In the simplest case two spectral printouts, one of the reference and the other of the analyte, can be overlaid on a light box and the spectral features related by eye. Overlaying spectra on the computer screen achieves the same objective.
When the spectrum of a substance being examined is compared with a reference spectrum, such as those in the British Pharmacopoeia, the positions and relative intensities of the absorption bands of the spectrum of the substance being examined should conform to those of the reference spectrum. When the two spectra are compared, care should be taken to allow for the possibility of differences in resolving power between the instrument on which the reference spectrum was prepared and the instrument used to examine the substance. It is good practice to run a spectrum of a polystyrene film on the same instrument to compare it with that recorded on the reference spectrum. The greatest variations through differences in resolving power are likely to occur in the region between 4000 and 2000 cm−1 (British Pharmacopoeia Commission 2002, p. A129).
When a chemical reference substance is available, the substance being examined and the chemical reference substance should be prepared by the same procedure before recording the spectra (see later under Polymorphism). The transmission minima in the spectrum obtained with the substance being examined should correspond in position and relative size to those in the spectrum obtained with the reference substance (British Pharmacopoeia Commission 2002, p. A128).
In recent years, IR spectral databases have been created and stored electronically in databases and/or libraries. The spectrum of the analyte is presented to the database and the computer attempts to match the spectrum with one already held in the database. A report is made of the best matches. The computer program lists the most likely hits in order of a closeness of fit. Many spectra compilations (databases) are private collections, held typically by individual pharmaceutical companies; some can be purchased and a few are in the public domain.
The number of compounds for which IR spectra have been measured is now massive. Potentially, the greater the number of spectra in a database, the greater is the probability of making a good match for the unknown sample. However, the probability of making a mismatch is also greater, as more spectra with fine differences are available for comparison. The computer is simply matching ‘pictures’ by the number of peaks, their positions and their relative intensities. The best the computer fitting can do is to indicate a mathematical similarity. It is important to qualify a computer search:
- A visual overlay of the test compound spectrum and the hit spectrum ensures that the search has not chosen a match that is mathematically acceptable, but chemically not acceptable.
- Knowledge of the class of a compound can help restrict the search to a more refined reference set (database).
- Other properties of the sample and the reference compound should match, such as chromatographic retention times, chemical and colour reactions and functional group assignments.
- The computer can only select spectra that are in its library and if the spectrum of the compound under investigation is absent, then it will select those that give the next–best fit.
- Different forms of the same compound give different IR spectra (different polymorphs, racemate and/or enantiomer, ionisation status, cations and anions).
- If the spectra have been recorded on different instruments, they may, superficially at least, appear very different. In this case a more detailed study of band frequencies and relative intensities must be undertaken.
If the matching procedure fails, and in cases where the type of compound is unknown or can only be allocated to a certain class (e.g. a phenothiazine or a barbiturate), reference may be made to the index of IR peaks in Part 3 of this book and to the information in the individual monographs. Comparison of the spectrum of the unknown with that of the suspected compound should either confirm or disprove the tentative identification. If the two spectra were recorded under similar conditions on the same type of instrument, they should be very similar in appearance. Some examples of the identification of drugs are given below.
Infra–red spectra of amfetamines
The IR spectra of amfetamine base and the hydrochloride have many similarities, but the hydrochloride spectrum shows much finer detail. The IR spectra of the hydrochloride and mandelate salts show differences because of the absorption of the mandelic acid. However, the spectra of the hydrochloride and sulfate salts are very similar since they both have inorganic anions. The only major difference is the absorption band caused by the sulfate at 1110 cm−1.
Infra–red spectra of barbiturates
Important derivatives of malonylurea (barbituric acid) have two substituents at position 5. Others are also substituted at position 1 and in others the oxygen atom attached to position 2 is replaced by sulfur to form thiobarbiturates.
