Page 378 spectrometry due to their simplicity, freedom from interferences and excellent precision. Fluorimetry is used much less extensively than absorptiometry because of the limited number of naturally fluorescing species, although its range of applications can be extended by forming fluorescent derivatives or complexes of non-fluorescent analytes. Quantitative applications predominate because, like absorption spectra, fluorescence spectra consist of only a few broad bands. It is inherently more selective and up to three orders of magnitude more sensitive than absorptiometry, detection limits extending down to ppb levels. This has made it particularly valuable for the determination of trace contaminants in foodstuffs and pharmaceuticals and for the determination of fluorescent substances in clinical and forensic samples. The range of possible analytes includes non-transition metals which form fluorescent neutral chelate complexes (charged complexes and those of transition metals do not fluoresce due to rapid relaxation by internal conversion and intersystem crossing), polynuclear aromatic hydrocarbons (PAH), lysergic acid diethylamide (LSD), penicillin, chlorophyll and other plant pigments, numerous alkaloids, steroids, some vitamins, amino acids, proteins and enzymes. Some examples are given in Table 9.5. 92— Infrared Spectrometry Summary Principles Absorption of electromagnetic radiation in the infrared region of the spectrum resulting in changes in the vibrational energy of molecules. Instrumentation Fourier transform spectrometer or double-beam spectrophotometer incorporating prism or grating monochromator, thermal or photon detector, alkali halide cells Applications Very widespread use, largely for the identification and structural analysis of organic materials; useful for quantitative analysis but less widely used than UV and visible spectrometry. Near infrared region used increasingly for industrial quality control, Disadvantages Difficult to analyse mixtures. Special cells required for aqueous samples. Absorption of radiation in the infrared region of the electromagnetic spectrum results in changes in the vibrational energy of molecules. Energy changes are typically 6 x 10° to 42 x 10°J mol, which corresponds to 250—Page 379 4000 cm! (2.540 um), although some occur between 4000 cm! and the beginning of the visible region at about 12500 cm- (0.8 um or 800 nm) in a region known as the near infrared (NIR). A molecule can absorb energy only if there is a net change in the dipole moment during a particular vibration, a condition fulfilled by virtually all polyatomic molecules. The absorption spectra of such molecules are often very complex and the underlying reason for this complexity is best understood by first considering the spectrum of a heteronuclear diatomic molecule in the gaseous state. Diatomic Molecules The classical analogy of a vibrating diatomic molecule is that of two weights connected by a spring. The potential energy of such a vibrating system is related to the displacement of the weights relative to each other along the axis of the spring. If the motion is simple harmonic the relation is given by E = 1/2 fe? where E denotes potential energy, x the displacement and f the force constant or stiffness of the spring. The equation describes a parabola, Figure 9.12, and shows that identical changes in potential energy occur on stretching or compressing the spring. A diatomic molecule behaves rather differently in that the forces of internuclear repulsion arising when the bond is compressed build up rapidly whilst, on stretching, the bond weakens and may finally disrupt. The resulting vibrational motion is anharmonic in nature and the mathematical relation between potential energy and displacement is necessarily more complex. The corresponding curve is modified in the manner shown in Figure 9.12. J simple hermonie anharmonic energy > Tniernudlarseparaion—> Figure 9.12 Vibrational energy levels of a diatomic molecule The concept of discrete or quantized energy levels can be superimposed on this diagram by representing them as a series of horizontal lines, the spacing of which becomes closer with increasing energy due to the anharmonic nature of the vibration. In quantum mechanical terms, these levels are labelled V = 0,1,2,3 ..., where Vis the vibrational quantum number, and the general mathematical expression for the potential energy of the system expressed in wavenumbers is given byPage 380 = (V+¥Poyen™ (9.16) ©, denoting the equilibrium vibration frequency and x, the