Spectroscopy refers to the interaction of component wavelengths or quanta of the electromagnetic spectrum with atoms and molecules. Quanta of energy are absorbed into the atom or molecule and induce excitation of the electrons surrounding the nucleus, normally the outermost electrons involved in forming chemical bonds in the atom or molecule. The excitation can be lost by emitting a photon of the same or lower energy as that absorbed initially, or by transfer into vibrational and rotational energy of the molecular structure, degrading to heat. The different wavelengths of the spectrum absorbed and the manner of detection of their absorbance or re-emission as another quantum define the different varieties of spectroscopy. Spectroscopic measurements are in essence measurements of the intensity and wavelength of radiative energy.
The spectroscopic methods described below are used as analytical chemical methods to probe molecular environments with specific wavelengths of light, often with the aim of measuring concentrations of specific components of a mixture.
Alternative excitation methods include thermal excitation and chemical reactions. The emission of black body radiation from a heated source is well known and analysis of the energy from such a source led to the quantum theory of energy, with energy defined as discrete packages or quanta, rather than as a continuum. Light emission from ceramics by flame heating leads to the glow of a gas mantle and the radiant heat of a gas fire. Electric lights function because the filament of tungsten is heated to emit a white light while fluorescent tubes operate by applying a high voltage to an inert gas such as neon. Temperature measurement by optical pyrometry depends on the emission of light from hot bodies. Chemical reactions giving rise to light emission include those used by glowworms and fireflies and the phosphorescence of the ocean observed at night.
Some analytical methods are termed spectroscopy although they are resonance methods involving absorption of energy. These include Mossbauer, electron and nuclear magnetic resonance spectroscopy. Mass and X-ray spectroscopies involve ionization of molecules and atoms. These are defined after those methods, which investigate molecular interactions with the electromagnetic spectrum.
The Electromagnetic Spectrum ranges from X-rays with a wavelength of 10−10 m, through vacuum ultraviolet, (10−8 m), ultraviolet (4 × 10−7 m), visible light (4.2 to 7.5 × 10−7 m), infrared (10−6 to 10−3 m) through microwaves (10−2 to 1 m) to radiowaves (102 to 104 m).
Atomic spectroscopy refers to the interaction between UV and visible light and the electron energy levels of atoms surrounding the nucleus. Electrons can be excited by various methods, such as collisional energy at high temperature, to energy levels higher than those occupied in the ground state and to different orbitals; emission spectra can be observed as the series of wavelengths emitted as the excited electrons move back to lower energy levels. Absorption spectra are observed if the atoms are irradiated with a light source giving a continuous spectrum, when those characteristic wavelengths corresponding to promoting an electron to a higher energy level are absorbed, giving a dark line on the continuum. Herzberg (1944) and Atkins (1986) describe the origins of atomic spectra. The observation of atomic spectra in light emitted from stars led to the understanding of stellar evolution and the formation of the elements 9see Greenwood and Earnshaw (1989), for a detailed discussion].
Molecular spectroscopy covers the interaction of electromagnetic radiation with molecules rather than atoms and normally involves UV and visible wavelengths. For absorbance spectroscopy, the experimental arrangement allows detection of the transmitted energy in the axis of the incoming radiation. For methods such as Fluorescence, Phosphorescence, and Raman spectroscopy, the emitted light is much weaker than the incident light, and emission is observed perpendicular to the incident beam.
UV-absorbance, UV-fluorescence and UV-phosphorescence spectroscopy are included within this group. In its simplest form, UV-absorbance indicates the presence of an absorber of UV light in the effluent from a chromatographic system by the attenuation of a beam of UV light of fixed wavelength. A diode array detector permits the simultaneous observation of the attenuation of many wavelengths as a compound or mixture passes through the detector. UV-absorbance spectroscopy normally involves the measurement of the intensity of the transmitted, attenuated light on passage through the sample or compound in comparison with the intensity of incident light. The absorbance at specific wavelengths is characteristic of the structure of the absorbing molecule, being determined by the energy levels of the outer electrons. Denney and Sinclair (1987) describe the technique.
UV-fluorescence spectroscopy follows from UV-absorbance where the absorbed energy or photon is emitted as a photon of lower energy than that absorbed. Absorbance of photons excites a singlet state electron in the ground vibrational state to an electronically excited state with various vibrational energy levels. Energy loss by collisions transfers electrons to the lowest vibrational level of the excited state. Fluorescence is observed when these electrons fall back to the various vibrational levels of the electronic ground state. The photons emitted as fluorescence are at longer wavelengths (lower energy, towards the visible region) than those absorbed. (See also Fluorescence.)
