In chemistry, spectrophotometry is the quantitative measurement of the reflection or transmission properties of a material as a function of wavelength.[2] 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.

Table top spectrophotometer
Beckman Ir-1 Spectrophotometer, ca. 1941
Beckman IR-1 Spectrophotometer, ca. 1941
Handheld spectrophotometer used in graphic industry.[1]
External video
Jacqueline Boytim discusses the development and early use of spectrophotometers, Chemical Heritage Foundation


Spectrophotometry is a tool that hinges on the quantitative analysis of molecules depending on how much light is absorbed by colored compounds. Spectrophotometry uses photometers, known as spectrophotometers, that can measure a light beam's intensity as a function of its color (wavelength). Important features of spectrophotometers are spectral bandwidth (the range of colors it can transmit through the test sample), the percentage of sample-transmission, the logarithmic range of sample-absorption, and sometimes a percentage of 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. Although many biochemicals are colored, as in, they absorb visible light and therefore can be measured by colorimetric procedures, even colorless biochemicals can often be converted to colored compounds suitable for chromogenic color-forming reactions to yield compounds suitable for colorimetric analysis.[3]:65 However, they can also be designed to measure the diffusivity on any of the listed light ranges that usually cover around 200 nm - 2500 nm 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.[4]

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 absorption of light is due to the interaction of light with the electronic and vibrational modes of molecules. Each type of molecule has an individual set of energy levels associated with the makeup of its chemical bonds and nuclei, and thus will absorb light of specific wavelengths, or energies, resulting in unique spectral properties.[5] This is based upon its specific and distinct makeup.

The use of spectrophotometers spans various scientific fields, such as physics, materials science, chemistry, biochemistry,Chemical Engineering, and molecular biology.[6] 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. Spectrophotometry is often used in measurements of enzyme activities, determinations of protein concentrations, determinations of enzymatic kinetic constants, and measurements of ligand binding reactions.[3]:65 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.

In astronomy, the term spectrophotometry refers to the measurement of the spectrum of a celestial object in which the flux scale of the spectrum is calibrated as a function of wavelength, usually by comparison with an observation of a spectrophotometric standard star, and corrected for the absorption of light by the Earth's atmosphere.[7]


Invented by Arnold O. Beckman in 1940, the spectrophotometer was created with the aid of his colleagues at his company National Technical Laboratories founded in 1935 which would become Beckman Instrument Company and ultimately Beckman Coulter. This would come as a solution to the previously created spectrophotometers which were unable to absorb the ultraviolet correctly. He would start with the invention of Model A where a glass prism was used to absorb the UV light. It would be found that this did not give satisfactory results, therefore in Model B, there was a shift from a glass to a quartz prism which allowed for better absorbance results. From there, Model C was born with an adjustment to the wavelength resolution which ended up having three units of it produced. The last and most popular model became Model D which is better recognized now as the DU spectrophotometer which contained the instrument case, hydrogen lamp with ultraviolent continuum and a better monochromator.[8] It was produced from 1941 to 1976 where the price for it in 1941 was US$723 (far-UV accessories were an option at additional cost). In the words of Nobel chemistry laureate Bruce Merrifield, it was "probably the most important instrument ever developed towards the advancement of bioscience."[9]

Once it became discontinued in 1976,[10] another company known as Hewlett-Packard created the first commercially available diode-assay spectrophotometer in 1979 known as the HP 8450A.[11] Diode-assay spectrophotometers differed from the original spectrophotometer created by Beckman because it was the first single-beam microprocessor-controlled spectrophotometer that scanned multiple wavelengths at a time in seconds. It irradiates the sample with polychromatic light which the sample absorbs depending on its properties. Then it is transmitted back by grating the photodiode array which detects the wavelength region of the spectrum.[12] Since then, the creation and implementation of spectrophotometry devices has increased immensely and has become one of the most innovative instruments of our time.


Single beam scanning spectrophotometer

There are two major classes of devices: single beam and double beam. A double beam spectrophotometer[13] 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 spectrophotometers 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 (scanning spectrophotometer) so that the detector can measure the light intensity at each wavelength (which will correspond to each "step"). Arrays of detectors (array spectrophotometer), 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. This technique is called Fourier transform infrared spectroscopy.

When making transmission measurements, the spectrophotometer quantitatively compares the fraction of light that passes through a reference solution and a test solution, then electronically compares the intensities of the two signals and computes the percentage of transmission of the sample compared to the reference standard. 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 through a rotating prism and outputs narrow bandwidths of this diffracted spectrum through a mechanical slit on the output side of the monochromator. These bandwidths 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. Most instruments will apply a logarithmic function to the linear transmittance ratio to calculate the 'absorbency' of the sample, a value which is proportional to the 'concentration' of the chemical being measured.