anharmonicity constant. This expression may be used to predict the appearance of the absorption spectrum of a diatomic molecule. At room temperature most molecules are in the ground vibrational state (V’— 0) so that only transitions from this level need be considered. The spectroscopic selection rule allows any change in the value of V, so for transitions originating in the ground state, Aeon = noel — (n+ 1}y,Jem™ (9.17) where n may be 1,2,3 Substitution of appropriate values of m into the equation leads to a set of expressions giving the wavenumber of each transition, e.g. the first three and their designations are Gipoe = Aeont = Fe (1-2) fundamental Gipce = Aty2 = 20, (1-346) first overtone A&o_3 = 30, (1—4y.) second overtone Spc where G,,., is the wavenumber of the spectroscopic absorption. The transition probability falls off rapidly with increasing m, and the spectrum therefore consists of two or three lines of diminishing intensity, each one being separated from the next by rather less than the value of 6,, Although basically a simple spectrum simultaneous rotational transitions produce a pattern of fine structure lines on either side of the vibrational transition. Rotational fine structure is not usually resolved for samples run in the liquid state or in solution. This is due to collisional broadening and the resulting spectrum would have the appearance shown in Figure 9.13. For a given molecule, and using the classical analogy considered earlier, the positions of the fundamental and hence the overtones, are determined by the atomic masses m, and m,, and the stiffness of the bond. These are related by the equation (0.18) t i & z fet 3 ovetone ond avenue Figure 9.13 Infrared spectrum of a diatomic ‘molecule in solution.Page 381 where fis the force constant of the bond and the reduced mass, given by (m,m,)i(m, + m,) It is important to note that the stiffer the bond or the lighter the atoms, the higher is the wavenumber of the fundamental and vice versa. Qualitative conclusions can therefore be reached from the relative positions of absorption bands in the infrared region (vide infra). Polyatomic Molecules For polyatomic molecules, whose spectra are usually recorded as liquids, solids or in solution, the complexity of the spectrum increases rapidly with the number of atoms JV in the molecule. The number of fundamentals, or normal modes of vibration is given by 3N - 6 and 3 — 5 for bent and linear molecules respectively. A normal mode of vibration is defined as the movement of all the atoms of the molecule in phase. In the case of polyatomies, the number of fundamentals includes both stretching and bending vibrations, the latter involving changes of bond angle. There are associated overtones with each fundamental and, in addition, other absorption bands may arise due to interactions between certain of the fundamentals and overtones. These are known as combination and Fermi resonance bands. The overall appearance of the spectrum is that of a large number of resolved and partially resolved bands of varying intensity (Figures 9.17 to 9.24). The most intense bands are fundamentals in which there is a large change of dipole moment during the vibration. Conversely, a band may be weak or absent if the dipole change is small or zero. Many bands can be assigned to the vibration of particular chemical groups within a molecule, the wavenumber being largely independent of the rest of the structure. Such characteristic vibration frequencies are of paramount importance in obtaining structural information and in the identification of unknown compounds. For the most part itis in the analysis of organic and organometallic compounds that infrared spectrometry has had its greatest impact. Characteristic Vibration Frequencies The occurrence of characteristic vibration or group frequencies can be explained in terms of relative masses and of force constants using the classical analogy of weights and springs as depicted in Figure 9.14, @— Mass Effect Ifthe masses 4, B, C, D.. . are all similar and the force constants f, f;,f,... are of the same magnitude, the vibrations of individual atoms are strongly coupled with the result that no band can be assigned solely to any particular group of atoms. If, however, the mass of atom A is considerably smaller than those of the other atoms, one mode of vibration will involve the stretching of the bond between A and the rest of the molecule. The systemPage 382 fe Suge pp fo ‘ pedi idan fh Figure 9.14 Classical anslogy of polyatomic molecule, can be considered as approximating