Spectrophotometry
Overview
 
In chemistry
Chemistry
Chemistry is the science of matter, especially its chemical reactions, but also its composition, structure and properties. Chemistry is concerned with atoms and their interactions with other atoms, and particularly with the properties of chemical bonds....

, spectrophotometry is the quantitative measurement of the reflection or transmission properties of a material as a function of wavelength. It is more specific than the general term electromagnetic spectroscopy in that spectrophotometry deals with visible
Visible spectrum
The visible spectrum is the portion of the electromagnetic spectrum that is visible to the human eye. Electromagnetic radiation in this range of wavelengths is called visible light or simply light. A typical human eye will respond to wavelengths from about 390 to 750 nm. In terms of...

 light, near-ultraviolet
Ultraviolet
Ultraviolet light is electromagnetic radiation with a wavelength shorter than that of visible light, but longer than X-rays, in the range 10 nm to 400 nm, and energies from 3 eV to 124 eV...

, and near-infrared
Infrared
Infrared light is electromagnetic radiation with a wavelength longer than that of visible light, measured from the nominal edge of visible red light at 0.74 micrometres , and extending conventionally to 300 µm...

, but does not cover time-resolved spectroscopic
Time-resolved spectroscopy
In physics and physical chemistry, time-resolved spectroscopy is the study of dynamic processes in materials or chemical compounds by means of spectroscopic techniques. Most often, processes are studied that occur after illumination of a material, but in principle, the technique can be applied to...

 techniques.

Spectrophotometry involves the use of a spectrophotometer.
Encyclopedia
In chemistry
Chemistry
Chemistry is the science of matter, especially its chemical reactions, but also its composition, structure and properties. Chemistry is concerned with atoms and their interactions with other atoms, and particularly with the properties of chemical bonds....

, spectrophotometry is the quantitative measurement of the reflection or transmission properties of a material as a function of wavelength. It is more specific than the general term electromagnetic spectroscopy in that spectrophotometry deals with visible
Visible spectrum
The visible spectrum is the portion of the electromagnetic spectrum that is visible to the human eye. Electromagnetic radiation in this range of wavelengths is called visible light or simply light. A typical human eye will respond to wavelengths from about 390 to 750 nm. In terms of...

 light, near-ultraviolet
Ultraviolet
Ultraviolet light is electromagnetic radiation with a wavelength shorter than that of visible light, but longer than X-rays, in the range 10 nm to 400 nm, and energies from 3 eV to 124 eV...

, and near-infrared
Infrared
Infrared light is electromagnetic radiation with a wavelength longer than that of visible light, measured from the nominal edge of visible red light at 0.74 micrometres , and extending conventionally to 300 µm...

, but does not cover time-resolved spectroscopic
Time-resolved spectroscopy
In physics and physical chemistry, time-resolved spectroscopy is the study of dynamic processes in materials or chemical compounds by means of spectroscopic techniques. Most often, processes are studied that occur after illumination of a material, but in principle, the technique can be applied to...

 techniques.

Spectrophotometry involves the use of a spectrophotometer. A spectrophotometer is a photometer
Photometer
In its widest sense, a photometer is an instrument for measuring light intensity or optical properties of solutions or surfaces. Photometers are used to measure:*Illuminance*Irradiance*Light absorption*Scattering of light*Reflection of light*Fluorescence...

 (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
Diffusivity
Diffusivity can refer to:*Diffusivity of heat*Diffusivity of mass:** Molecular diffusivity ** Eddy diffusivity*Momentum diffusivity...

 on any of the listed light ranges that usually cover around 200nm - 2500nm using different controls and calibrations. Within these ranges of light, calibrations are needed on the machine using standards that vary in type depending on the wavelength
Wavelength
In physics, the wavelength of a sinusoidal wave is the spatial period of the wave—the distance over which the wave's shape repeats.It is usually determined by considering the distance between consecutive corresponding points of the same phase, such as crests, troughs, or zero crossings, and is a...

 of the photometric determination.

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
Physics
Physics is a natural science that involves the study of matter and its motion through spacetime, along with related concepts such as energy and force. More broadly, it is the general analysis of nature, conducted in order to understand how the universe behaves.Physics is one of the oldest academic...

