vector CP Before the pulse we have more hnuciel oriented in the up than in the down direction (net ‘magnetization in z direction) After the pulse we nave more nuclei oriented to the right than to the left (net magnetization is now Inthe x' direction) This magnetization is rotating at the Larmor prcession frequency, and itis this oscillating magnetic field that is detected by the NMR spectrometer receiver coils Figure 8.1-2. Symbolic presentation of nuclear spins in response to a 90° pulse. The population difference between the a and B spin states is greatly exaggerated Measurement of T; by Inversion-Recovery Measurement of T,. A common method is the 5T;-n-T-n/2-FID pulse sequence. A series of NMR spectra are measured in which the spins are inverted with a n pulse, followed by a variable waiting period T (Figure 8.1-3). The degree to which the signal has returned to its equilibrium value during the waiting period 1 for each sequence is measured, and plotted as a first order rate process. A sample set of spectra for such an experiment is given in Figure 8.1-4. hitpswww' chem wise-edulareasitelehinmv/08-tech-01-0laxhim aia2iero017 8-1 Relaxation in NMR Spectroscopy jo. | ow | ow - | | 4 te = Tyin(2) a2 FID increasing / delay period % w2 FID | @. LL % w2 FID [ jure 8.1-3, Pulse sequence for a T; determination by the inversion-recovery method. ce + ey . Figure 8.1-4. Example of an inversion-recovery T determination. From "13C NMR Spectroscopy, High Resolution Methods and Applications in Organic Chemistry and Biochemistry," E. Breitmaier and W. Voelter, Verlag Chemie, Weinheim, 1987, page 52. hitpsww'chom.wise.odularoasitelehinmv/08-tech-01-elaxhim sie2iero017 8-1 Relaxation in NMR Spectroscopy Relaxation Mechanisms Spontaneous T; relaxation of spin ¥ nuclei, i.e., relaxation in the absence of external influences, is essentially absent, For T; relaxation to occur there must be magnetic field fluctuations in the x,y direction, Such fluctuations are most effective when they occur at the Larmor precession frequency (vg). 7; relaxation is thus field dependent, since vg varies with the field. Tz relaxation is caused by fluctuations in any direction, The principal source of fluctuating magnetic fields in most molecules is molecular motion. We can define a correlation time T¢ for a molecule (assuming it behaves more or less spherically). This is the average time it takes the molecule to rotate through one radian. The correlation time for small molecules is of the order of 10"! sec in solution (longer in viscous solvents). Since for a proton at 300 MHz Vo = 108, most molecules below molecular weight of 1000 are moving too rapidly for effective relaxation (Figure 8.1-1). There are several mechanisms by which molecular motions can influence nuclear relaxation: direct interactions with nearby magnetic nuclei (DD), chemical shift effects (CSA), quadrupole-electric field gradient interaction (QR) and rapid modulation of J-coupling (SC). In addition to molecular motion, rotational transitions can also be the source of fluctuating magnetic fields (SR). Below are summarized the pricipal relaxation mechanisms (more or less in inverse order of importance). 1. Spin Rotation (SR). A local magnetic field is generated by the circular motion of electrons in 2 rapidly rotating molecule (or part of a molecule, such as a methyl group). {8 Bchanges as the rate of rotation changes The magnitude of this field changes when the rotational energy levels change as a result of molecular collisions. These changes, if they occur at the Larmor precession frequency, can cause relaxation of nearby nuclei. The correlation time for SR relaxation is not molecular motion, but the lifetime of rotational quantum states. Characteristic features: -Small molecules, or freely spinning portions of larger molecules without other efficient relaxation mechanisms. -Better relaxation (shorter Ti) at higher temperatures % nuclei only, and only for nuclei which are not at the center of tetrahedral or octahedral symmetry. Spin ¥2 nuclei can be considered to have spherical charge distributions, but for spin >¥ nuclei the charge distribution has the shape of an oblate or prolate spheroid. Electric field gradients in such molecules exert a torque on the quadrupolar nuclei. Tumbling of the molecule can then initiate transitions among the spin states (there is "friction" between the nucleus and the surrounding electrons, the "quadrupole coupling constant" €2Qq;/h). The effectiveness of this relaxation mechanism is critically dependent on this coupling. If Q is small (as for 2H and SLi, both with = 1) the nucleus behaves almost like a spin V2 nucleus (i.e. there is only a small deviation from spherical symmetry), if itis large the nucleus can have very short Ty, and observation can be very difficult, and spin-spin splitting to the nucleus cannot be observed hitpswww' chem wise-edulareasitelehinmv/08-tech-01-0laxhim sina2iero017 8-1 Relaxation in NMR Spectroscopy Quadrupole moment (0 for | = 1/2 nuclei) Electric fleld gradient (0 for tetrahedral symm) Correlation time - molecular motion | ‘Asymmetry of electric field \ 1. _ 3p +3 ( yer, 10 P(21-1) v= (@,y-GetV/g.z (asymmetry parameter) = electric field gradient Q = electric quadrupole moment T.