In short, the sequence of events in a scanning spectrophotometer is as follows:

  1. The light source is shone into a monochromator, diffracted into a rainbow, and split into two beams. It is then scanned through the sample and the reference solutions.
  2. Fractions of the incident wavelengths are transmitted through, or reflected from, the sample and the reference.
  3. The resultant light strikes the photodetector device, which compares the relative intensity of the two beams.
  4. Electronic circuits convert the relative currents into linear transmission percentages and/or absorbance/concentration values.

In an array spectrophotometer, the sequence is as follows:[14]

  1. The light source is shone into the sample and focused into a slit
  2. The transmitted light is refracted into a rainbow with the reflection grating
  3. The resulting light strikes the photodetector device which compares the intensity of the beam
  4. Electronic circuits convert the relative currents into linear transmission percentages and/or absorbance/concentration values

Many older spectrophotometers must be calibrated by a procedure known as "zeroing", to balance the null current output of the two beams at the detector. The transmission of a reference substance is set as a baseline (datum) value, so the transmission of all other substances are recorded relative to the initial "zeroed" substance. The spectrophotometer then converts the transmission ratio into 'absorbency', the concentration of specific components of the test sample relative to the initial substance.[6]

Applications in biochemistry

Spectrophotometry is an important technique used in many biochemical experiments that involve DNA, RNA, and protein isolation, enzyme kinetics and biochemical analyses.[15] Since samples in these applications are not readily available in large quantities, they are especially suited to being analyzed in this non-destructive technique. In addition, precious sample can be saved by utilizing a micro-volume platform where as little as 1uL of sample is required for complete analyses.[16] A brief explanation of the procedure of spectrophotometry includes comparing the absorbency of a blank sample that does not contain a colored compound to a sample that contains a colored compound. This coloring can be accomplished by either a dye such as Coomasie Brilliant Blue G-250 dye measured at 595 nm or by an enzymatic reaction as seen between β-galactosidase and ONPG (turns sample yellow) measured at 420 nm.[3]:21-119 The spectrophotometer is used to measure colored compounds in the visible region of light (between 350 nm and 800 nm),[3]:65 thus it can be used to find more information about the substance being studied. In biochemical experiments, a chemical and/or physical property is chosen and the procedure that is used is specific to that property in order to derive more information about the sample, such as the quantity, purity, enzyme activity, etc. Spectrophotometry can be used for a number of techniques such as determining optimal wavelength absorbance of samples, determining optimal pH for absorbance of samples, determining concentrations of unknown samples, and determining the pKa of various samples.[3]:21-119 Spectrophotometry is also a helpful process for protein purification [17] and can also be used as a method to create optical assays of a compound. Spectrophotometric data can also be used in conjunction with the Beer-Lambert Equation, , in order to determine various relationships between transmittance and concentration, and absorbance and concentration.[3]:21-119 Because a spectrophotometer measures the wavelength of a compound through its color, a dye binding substance can be added so that it can undergo a color change and be measured.[18] It is possible to know the concentrations of a two component mixture using the absorption spectra of the standard solutions of each component. To do this, it is necessary to know the extinction coefficient of this mixture at two wave lengths and the extinction coefficients of solutions that contain the known weights of the two components.[19] Spectrophotometers have been developed and improved over decades and have been widely used among chemists. Additionally, Spectrophotometers are specialized to measure either UV or Visible light wavelength absorbance values.[3]:21-119 It is considered to be a highly accurate instrument that is also very sensitive and therefore extremely precise, especially in determining color change.[20] This method is also convenient for use in laboratory experiments because it is an inexpensive and relatively simple process.

UV-visible spectrophotometry

Most 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. The concentration of a protein can be estimated by measuring the OD at 280 nm due to the presence of tryptophan, tyrosine and phenylalanine. This method is not very accurate since the composition of proteins varies greatly and proteins with none of these amino acids do not have maximum absorption at 280 nm. Nucleic acid contamination can also interfere. This method requires a spectrophotometer capable of measuring in the UV region with quartz cuvettes.[3]:135

Ultraviolet-visible (UV-vis) spectroscopy involves energy levels that excite electronic transitions. Absorption of UV-vis light excites molecules that are in ground-states to their excited-states.[5]

Visible region 400–700 nm spectrophotometry is used extensively in colorimetry science. It is a known fact that it operates best at the range of 0.2-0.8 O.D. 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.

