Craig L. Van Antwerp' Juniata College Huntingdon, Pennsylvania 16652
Experimental Determination of the Nuclear Overhauser Effect
Since the intramolecular nuclear Overhauser effect (NOE) was first descrihed by Anet and Bourn (I) in 1965, it bas reneatedlv been shown t o be a n invaluable tool in the dete;minatik of both t h e structure a n d conformation of oreanic c o m ~ o u n d s(2). T h e N O E enhancement is t h e amount by w h k h t h e intensity of a n nmr proton signal changes when t h e peak of a nearby proton is irradiated. It is the strong dependence of this enhancement on the distance between the two protons, a n d hence on the spatial orientation of t h e molecule, t h a t permits elucidation of such detailed structural information. Since most n m r spectrometers in general uie are equipped for double resonance o ~ e r a t i o n .the NOE can find rewardinn- a -. p~lications in unde&aduat'e laboratories or research experiences. T h e NOE can b e observed hv following t h e same basic procedure a s for spin-spin decoupling experiments followed by integration of the peaks of interest. Because of the quantitative nature of NOE measurements, however, it is necessary t o ohserve certain precautions in order t o obtain useful results. In spite of t h e considerable number of papers which have appeared in the last decade descrihing the results of NOE experiments, relatively few seem t o give a detailed description of the experimental procedure involved (3).This paper will discuss procedures t h a t were used t o obtain experimental N O E enhancements on a commercial student spectrometer.2 Applications of NOE results have been reviewed by Kennewell (2). Preparation of the Sample Because t h e N O E involves t h e relaxation processes of the various protons, care must he taken i n preparing the samnles t o exclude a n . v snecies t h a t will contribute t o in. termolecular relaxation. As with routine nmr work, viscous or turbid samnles must he avoided. T h e concentration of t h e compo&d being studied should he less than 10% (v/v) a n d preferably less t h a n 5% (v/v) in a nonmagnetic or low-magnetic solvent such a s CS2, CC14, OCS, SO*, or deuterated compounds (4). Careful degassing of the sample by several freeze-pump-thaw cycles uhder vacuum3 is strongly recommended (5). A study in this laboratory an 3,3-dimethylacrylic acid (Compound I) indicat-
ed that degassing the sample increased the apparent NOE enhancement of H-1 upon irradiation of the A methyl group by approximately 22%. besides improving the overall resolution of the spectrum. The sample can be degassed successfully in an nmr tube joined directly to a high vacuum system, or, if an additional amount of degassed sample is desired for further study, the degassing can be done in the apparatus shown in the figure. An Oring joint is attached to a stopcock leading to an inverted Y ; one 638 /Journal of Chemical Education
Thedegassing apparatus.
arm is the nmr tube, the other arm holds the sample and a hoiling chip. The sample is frozen in liquid nitrogen and the system evacuated. After minimum pressure is obtained, the stopcoek to the vacuum system is closed and the Nz bath removed, allowing the solution to melt. Mild boiling can be expected during the melting process. After the melting is complete and the sample approaches room temperature, the Nz bath is replaced to freeze the sample again and the stopcock is opened. After pumping far a few minutes, the process is repeated. Finally the stopcock is closed and the Y assembly disconnected from the vacuum system at the O-ring joint. After the assembly is removed from the liquid nitrogen and allowed to reach room temperature, some of the liquid solution is poured from the boiling tube into the nmr tube. Both arms are then frozen and the nrnr tube heated and sealed at the constriction and pulled from the rest of the assembly. In addition to the degassed sample in the nmr tube, this apparatus also furnishes a degassed solution of identical composition which is available for further analysis. After careful cleaning, the apparatus can be reused by simply attaching another nmr tube. Irradiating the Desired Peak NOE experiments are done in the frequency sweep made of double resonance, in which the irradiating frequency is held fixed at a chosen mint in the wectmm4 while the s ~ e c t m mis beine swept by the ohserving frequency. Adlustmp: the irradiating beam to the correct frequency rs done in the same manner ns in decoupling experiments, i.e., the recorder pen is positioned over the peak to be irradiated and the frequency is adjusted for minimum pen motion between the two areas of intense pen beat. If a numher of experiments are to he N n on the same sample, reproducibilitv of the irradiatine.. freouencv . , can be im~rovedusine an auxd u r y frequency counter by adjusting the t'rcqueney in the usual manner, then placing the pen over some reference point ( e g . 0.W 6) and noting the frequency differenceon the counter. It has been shown ( 6 ) that the magnitude of the NOE enhaneement depends strongly on both the accuracy of the irradiating frequency and the power level of the irradiating beam. Careful adjustment of bath of these parameters may he necessary to maximize the enhancement. ~
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This project was supported by the National Science Foundation Undergraduate Research Program. 'Present address: Chemistry Department, Stanford University, Stanford, Calif. 94305. The procedures described herein were used to determine NOE enhancements on the Varian T-60 spectrometer. If this is impractical, bubbling gaseous nitrogen through the sample andsealing the nmr tubeunder nitrogen is adequate. On the T-60, overtones of the resonant frequency beat occur at +60 Hz from the frequency of the irradiating beam. Because any peaks so positioned may be distorted by the overtone resonance, observations and integrals made in this region may be unreliable.
