The NMR Sample

All samples for NMR analysis must be soluble in one of the commercially available deuterated NMR solvents (see Table 11.2). When this is not 

For C-NMR, because of the 1.1% natural abundance, the same sample would require an overnight run to produce a reasonable spectrum. Clearly, for C-NMR, the more concentrated the solution, the shorter the analysis time, e.g. 0.3 g of neat pyridine (with just a few drops of deuterated solvent added) produces an excellent C-NMR spectrum within 10 min. 

Samples are made up in 5 mm (overall diameter) NMR tubes (typical for work) or 10 mm tubes (typical for multinuclear NMR) and 

Advances in NMR software have now made the process of actually taking an NMR spectrum so simple that just a few simple electronic commands will nowadays produce a basic H/ C-NMR spectrum. 

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When two equivalents of pyridine were added to the nmr sample and the probe heated to 80° C, the enol formate 61 decreased and phenyl cyclopropyl ketone 58 appeared at a rate approximately ten times faster than in the previous buffered system. The observation of intermediate 61 and the kinetic results, together with the observed induction periods, are consistent with the idea that some and perhaps all of the rearranged product ketone in the solvolysis of this system arises via double-bond participation in 61 rather than triple-bond participation and a vinyl cation (80). 

Transient Time-domain signal (FID) acquired in an FT experiment. Transmitter Coil of wire and accompanying electronics from which Rf energy is applied to the NMR sample. 

The best and easiest way to implement such an experiment is to use adiabatic inversion pulses, in order to introduce heterogeneity for evolution under 13C-1H scalar or residual dipolar couplings by means of a frequency-swept 180° pulse on 13C that inverts 13C nuclei at different positions in the NMR sample at different times (Figure 13) 40,45 This filter is robust with respect to pulse miscalibration and operates efficiently without the need to cycle the phases of pulses that otherwise is a common feature of non-destructive LPJFs. 

Because most common solvents, including water, contain protons, and most NMR analyses involve the measurement of protons, a solvent without protons is generally used in NMR spectroscopy. Commonly, solvents in which the hydrogen atoms are replaced with deuterium (i.e., solvents that have been deuterated) are used, the most common being deuterochloroform. In addition, an internal standard, most commonly tetramethylsilane (TMS), is added to the sample in the NMR sample tube (see Figure 14.3, D) and all absorption features are recorded relative to the absorption due to TMS. 

Labile protons can always be positively identified by in situ exchange with D2O. In practice, a normal H NMR spectmm is recorded then deuterium exchange of labile protons is achieved by simply adding a drop of deuterated water (D2O) to the NMR sample. Labile protons in -OH, -COOH, -NH2 and -SH groups exchange rapidly for deuterons in D2O and the H NMR is recorded again. Since deuterium is invisible in the iH NMR spectmm, labile protons disappear from the NMR spectmm and can be readily identified by comparison of the spectra before and after D2O is addition. 

Agostic interactions, i. e., the three-center bonds related to structure 51 [26, 44-49], were noted earlier by Green and Brookhart and have been cited above in the methoxycarbonylation chemistry (Figure 1.9). These bonds are often characterized by low frequency (hydride-like) proton chemical shifts, and/or substantially reduced /( C, H) values. Often, it is necessary to cool the NMR sample in order to freeze the equilibrium. Complex 52 represents a nice example of an agostic C-H bond, with relevance to polymerization chemistry [47]. 

As expected for the icosahedral symmetry, the NMR spectrum of Cjq (Figure 1.24) shows one signal, at 5 = 143.2 [25]. Since the amount of fullerene in the NMR samples due to low solubility is small and the spin-lattice relaxation... 

The H-NMR peaks sometimes broadened due to a trace of impurities. The impurities can be removed from the NMR sample by passing through alumina... 

Monitor target integrity in the NMR sample by comparing the protein background signal with time. 

The protein stock concentration should be in the NMR running buffer at concentrations slightly higher than what is used in the NMR samples. Alternatively, if the protein stability is better in a different buffer, the protein could be stored at high concentration (80 iM or higher) and a small aliquot diluted into the NMR running buffer for sample preparation. 

Data provided by the NMR sample tube producers Wilmad and Norell. 

Dry the sample, pipet tips, and the NMR sample tube plug 2 hr in a desiccator under vacuum at room temperature. 

Plug the NMR sample tube and seal with Parafilm. 

If it hasn t been done already, prepare the NMR sample, set the temperature, and read a shim file as described (see Basic Protocol, steps 1 to 4). 

In practice a small amount of TMS (<1%) is added to the NMR sample, the TMS signal is set at 0 ppm, and the protons of the sample are then measured in parts per million relative to TMS. The choice of TMS as a standard is useful because nearly all other protons absorb at frequencies lower titan TMS. It is routine to present NMR spectra with low frequency on the left and high frequency on the right (Figure 11.5). Thus the TMS signal defines 5=0 ppm on the right side... 

The reactivity of the incarcerated cyclobutadiene was probed by warming the NMR sample to 220°C for 5 min. This resulted in the formation of free cyclooctatetraene (6.108), clearly from ejection of the cyclobutadiene from the cavity and its subsequent dimerisation via 6.107. Reaction with 02 (which is able to enter the cavity of 6.101 gave incarcerated malealdehyde (6.109). 

Spin the columns for 2 min at 735g. Collect the NMR sample from the bottom of the microcentrifuge tubes (see Note 7). Adjust the sample volume to 225 pL with NMR sample buffer. Add 25 pL of 10 mMDSS (chemical shift internal standard) dissolved in D20. 

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