METTLER TOLEDO UV5Nano Micro-Volume Spectrophotometer

Samples are usually prepared in cuvettes; depending on the region of interest, they may be constructed of glass, plastic (visible spectrum region of interest), or quartz (Far UV spectrum region of interest). Some applications require small volume measurements which can be performed with micro-volume platforms.


Experimental Application

As described in the applications section, spectrophotometry can be used in both qualitative and quantitative analysis of DNA, RNA, and proteins. Qualitative analysis can be used and spectrophotometers are used to record spectra of compounds by scanning broad wavelength regions to determine the absorbance properties (the intensity of the color) of the compound at each wavelength.[5] One experiment that can demonstrate the various uses that visible spectrophotometry can have is the separation of β-galactosidase from a mixture of various proteins. Largely, spectrophotometry is best used to help quantify the amount of purification your sample has undergone relative to total protein concentration. By running an affinity chromatography, B-Galactosidase can be isolated and tested by reacting collected samples with ONPG and determining if the sample turns yellow.[3]:21-119 Following this testing the sample at 420 nm for specific interaction with ONPG and at 595 for a Bradford Assay the amount of purification can be assessed quantitatively.[3]:21-119 In addition to this spectrophotometry can be used in tandem with other techniques such as SDS-Page electrophoresis in order to purify and isolate various protein samples.

IR spectrophotometry

Spectrophotometers designed for the 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. Applications may include evaluation and categorization of 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


  1. ^ ISO 12647-2: Graphic technology — Process control for the production of halftone colour separations, proof and production prints — Part 2: Offset lithographic processes. Geneva: International Organization for Standardization. 2013. p. 13.
  2. ^ a b Allen, DW; Cooksey, C; Tsai, BK (Nov 13, 2009). "Spectrophotometry". NIST. Retrieved Dec 23, 2018.
  3. ^ a b c d e f g h i j Ninfa AJ, Ballou DP, Benore M (2010). Fundamental Laboratory Approaches for Biochemistry and Biotechnology (2nd ed.). Hoboken: Wiley & Sons. ISBN 9780470087664. OCLC 488246403.
  4. ^ Schwedt G (1997). The essential guide to analytical chemistry. Translated by Brooks H. Chichester, NY: Wiley. pp. 16–17. ISBN 9780471974123. OCLC 36543293.
  5. ^ a b c Ninfa AJ, Ballou DP (2004). Fundamental laboratory approaches for biochemistry and biotechnology. Hoboken: Wiley. p. 66. ISBN 9781891786006. OCLC 633862582.
  6. ^ a b Rendina G (1976). Experimental Methods in Modern Biochemistry. Philadelphia, PA: W. B. Saunders Company. pp. 46–55. ISBN 0721675506. OCLC 147990.
  7. ^ Oke, J. B.; Gunn, J. E. (1983). "Secondary standard stars for absolute spectrophotometry". The Astrophysical Journal. 266: 713. Bibcode:1983ApJ...266..713O. doi:10.1086/160817.
  8. ^ Ishani, G (2006). "The first commercial UV-vis spectrophotometer". The Scientist. p. 100. Retrieved Dec 23, 2018 – via Science In Context.
  9. ^ Simoni, RD; Hill, RL; Vaughan, M; Tabor, H (Dec 5, 2003). "A Classic Instrument: The Beckman DU Spectrophotometer and Its Inventor, Arnold O. Beckman". J. Biol. Chem. 278 (49): e1. ISSN 1083-351X.
  10. ^ Beckman, A. O.; Gallaway, W. S.; Kaye, W.; Ulrich, W. F. (March 1977). "History of spectrophotometry at Beckman Instruments, Inc". Analytical Chemistry. 49 (3): 280A–300A. doi:10.1021/ac50011a001.
  11. ^ "Hewlett Packard: Compound Identification with HP 8450 A UV Visible Spectrophotometer". Analytical Chemistry. 51 (12): 1188A–1189A. 1979-10-01. doi:10.1021/ac50048a728. ISSN 0003-2700.
  12. ^ Ninfa AJ, Ballou DP, Benore M (2015). Fundamental Laboratory Approaches for Biochemistry and Biotechnology (3, rev. ed.). Hoboken, NJ: Wiley & Sons. p. 77. ISBN 9780470924525. OCLC 915641828.
  13. ^ "Fully Automatic Double Beam - Atomic Absorption Spectrophotometer (AA 8000)". Laboratory Equipment. Labindia Analytical Instruments Pvt. Ltd.
  14. ^ "Spectrophotometry Applications and Fundamentals". www.mt.com. Mettler-Toledo International Inc. Retrieved Jul 4, 2018.
  15. ^ Trumbo, Toni A.; Schultz, Emeric; Borland, Michael G.; Pugh, Michael Eugene (April 27, 2013). "Applied Spectrophotometry: Analysis of a Biochemical Mixture". Biochemistry and Molecular Biology Education. 41 (4): 242–50. doi:10.1002/bmb.20694. PMID 23625877.
  16. ^ "FastTrack™ UV/VIS Spectroscopy" (PDF). www.mt.com. Mettler-Toledo AG, Analytical. 2016. Retrieved Dec 23, 2018.
  17. ^ Cortez, C.; Szepaniuk, A.; Gomes da Silva, L. (May 1, 2010). "Exploring Proteins Purification Techniques Animations as Tools for the Biochemistry Teaching". Journal of Biochemistry Education. 8 (2): 12. doi:10.16923/reb.v8i2.215.
  18. ^ Garrett RH, Grisham CM (2013). Biochemistry. Belmont, CA: Cengage. p. 106. ISBN 978-1133106296. OCLC 801650341.
  19. ^ Holiday, Ensor Roslyn (May 27, 1936). "Spectrophotometry of proteins". Biochemical Journal. 30 (10): 1795–1803. doi:10.1042/bj0301795. PMC 1263262. PMID 16746224.
  20. ^ Mavrodineanu R, Schultz JI, Menis O, eds. (1973). Accuracy in Spectrophotometry and Luminescence Measurements: Proceedings. Washington, D.C.: U.S. National Bureau of Standards. p. 2. OCLC 920079.