Determination of Intensity Changes Most NOE results described in the literature are obtained by first integrating the peak of interest with the irradiating beam moved off resonance (i.e., the irradiating frequency is adjusted so that i t is not near any proton signal). The irradiating beam is then moved to the frequency of the peak to be irradiated. The intensity of the peak of interest is again measured, and the two intensity measurements are compared. Another method-possibly more precise because of inherent compensation for possible instrumental errors-is based on comparing the intensity of the peak of interest with the intensity of some nearby peak which is assumed to be unaffected by the NOE. Colson, et. al. (7),utilized this technique in a NOE study of compound II. Upon irradiation of the methyl protons, the enhancement of the axial C-7 proton signal relative to that of the unaffected equatorial C-7 proton was found to be 27%. Use of an intramoleeular reference peak must be applied very judiciously, however, because unexpected NOE interactions could give misleading results. This problem is eliminated when the "standard peak" belongs t o the protons of a different compound--one which, when added to the sample, will not interact with the compound being investigated. The added reference compound should he chosen so that its signal, which is t o be used as a standard, is close to the peak of interest and no other peaks are introduced that will interfere with the NOE experiments. Benzene (6 = 7.24) was used successfully in this laboratory in an experiment to determine the NOE of a vinyl proton (6 = 5.63). Depending on the chemical shift of the peak of interest, other useful standards may include T M S (6 = 0.00), cyclohexane (6 = 1.42), dichloroethane (6 = 3.69), and dichloromethane (6 = 5.28). T o reduce intermolecular relaxation, the concentration of the standard compound should he as low as is consistent with an adequate peak intensity. Comparing 5 When time averaging is used, a magnetic field lock is recommended (8). The field stability of the standard T-60 is adequate for obtaining data at a high level of significance.
the ratio (01s)of the intensity of the peak being observed to the intensity of the standard peak when the irradiating beam is off resonance with that ratio when the desired peak is being irradiated will give the magnitude of the NOE because the intensity of the standard peak is assumed to remain constant. Care must be exercised in obtaining integration values. Baseline drift may be a serious prohlem because of the relatively poor signal-to-noise ratio resulting both from the low concentration of the sample and from the simultaneous use of the second beam. Also, i t is important to keep the power level of the observing beam low enough so that saturation of the ~ e a kheine observed does not occur during the sweep. Unless time averaeine of the simal is available5, any single determinationuof enhancement is likely to be unreliable, so a number of trials must be run. When performing a series of electronic integrations, it is necessary to allow enoueh time between sweeps for the total relaxation of interest. A 20-30-sec pause between of the integration sweeps is probably sufficient in most cases. The results and error limits are obtained by a standard statistical treatment of the data (9) and are usually expressed in terms of the percent change of intensity of the peak of interest. Acknowledgment The author wishes to express his appreciation to Dr. William E. Russey and Dr. Paul D. Schettler for much assistance and valuable advice. Literature Cited
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(5, Reiennce (4, SS, (6)Sehirmer, R. E.. Noggie. J. H.. Davis, J. P..and Hart, P.A., J. Amen Chom. Soe.. 92,326611970). (7) Cobon. J. G.,Lsmbury, P. T.. and Saeue. F. D.. J Amer C h m Soe. 89. 4981 114fm , , (8) h i e n n e e (41. (91 See. for exsmpb, W i h n . E. 8.. "An Tntdurtion tn Sci~ntiRcU P L P I I ) ~ C ~ , " MeCraw-Hill BmkCompany, New York. 1952. pp. 237-40.254-5.
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Volume 50. Number 9, September 7973 / 639