= molecular correlation time (molecular or segmental rotation) |= nuclear spin In molecules with tetrahedral or octahedral symmetry the electric field gradient is small or zero, and T; values can be long enough to obtain acceptable spectra (e.g., ClOq” for 35CI, S04"? or RpSO for 33), “B 13/2 Q= 0.036 “MN 14 Q = 0016 NaBH T, = 11sec NHS CrinH,O — T, = 50sec BEL T, = 0.01 sec CHsCeNiIn HzO T; = 0.022 sec However when Q is large enough, even nuclel in a formally symmetric environment can show fast QR. For example the spherically symmetrical iodide ion (I = 5/2) has such a large quadrupole moment (Q = 0.79) that line widths of from 1200 to >50,000 Hz are seen in various solvents due to transient asymmetric solvation, Quadrupolar relaxation of a nucleus can also have effects on nearby magnetic nuclei, since rapid relaxation can either broaden or entirely remove J-coupling between the two nuclel. This effect is especially common for #4N-x and 11B-X groups. Such broadening is actually a T effect at the X nucleus (interchange of spin states), It is often difficult to observe carbons or protons directly bonded to boron because of this kind of T2 broadening. Rapid quadrupolar relaxation of Cl, Br and I nuclei is the reason we don't normally see any indication of coupling between these nuclei and adjacent carbons or protons (these nuclei are self-decoupled). + only for nuclei with I > 1/2. + faster relaxation at lower temperatures in solution. + there must be a permanent (or transient) electric field gradient across the nucleus (i.e., QR is much less effective in molecules in which the nucleus is at a center of tetrahedral or actahedral symmetry). for nuclei with a large electric quadrupole moment, this mechanism is so effective that only KHz wide lines can be observed, and no J coupling to other nuclei can be detected. + For most quadrupolar nuclei no NOE effects are seen. 4, Scalar Relaxation (SC). Scalar (J) coupling of a nucleus ¥ to a second quadrupolar nucleus X can provide a relaxation mechanism for Y if X is undergoing very rapid T; relaxation, Under these conditions Y is subject to a fluctuating magnetic field because of the rapid spin reorientation of X. For this mechanism to be very effective, the Larmor precession frequencies of X and Y must be very close together (i.e., wx - wy must be small). hitpswww' chem wise-edulareasitelehinmv/08-tech-01-0laxhim eine2iero017 8-1 Relaxation in NMR Spectroscopy Rysey = <= FF ety) ——2 Tse) 3 1+ (x - @y)? toc? Rog ~ Scalar relaxation rate of ¥ (spin 1/2) caused by X (quadrupolar nucleus) J = coupling constant between X and Y tse = Tyan) (1/rate of quadrupolar relaxation of X) armor precession frequencies of X and Y pin of X Because of the stringent requirement that the Larmor precession frequencies of the two nuclei involved be close together, this effect is very rare, The unusually short T; values for bromine-bearing carbons are believed to be caused by SC relaxation, On a 200 MHz spectrometer, the Larmor precession frequency of 13C is 50.28 MHz, that of 79Br (I = 3/2), 50.18 MHz, T, Of CHCl, = 32.4 sec T, of CHBr, = 1.65 sec Note that the SC T; relaxation process is quite distinct from the broadening of ¥ caused by partially coalesced J coupling between ¥ and X that occurs when Tix) has values in the range of 1/(nJc.x) (as is often seen for spin 2 nuclei attached to 74N and 148). Such broadening is a T2 process in ¥ (swapping of spin states). 5. Dipole-Dipole Relaxation (DD). Coupling between magnetic nuclei is of two types: the scalar (J) coupling is the result of polarization of the electrons by the nuclear spins. The direct dipole- dipole coupling interaction is very large (often kilohertz) and depends principally on the distance between nuclei and the angular relationship between the magnetic field and the internuclear vectors. This coupling is not seen in mobile solutions because it is averaged to zero by tumbling of the molecule. However, as the molecule tumbles in solution the dipole-dipole coupling is constantly changing as the vector relationships change. This creates a fluctuating magnetic field at each nucleus. To the extent that these fluctuations occur at the Larmor precession frequency, they can cause nuclear relaxation of nearby nuclei. Since the proton has the highest magnetic dipole of common nuclei, it is the most effective nucleus for causing DD relaxation. DD relaxation is the principal relaxation pathway for protons in molecules containing contiguous protons, and for carbons with directly attached protons, The correlation time for DD relaxation is molecular (or, more accurately, segmental) motion. The equation governing DD relaxation of an X nucleus by nearby protons in the region of fast molecular motion (extreme narrowing region): Rio) Treo .. the correlation time (average time for the H-X vector to rotate 1 radian) ‘x Ya gyomagnetic ratios fic distance between H and the X