, materials science
Materials science
Materials science is an interdisciplinary field applying the properties of matter to various areas of science and engineering. This scientific field investigates the relationship between the structure of materials at atomic or molecular scales and their macroscopic properties. It incorporates...

, chemistry
Chemistry
Chemistry is the science of matter, especially its chemical reactions, but also its composition, structure and properties. Chemistry is concerned with atoms and their interactions with other atoms, and particularly with the properties of chemical bonds....

, biochemistry
Biochemistry
Biochemistry, sometimes called biological chemistry, is the study of chemical processes in living organisms, including, but not limited to, living matter. Biochemistry governs all living organisms and living processes...

, and molecular biology
Molecular biology
Molecular biology is the branch of biology that deals with the molecular basis of biological activity. This field overlaps with other areas of biology and chemistry, particularly genetics and biochemistry...

. 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.

Design

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
Monochromator
A monochromator is an optical device that transmits a mechanically selectable narrow band of wavelengths of light or other radiation chosen from a wider range of wavelengths available at the input...

 containing a diffraction grating
Diffraction grating
In optics, a diffraction grating is an optical component with a periodic structure, which splits and diffracts light into several beams travelling in different directions. The directions of these beams depend on the spacing of the grating and the wavelength of the light so that the grating acts as...

 to produce the analytical spectrum. The grating can either be movable or fixed. If a single detector, such as a photomultiplier tube or photodiode
Photodiode
A photodiode is a type of photodetector capable of converting light into either current or voltage, depending upon the mode of operation.The common, traditional solar cell used to generateelectric solar power is a large area 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
Fourier transform
In mathematics, Fourier analysis is a subject area which grew from the study of Fourier series. The subject began with the study of the way general functions may be represented by sums of simpler trigonometric functions...

 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
Photodiode
A photodiode is a type of photodetector capable of converting light into either current or voltage, depending upon the mode of operation.The common, traditional solar cell used to generateelectric solar power is a large area photodiode....

, charge coupled device or other light sensor
Photodetector
Photosensors or photodetectors are sensors of light or other electromagnetic energy. There are several varieties:*Active pixel sensors are image sensors consisting of an integrated circuit that contains an array of pixel sensors, each pixel containing a both a light sensor and an active amplifier...

. The transmittance
Transmittance
In optics and spectroscopy, transmittance is the fraction of incident light at a specified wavelength that passes through a sample. A related term is absorptance, or absorption factor, which is the fraction of radiation absorbed by a sample at a specified wavelength...

 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:
  1. The light source is imaged upon the sample
  2. A fraction of the light is transmitted or reflected from the sample
  3. The light from the sample is imaged upon the entrance slit of the monochromator
  4. The monochromator separates the wavelengths of light and focuses each of them onto the photodetector sequentially


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).

UV-visible spectrophotometry

The most common spectrophotometers are used in the UV and visible
Visible spectrum
The visible spectrum is the portion of the electromagnetic spectrum that is visible to the human eye. Electromagnetic radiation in this range of wavelengths is called visible light or simply light. A typical human eye will respond to wavelengths from about 390 to 750 nm. In terms of...

 regions of the spectrum, and some of these instruments also operate into the near-infrared
Infrared
Infrared light is electromagnetic radiation with a wavelength longer than that of visible light, measured from the nominal edge of visible red light at 0.74 micrometres , and extending conventionally to 300 µm...

 region as well.

Visible region 400–700 nm spectrophotometry is used extensively in colorimetry
Colorimetry
Colorimetry is "the science and technology used to quantify and describe physically the human color perception."It is similar to spectrophotometry, but is distinguished by its interest in reducing spectra to the physical correlates of color perception, most often the CIE 1931 XYZ color space...

 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
Beer-Lambert law
In optics, the Beer–Lambert law, also known as Beer's law or the Lambert–Beer law or the Beer–Lambert–Bouguer law relates the absorption of light to the properties of the material through which the light is travelling.-Equations:The law states that there is a logarithmic dependence between the...

