Optical spectroscopy

Electrons exist in energy levels within an atom. These levels have well defined energies and electrons moving between them must absorb or emit energy equal to the difference between them. In optical spectroscopy, the energy absorbed to move an electron to a more energetic level and/or the energy emitted as the electron moves to a lower energy level is in the form of a photon (a particle of light). Because this energy is well-defined, an atom's identity can be found by the energy of this transition. The wavelength of light can be related to its energy. It is usually easier to measure the wavelength of light than to directly measure its energy. Optical spectroscopy can be further divided into absorption, emission, and fluorescence.

Atomic absorption spectroscopy

Atomic absorption spectrometer In analytical chemistry, atomic absorption spectroscopy is a technique for determining the concentration of a particular metal element in a sample. The technique can be used to analyze the concentration of over 70 different metals in a solution. Principles The technique makes use of absorption spectrometry to assess the concentration of an analyte in a sample. It relies therefore heavily on Beer-Lambert law. In short, the electrons of the atoms in the atomizer can be promoted to higher orbitals for a short amount of time by absorbing a set quantity of energy (i.e. light of a given wavelength). This amount of energy (or wavelength) is specific to a particular electron transition in a particular element, and in general, each wavelength corresponds to only one element. This gives the technique its elemental selectivity. As the quantity of energy (the power) put into the flame is known, and the quantity remaining at the other side (at the detector) can be measured, it is possible, from Beer-Lambert law, to calculate how many of these transitions took place, and thus get a signal that is proportional to the concentration of the element being measured.

Instrumentation

Atomic absorption spectrometer block diagram In order to analyze a sample for its atomic constituents, it has to be atomized. The sample should then be illuminated by light. The light transmitted is finally measured by a detector. In order to reduce the effect of emission from the atomizer (e.g. the black body radiation) or the environment, a spectrometer is normally used between the atomizer and the detector. Types of Atomizer The technique typically makes use of a flame to atomize the sample,[3] but other atomizers such as a graphite furnace[4] or plasmas, primarily inductively coupled plasmas, are also used.[5] When a flame is used it is laterally long (usually 10 cm) and not deep. The height of the flame above the burner head can be controlled by adjusting the flow of the fuel mixture. A beam of light passes through this flame at its longest axis (the lateral axis) and hits a detector. Analysis of liquids A liquid sample is normally turned into an atomic gas in three steps: 1. Desolvation (Drying) the liquid solvent is evaporated, and the dry sample remains 2. Vaporization (Ashing) the solid sample vaporises to a gas 3. Atomization the compounds making up the sample are broken into free atoms. Radiation Sources The radiation source chosen has a spectral width narrower than that of the atomic transitions. Hollow cathode lamps Hollow cathode lamps are the most common radiation source in atomic absorption spectroscopy. Inside the lamp, filled with argon or neon gas, is a cylindrical metal cathode containing the metal for excitation, and an anode. When a high voltage is applied across the anode and cathode, gas particles are ionized. As voltage is increased, gaseous ions acquire enough energy to eject metal atoms from the cathode. Some of these atoms are in an excited states and emit light with the frequency characteristic to the metal[6]. Many modern hollow cathode lamps are selective for several metals.

Diode lasers Atomic absorption spectroscopy can also be performed by lasers, primarily diode lasers because of their good properties for laser absorption spectrometry. The technique is then either referred to as diode laser atomic absorption spectrometry (DLAAS or DLAS),[8] or, since wavelength modulation most often is employed, wavelength modulation absorption spectrometry.

Fluorescence spectroscopy
Fluorescence spectroscopy aka fluorometry or spectrofluorometry, is a type of electromagnetic spectroscopy which analyzes fluorescence from a sample. It involves using a beam of light, usually ultraviolet light, that excites the electrons in molecules of certain compounds and causes them to emit light of a lower energy, typically, but not necessarily, visible light. Devices that measure fluorescence are called fluorometers or fluorimeters. Theory Molecules have various states referred to as energy levels. Fluorescence spectroscopy is primarily concerned with electronic and vibrational states. Generally, the species being examined will have a ground electronic state (a low energy state) of interest, and an excited electronic state of higher energy. Within each of these electronic states are various vibrational states. In fluorescence spectroscopy, the species is first excited, by absorbing a photon, from its ground electronic state to one of the various vibrational states in the excited electronic state. Collisions with other molecules cause the excited molecule to lose vibrational energy until it reaches the lowest vibrational state of the excited electronic state. The molecule then drops down to one of the various vibrational levels of the ground electronic state again, emitting a photon in the process. As molecules may drop down into any of several vibrational levels in the ground state, the emitted photons will have different energies, and thus frequencies. Therefore, by analysing the different frequencies of light emitted in fluorescent spectroscopy, along with their relative intensities, the structure of the different vibrational levels can be determined. In a typical experiment, the different frequencies of fluorescent light emitted by a sample are measured, holding the excitation light at a constant wavelength. This is called an emission spectrum. An excitation spectrum is measured by recording a number of emission spectra using different wavelengths of excitation light. Instrumentation Two general types of instruments exist: Filter fluorometers use filters to isolate the incident light and fluorescent light. Spectrofluorometers use diffraction grating monochromators to isolate the incident light and fluorescent light.

