NUCLEAR MAGNETIC RESONANCE V.6.23.

V.6.23. NUCLEAR MAGNETIC RESONANCE SPECTROMETRY

Nuclear magnetic resonance (NMR) spectrometry is based on the fact that nuclei such as IH, 13C, 19F, 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 0 wavelengths at radio frequencies. The determination of these frequencies may be made either by sequen- tial 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).

Aproton magnetic resonance spectrum appears as a set of signals which correspond to protons and are characteristic of their nuclear and elec- tronic environment within the molecule. The separation between a given signal and that of a reference compound is called a chemical shift (6) and is expressed in parts per million (ppm); it characterises 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 ema- nating from adjacent nuclei, particularly from other protons within two to five valence bonds. The intensity of each sianal. determined from the area under the signal, is proportional to the number of equivalent O NOT CURRENT Apparatus. - A nuclear magnetic resonance spectrometer for con- tinuous-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 ad- @ ditionally equipped with a pulse transmitter and a computer for the acquisition, storage and mathematical transformation of the data into a conventional spectrum.

Use a nuclear magnetic resonance spectrometer operating at not less than 60 MHz for IH. Unless otherwise prescribed, follow the instructions of the manufacturer. Before recording the spectrum, verify that: V.6.23.-2 NUCLEAR MAGNETIC RESONANCE

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 the band at 6 7.33 ppm or at 6 7.51 ppm of the symmetrical multipIet of a 20 per cent VjV solution of dichlorobenzene R in deuterated acetone R, - or the band at 6 0.00 ppm of a 5 per cent VIV solution of tetra- methylsilane R in deuterated chloroform R; O 2) The signal-to-noise ratio (SjN),measured over the range from 6 2 ppm to 6 5 ppm on the spectrum obtained with a 1 per cent V/V solution of ethylbenzene R in carbon tetrachloride R, is at least 25:l. This ratio is calculated as the mean of five successive determinations from the expression A S/N 2.5- H

where

A = amplitude, measured in millimetres, of the largest peak of the methylene quartet of ethylbenzene centred at 6 2.65 ppm. The amplitude is measured from a base line 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.

H = peak to peak amplitude of the base line noise measured in millimetres obtained between 6 4 ppm and 6 5 ppm. 3) The amplitude of spinning side bands is not greater than 2 per cent of the sample peak height in a tube rotating at a speed appropriate for the spectrometer used. 4) For quantitative measurements verify the repeatability of the inte- grator responses, using a 5 per cent VjV solution of ethylbenzene R @ NOTin carbon tetrachloride CURRENT R. Carry out five successive scans 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 to be examined as prescribed and filter; the solution must be clear. Use a chemical shift internal reference compound, which, unless otherwise prescribed, is a solution containing 0.5 per cent V/V to 1.0 per cent VIV of tetramethylsilane R (TMS) in deuterated organic solvents or 0.5 per cent m/V to'1.O per cent m/V of the sodium salt of tetradeuteriodimethylsilapentanoic acid R (TSP) in deuterium oxide R. Take the necessary quantity and record the spectrum. NUCLEAR MAGNETIC RESONANCE V.6.23.-3

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 which avoids saturation of the signals. Adjust the controls of the spectrometer so that the strongest peak in the spectrum of the substance to be examined occupies almost the whole of the scale on the recorder chart and that the signal of the internal reference compound corresponds to a chemical shift of 6 0.00 ppm. Record the spectrum over the prescribed spectral width and, unless otherwise specified, at a sweep rate of not more than 2 Hz per second. Record the integral spectrum over the same spectral width and at a suitable sweep rate according to the instrument used. When quantitative measurements are required, these should be obtained as prescribed.

PULSED SPECTROMETRY

Set the spectrometer controls, e.g. 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.

NOTVII.l.l. CURRENT REAGENTS Deuterated acetone. - C$H60 (Mr 64.1). (2Hs)Ácetone. A clear, colourless liquid, with a characteristic odour, miscible with water, with dimethylformamide, with chloroform, with ethanol, with ether and with methanol. d$: about 0.8732 n : about 1.3565 bp : about 55 "C. The degree of deuteration is not less than 99.5 per cent.

Water and deuterium oxide: not more than 0.1 per cent. V.6.23.-4 NUCLEAR MAGNETIC RESONANCE

Deuterated chloroform. - CZHC13 (Mr 120.4). (2H)Chloroform. clear, colourless liquid with a characteristicodour, slightly soluble in water, miscible Awith acetone, with alcohol and with ether. It may be stabilised over silver foil. dzt: about 1.506 n E: about 1.445 bp : about 60 "C.

The degree of deuteration is not less than 99.1 per cent. Water and deuterium oxide: not more than 0.05 per cent.

Deuterium oxide. - 2H20 (Mr 20.03). Deuterated water. d;:: about 1.107 n E: about 1.328 bp : about 101 "C. The degree of deuteration is not less than 99.7 per cent.

Deuterated dimethyl sulphoxide. - C22HsOS (Mr 84.2). (ZH6)Dimethyl sulphoxide. very hygroscopic liquid, practically colourless and odourless, viscous, soluble in Awater, in acetone, in ethanol, in ether and in chloroform. di:: about 1.18 mp: about 20 "C. The degree of deuteration is not less than 99.8 per cent. Water and deuterium oxide: not more than 0.1 per cent.

Ethylbenzene. - C8Hlo íM, Contains not less than per cent m/m of C8H10, determined by gas chromatography.106.2). 99.5 A clear, colourless liquid, practically insoluble in water, soluble in acetone, in alcohol and in chloroform. di::NOT about 0.866 CURRENT n 2: about 1.496 bp: about 135 OC.

Sodium tetradeuteriodimethylsilapentanoate. - CSH92HdNa02Si (Mr 172.3). TSP. Sodium (2,2,3,3-2H&4,4-dimethyl-4-silapentanoate. A white crystalline powder, freely soluble in water, in ethanol and in methanol. mp: about 300 "C. The degree of deuteration is not less than 99 per cent. Water und deuterium oxide: not more than 0.5 per cent. NUCLEAR MAGNETIC RESONANCE V.6.23.-5

Tetramethylsilane. - C4HizSi (M, 88.2). TMS. A clear, colourless liquid, very slightly soluble in water, soluble in acetone, in alcohol and in chloroform, d::: about 0.64 ng:about 1.358 bp : about 26 "C. Tetramethylsilane, when used in nuclear magnetic resonance spectrometry, also complies with the following additional requirement. In the nuclear magnetic resonance spectrum of an approximately 10 per cent VIV solution of tetramethylsilane in deuterated chloroform R, the intensity of any foreign signal, excluding those due to spinning side bands and to chloroform, is not greater than the intensity of the C-13 satellite signais located at a distance of 59.1 Hz on each side of the principal signal of tetramethylsilane.

NOT CURRENT NOT CURRENT VI.1. MATERIALS USED FOR THE MANUFACTURE OF CONTAINERS

The materials described below are used for the manufacture of con- tainers for pharmaceutical use. Materials other than those described in the Pharmacopoeia may be used subject to approval in each case by the national authority respon- .sible for the licensing for sale of the preparation in the container.

NOT CURRENT