In chemistry, spectrophotometry is the quantitative measurement of the reflection or transmission properties of a material as a function of wavelength.[1] It is more specific than the general term electromagnetic spectroscopy in that spectrophotometry deals with visible light, near-ultraviolet, and near-infrared, but does not cover time-resolved spectroscopic techniques.
Spectrophotometry involves the use of a spectrophotometer. A spectrophotometer is a photometer (a device for measuring light intensity) that can measure intensity as a function of the light source wavelength. Important features of spectrophotometers are spectral bandwidth and linear range of absorption or reflectance measurement.
A spectrophotometer is commonly used for the measurement of transmittance or reflectance of solutions, transparent or opaque solids, such as polished glass, or gases. However they can also be designed to measure the diffusivity on any of the listed light ranges that usually cover around 200nm - 2500nm using different controls and calibrations.[2] Within these ranges of light, calibrations are needed on the machine using standards that vary in type depending on the wavelength of the photometric determination.[3]
An example of an experiment in which spectrophotometry is used is the determination of the equilibrium constant of a solution. A certain chemical reaction within a solution may occur in a forward and reverse direction where reactants form products and products break down into reactants. At some point, this chemical reaction will reach a point of balance called an equilibrium point. In order to determine the respective concentrations of reactants and products at this point, the light transmittance of the solution can be tested using spectrophotometry. The amount of light that passes through the solution is indicative of the concentration of certain chemicals that do not allow light to pass through.
The use of spectrophotometers spans various scientific fields, such as physics, materials science, chemistry, biochemistry, and molecular biology.[4] They are widely used in many industries including semiconductors, laser and optical manufacturing, printing and forensic examination, as well in laboratories for the study of chemical substances. Ultimately, a spectrophotometer is able to determine, depending on the control or calibration, what substances are present in a target and exactly how much through calculations of observed wavelengths.
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There are two major classes of devices: single beam and double beam. A double beam spectrophotometer compares the light intensity between two light paths, one path containing a reference sample and the other the test sample. A single beam spectrophotometer measures the relative light intensity of the beam before and after a test sample is inserted. Although comparison measurements from double beam instruments are easier and more stable, single beam instruments can have a larger dynamic range and are optically simpler and more compact. Additionally, some specialized instruments, such as spectrophotometer built onto microscopes or telescopes, are single beam instruments due to practicality.
Historically, spectrophotometers use a monochromator containing a diffraction grating to produce the analytical spectrum. The grating can either be movable or fixed. If a single detector, such as a photomultiplier tube or photodiode is used, the grating can be scanned stepwise so that the detector can measure the light intensity at each wavelength (which will correspond to each "step"). Arrays of detectors, such as charge coupled devices (CCD) or photodiode arrays (PDA) can also be used. In such systems, the grating is fixed and the intensity of each wavelength of light is measured by a different detector in the array. Additionally, most modern mid-infrared spectrophotometers use a Fourier transform technique to acquire the spectral information. The technique is called Fourier Transform Infrared.
When making transmission measurements, the spectrophotometer quantitatively compares the fraction of light that passes through a reference solution and a test solution. For reflectance measurements, the spectrophotometer quantitatively compares the fraction of light that reflects from the reference and test samples. Light from the source lamp is passed through a monochromator, which diffracts the light into a "rainbow" of wavelengths and outputs narrow bandwidths of this diffracted spectrum. Discrete frequencies are transmitted through the test sample. Then the photon flux density (watts per metre squared usually) of the transmitted or reflected light is measured with a photodiode, charge coupled device or other light sensor. The transmittance or reflectance value for each wavelength of the test sample is then compared with the transmission (or reflectance) values from the reference sample.
In short, the sequence of events in a modern spectrophotometer is as follows:
Many older spectrophotometers must be calibrated by a procedure known as "zeroing." The absorbancy of a reference substance is set as a baseline value, so the absorbancies of all other substances are recorded relative to the initial "zeroed" substance. The spectrophotometer then displays % absorbancy (the amount of light absorbed relative to the initial substance).[4]
The most common spectrophotometers are used in the UV and visible regions of the spectrum, and some of these instruments also operate into the near-infrared region as well.
Visible region 400–700 nm spectrophotometry is used extensively in colorimetry science. Ink manufacturers, printing companies, textiles vendors, and many more, need the data provided through colorimetry. They take readings in the region of every 5–20 nanometers along the visible region, and produce a spectral reflectance curve or a data stream for alternative presentations. These curves can be used to test a new batch of colorant to check if it makes a match to specifications e.g., ISO printing standards.
Traditional visible region spectrophotometers cannot detect if a colorant or the base material has fluorescence. This can make it difficult to manage color issues if for example one or more of the printing inks is fluorescent. Where a colorant contains fluorescence, a bi-spectral fluorescent spectrophotometer is used. There are two major setups for visual spectrum spectrophotometers, d/8 (spherical) and 0/45. The names are due to the geometry of the light source, observer and interior of the measurement chamber. Scientists use this instrument to measure the amount of compounds in a sample. If the compound is more concentrated more light will be absorbed by the sample; within small ranges, the Beer-Lambert law holds and the absorbance between samples vary with concentration linearly. In the case of printing measurements two alternative settings are commonly used- without/with uv filter to control better the effect of uv brighteners within the paper stock.
Samples are usually prepared in cuvettes; depending on the region of interest, they may be constructed of glass, plastic, or quartz. Small sample amounts (starting with 0.3 µl) of biological samples (DNA, RNA, Protein) can be quantified cuvetteless by pipetting the sample directly on the measuring window of a specialized NanoPhotometer.[5]
Multiple biological applications (e.g. DNA microarray experiment, array CGH, qPCR, Nucleic acid and protein labelling) imply quantitative and qualitative nucleic acid and protein quantification with minimal sample volumes. Specialized nano-volume photometer offer the possibility to determine sample concentrations cuvetteless with submicroliter volumes. A drop of sample (0.03 µl to 5 µl) is pipetted directly onto the measuring window of the photometer. In the measuring chamber the sample is squeezed to defined path lengths ranging from 0.04 mm up to 2 mm. Due to this reduction of the optical pathlength samples are diluted automatically (virtual dilution factor: 1/250 to 1/5) in comparison to standard cuvette measurements (path length 1 cm). Because the measurements are processed with undiluted samples, the samples can be retrieved after the measurement for further processing.
Spectrophotometers designed for the main infrared region are quite different because of the technical requirements of measurement in that region. One major factor is the type of photosensors that are available for different spectral regions, but infrared measurement is also challenging because virtually everything emits IR light as thermal radiation, especially at wavelengths beyond about 5 μm.
Another complication is that quite a few materials such as glass and plastic absorb infrared light, making it incompatible as an optical medium. Ideal optical materials are salts, which do not absorb strongly. Samples for IR spectrophotometry may be smeared between two discs of potassium bromide or ground with potassium bromide and pressed into a pellet. Where aqueous solutions are to be measured, insoluble silver chloride is used to construct the cell.
Spectroradiometers, which operate almost like the visible region spectrophotometers, are designed to measure the spectral density of illuminants in order to evaluate and categorize lighting for sales by the manufacturer, or for the customers to confirm the lamp they decided to purchase is within their specifications. Components:
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