A routine application of UV-fluorescence is the use of "whiteners" in washing powders; these compounds absorb any available UV light and emit in the blue end of the visible spectrum, enhancing the brightness or "cleanliness". Applications include the quantitative analysis of specific compounds at low abundance in the environment and in clinical studies where a fluorescent tag may be added to tissues to reveal the onset of disease [Rendell (1987)].
UV-Phosphorescence spectroscopy is similar in mechanism to UV-fluorescence except that the energy of the singlet excited state transfers by intersystem crossing to a triplet state, of lower energy than the singlet excited state, and subsequently emits photons as the electrons fall back to the ground state. The intersystem transfer is a forbidden transfer and occurs on a much longer time scale than fluorescence. Consequently, the emission of light can be separated from the illumination in time by having a rotating shutter to deflect the incident radiation while observing the emitted radiation. The sample can be cooled in liquid nitrogen to remove some of the vibrational and collisional modes of loss of energy and enhance the phosphorescence [Rendell (1987)]. (See also Phosphorescence.)
Many chemical reactions occur with visible color changes, for example, the addition of water to anhydrous copper sulfate produces a blue color, acid-base titrations often use an indicator which exhibits a change of color with a change in pH such as phenolphthalein (colorless to red), litmus (blue to red). The breathalyzer used to monitor ethanol in the expired air from the lungs relies on a color change from yellow to green; for a positive indication, a separate test for ethanol is made on a blood sample using gas chromatography. These color changes accompany changes in the atomic or molecular structure of the colored compound. They are of particular importance in clinical and forensic analysis where rapid or continuous analyses may be required to observe and determine changes in bodily fluids such as urine and blood. Air pollutants such as formaldehyde from car exhausts and combustion sources can be monitored by forming a complex in concentrated sulfuric acid solution which is blue and specific to formaldehyde, enabling its estimation at the parts per million level.
The same principles for obtaining spectra apply as in UV spectroscopy; for the establishment of an analytical method, a wavelength is chosen which is strongly absorbed by the analyte but not absorbed by other components of the mixture. Denney and Sinclair (1987) describe the technique.
This use of spectroscopy developed over the last 30 years or so from an attempt to develop a rapid method for the analysis of moisture in grain. Instruments featuring low resolution and high signal to noise ratios were developed to allow rapid scanning. Current uses of the technique concentrate mainly in the analysis of agricultural samples such as the measurement of protein in wheat. The advantages are simple sample preparation, speed and precision of analysis, and the ability to analyze for several constituents at the same time. The chief disadvantages are the dependence on a calibration procedure and the need for dedicated instrumentation. The technique is described in Creaser and Davies (1988).
Molecular vibrational modes are examined by infrared methods. The radiation requires the use of materials such as potassium bromide for the lenses and optical systems. For a vibrational mode to be infrared active, the motion corresponding to a normal mode should be accompanied by a change of dipole moment. Rotational effects in liquid or solid samples cannot be distinguished, but lead to line broadening of vibrational modes, in effect blurring the rotational structure. In chemical analysis, this blurring leads to a useful simplification since the vibrational spectra give rise to characteristic frequencies of absorption, with intensities transferable between molecules. Vibrational modes can be assigned in an unknown spectrum from a table of values from standard molecules. The advent of IR-microscopy has enabled the examination of small samples without the need to pelletize the sample in KBr [Atkins (1986)].
Raman spectroscopy permits the examination of vibrational and rotational energy levels by light scattering. Photons in a monochromatic light beam collide with the molecules and are scattered with lower or higher energy, in a different direction than the incident beam. The scattered photons give rise to Stokes radiation (lower energy) and anti-Stokes radiation (higher energy). All linear molecules and diatomic molecules, homo- or heteronuclear, are active in rotational Raman. Normal modes of vibration are Raman active if the polarizability changes during the vibration; if the molecule has a center of symmetry, no vibrational modes can be active in both Raman and Infrared [Atkins (1986)].
