Although chemical tests provide evidence for the presence of carboxyl and phenol groups in salicylic acid, instrumental techniques are the basis of modern analysis.
A mass spectrometer at its simplest level records what happens when a sample is bombarded with high energy electrons. There are a number of stages in the process.
The mass spectrum that is produced is characteristic of that particular sample. The mass peaks provide the information that enables identification of the sample from the molecular ion peak and from the characteristic fragmentation pattern.
Mass spectra give characteristic isotope patterns according to mass and natural abundance. For example, the two isotopes of chlorine have mass numbers of 35 and 37 respectively and they occur naturally in the ratio 76:24. This is shewn by two peaks differing by two mass units in the approximate ratio 3:1, as shewn below.
If we look at a typical mass spectrum for a compound such as benzoic acid, we see that it is quite complex.
The heaviest ion (m/z = 122) is the one corresponding to benzoic acid with one electron removed. This is called the molecular ion (or parent ion). The other ions reaching the detector are the result of fragmentation. The most abundant ion gives the strongest detector signal which is set to 100% in the spectrum. This is referred to as the base peak and the intensities of all the other peaks are expressed as a percentage fraction of its value. For monosubstituted aromatics a peak is expected at m/z = 77 corresponding to C6H5+. This peak is often observed and is present in the spectrum of benzoic acid. However, bond breaking occurs more frequently one bond away from the benzene ring (see fragment at 105).
One isotope which can be useful in working out mass spectra of more complicated molecules is 13C. This accounts for only 1.1% of a sample of carbon atoms. However, if there are ten carbon atoms in a compound there is an 11% chance of one atom being 13C. This would make the molecular ion heavier by 1 unit. This accounts for small peaks to the right of each major peak at 51, 77, 105 and 122.
Infra-red spectroscopy is also known as vibrational spectroscopy since the spectra observed correspond to vibrational transitions within molecules, resulting from the absorption of radiation in the infra-red region. The infra-red region of the electromagnetic spectrum lies between the red end of the visible spectrum and the beginning of the microwave region. Conventional infra-red spectroscopy is concerned with the wavelength region 2.5μm to 25μm. The infra-red spectrum is a plot of % transmission against wavenumber. The wavenumber scale is the reciprocal of wavelength and has the units cm-1. Wavenumber is directly proportional to the energy of radiation.
When infra-red radiation is passed through a molecular compound, the molecules absorb radiation with just the right energy to cause the bonds to vibrate, twist, bend or stretch more rapidly. The energy required depends in each case on the atoms involved and the nature of the bonds. For example, bonds between light atoms vibrate at higher frequencies (more rapidly) than those between heavy atoms. Multiple covalent bonds vibrate at higher frequencies than single covalent bonds.
When the full spectrum of infra-red radiation is passed through a compound, an absorption spectrum is obtained. The spectrum of an unknown compound can offer clues about its structural formula. This due to certain bonds and functional groups absorbing infra-red radiation of a characteristic wavenumber. For example the carbonyl group, C = O, absorbs strongly at about 1680 - 1750 cm-1. The precise position of an absorption depends on the environment of the bond in the molecule, so we can only quote wavenumber regions in which we can expect absorptions to arise.
The infra-red spectrum below is for benzoic acid, C6H5CO2H.
Many atomic nucleii spin about an axis. Since they are associated with an electric charge, this spin gives rise to a magnetic field and these nucleii can be looked upon as tiny bar magnets. Not all nucleii have this spin. The most commonly studied nucleus is hydrogen which is, of course, the proton.
If a compound containing hydrogen atoms is placed in a magnetic field the protons can take up two positions. In one of these (the low energy position) the magnetic field of the proton lines up with the applied magnetic field; in the other (high energy position) the magnetic fields oppose one another. The quantum of energy separating these two states is proportional to the magnetic field strength and because E1 - E2 = hν the corresponding frequency, ν, is also proportional to the magnetic field strength.
The magnetic field strengths used in practice are quite large and the frequencies involved are in the radio-wave region.
The energy needed for the nuclear 'magnet' to move to a higher energy level depends on the strength of the magnetic field. This is not quite the same as the field being applied by the instrument because the electrons associated with the neighbouring atoms and groups in the molecule give rise to tiny magnetic fields of their own.
These local fields are usually opposed to the external field. The overall field experienced by a proton is therefore slightly smaller than the external field, depending on the local field from the surrounding part of the molecule.
Hence for every type of molecular arrangement, there is a very slightly different magnetic field. The hydrogen nucleii in these different environments have different energy gaps ΔE between their high and low levels, and so absorb different frequencies of radiation. They therefore give different n.m.r. peaks and we can find out how many hydrogen atoms of different types there are in a molecule. The diagram below shews a low resolution spectrum for ethanol.
Ethanol has three types of protons; one in the -OH group, one in the CH2 group and one in the CH3 group. The molecule will emit three frequencies in an n.m.r. spectrum. The relative areas under the peaks are in the ratio 1:2:3, i.e. representing 1H, 2H, 3H in the three groups.
As well as the spectrum trace, there is an integrated spectrum trace. This goes upwards in steps which are proportional to the areas of the peaks and tells us how many protons are absorbing each time. This again is in the ratio 1:2:3.
Also shewn on the spectrum is the absorption of tetramethylsilanes, TMS. This is used as the reference. The extent to which a signal differs from TMS is called its chemical shift. TMS is set at a chemical shift at 0 and the shifts of other types of protons can be found from reference tables.
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