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2.9 NUCLEAR MAGNETIC RESONANCE SPECTROMETRY

      Nuclear magnetic resonance (NMR) spectrometry is based on the fact that nuclei such as ¹ H, ¹³C, ¹⁹F and ³¹P possess a permanent nuclear magnetic moment. When placed in an external magnetic field, (main field), they take certain well-defined orientations with respect to the direction of this field which correspond to distinct energy levels. For a given field value, transitions between neighbouring energy levels take place due to absorption of electromagnetic radiation of characteristic wavelengths at radio frequencies.

      The determination of these frequencies may be made either by sequential search of the resonance conditions (continuous-wave spectrometry) or by simultaneous excitation of all transitions with a multifrequency pulse followed by computer analysis of the free-induction decay of the irradiation emitted as the system returns to the initial state (pulsed spectrometry).

      A proton magnetic resonance spectrum appears as a set of signals which correspond to protons and are characteristic of their nuclear and electronic environment within the molecule. The separation between a given signal and that of a reference compound is called a chemical shift (δ) and is expressed in parts per million (ppm); it characterizes the kind of proton in terms of electronic environment. Signals are frequently split into groups of related peaks, called doublets, triplets... multiplets; this splitting is due to the presence of permanent magnetic fields emanating from adjacent nuclei, particularly from other protons within two to five valence bonds. The intensity of each signal, determined from the area under the signal, is proportional to the number of equivalent protons.

Apparatus

      A nuclear magnetic resonance spectrometer for continuous wave spectrometry consists of a magnet, a low-frequency sweep generator, a sample holder, a radio-frequency transmitter and receiver, a recorder and an electronic integrator. A pulsed spectrometer is additionally equipped with a pulse transmitter and a computer for the acquisition, storage and mathematical transformation of the data into a conventional spectrum.

      Unless otherwise directed in the monograph, use a nuclear magnetic resonance spectrometer operating at not less than 60 MHz in accordance with the manufacturer’s instructions and the following operating conditions

      Before recording the spectrum, verify the following.

      (1) the resolution is equal to 0.5 Hz or less by measuring the peak width at half-height using an adequate scale expansion of either (i) the band at δ7.33 ppm or at δ7.51 ppm of the symmetrical multiplet of a 20 per cent v/v solution of 1,2-dichlorobenzene in deuterated acetone or (ii) the band at δ0.00 ppm of a 5 per cent v/v solution of tetramethylsilane in deuterochloroform.

      (2) the signal-to-noise ratio (S/N), measured over the range from δ2 to 5 ppm on the spectrum obtained with a 1 per cent v/v solution of ethylbenzene in deuterochloroform, is at least 25:1. This ratio is calculated as the mean of five successive determinations from the formula:

S/N = 2.5A/H,

where A is the amplitude, measured in millimetres, of the largest peak of the methylene quartet of ethylbenzene centred at δ2.65 ppm and H is the peak-topeak amplitude of the baseline noise measured in millimetres obtained between δ4 and δ5 ppm. The amplitude is measured from a baseline constructed from the centre of the noise on either side of this quartet and at a distance of at least 1 ppm from its centre.

      (3) The amplitude of spinning side bands is not more than 2 per cent of the sample peak height in a tube rotating at a speed appropriated for the spectrometer used.

      (4) For quantitative measurements verify the repeatability of the integrator responses using a 5 per cent v/v solution of ethylbenzene in deuterochloroform. Carry out five successive scan of the protons of the phenyl and ethyl groups and determine the mean of the values obtained. None of the individual values differs by more than 2.5 per cent from the mean.

Method

      Dissolve the substance being examined as prescribed in the monograph and filter; the solution must be clear. Unless otherwise specified in the monograph, an internal chemical shift standard should be used; for solutions in deuterated organic solvents, 0.5 to 1.0 per cent v/v of tetramethylsilane may be added; for solutions in deuterium oxide, 0.5 to 1.0 per cent w/v of sodium tetradeuterodimethylsilapentanoate may be added. Take the necessary quantity and record the spectrum.

CONTINUOUS WAVE SPECTROMETRY Adjust the spectrometer so that it is operating as closely as possible in the pure absorption mode and use a radio frequency setting that avoids saturation of the signals. Adjust the spectrometer controls so that the height of the strongest peak of the spectrum of the substance being examined reaches almost to full-scale deflection and the peak associated with the internal reference standard registers on the chart at δ0.00 ppm. Record the spectrum over the prescribed spectral width, using a scan speed of not more than 2 Hz per second unless otherwise specified. It is advisable to record an integral spectrum over the same spectral range, using a suitable scan speed.

      Directions for quantitative measurements are given in individual monographs.

PULSED SPECTROMETRY Set the spectrometer controls, for example pulse flip angle, pulse amplitude, pulse interval, spectral width, number of data points (resolution) and data acquisition rate, as indicated in the manufacturer’s instructions and collect the necessary number of free induction decays. After mathematical transformation of the data by the computer, adjust the phase control in order to obtain as far as possible a pure absorption spectrum and calibrate the spectrum relative to the resonance frequency of the chemical shift internal reference compound. Display the spectrum stored in the computer on a suitable output device and, for quantitative measurements, process the integral according to the facility of the instrument.