nucleus being relaxed. DD relaxation shows a very strong distance dependence, and operates most effectively between directly bonded nuclei, At the usual spectrometer frequencies, small molecules (MW <1000) are tumbling too fast for the most effective relaxation (1; is too short). Thus the more rapidly @ molecule or part of a molecule tumbles, the less effective DD relaxation is, and the longer T, becomes. Large molecules (e.g. proteins) are usually moving too slowly (T¢ is too long), and they have the opposite hitpswww' chem wise-edulareasitelehinmv/08-tech-01-0laxhim soit2iero017 8-1 Relaxation in NMR Spectroscopy relationship between molecular motion and T; ({.e., relaxation is more effective when the molecule moves faster). For 13C NMR, dipole relaxation by directly attached protons (if any are present) is the principal relaxation mechanism. Thus quaternary carbons will have long relaxation times (hence their low intensity under normal conditions of spectrum acquisition due to saturation). CH groups will have shorter values, and CH2 groups still shorter by approximately a factor of two. This can be seen in the 13¢ T; values of adamantane, and the carbons in the ring framework of cholesteryl chloride. The side chain and methyl carbons have separate (faster) motions, and thus have longer T; values. sess a ag = us och sos T, values of cholesteryl chloride Effects of Anisotropic Motion Anisotropic Motion: Long thin molecules do not move isotropically in solution (i.e., T¢ will be different for rotation around different axes of the molecule), so that relaxation times can be quite different for carbons which are on the long axis, compared to those off it. A nice example is diphenyldiacetylene. The para carbons have relatively short relaxation time (1.1 s) whereas for the ortho and meta carbons 7; is about five times as long. This is because rotation around the long axis is very fast, and causes inefficient relaxation for the off-axis carbons. The para carbons change their relationship with the attached hydrogen only when rotating around an axis perpendicular to the acetylenes, a much slower process. This effect can sometimes be used to assign carbon signals. Note the unusually long @11 of the central acetylene carbons - the result of being very far from any protons. Segmental Motion: Different parts of molecules can have different correlation times, and consequently Ty values. For 1-bromodecane, the T; values are uniform down the chain except near the ends, where local conformational motions are somewhat faster. On the other hand, in 1-decanol the T values become progressively shorter closer to the OH group, probably a consequence of "anchoring" of the OH end by intermolecular hydrogen bonding. You can also see the effects of segmental motion in the chlolesteryl chloride T; values - all of ring CH are ca 0.5 sec, the CHz 0.25 sec - that part of the molecule moves as a unit. However, in the side chain the T; values become longer, up to 0.67 sec for the last CH and 1.8 sec for the last CH, because the side chain has additional flexibility, and thus a shorter correlation time, Severe steric himdrance for this Ho 27 9M td a i Ha o> CH, greatly reduces rotation 2 rate, and thus a shorter T, for C o7 08 o8 11 22 YY ‘S 1 ik 2 ye os 08 08 18 31 Effect of Segmental motion (ACS 1971, 93, 1558 Gaiee Methyl groups show highly variable 7; values. There are now three directly attached protons and thus one would expect 1/3 the T, compared to CH carbons. However, unless the CH3 group is very sterically hindered the local motions are much more rapid than the rest of the molecule, and thus the hitpswww' chem wise-edulareasitelehinmv/08-tech-01-0laxhim sine2iero017 8-1 Relaxation in NMR Spectroscopy carbon undergoes less effective DD relaxation. This may be in part offset by SR relaxation, which becomes more effective at higher rates of rotation Relaxation by Oxygen. The unpaired spins on 0) make it capable of causing proton and carbon relaxation by a dipolar interaction. The effect is not large, but can be easily seen for the quaternary carbons of phenylacetylene - e.g. the ipso carbon T; increases from 56 to 107 sec if the dissolved ‘oxygen is removed. This can be done by vaccum techniques, or more simply by bubbling Nz or argon through the sample for a minute or two. Normally O2 makes a contribution at the level of T; ca 50 to 100 sec. Since most T; values are less than 20 sec, O2 does not normally have much of an effect, Only very long T; values (>>20 sec) are significantly affected by oxygen. It is important to remove dissolved oxygen from samples for NMR experiments where long T; values are important (e.g. in multipulse/2D experiments where long delay times or used), or where proton- proton DD relaxation must be maximized (e.g. in the NOE experiment discussed in Section 8.2) 132132 “ou 53.85 DY 3293 _ jar Atmospheric O, Degassed Effect of DD relaxation by oxygen © 2016 Hans J. Reich, All Rights Reserved Web page created by winett. Last updated 03/22/2016 15:30:21 hitpswww' chem wise-edulareasitelehinmv/08-tech-01-0laxhim rane