 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 cuvette
Cuvette
A cuvette is a small tube of circular or square cross section, sealed at one end, made of plastic, glass, or fused quartz and designed to hold samples for spectroscopic experiments. The best cuvettes are as clear as possible, without impurities that might affect a spectroscopic reading...

s; depending on the region of interest, they may be constructed of glass
Glass
Glass is an amorphous solid material. Glasses are typically brittle and optically transparent.The most familiar type of glass, used for centuries in windows and drinking vessels, is soda-lime glass, composed of about 75% silica plus Na2O, CaO, and several minor additives...

, plastic
Plastic
A plastic material is any of a wide range of synthetic or semi-synthetic organic solids used in the manufacture of industrial products. Plastics are typically polymers of high molecular mass, and may contain other substances to improve performance and/or reduce production costs...

, or quartz. Small sample amounts (starting with 0.3 µl) of biological samples (DNA
DNA
Deoxyribonucleic acid is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms . The DNA segments that carry this genetic information are called genes, but other DNA sequences have structural purposes, or are involved in...

, RNA
RNA
Ribonucleic acid , or RNA, is one of the three major macromolecules that are essential for all known forms of life....

, Protein
Protein
Proteins are biochemical compounds consisting of one or more polypeptides typically folded into a globular or fibrous form, facilitating a biological function. A polypeptide is a single linear polymer chain of amino acids bonded together by peptide bonds between the carboxyl and amino groups of...

) can be quantified cuvetteless by pipetting the sample directly on the measuring window of a specialized NanoPhotometer.

Nano UV-visible spectrophotometry

Multiple biological applications (e.g. DNA microarray experiment
DNA microarray experiment
This is an example of a DNA microarray experiment, detailing a particular case to better explain DNA microarray experiments, while enumerating possible alternatives.# The two samples to be compared are grown/acquired...

, array CGH, qPCR, Nucleic acid and protein labelling
Cyanine
Cyanine is a non-systematic name of a synthetic dye family belonging to polymethine group. Cyanines have many uses as fluorescent dyes, particularly in biomedical imaging...

) 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.

IR spectrophotometry

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
Potassium bromide
Potassium bromide is a salt, widely used as an anticonvulsant and a sedative in the late 19th and early 20th centuries, with over-the-counter use extending to 1975 in the United States. Its action is due to the bromide ion...

 or ground with potassium bromide and pressed into a pellet. Where aqueous solutions are to be measured, insoluble silver chloride
Silver chloride
Silver chloride is a chemical compound with the chemical formula AgCl. This white crystalline solid is well known for its low solubility in water . Upon illumination or heating, silver chloride converts to silver , which is signalled by greyish or purplish coloration to some samples...

 is used to construct the cell.

Spectroradiometers

Spectroradiometer
Spectroradiometer
Spectroradiometers are designed to measure the spectral power distributions of illuminants. They operate almost like spectrophotometers in the visible region...

s, which operate almost like the visible region spectrophotometers, are designed to measure the spectral density
Spectral density
In statistical signal processing and physics, the spectral density, power spectral density , or energy spectral density , is a positive real function of a frequency variable associated with a stationary stochastic process, or a deterministic function of time, which has dimensions of power per hertz...

 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:
  1. The light source shines onto or through the sample.
  2. The sample transmits or reflects light.
  3. The detector detects how much light was reflected from or transmitted through the sample.
  4. The detector then converts how much light the sample transmitted or reflected into a number.

See also

  • Atomic Absorption Spectrophotometry
  • Atomic emission spectroscopy
    Atomic emission spectroscopy
    Atomic emission spectroscopy 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...

  • Inductively coupled plasma atomic emission spectroscopy
  • Inductively coupled plasma mass spectrometry
  • Spectroradiometry
  • Slope spectroscopy
    Slope Spectroscopy
    Slope spectroscopy is a spectroscopic technique that is used to quantify concentrations of various compounds, proteins and antibodies in which the operating path length is varied and the absorbance is measured.- Equations :...

The source of this article is wikipedia, the free encyclopedia.  The text of this article is licensed under the GFDL.
 
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