The barbiturates can be classified chemically into three classes: 5,5–disubstituted barbituric acids, 1,5,5–trisubstituted barbituric acids and 5,5–disubstituted thiobarbituric acids. These classes can be further divided depending on whether the substituents in position 5 are alkyl, alkenyl, aryl or cycloalkenyl. In most common barbiturates, one of the 5–substituents is either ethyl or allyl and the other is either a straight- or branched–chain alkyl or alkenyl group with five or fewer carbon atoms. Some barbiturates are available as sodium salts. The IR spectrum of a barbiturate therefore depends on the class of compound, the nature of the substituents and whether it is the free acid or the sodium salt.
With the exception of phenobarbital and barbituric acid, the free barbiturates do not absorb appreciably above 3300 cm−1 (e.g. barbital), a feature that distinguishes them from the ureides; a weak band of unknown origin sometimes occurs between 3500 and 3400 cm−1. All the barbiturates have two bands, which occur near 3200 and 3100 cm−1 and are caused by N–H stretching vibrations. In the 5,5–disubstituted compounds, the relative intensities of the two bands are similar, although that at 3100 cm−1 is usually slightly less intense. In compounds substituted on the nitrogen atom at position 1, the intensity of the band at 3100 cm−1 may be greatly reduced and is often present only as a shoulder on the band at 3200 cm−1, e.g. metharbital. Methylphenobarbital appears to be an exception in that the band at 3100 cm−1 is the most intense one in the region. A similar phenomenon occurs with the sodium salts, since here again one of the hydrogen atoms in either position 1 or 3 has been replaced.
A series of up to four medium–to–intense bands occurs in the region 3000 to 2800 cm−1, and is caused by alkyl C–H stretching vibrations of the substituents in positions 1 and 5. The intensity of the bands gives a very approximate indication of the number of C–H bonds and hence the number of carbon atoms in the chain. This does not appear to apply to the sodium salts, in which the band that occurs at 3000 to 2950 cm−1 is usually increased in intensity, compared to that of the free acid, and becomes the strongest band. Compare, for example, the spectra of barbital and barbital sodium.
The barbiturates have up to three strong bands in the region 1765 to 1670 cm−1, which result from C=O stretching vibrations. Knowledge of the origin of these bands helps to understand the differences in the spectra of the various types of barbiturate.
In symmetrical molecules, the three bands are all of similar intensity. In asymmetrical molecules, the band at the highest frequency is often less intense than the other two, particularly so when the molecule is substituted in position 1. The sodium salts of the barbiturates have only two bands in this region, since the molecule is no longer symmetrical, and these occur at a lower frequency, between 1700 and 1650 cm−1. In addition, a broad strong band occurs between 1600 and 1550 cm−1; the free barbiturates show practically no absorption in this region. The sodium salts of the thiobarbiturates exhibit only the lowest of the three C=O vibrations in the region 1700 to 1680 cm−1. They do, however, exhibit the broad, strong band that occurs between 1650 and 1600 cm−1. Therefore, the number, position and intensity of the bands between 1800 and 1500 cm−1 give a very good indication of whether the barbiturate is the free acid, the salt or a thiobarbiturate.
Most barbiturates have a number of strong bands between 1460 and 1250 cm−1, and some of these result from C–H deformation and C–N stretching vibrations. The sodium salts of the thiobarbiturates have a broad strong band between 1500 and 1480 cm−1, which is believed to be caused by C–N stretching vibrations of the carbon atom attached to sulfur. This band is not present in the ordinary barbiturates and therefore provides another way to distinguish those that contain sulfur. Many barbiturates exhibit a few weak–to–medium intensity bands in the region 1150 to 900 cm−1. The 1–substituted barbiturates exhibit a greater number of sharp bands of medium intensity. Those compounds that contain an allyl group exhibit bands at about 1000 to 960 cm−1, which probably result from C–H deformation vibrations. The sodium salts of the thiobarbiturates show a band of medium intensity between 1020 and 1000 cm−1. Finally, many barbiturates, but not the thiobarbiturates, exhibit a broad band of medium–to–strong intensity between 900 and 800 cm−1.
Infra–red spectra of aspirin, Nujol and paracetamol
The spectra of aspirin, Nujol and paracetamol, which illustrates the differentiation of N–H, O–H, ester, carboxylic acid and amide groups. In particular, the effect of Nujol on the drug spectra is apparent.