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UV-VIS and NIR spectrophotometers use ultraviolet, visible, and near-infrared light to excite a sample and determine it’s unique optical properties, such as reflectance, absorbance, and transmittance.
These instruments employ UV/VIS/NIR spectroscopy, a detection and characterization method, in order to obtain these distinct optical properties, providing both qualitative and quantitative data of the sample.
Combined, these three wavelengths allow the spectrometer to operate in an optical range from 100 to above 3000 nanometers (nm).
Ultraviolet (UV) light typically has a wavelength range of 100 to 400 nm, and visible light ranges from 400 to 800 nm. Where the range truly extends is in the near-infrared, where wavelengths range from 800 to 3200 nm.
UV-VIS and NIR spectroscopic techniques are useful in a number of ways, and can be used separately or in combination.
On it’s own, UV-VIS spectroscopy only uses the UV and visible portion of the electromagnetic spectrum to look at properties of a substance, observing how matter interacts with either UV or visible light, whether it is through absorption or reflection.
Interactions in this portion of the spectrum result in organic molecules undergoing molecular electronic transition. This is to say that these interactions result in electrons in the molecules becoming energized or excited.
For visible light specifically, the reflection of light results in humans ability to see color. An important law that is used in the application of UV/Vis spectroscopy is the Beer-Lambert law which states that the higher the concentration of a molecule, the more UV/Vis radiation it will absorb.
The structural component responsible for this absorption is called a chromophore. Due to the linear relationship of this law, UV-Vis spectroscopy is an important tool in chemistry for the quantitative determination of analytes in a compound.
Near-infrared spectroscopy (NIRS), when used on its own, is a non-destructive analytical technique that measures how samples absorb NIR light to observe their properties. While UV-Vis observes energy transitions, NIRS is generally used to look at vibrational transitions.
In principle, NIRS shoots a sample with a broad-spectrum of NIR light and analyzes what wavelengths are absorbed, reflected, or scattered by the material. This data can then be used to determine properties about the bonds of the molecules that are in the object of interest.
Bond vibrations between hydrogen and other elements such as oxygen (OH), carbon (CH), and nitrogen (NH) are seen as NIR absorbance bands.
The spectral bands that NIRS gathers are wide which can make their interpretation more complex than other spectroscopy methods, however, their ability to penetrate deep into samples makes them a useful tool.
UV-Vis and NIR spectrometer setups differ in many important ways, but their basic configuration consists of a light source, a filter, and a detector.
They may also have a dispersion unit that makes analyses at different wavelengths possible. The filters that are used are employed to limit or select specific wavelengths that you wish to observe. Depending on what properties are being looked for in the sample, the specific instrumentation used can vary.
For UV-VIS and NIR spectroscopy, specific light sources are used, however, these light sources are also commonly found in other types of spectrophotometers. They include halogen lamps, deuterium lamps, xenon arc and flash lamps, as well as low-pressure mercury lamps.
In the case of NIR spectrometers and spectrophotometers, tungsten and tungsten-halogen lamps or bulbs may be used as well, along with some narrowband NIR LEDs, NIR lasers, NIR laser diodes, and a range of other NIR light sources available.
The light source creates the beam of light used to radiate the sample within the spectrophotometer, which is held within a cuvette of specific outer and inner dimensions. The transparent cuvette is typically made of optical glass, transparent plastic, or quartz.
Furthermore, Spectrophotometers can be either single beam, double beam, or split beam. Single beam devices use one beam of light to radiate the sample, while double beam devices use two beams of light.
When using a single beam device, only one cuvette is required. However, this means the end-user must switch between the sample cuvette and a reference cuvette to perform calibration. Double beam spectrophotometers provide increased ease-of-use and high-throughput capabilities by removing the need to manually switch cuvettes during experimentation and making up for the loss of light intensity as the beams pass through both cuvettes.
Split-beam systems are similar to double beam, however, as the name suggests, they split the beam by rapidly alternating between the reference cuvette and the sample cuvette.
For UV/Vis spectroscopy, there are a few common types of detectors used:
Barrier layer cell or photovoltaic cells
They consist of two thin metallic layers, often copper or iron, where one acts as a base plate and the other an electrode. A semiconductor, such as selenium, is deposited on them, and finally, a thin layer of either silver or gold covers them. When radiation hits the semiconductor, the electrons move through the metal layer creating a current that can be measured.