to a diatomic molecule A-X, and the wavenumber of the vibration is then given by apegelll eam Hence, to a good approximation 1 (fax) ov-x = pie (238) and the wavenumber of the vibration is therefore independent of the masses of the other atoms in the molecule. This type of stretching vibration is exemplified by hydrogen atoms linked to carbon, oxygen, nitrogen or sulphur, and results in absorption bands around 3000 cm-!, The wavenumber ranges of a number of such groups are given in Table 9.6(a). Also shown in the table are the wavenumbers of the bending modes, and it should be noted that the energies involved, and hence the wavenumbers, are significantly lower than those of the corresponding stretching vibrations. O— Force Constant Effect Considering the same model as that shown in Figure 9.14, if the force constant of the AB bond is significantly higher than those for the rest of the molecule, one stretching vibration will oceur at a higher wavenumber as 6, is directly proportional to f, 4. This situation arises in molecules with unsaturated groups such as C ‘Cand C—O because the magnitude of the force constant increases significantly with bond order. Examples of the wavenumber ranges for some unsaturated and saturated groups are compared in Table 9.6(b). Differences between the force constants of singlePage 383 ‘Table 9.6 Wavenumbers of characteristic vibrations (@) XH Vibrations XH Streteh/om™ Bend/em™ ca 2700-3300 1200-1500 600-900 on 3000-3700 1200-1500 NOH 3000-3700 1500-1700 SoH 2550-2600 700-900 (b) Saturated and unsaturated groups Stretehiem™! 400-1300 1550-1900 2100-2400 bonds are not sufficiently great to enable characteristic group frequencies to be identified. Prominent ‘bands can be observed, however, for groups such as C—O where the dipole change during the vibration is large. For groups with multiple bonds the change in dipole and hence the intensity of the band is determined by the position of the group in the molecule. Thus little or no absorption due to the multiple bond is observed for symmetrically substituted alkenes or alkynes, whereas a terminal unsaturated group gives rise to a strong absorption, Factors Affecting Group Frequencies Hydrogen Bonding Compounds containing proton donor groups such as O—H and N—H can be involved in intra- or intermolecular hydrogen bonding in the presence of proton acceptors, e.g. O,N, halogens, CC. The stiffiness of the X—H bond is thereby lessened, resulting in a lowering of the stretching frequency, and the band broadens and often intensifies. Conversely, the frequency of the bending mode is raised but the effect is much less pronounced. /ntermolecular hydrogen bonding is suppressed at elevated temperatures but itis favoured by 2 high solute concentration. Intramolecular rydrogen bonding is also reduced at elevated temperatures but it is unaffected by changes in solute concentration. These effects are particularly significant in the spectra of alcohols, phenols, carboxylic acids and amines. Adjacent Groups The wealth of structural information that can be obtained from infrared spectra arises largely from differences in band position, shape and intensityPage 384 that are observed when comparing similar but not identical compounds. Predictable shifts occur on altering the substituents or structure adjacent to a group giving rise to a particular absorption. These can be explained in terms of an inductive effect, a mesomeric or resonance effect and steric factors such as ring strain. The effects are typified by the carbonyl group, whose stretching frequeney occurs within the range 1650-1850 cm, Shifts are usually related to the position of the carbonyl band in pure acetone (1715 cm-"). The proximity of electron withdrawing substituents causes an inductive withdrawal of electron density from around the oxygen atom thereby shortening the bond (Figure 9.15). This increases the force constant and hence the wavenumber of the vibration (Table 9.7). If the adjacent group contains a weakly electronegative atom with lone-pair electrons or electrons occupying m orbitals, resonance interaction with electrons in the carbonyl group leads to a reduction in bond order (Figure 9.15). The correspondingly smaller force constant results in a lower value for the wavenumber of the vibration, (Table 9.7). Steric hindrance may inhibit the resonance effect by reducing the coplanarity of a conjugated system whilst carbonyl groups which form part of a strained ring system absorb at high wavenumbers because of the constraint imposed upon the carbon atom (Table 