Table 2.Solvents Commonly Used for Proton NMR

Some solvents (e.g.,D2Oor CD3OD)enter into fast exchange reactions with protons and may eliminate resonance signals from –COOH,–OH,and –NH2structural groups.The protons in alcohols and amines do not take part in rapid exchange unless catalyzed by small concentrations of acid or base,except in the presence of D2Oand some other solvents (e.g.,CD3OD).

For 19F NMR,most solvents used in proton NMRmay be employed,the most common ones being CHCl3,CCl4,H2O,CS2,aqueous acids and bases,and dimethylacetamide.In general,any nonfluorinated solvent may be used,provided that it is of spectral quality.Obviously,there is no interference from the protonated functional groups of the solvent.However,unless they are decoupled,protonated functional groups on the 19F-containing specimen will provide J-coupling.

The NMRspecimen tubes must meet narrow tolerance specifications in diameter,wall thickness,concentricity,and camber.The most widely used tubes have a 5-or 10-mm outside diameter and a length of between 15and 20cm.Microtubes are available for the analysis of small amounts of specimen.

Instrument adjustments are made before each experiment.The spinning rate of the specimen tube is adjusted so that spinning side bands do not interfere with the peaks of interest and the vortex does not extend beyond the coils in the probe.To optimize the instrument performance,the magnetic shim gradients on FT-NMRspectrometers are adjusted.In adjusting resolution on CWspectrometers,a good indicator is the definite “ringing”of the TMSpeak.The phenomenon of ringing is the oscillation of the recorder trace after the magnetic field has passed through a resonance frequency.Ringing,evident on a number of the peaks in Figures 5and 6,arises during rapid scans and decays exponentially to the baseline value.

Fig.6.Continuous wave proton spectrum of ethyl ether.

Figure 7clearly indicates the absence,in an FTexperiment,of the ringing phenomenon.Ringing will not appear because the spectrum obtained is the result of analysis of the FIDby Fourier transformation and not a magnetic field or frequency sweep through the individual resonance positions.

Fig.7.Proton NMRspectrum of ethyl ether in deuterated chloroform.

With proton CWinstruments the spectrum is scanned from 0ppm to about 10ppm with a scan time of about 1to 5minutes.The amplification is adjusted so that all peaks remain on scale.If the response is low at reasonable amplitude,the radio-frequency power is increased to obtain the highest possible peak response without peak broadening.After the initial scan,the presence of peaks downfield of 10ppm is quickly checked by off-setting the instrument response by about 5ppm.With CWinstrumentation,it is common for the TMSpeak to shift slightly during an extended scan.The extent of the shift is usually obtained by comparing the relative positions of another peak in the initial scan with the same peak in the offset scan.

The operation of an FT-NMRspectrometer is a much more elaborate experiment.The computer serves to control the spectrometer,to program the experiment,and to store and process the data.Programming the experiment involves setting values for a large number of variables including the spectral width to be examined,the duration (“width”)of the excitation pulse,the time interval over which data will be acquired,the number of transients to be accumulated,and the delay between one acquisition and the next.The analysis time for one transient is in the order of seconds.The number of transients is a function of the specimen concentration,the type of nucleus,and the objective of the experiment.At the end of the experiment,the FIDsignal is stored in digitized form in the computer memory and is displayed on the video screen.The signal can be processed mathematically to enhance either the resolution or the sensitivity,and it can be Fourier-transformed into a frequency-domain spectrum.The instrument provides a plot of the spectrum.The integration routine,accessed through keyboard commands,results in a stepped-line plot.Considerably more accurate integrals are obtained if the signals or regions of interest are separately integrated.

FT-NMRspectrometers may yield qualitative and quantitative data from the same experiment,but this is seldom done in practice.In quantitative FTexperiments,special precautions must be taken for the signal areas to be proportional to the number of protons.The delays between pulses must be long enough to allow complete relaxation of all excited nuclei.This results in a considerable increase in analysis time and in some loss of resolution.Qualitative analysis is usually performed in nonquantitative conditions,with the design of the experiment directed to fast analysis with maximum resolution or sensitivity.

NMRspectroscopy has been used for a wide range of applications such as structure elucidation;thermodynamic,kinetic,and mechanistic studies;and quantitative analysis.Some of these applications are beyond the scope of compendial methods.