Phototubes or photoemissive cells
An evacuated glass bulb is used and inside it, a cathode is placed along with an anode. The photocathode is coated with a light-sensitive layer like potassium oxide or silver oxide. When radiation hits the cathode, electrons are emitted and flow to the anode which creates an observable current.
Photomultipliers
These detectors use a vacuum tube, a fixed emissive cathode, many dynodes, and an anode. When the radiation’s electrons hit the first dynode, more electrons are emitted which strike the second dynode, which then emits even more electrons. This continues until the electrons are gathered by the anode and the resulting current is looked at.
NIRS Detectors
Detectors for NIRS are commonly based on silicon, lead-sulfate (PbS), and indium gallium arsenide (InGaAs) photoconductive material. For analysis of short NIR wavelengths anything under 1000 nm, silicon-based charge-coupled devices, or CCD, are used. Both InGaAs and PbS are widely used due to their high response speed, however, they are not as sensitive as CCDs.
Monochromators are a type of optical device used in spectrophotometers to filter for specific wavelengths of light using a few different filtering methods. In this case, the monochromator filters for UV, visible, and near-infrared light.
Spectrophotometers available today are generally categorized into two types: the single or double monochromator type. Single types have a single monochromator, while double types have two monochromators. The purpose of having a secondary monochromator device is to further filter the monochromatic light split by the first monochromator, creating purer monochromatic light with reduced stray light (any light generated at wavelengths other than the target wavelength).
This device, which operate similarly to various optical filters, consist of an entrance slit, collimating lens, dispersing device, a focusing lens, and an exit slit.
Furthermore, It can be characterized as either an interference or absorption filter. In an interference filter, a dielectric layer is used and only specific in-phase wavelengths can be reflected and recorded. In an absorption filter, specific colored plates are used to absorb certain wavelengths, leaving only the spectra that is to be observed.
Two common filters used in NIRS include interference filters and acousto-optic tunable filters (AOTF). Interference filters, also called Fabry-Pérot filters, are narrow bandpass filters that use mirrors and a resonance cavity to filter out unwanted wavelengths that pass through it. AOTFs, also called Bragg cells, use sound waves to shift or diffract unwanted frequencies of light.
In UV-Vis spectroscopy, the dispersive devices used are prisms and grating. NIRS primarily uses grating as a dispersion technique. Prisms are semi-transparent in nature and made from glass, quartz, fused silica, or other transparent material.
Depending on the geometry and the substance used, prisms are able to bend the light that passes through them. If white light passed through a glass prism the resulting bent light would be a rainbow.
Grating disperses light either by diffraction or transmission. Transmission grating uses a series of grooves at specific angles that are able to refract the incoming light. On the opposite side of the grating, the light is dispersed at a fixed angle.
Diffraction grating is similar, except the incoming light is reflected. The incoming polychromatic light bounces off of the grating and diffracts into multiple monochromatic beams. One of these beams can then be isolated by angling it through an exit slit.
Interferometry superimposes electromagnetic waves to interfere with the incoming waves so that they may be analyzed.
An alternative to light diffraction, interferometers use beam splitters and measure the intensities of individual NIR wavelengths. The polychromatic wave is split into two beams, both of which are analyzed for NIR wavelengths. The minor offset between the two beams readouts is called an interferogram.
Fourier transform near-infrared spectrometers, which utilize NIRS, employ an interferometer to detect signals.
NIRS has many applications across a variety of fields, including the medical profession. Their applications are usually reserved for analyzing the oxygen saturation of hemoglobin within the microcirculation.
When used to look specifically at the oxygenation levels going to the brain, the technique is referred to as cerebral NIRS. The brain is one of, if not the most important organs in your body, and being able to non-invasively examine it is extremely important.
When there is internal bleeding in the skull blood tends to pool in the affected area. Cerebral NIRS would be able to detect this quickly and without invasive procedures.
Additionally, as different parts of the brain become active the oxygen levels in those parts change. Cerebral NIRS would be a smaller, easier to use, and cheaper device than current solutions.
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