9.7). x / é Hy © Figure 9.15 Effect of substituents of the carbonyl bond. (a) Inductive effect, X = H, OH, OR, halogen, ce effect, Y = NH,, NHR, NR, phenyl. (b) Resona ‘Table 9.7 Effect of substituents on the wavenutmber of the carbonyl stretching vibration ‘Compound C=0 Stretehiem~! saturated ketones 1710-1720 carboxylic acids, monomer 1750-1770 carboxylic acids, dimer 1710-1720 saturated esters 1728-1745 acid chlorides 1790-1815 aliphatic amides 1630-1700 aromatic ketones 1680-1700 aromatic and a, ounsaturated esters 1715-1730 ‘lactones 1760-1780Page 385 Coupled Vibrations Groups in which two or more atoms of an element are bonded to a common atom are mechanically coupled resulting in a band which is generally resolved into two components. These correspond to symmetric and asymmetric modes of vibration. They also execute a variety of bending vibrations each of which may be observed at a characteristic wavenumber. Examples include CH,, CH,, NH,, NO, and COO-. The stretching and bending modes of methylene group are illustrated in Figure 9.16. It should be noted that asymmetric vibrations occur at a higher wavenumber than symmetric and that the absorption is more intense. Another form of vibrational coupling occurs between the C—H groups in both aromatic and alkene compounds resulting in bands which are characteristic of the substitution pattern. In the case of out-of-plane bending, they are of a high intensity and are especially useful for diagnostic purposes (vide infra). symmetric asymmetric ad =n ae ay x y¥ xX ¥ inplanebending modes ‘out-of plane tending modes Figure 9.16 Stretching and bending modes of a methylene group, Qualitative Analysis - The Identification of Structural Features The value of infrared spectrometry as a means of identification of unknown compounds and to investigate structural features is immense. Spectra are used in an empirical manner by comparison of samples with known materials and by reference to charts of group frequencies. A simplified correlation chart is shown in Table 9.8. The interpretation of infrared spectra is best considered by discussing the prominent features of a representative series of compounds. The spectrum of a saturated hydrocarbon, n-decane is shown in Figure 9.17. Four prominent bands arise from C—H stretching and bending vibrations: asymmetric and symmetric C—H stretching (~ 2900 cm~ °), CH, bending and asymmetric CH, bending (~ 1460 cm~!), symmetric CH, bending (~ 1380 em-}) and rocking of all the CH, groups in the chain (~ 720 cm~). The position of the latter depends upon chain length and is not observed if there are fewer than four methylene groups together. The CH stretching bands may be further resolved into components due toPage 386 ‘Table 9.8 Simplified infrared spectral correlation CH a a LKANES| soaps lacrenes| = | ‘S] YEH raf cr a | = PAE neapeeagytigioN VES eran oa aS poets Hester fear evens] oo im lLacTones ibRes Reo 1 Poe coma E Jasion ct vee) ven Sh siete (table continued on next page)Page 387 Contd. ‘Table 9.8 [ecuoes (ares rooms (souretes) IMINES CH,N&O Sh a0 os ra tines ef neon eco Sars] e we rete Pa existe iL pane harearesPage 388 “| | | & 0 4009 350039002500 2000 1800 1e00 1400 1290 1000 BO «25 ‘wavenumber (om!) Figure 9.17 Infrared spectrum of n-decane. CH, and CH, groups. It should be noted that no bands are observed above 3000 cm~, indicating the absence of an aromatic, alkene or alkyne structure. Contrast this spectrum with those of 1,2-dimethylbenzene (Figure 9.18) and 1-octene (Figure 9.19). In both spectra, additional CH stretching vibrations appear above 3000 m-!, and characteristic C—H out-of-plane bending vibrations are seen between 650 and 1000 em-t, The bands at 910 and 990 errr! in the spectrum of I-octene signify the presence of a vinyl group. This is confirmed by a sharp band at 1640 cm-* due to stretching of the alkene double bond. The spectrum of 1,2-dimethylbenzene (0- xylene) is more complex and includes skeletal vibrations of the ring and C—H in-plane bending between 1000 and 1600 em, together with weak overtone bands in the region between 1600 and 2000 em, The presence of 1,2-disubstitution is apparent from the single strong band at 740 em~! due to C-H out-of-plane bending Compare, also, the spectrum of n-decane with those of n-propyl alcohol, (Figure 9.20) and n-undecanal (Figure 9.21). The spectrum of the alcohol is characterized by vibrations due to the O—H and C—O groups. The O—H Notes to Table 9.8 The charts are arranged in order of increasing element content, Corzelations given in one chart (e.g. those for CH, and CH.) are not repeated in subsequent charts, The frequency limits within which the band of a particular grouping is usually found are indicated by the black strips and extensions of the range to include unusual examples are shown as thin lines, eg, relative intensities are given in a very approximate fashion (see below), Both the position and the intensity of some absorptions are dependent on state, solvent, elc., and the actual frequency quoted is that most commonly observed. Abbreviations = stretch (Vis also used for quoting an absorption frequency, ¢.g. V,., 1700 em); 6 = in-plane bend; y= out-of-plane bend; p = rock; = wag; « = owist; sym = symmetrical; antisym = antisymmetrically (asymmetrie); var. = variable (in frequency); br ~ broad; w = weak (in absorption); m = medium; s~ strong; v.w. = very Weak; vs ~ very strong; w—m ‘weak to medium (ie. typical intensity range for that particular band) Het a = em atePage 389 wavenumber (en! 1:00 i i ° Sr ‘oxyiene (thin firm) wavelength ym Figure 9.18 Infrared spectrum of 1,2-dimethylbenzene, stretching band at ~ 3350 cm-' is broadened by hydrogen bonding whilst the strong band at ~ 1070 cm-! is due to a complex C—O stretching vibration. In the aldehyde a strong band assigned to stretching of the carbonyl group appears at ~ 1730 em and a sharp peak due to stretching of the aldehydic C—H (~ 2720 em) serves to differentiate it from other types of carbonyl compound. The spectrum of a carboxylic acid can be compared with that of an ester in Figures 9.22 and 9.23. Such acids exist primarily as dimers, and the strongly hydrogen bonded structures give rise to a very broad O—H stretching band between 2500 and 3500 em and another at around 940 em” due to O—H out- of-plane bending. An intense carbonyl stretching band occurs near 1710 em~, similar to that of an aliphatic ketone because of hydrogen bonding, and the C—O stretch appears as a broad absorption between 1200 and 1300 em~!. The ester can be distinguished by the absence of O—H bands around 3000 and 940 cm" and the shift of the carbonyl band to ~ 1740 cm”! caused by replacement of the acidic proton with an alkyl group. (N.B. carbonyl absorption in a monomer acid occurs at ~ 1760 cnr.) Furthermore the C—O stretching band wavenumber fem“*) 1500 (20) 100) $00 o_o, sw Berni ‘0 Sw £ a 3 40} i cH ict cc Ba ol wbuirles aaa anata vaveleret an Figure 919 Infrared spectrum of 1 -octene.Page 390 109 aa] | Md a 20 a 000 3000 Ton Tae OS TOO svaxenvmber (em) Figure 9.20 Infrared spectrum of n-propyl alcohol. wavenumber fem") som 2000500 oat too 700 7 Teepe MT eT or par 1 ao CA, (CH2)-CK 4 ao 4 oll DL abesusassalsal rr a ‘decanal(thin fim) wavelength, yum Figure 9.21 Infrared spectrum of n-undecanal, ‘wavenumber (er!) 1 » a) a | ° rr a a nonanok acd hi liuidilm) —_vavelergth ym Figure 9.22 Infrared spectrum of n-nonanoie acid. between 1200 and 1300 cm-' is considerably intensified. It should be noted that in the spectra of acids the CH stretching band is frequently observed as a sharp peak above the broad O—H band, The final spectrum (Figure 9.24) is that of p-nitroaniline. Although complex it is immediately recognizable as being aromatic (C-H abovePage 391 4008 300 2000 18001400025 vwavenember jem") Figure 9.23 Infrared spectrum of n-propyl acctate 3000 cm-!), skeletal and overtone bands between 1400 and 2000 em! and a C—H out-of-plane bending vibrations at 835 cm-! indicating p-substitution). Asymmetric and symmetric N—O stretching bands are very prominent at 1475 and 1310 cnr, whilst evidence for an amine group is given by the doublet NH stretching band near 3400 cm-! and the in-plane N—H bending band near 1600 cm-. A strong C—N stretching vibration overlaps the symmetric N~O band near 1300 cm-!, Some further examples of infrared spectra are given in the section on the combined use of spectral data. pritroasilite O.NCH.NH, sw. (3813,mp. 15-15" ‘avenumber em“* (0 00250 200 200010100 fa fd 8 a a ay) Ql 23 8 ST OW DS ta 1S TO wavelength, am Figure 9.24 Infrared spectrum of p-niteoaniline Quantitative Analysis Absorption in the infrared region of the spectrum can be expected to obey the Beer-Lambert law (p. 357) for bands which are well resolved and in the absence of chemical effects such as hydrogen bonding. It is important that the slit width of the instrument is kept constant for a series of measurements as band intensities are highly sensitive to changes. This is because slit widths used in infrared spectrometry are of the same order of magnitude as bandwidths. A commonly used method for