All five characteristics of the signal—chemical shift,multiplicity,line width,coupling constants,and relative intensity—contribute analytical information.

For unknown specimens,NMRanalysis,usually coupled with other analytical techniques,is a powerful tool for structure elucidation.Chemical shifts provide information on the chemical environment of the nuclei.Extensive literature is available with correlation charts and rules for predicting chemical shifts.The multiplicity of the signals provides important stereochemical information.Mutual signal splitting of functional groups indicates close proximity.The magnitude of the coupling constant,J,between residual protons on substituted aromatic,olefinic,or cycloalkyl structures is used to identify the relative position of the substituents.

Several special techniques (double resonance,chemical exchange,use of shift reagents,two-dimensional analysis,etc.)are available to simplify some of the more complex spectra,to identify certain functional groups,and to determine coupling correlations.

Double resonance,or spin decoupling,is a technique that removes the coupling between nuclei and thus simplifies the spectrum and identifies the components in a coupling relationship.For example,in a simple two-proton system,generally designated an AXsystem (see Figure 4),each proton appears as a doublet.If a strong radio-frequency field is introduced at the frequency of X,while the normal radio-frequency field is maintained at the frequency that causes Ato resonate,the coupling between Aand Xis removed (homonuclear decoupling).Ais no longer split,but instead appears as a singlet.Routine 13Cspectra are obtained under proton decoupling conditions that remove all heteronuclear 13C-1Hcouplings.As a result of this decoupling,the carbon signals appear as singlets,unless other nuclei that are not decoupled are present (e.g.,19F,31P).

Functional groups containing exchangeable protons bound to hetero-atoms such as –OH,–NH2,or –COOHgroups may be identified by taking advantage of the rapid exchange of these protons with D2O.To determine the presence and position of these groups,scan the test substance in CDCl3or DMSO-d6,then add a few drops of D2Oto the specimen tube,shake,and scan again.The resonance peaks from these groups collapse in the second scan and are replaced by the HDOsinglet between 4.7and 5.0ppm.

This chemical exchangeis an example of the effect of intermolecular and intramolecular rate processes on NMRspectra.If a proton can experience different environments by virtue of such a process (tautomerism,rotation about a bond,exchange equilibria,ring inversion,etc.),the appearance of the spectrum will be a function of the rate of the process.Slow processes (on an NMRtime scale)result in more than one signal,fast processes average these signals to one line,and intermediate processes produce broad signals.

The software of modern FT-NMRspectrometers allows for sequences of pulses much more complex than the repetitive accumulation of transients described above.Such experiments include homonuclear or heteronuclear two-dimensional analysis,which determines the correlation of couplings and may simplify the interpretation of otherwise complex spectra.

If the two signals originate from two functional groups of the same molecule,the equation can be simplified to

in which n1and n2are the number of protons in the respective functional groups.

If the two signals originate from different molecular species,

where m1and m2are the numbers of moles;W1and W2are the masses;andM1andM2are the molecular weights of compounds 1and 2,respectively.

Examination of Equations 2and 3shows that NMRquantitative analysis can be performed in an absolute or relative manner.In the absolute method,an internal standard is added to the specimen and a resonance peak area arising from the test substance is compared with a resonance peak area from the internal standard.If both test substance and internal standard are accurately weighed,the absolute purity of the substance may be calculated.Agood internal standard has the following properties:it presents a reference resonance peak,preferably a singlet,at a field position removed from all specimen peaks;it is soluble in the analytical solvent;its proton equivalent weight,i.e.,the molecular weight divided by the number of protons giving rise to the reference peak,is low;and it does not interact with the compound being tested.Typical examples of useful standards are 1,2,4,5-tetrachlorobenzene,1,4-dinitrobenzene,benzyl benzoate,and maleic acid.The choice of a standard will be dictated by the spectrum of the specimen.

The relative method may be used to determine the molar fraction of an impurity in a test substance (or of the components in a mixture)as calculated by Equation 3.

Quantitative analysis,as well as detection of trace impurities,is markedly improved with modern instrumentation.Stronger magnetic fields and the ability to accumulate and/or average signals over long periods of time greatly enhance the sensitivity of the method.