Two-dimensional NMR. Two-dimensional (2D) NMR spectroscopy was first proposed in 1971, but its full power was not realized until 1980.

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1 Two-dimensional NMR Two-dimensional (D) NMR spectroscop was first proposed in 97, but its full power was not realied until 980. D pulse sequences consist of a preparation period (frequentl a 90 pulse), an evolution period (t) during which some modulation occurs due to the interaction of interest, followed b a detection period (t). Preparation Evolution Miing period Detection FD t t m t A series of FDs are recorded while the value of t is incremented in a regular manner from one eperiment to another (i.e., FDs Z(t,t ) are acquired). Fourier transformation of the t time domain gives us a set of spectra as a function of the t step sie. A second Fourier transformation of t time domain gives a D spectrum revealing the frequencies of the modulation that were occurring during the t evolution period. D NMR spectroscop can be used to probe specific NMR interactions selectivel. preading out the spectrum in two dimensions has the advantage for large molecules that it removes much peak overlap. While the simple vector model ma be used to eplain some D eperiments, it is unable to eplain D techniques. NOEY One of the simplest D eperiments t RD t m t Preparation Evolution Miing Detection Can be pictured in terms of the vector model.

2 The amplitude of the signal in the Z(t, F ) spectra (after the first Fourier transformation of Z(t, t )) varies regularl as a cosine function of t. Wh? Consider a two-spin sstem, (both spin-/), J = 0. Transmitter frequenc, B 90, t B t t angles 90 -cos t -cos t The eperiment is designed to record onl the components of the magnetiation evolved during the miing period m. The component is rejected using a suitable phase-ccling scheme. A third 90 pulse converts -components into transverse signal for detection. The components are said to be frequenc labelled.

3 m = 0: a second FT with respect to t etracts the frequenc of the amplitude modulation of Z(t, F ): Diagonal peaks F F m 0: useful information can be obtained if the two spins echange magnetiation during m, either b cross-relaation or b chemical echange. When t is such that t = /, the component of has ero intensit at the start of m, while the corresponding vector is still largel inverted. This is similar to the situation at the start of the in a normal D transient NOE eperiment (one spin is completel inverted, while the other is at equilibrium). Cross-relaation during m results in a transient NOE enhancement at, similar to that epected in the D eperiment. The sie and direction of these enhancements depend on the difference in the values of the components of and at the start of m. These values in turn depend on the etent of precession that and underwent during t.

4 B the end of the miing time m, the intensit of the vector, which was initiall given b -cos t, acquires an additional dependence on cos t. This results in a major peak (diagonal peak) at F = and a minor peak (cross-peak) at F = on Fourier transformation with respect to t. The presence of crosspeaks in NOEY spectra indicates that the proton spin sites are close in space (< 5 Å). Other D pectra COY - Homonuclear COrrelation pectroscopy is based on the pulse sequence: 90 t 90 acquire (t ) Double Fourier transformation gives a D spectrum with the normal one-dimensional spectrum along the diagonal, while cross-peaks indicate J-couplings between nuclei. J J F F There are man variants of the COY sequence. For instance, the pulse sequence can be modified to emphasie long-range J-couplings. dentifing cross-peaks close to the diagonal can be a major problem, but methods eist to reduce (or eliminate) diagonal peak intensit (e.g. Double-Quantum Filtered COY). ( H, H)-COY Br H N H N 6 7 F / H 9 F / H

5 HETCOR - HETeronuclear CORrelation coupling eperiments are also straightforward. n the case of C/ H correlation, the cross-sections through the two aes reveal the normal one-dimensional C and H spectra, while the D peaks reveal which carbons are bonded to which hdrogens. This simplifies considerabl assignment of peaks. J CH CH -CH -CH(OH)-CH H / ppm C / ppm NADEQUATE - correlates through the coupling between dilute nuclei. This enables, for eample, the entire carbon skeleton to be determined in those cases where H and C- H J-couplings are not sufficient to solve the problem. t is a powerful technique, but time-consuming. J CC CH -CH -CH -CH -OH C / ppm

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7 J-resolved pectroscop - both homonuclear and heteronuclear versions eist. The pulse sequence is based on a simple spin-echo tpe eperiment. One dimension gives the spectrum with chemical shift information onl (i.e., all J-couplings removed), while the other dimension shows onl J-coupling information. J CH CH -CH -CH(OH)-CH J CH / H C / ppm D EXY - gives cross-peaks between sites that are in slow chemical echange; this enables dnamic information to be obtained when in the slow-echange limit on the chemical shift timescale. a-me, 7. ppm eq-me,.5 ppm D C- C EXY Methlccloheane, C 6 H CH, in CDCl /CFCl (: v/v) at 00 K. The observed cross-peaks are due to slow echange between two chair conformers of C 6 H CH with aial (population ~%) and equatorial (~99%) orientations of the methl group.