Use of Relaxation Reagents

This section includes three topics not easily classified elsewhere, namely the use of relaxation reagents, the measurement of magnetic moments in solution, and the use of paramagnetic ions for studying surface phenomena. 

The addition of paramagnetic species, such as the metal ions Cu ", Mn, or CF", can have dramatic effects on both the observed spectmm and the relaxation behavior of a molecule. The added ion reduces nuclear relaxation times, and permitting more rapid data collection. In addition, faster relaxation rates minimize NOE effects in the spectra, which can be useful in obtaining quantitative intensity data. The most widely used reagent for this purpose is chromium acetylacetonate [13681 -82-8] known as Cr(acac)2. Practically speaking, the use of such reagents requires care, because at... 

The study of the fiillerenes by C NMR spectroscopy is not straightforward. C has a low natural isotopic abundance (1.11%) and a low relative receptivity. In addition, the carbon atoms in fiillerenes have relatively long relaxation times and spectra for the higher, less symmetric fiillerenes can only be obtained within a reasonable time by using paramagnetic relaxation reagents, typically Cr(acac)3. 

Rates of nuclear relaxations can be altered by the deliberate addition to the solutions used for NMR spectroscopy of species containing paramagnetic nuclei. The usual relaxation agent employed with substrates dissolved in organic solvents is the chelate chromium tris(acetylacetonate) (Cr(acac)3), [Me C6 CH C(6)Me]3Cr, i.e. chromium(III)-2,4-pentane-dionate. It acts to reduce values of via dipole-dipole interactions, leaving chemical shift values unaltered. Cr(acac)3, when used at concentrations of about 0.05 M, substantially reduces values of T, particularly those for quaternary nuclei simultaneously it removes the nuclear Overhauser enhancement from proton-decoupled and other resonances. Use of this reagent could therefore considerably shorten the times required for quantitative NMR measurements. 

It is important to avoid saturation of the signal during pulse width calibration. The Bloch equations predict that a delay of 5 1] will be required for complete restoration to the equilibrium state. It is therefore advisable to determine the 1] values an approximate determination may be made quickly by using the inversion-recovery sequence (see next paragraph). The protons of the sample on which the pulse widths are being determined should have relaxation times of less than a second, to avoid unnecessary delays in pulse width calibration. If the sample has protons with longer relaxation times, then it may be advisable to add a small quantity of a relaxation reagent, such as Cr(acac) or Gkl(FOD)3, to induce the nuclei to relax more quickly. 

Figure I. 39.7 MHz Si NMR spectra of PSQ and APSQ obtained from PSQ-B in acetone-d6. Chromium acetylacetonate was used as a relaxation reagent, and transients were 5000. PSQ-A (Mw = 900) and PSQ-B (Mw = 9500) were purchased from Owens-Illinois and Petrarch Systems, respectively.

The steady-state free-procession (SSFP) technique was used with no NOE, and in the case of aniline the T of the 15N was reduced by addition of chromium acetylacetate as a relaxation reagent to the sample. The 15N spectrum of aniline showed a single broad line (Avi/2 = 42 Hz) in which, because of proton exchange125,126, there is no evidence of N H coupling (J = 80 Hz). 

Each of the physical phenomena - , including spin-lattice relaxation and NOE experiments and the resulting NMR parameters, is discussed. By presenting selected examples, it is shown how information from these parameters can be used for the determination of relative configuration. The practice of recording NMR spectra in the presence of auxiliary reagents is also demonstrated (Section 4.1.1.4.). 

A detailed understanding of the many factors involved when [Cr(acac)3] is used as a spin relaxant is required. It has been shown that line widths are affected by the reagent.779 If temperature dependent features are to be studied, it was concluded that die ratio of relaxant to substrate must be kept as low as possible. Caution in the use of [Cr(acac)3] as a spin relaxant was also emphasized in a study of cholesterol chloride.780 Despite the problems with the use of this complex as a spin relaxant, it remains widely used.779,780... 

Quantitative measurements in NMR are based on the area of the signals present in the spectrum. Signal areas can be produced as numerical values proportional to the area or, on less modern instruments, from the integration plots that are superimposed on the spectrum (Fig. 9.1). For the proton, the precision obtained in area measurements does not exceed l % even if continuous wave instruments are used at slow scanning speeds. In l3C NMR, it is preferable to add a relaxation reagent in order to avoid saturation related to relaxation times that alter the intensity of the signal. Using the molar ratios that are easily accessible from the spectrum, it is possible to deduce concentrations. 

An alternative method of overcoming the time delay of mixing is to use a relaxation method. An equilibrium mixture of reagents is preincubated and the equilibrium is perturbed by an external influence. The rate of return, or relaxation, to equilibrium is then measured. The most common procedure for this is temperature jump (Figure 4.6).13 A solution is incubated in an absorbance or fluorescence cell and its temperature is raised through 5 to 10°C in less than a microsecond by the discharge of a capacitor (or, in more recent developments, in 10 to 100 ns by the discharge of an infrared laser). If the equilibrium involves an... 

Using single-frequency and noise-modulated resonance and off-resonance proton decoupling, 7] relaxation time measurements, relaxation reagents like Gd (fod)3 and specifically deuterated compounds, all the carbons in retinal isomers, the model compounds a-and /i-ionone, and vitamin A and its isomers [165, 555-557] were assigned. The olefinic ring carbons (C-5 and C-6) could be identified on the assumption that the 13C relaxation times are dominated by intramolecular dipole-dipole interactions with neighboring protons and that the same rotational correlation time characterizes the interactions for both carbons. Consequently the ratio of T/s for C-5 and C-6 can be estimated from eq. (5.1)... 

The study of nucleic acid bases by NMR has been reported in a number of monographs (/), but very little data is available on the, 3C and, 5N NMR chemical shift tensors in these compounds. The low sensitivity of NMR spectroscopy and the long relaxation times exhibited by many of these compounds have posed the main impediments for these studies. The use of sample doping with free radical relaxation reagents, to reduce the relaxation times facilitating 2D multiple pulse experiment (2, 3), enables one to measure and analyze the principal values of the chemical shift tensors in natural abundance samples. In previous papers from this laboratory we have presented, 5N NMR chemical shift principal values for adenine, guanine, cytosine, thymine and uracil (4, 5). 

It is convenient to divide the subject into four sections. The first two, on chemical shift reagents and on relaxation studies, deal with techniques of line assignments and other facets of steroid behavior. The third, on substituent effects, lays the background for predicting steroid 13C chemical shifts and interactions of substituents with the steroid framework. In the fourth section the use of 13C NMR to solve problems in steroid stereochemistry is discussed. All chemical shift data are reported on the delta scale. 

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