establishing the blank or background absorbance is to record the spectrum in the vicinity of the band of interestPage 392 and to construct a baseline (Figure 9.25) for all standards and samples. The baseline absorbance is, measured at the point where a perpendicular line from the peak maximum intersects the baseline and this absorbance is subtracted from the peak absorbance in all cases Figure 9.25 Construction of a base! quantitative analysis. e for ‘Most cells used in infrared spectrometry have sodium chloride windows and the path length is likely to vary with use because of corrosion. For quantitative work, therefore, the same cell should be used for samples and standards. In general, quantitative analysis in the infrared region of the spectrum is not practised as widely as in the ultraviolet and visible regions, partly because of the additional care necessary to obtain reliable results and partly because the technique is generally considered to be less sensitive and less precise; a precision of 3-8% can be expected. Sampling Procedures Solid samples can be handled in a number of ways. A mull or paste is made by grinding a few milligrams of sample in an agate mortar with a drop of a liquid paraffin (e.g. Nujol) or hexachlorobutadiene. The paste is formed into a thin film by compressing it between sodium chloride or potassium bromide plates which are mounted in a suitable sample holder. A complete spectrum of the sample can be obtained from separate mulls in Nujol and hexachlorobutadiene. The C—H bands in the former obscure the sample bands in that region but these can be observed in the spectrum of the chlorinated butadiene mull. An alternative procedure is to grind a few milligrams of sample with about 200 mg of dried potassium bromide and to compress the mixture into a thin, homogeneous disk using a hydraulic press. Liquid samples can be examined neat as a thin film between sodium chloride or potassium bromide plates or as a solution in a cell of path-length 0.110 mm. Three solvents in widespread use, and which are largely transparent in the infrared region, are carbon tetrachloride, chloroform and carbon disulphide. Spectra of the surface of solids and liquid samples can also be obtained either by attenuated total reflectance (ATR) or by diffuse reflectance. The former involves placing the sample in contact with thePage 393 sides of an optically flat thin prismatic crystal of thallium bromide/iodide (KRS-5) through which radiation from the source is passed. By directing the beam into one end of the crystal at the correct, angle of incidence it can be made to undergo multiple internal reflections before emerging from the other end and passing through the spectrometer to the detector. At cach reflection, the beam penetrates the surface of the sample to a depth of a few micrometres. The intensity of the beam is attenuated according to the absorption characteristics of the sample so enabling an absorption spectrum to be recorded. Diffuse reflectance spectra are obtained using a specially designed curved or plain mirror system to collect the radiation diffusely reflected from the surface of a sample over a wide solid angle and focusing it onto the detector. It is particularly suitable for powdered solid samples, which are simply placed in a small cup, and for liquids or gases adsorbed onto the surface of a non-absorbing powdered substrate such as alkali halide. As little as a few micrograms of material can give an acceptable spectrum. Infrared microscopy combines an optical microscope with an FT-IR spectrometer enabling pico- to femtogram (10-—10-# g) quantities of substances to be characterized or very small areas of larger samples to be analysed. Beam-condensing optics focus the radiation onto an area of the sample identified using the optical microscope and either reflectance or transmittance spectra can be recorded. The highly-sensitive MCT detector (p. 283) is normally used as its size can be matched to that of the radiation beam to maximize its response. Gaseous samples require long path length cells to produce absorption bands of reasonable intensity; up to several metres of optical path are obtainable from cells incorporating mirrors which produce multiple reflections. For GC-IR, light pipes provide the best sensitivity (p. 117). Near Infrared Spectrometry Overtones and combination bands from CH, OH and NH vibrations are found in the near infrared region (NIR) between 4000 cm~ and about 12500 em (2500 nm to about 800 nm). Although these are weak bands (low molar absorptivities), sensitivity