In X-ray photo-electron spectroscopy, the incident photon, an X-ray, is of sufficient energy to eject an electron from the core of the electron structure of an atom. These inner electrons are not much affected by changes in energy levels of the outer or valence electrons, which reflect the molecular context of the atom. Hence the observed inner shell ionization energies are a property of the atom and characteristic of the elements present in a mixture or alloy. An alternative name is ESCA (electron spectroscopy for chemical analysis). The technique is limited to solid samples and to their surfaces because the emitted electrons can only escape from surface layers [Atkins (1986)].
Nuclei of atoms with spin 1/2 possess magnetic moments, with discrete energy levels when placed in a magnetic field. If the sample is bathed in radiation of an appropriate frequency to come into resonance with the energy separations of the nucleus, then there is a strong coupling between the nuclear spin and the radiation, with absorption of the radiation. NMR is the study of the properties of molecules containing magnetic nuclei by observing the magnetic fields at which they come into resonance with an applied radiofrequency field. The resonance frequency depends on the local electron structure of the atomic environment and reveals chemical interactions; the effect is called the chemical shift of the magnetic resonance. To ensure homogeneity of the magnetic field, the sample is rotated rapidly within the magnetic field. The most commonly studied nuclei are hydrogen, carbon (13C) and phosphorus (31P) although other nuclei can be studied. Solids and liquids are examined as complex mixtures, although only pure compounds were allowed near spectrometers in the early days. Fourier transform techniques have been applied with many different techniques of pulsing the applied fields [Atkins (1986)].
Also termed electron paramagnetic resonance, this method is the study of molecules containing unpaired electrons by observation of the magnetic fields at which they come into resonance with monochromatic radiation. The appropriate radiation is in the X-band microwave region. Free radicals and charge transfer complexes are the type of molecular formations, which can be studied, either stable in mixtures or formed by radiation.
This method uses gamma rays, photons at the extreme end of the electromagnetic spectrum, and depends on the resonant absorption of the photon by the nucleus of the target atom. The decay of 57Co to 57Fe in an excited state occurs slowly, with a half life of 270 days. The excited Fe atom decays rapidly to its ground state, emitting a gamma ray. When this gamma ray strikes another 57Fe atom in the ground state, it is absorbed resonantly if its energy matches the energy difference between the excited state and the ground state of the target Fe atom. When the excited atom emits a photon, the atom recoils because the photon possesses a significant linear momentum, giving a Doppler shift to the photon velocity. If the emitter and target Fe atoms are held in rigid foils, the recoil is taken up by the whole crystal and the Doppler shift is very small. In consequence, the energy of the photon can be modified by moving the source foil at a known velocity over a range of a few mm/sec. The target undergoes resonant absorption and the quadrupole moment of the iron nucleus splits the signal; the change in position of the resonance, called the isomer shift, is a function of the electrostatic interaction with the surrounding electrons, which changes with chemical environment of the iron. The importance of the technique lies in the many biological and technological occurrences of iron, such as plant proteins, blood and rust. 119Sn also can be examined by Mössbauer spectroscopy .
Ionization methods involving photons may be classed as spectroscopy since the interaction with the outer electrons of molecules in the gas phase or solid state involves excitation of an electron to an infinite distance, leaving a positively charged ion (photoionization). In the solid state, the technique is known as laser desorption ionization or matrix assisted laser desorption; the mechanism of ion formation results in the rapid transfer of intact molecule ions into the vapor phase.
Mass spectroscopy is a general term for mass spectroscopy using photoplates for detection of all ion beams (m/z values) simultaneously. However, where ion detection is by the sequential focusing of selected ion beams onto an electron multiplier or similar detector, the technique is normally referred to as mass spectrometry.
Analytical Applications of Spectroscopy, (1988) C. S. Creaser and A. M. C. Davies, Eds., Royal Soc. Chem., London, UK.
Atkins, P. W. (1989) Physical Chemistry, 3rd. edn., Oxford University Press, Oxford, UK.
Denney, R. C. and Sinclair, R. (1987) Visible and ultraviolet spectroscopy, Analytical Chemistry by Open Learning (ACOL), John Wiley and Sons, Chichester, UK.
Greenwood, N. N. and Earnshaw, A. (1984) Chemistry of the Elements, Pergamon Press, Oxford, UK. reprinted 1989.
Herzberg, G. (1944) Atomic Spectra and Atomic Structure, 2nd edn., Dover Publications, New York.
Rendell, D. (1987) Fluorescence and phosphorescence spectroscopy, Analytical Chemistry by Open Learning (ACOL), John Wiley and Sons, Chichester, UK.