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For nearly 200 years, optical spectroscopy has been used in a wide range of disciplines. These include physics, biology, chemistry, medicine, and materials science. Sub-disciplines of these fields that also make use of spectroscopy include: astronomy, organic chemistry, and nanotechnology. Using optical spectroscopy, elements and molecules can be detected in any aggregation state both qualitatively and quantitatively. Information about the change over time of the bonds in a compound can also be obtained. Unlike other analytical procedures the samples do not need time-consuming preparation.
Emission Spectroscopy
Fluorescence
Fluorescence is a phenomenon wherein the absorption of a photon triggers the release of another photon at a longer wavelength. The energy difference ends up as molecular vibration or heat. With fluorescence measurements it is necessary to distinguish between the excitation spectrum and the emission spectrum. In measuring the excitation spectrum, we are comparing the amount of visible light in relation to the total electromagnetic radiation absorbed by a body for given frequencies of exciting light. To measure an excitation spectrum, the fluorescent sample is illuminated with different wavelengths one after another. The intensity of the fluorescent radiation is measured at a fixed wavelength. The intensity measured at this wavelength is applied at the excitation wavelength. Reference graphs are then used to determine the wavelength at which the spectrum was taken. If an emission spectrum is being measured, the fluorescent sample is illuminated with a fixed wavelength. The sample emits fluorescent radiation over a wide range of wavelengths. In this case the intensity emitted is applied at the particular emission wavelength. Reference graphs are then used that show the excitation wavelengths.
In biology, materials which are said to be fluorescent include: heme, flavin, retinal, and phytochrome as well as the aromatic amino acids phenylalanine, tyrosine, and tryptophan. Apart from these, metabolites such as porphine, carbohydrates, pigments and coenzymes can also fluoresce. If the fluorescence is limited, then markers which are suitable for coloring certain cell regions or organelles can sometimes be employed. Among these markers are: acridine orange derivative, rhodamine 123, doxycycline, and 1-anilinonaphthalene sulfonate. Other fluorescent materials include fluorescein and naphthol. Metals can also be excited to fluorescence using complexing agents.
Light Source Measurement
One area where emission spectroscopy is commonly used is light source measurement. In this kind of measurement, the goal is not to evaluate certain elements. The measurements are used for the evaluation of light sources with regard to physiological criteria (workplace illumination, solar panels, LED, Computer Monitors). Emission spectroscopy can also be used for production oversight (e.g. monitoring UV lamps used for hardening).
Absorption
To measure the absorption of a substance, a light source, sample holder and a spectrometer are necessary. First, the radiation intensity and spectral diffusion of the light source are measured. This reading is the reference measurement. The sample is then fixed into the holder. The spectrometer is set to absorption measurement. A spectrum is then taken. The absorption by the wavelength is then displayed.
For materials in a water solution or as a gas, the Lambert-Beersch Law applies:
Ι = Ι0eα(λ)xc
Where Ι0 is the reduced intensity of the light without the sample in the optical path. Ι is the weakened light intensity after the sample has been measured, c stands for material concentration, x for sample thickness and α for the natural molar extinction coefficient. In decimal form with the decimal molar extinction coefficient ε(λ) = α(λ)· 0.4343
log [ Ι / Ι0 ] = ε(λ)xc = A(λ)
From this it follows that the optical density is directly proportional to the concentration of the material. In its simple form this law only applies to monochromatic radiation. When measuring cloudy samples, a sensible choice of reference measurements must be made.
Both absorption and reflection measurement values change as a result of the dispersion of the radiation. In order to get real spectra, the reference measurements in the observed wavelength range must be transparent but display a comparable dispersion.
Reflection
The reflection of light is the most common measurement method in spectroscopy.Applications and measurement equipment vary widely. Depending on the surface, the reflected light can be diffuse or specular or a mixture of both. The reflectance (ratio of incident light to reflected light) can be measured, and so can changes in the wavelength. Additionally, the interference of two reflections can also be the object of interest.
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The sample reflects the radiation diffusely; the directly reflected radiation is coupled out through the gloss trap. Only the diffuse reflection is coupled into the optical fiber. The reference measurement is measured against a comparable strongly diffusereflecting white reference standard. The spectrometer is reset to reflection measurement. The reflectance from the wavelength can be read off on the spectrometer.
Transmission
The setup for measuring the transmission of a material is identical to the setup for absorption. First the radiation intensity and special diffusion of the light source are measured. This reading is the reference measurement. The sample is then fixed into the holder. The spectrometer is set to transmission measurement. A spectrum is then taken. Depending on the setting, the transmission can be displayed as a percentage or transmittance from the wavelength.
UV Spectroscopy
UV spectroscopy comprises a separate field, since it places special demands on the spectrometer. The cover glass in the sensor must be made of UV permeable quartz glass (Suprasil) and the sensor must be able to detect UV radiation (see sensors). UV Spectroscopy is primarily executed as absorption and fluorescence spectroscopy. Using UV Spectroscopy, nitrite can be detected in drinking water without having to resort to chemical reagents. Other detectable substances are: nitrate, bisulfite, nitrogen, phosphorus, benzene.
Infrared Spectroscopy
As with UV spectroscopy infrared spectroscopy is also a separate field due to the special demands it places on the spectrometer. This kind of spectroscopy has become extremely important because the molecular vibrations have an effect on the spectrum in infrared. Nearly all organic materials exhibit specific absorptions in the near infrared. NIR can detect differences between polymers in plastic screening, the water content of, for example, fruit or grain. NIR can detect octane, caffeine, salicylic acid, or nicotine content. Blood values such as cholesterol, glucose, and oxygen, can also be determined noninvasively. Additionally, the salt content of sea water can also be determined using NIR spectroscopy.
Laser Spectroscopy
This analytical procedure is similar to atom spectroscopy. A laser is used as a light source. The laser is used both as a light source for excitation as well as for illuminating the sample for absorption measurement. If the sample is excited by the laser, the sample itself begins to emit radiation. The spectrometer records the radiation at a right angle to the direction of the radiation excitations. If the sample absorbs the laser beam, the sample is placed between the laser and the spectrometer.
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