can be enhanced by the use of more powerful radiation sources and more sensitive detectors. NIR spectra are generally lacking in prominent features, consisting largely of broad overlapping bands of varying intensities that are strongly affected by H- bonding. Some sharper bands may also appear, particularly in the region between 4000 and 6000 cm-! (2500-1667 nm) (Figure 9.26(a) and (b)). Band positions, profiles and intensities are highly dependent on the physical characteristics of the sample, especially particle size and moisture content. The comparison of spectra for identification purposes and calibration procedures for quantitative analysis, are both more complicated than in the mid-infrared region, being largely empirical in approach However, the development of Fourier transform (FT) instruments (pp. 281,Page 394 © Absorbance om 109 ‘000 a500 ego Tos aw eto se SO SOY «SBD » 7500 Figure 9.26 (a) Typical NIR spectrum of ground whest. (b) NIR spectra of a chiral pharmaceutical compound. 282, 356) and chemometric software packages (p. 525) enable data to be collected and processed in seconds. The basis of the complex computations required is multivariate statistics, a branch of chemometries employing such mathematical and statistical techniques as multiple linear regression (MLR) principal component analysis (PCA) and partial least squares (PLS) regression. However, a description of these techniques is beyond the scopePage 395 of this text. Unknowns can be identified by matching their spectra with stored library spectra, by a process of pattern recognition. Quantitative analysis at a single wavelength is not normally possible using the Beer—Lambert law (p. 357) as almost all NIR spectra are recorded in diffuse reflectance rather than transmittance mode. Diffuse reflectance is a complex phenomenon that is highly dependent on the physical and chemical characteristics of the sample and does not have a simple relation with sample concentration. For calibration purposes, it is necessary to record the reflectance spectra of a series of standards of known composition over a particular wavelength (wavenumber) range. Reflected intensities measured at multiple wavelengths selected by the software are used in conjunction with concentrations to generate sets of equations. Empirical coefficients evaluated from these equations are then used to calculate the concentrations of specified sample components after recording reflected intensities from the samples at the same wavelengths. In effect, a mathematical model of the reflectance spectrum of each standard is computed. These models are then combined and refined by a learning process so as to generate the best set of calibration data to quantify samples run under the same conditions. Detection limits are of the order of 1%, although levels down to around 0.1% have been reported. Applications of Infrared Spectrometry The unique appearance of an infrared spectrum has resulted in the extensive use of infrared spectrometry to characterize such materials as natural products, polymers, detergents, lubricants, fats and resins, Itis of particular value to the petroleum and polymer industries, to drug manufacturers and to producers of organic chemicals. Quantitative applications include the quality control of additives in fuel and lubricant blends and to assess the extent of chemical changes in various products due to ageing and use. Non-dispersive infrared analysers are used to monitor gas streams in industrial processes and atmospheric pollution. The instruments are generally portable and robust, consisting only of a radiation source, reference and sample cells and a detector filled with the gas which is to be monitored The original applications of NIR were in the food and agricultural industries where the routine determination of the moisture content of foodstuffs, the protein content of grain and the fat content of edible oils and meats at the 1% level and above are typical examples. The range of industries now using the technique is much wider and includes pharmaceutical, polymer, adhesives and textile companies. The first in particular are employing NIR spectrometry for the quality control of raw materials and intermediates and to check on actives and excipients in formulated products. Figure 9.26(b) demonstrates that even subtle differences between the NIR spectra of enantiomers can be detected Instrumentation incorporating fibre optics are utilized for remote sensing in process streams, and. identity checks on products in glass vials, plastic