Schematic of a fluorometer with 90 geometry utilizing a Xe light source Both types utilize the following scheme: The light from an excitation source passes through a filter or monochromator, and strikes the sample. A proportion of the incident light is absorbed by the sample, and some of the molecules in the sample fluoresce. The fluorescent light is emitted in all directions. Some of this fluorescent light passes through a second filter or monochromator and reaches a detector, which is usually placed at 90 to the incident light beam to minimize the risk of transmitted or reflected incident light reaching the detector. Various light sources may be used as excitation sources, including lasers, photodiodes, and lamps; xenon arcs and mercury-vapor lamps in particular. A laser only emits light of high irradiance at a very narrow wavelength interval, typically under 0.01 nm, which makes an excitation monochromator or filter unnecessary. The disadvantage of this method is that the wavelength of a laser cannot be changed by much. A mercury vapor lamp is a line lamp, meaning it emits light near peak wavelengths. By contrast, a xenon arc has a continuous emission spectrum with nearly constant intensity in the range from 300-800 nm and a sufficient irradiance for measurements down to just above 200 nm. Filters and/or monochromators may be used in fluorimeters. A monochromator transmits light of an adjustable wavelength with an adjustable tolerance. The most common type of monochromator utilizes a diffraction grating, that is, collimated light illuminates a grating and exits with a different angle depending on the wavelength. The monochromator can then be adjusted to select which wavelengths to transmit. For allowing anisotropy measurements the addition of two polarization filters are necessary: One after the excitation monochromator or filter, and one before the emission monochromator or filter. As mentioned before, the fluorescence is most often measured at a 90 angle relative to the excitation light. This geometry is used instead of placing the sensor at the line of the excitation light at a 180 angle in order to avoid interference of the transmitted excitation light. No monochromator is perfect and it will transmit some stray light, that is, light with other wavelengths than the targeted. An ideal monochromator would only transmit light in the specified range and have a high wavelengthindependent transmission. When measuring at a 90 angle, only the light scattered by the sample causes stray light. This results in a better signal-to-noise ratio, and lowers the detection limit by approximately a factor 10000, when compared to the 180 geometry. Furthermore, the fluorescence can also be measured from the front, which is often done for turbid or opaque samples.

The detector can either be single-channeled or multichanneled. The single-channeled detector can only detect the intensity of one wavelength at a time, while the multichanneled detects the intensity at all wavelengths simultaneously, making the emission monochromator or filter unnecessary. The different types of detectors have both advantages and disadvantages. The most versatile fluorimeters with dual monochromators and a continuous excitation light source can record both an excitation spectrum and a fluorescence spectrum. When measuring fluorescence spectra, the wavelength of the excitation light is kept constant, preferably at a wavelength of high absorption, and the emission monochromator scans the spectrum. For measuring excitation spectra, the wavelength passing though the emission filter or monochromator is kept constant and the excitation monochromator is scanning. The excitation spectrum generally is identical to the absorption spectrum as the fluorescence intensity is proportional to the absorption. Tryptophan Fluorescence The fluorescence of a folded protein is a mixture of the fluorescence from individual aromatic residues. Most of the intrinsic fluorescence emissions of a folded protein are due to excitation of tryptophan residues, with some emissions due to tyrosine and phenylalanine; but disulfide bonds also have appreciable absorption in this wavelength range. Typically, tryptophan has a wavelength of maximum absorption of 280 nm and an emission peak that is solvatochromic, ranging from ca. 300 to 350 nm depending in the polarity of the local environment Hence, protein fluorescence may be used as a diagnostic of the conformational state of a protein.[8] Furthermore, tryptophan fluorescence is strongly influenced by the proximity of other residues (i.e., nearby protonated groups such as Asp or Glu can cause quenching of Trp fluorescence). Also, energy transfer between tryptophan and the other fluorescent amino acids is possible, which would affect the analysis, especially in cases where the Frster acidic approach is taken. In addition, tryptophan is a relatively rare amino acid; many proteins contain only one or a few tryptophan residues. Therefore, tryptophan fluorescence can be a very sensitive measurement of the conformational state of individual tryptophan residues. The advantage compared to extrinsic probes is that the protein itself is not changed. The use of intrinsic fluorescence for the study of protein conformation is in practice limited to cases with few (or perhaps only one) tryptophan residues, since each experiences a different local environment, which gives rise to different emission spectra. Applications Fluorescence spectroscopy is used in, among others, biochemical, medical, and chemical research fields for analyzing organic compounds. There has also been a report of its use in differentiating malignant, bashful skin tumors from benign. Fluorescence can also be used to redirect photons.

Atomic emission spectroscopy

Atomic emission spectroscopy (AES) is a method of chemical analysis that uses the intensity of light emitted from a flame, plasma, arc, or spark at a particular wavelength to determine the quantity of an

element in a sample. The wavelength of the atomic spectral line gives the identity of the element while the intensity of the emitted light is proportional to the number of atoms of the element.

Flame emission spectroscopy

A flame during the assessment of calcium ions in a flame photometer A sample of a material (analyte) is brought into the flame as a gas or sprayed solution. The heat from the flame evaporates the solvent and breaks chemical bonds to create free atoms. The thermal energy also excites the atoms into excited electronic states that subsequently emit light when they return to the ground electronic state. Each element emits light at a characteristic wavelength, which is dispersed by a grating or prism and detected in the spectrometer. A frequent application of the emission measurement with the flame is the regulation of alkali metals for pharmaceutical analytics.

Inductively coupled plasma atomic emission spectroscopy

Inductively coupled plasma atomic emission spectroscopy (ICP-AES) uses an inductively coupled plasma to produce excited atoms and ions that emit electromagnetic radiation at wavelengths characteristic of a particular element. Advantages of ICP-AES are excellent limit of detection and linear dynamic range, multi-element capability low chemical interference and a stable and reproducible signal. Disadvantages are spectral interferences (many emission lines), cost and operating expense and the fact that samples typically must be in solution. Spark and arc atomic emission spectroscopy Spark or arc atomic emission spectroscopy is used for the analysis of metallic elements in solid samples. For non-conductive materials, the sample is ground with graphite powder to make it conductive. In traditional arc spectroscopy methods, a sample of the solid was commonly ground up and destroyed during analysis. An electric arc or spark is passed through the sample, heating it to a high temperature to excite the atoms within it. The excited analyte atoms emit light at characteristic wavelengths that can be dispersed with a monochromator and detected. As the spark or arc conditions are typically not well controlled, the analysis for the elements in the sample is qualitative. However, modern spark sources with controlled discharges under an argon atmosphere can be considered quantitative. Both qualitative and quantitative spark analysis are widely used for production quality control in foundries and steel mills.

References
^ Rendell, D. (1987). Fluorescence and Phosphorescence. Crown ^ Sharma, A. and Schulman, S. G. (1999). Introduction to Fluorescence Spectroscopy. Wiley interscience. ^ Gauglitz, G. and Vo-Dinh, T. (2003). Handbook of spectroscopy. Wiley-VCH. ^ Lakowicz, J. R. (1999). Principles of Fluorescence Spectroscopy. Kluwer Academic / Plenum Publishers ^ Sperling, Michael B.; Welz, Bernhard (1999). Atomic Absorption Spectrometry. Weinheim: Wiley-VCH. ISBN 3-527-28571-7. ^ L vov, B. V. (2005), "Fifty years of atomic absorption spectrometry", Journal of Analytical Chemistry 60: 382, doi:10.1007/s10809-005-0103-0 ^ C. T. J. Alkemade, T. Hollander, W. Snelleman and P. J. T. Zeegers, Metal Vapours in Flames, Pergamon Press, Oxford (1982). ^ Sthlavsk A (April 1973). "[The use of spectrum analytical methods in drug analysis. 1. Determination of alkaline metals using emission flame photometry]" (in German). Pharmazie 28 (4): 238 9. PMID 4716605. ^ Stefnsson A, Gunnarsson I, Giroud N (2007). "New methods for the direct determination of dissolved inorganic, organic and total carbon in natural waters by Reagent-Free Ion Chromatography and inductively coupled plasma atomic emission spectrometry". Anal. Chim. Acta 582 (1): 69 74. doi:10.1016/j.aca.2006.09.001. PMID 17386476.