External links

Absorption (chemistry)

In chemistry, absorption is a physical or chemical phenomenon or a process in which atoms, molecules or ions enter some bulk phase – liquid or solid material. This is a different process from adsorption, since molecules undergoing absorption are taken up by the volume, not by the surface (as in the case for adsorption). A more general term is sorption, which covers absorption, adsorption, and ion exchange. Absorption is a condition in which something takes in another substance.In many processes important in technology, the chemical absorption is used in place of the physical process, e.g., absorption of carbon dioxide by sodium hydroxide – such acid-base processes do not follow the Nernst partition law.

For some examples of this effect, see liquid-liquid extraction. It is possible to extract from one liquid phase to another a solute without a chemical reaction. Examples of such solutes are noble gases and osmium tetroxide.The process of absorption means that a substance captures and transforms energy. The absorbent distributes the material it captures throughout whole and adsorbent only distributes it through the surface.

The process of gas or liquid which penetrate into the body of adsorbent is commonly known as absorption.

Atomic absorption spectroscopy

Atomic absorption spectroscopy (AAS) and atomic emission spectroscopy (AES) is a spectroanalytical procedure for the quantitative determination of chemical elements using the absorption of optical radiation (light) by free atoms in the gaseous state. Atomic absorption spectroscopy is based on absorption of light by free metallic ions.

In analytical chemistry the technique is used for determining the concentration of a particular element (the analyte) in a sample to be analyzed. AAS can be used to determine over 70 different elements in solution, or directly in solid samples via electrothermal vaporization, and is used in pharmacology, biophysics,

archaeology and toxicology research.

Atomic emmission spectroscopy was first used as an analytical technique, and the underlying principles were established in the second half of the 19th century by Robert Wilhelm Bunsen and Gustav Robert Kirchhoff, both professors at the University of Heidelberg, Germany.The modern form of AAS was largely developed during the 1950s by a team of Australian chemists. They were led by Sir Alan Walsh at the Commonwealth Scientific and Industrial Research Organisation (CSIRO), Division of Chemical Physics, in Melbourne, Australia.Atomic absorption spectrometry has many uses in different areas of chemistry such as clinical analysis of metals in biological fluids and tissues such as whole blood, plasma, urine, saliva, brain tissue, liver, hair, muscle tissue, semen, in some pharmaceutical manufacturing processes, minute quantities of a catalyst that remain in the final drug product, and analyzing water for its metal content.

Beer–Lambert law

The Beer–Lambert law, also known as Beer's law, the Lambert–Beer law, or the Beer–Lambert–Bouguer law relates the attenuation of light to the properties of the material through which the light is travelling. The law is commonly applied to chemical analysis measurements and used in understanding attenuation in physical optics, for photons, neutrons, or rarefied gases. In mathematical physics, this law arises as a solution of the BGK equation.

Cell counting

Cell counting is any of various methods for the counting or similar quantification of cells in the life sciences, including medical diagnosis and treatment. It is an important subset of cytometry, with applications in research and clinical practice. For example, the complete blood count can help a physician to determine why a patient feels unwell and what to do to help. Cell counts within liquid media (such as blood, plasma, lymph, or laboratory rinsate) are usually expressed as a number of cells per unit of volume, thus expressing a concentration (for example, 5,000 cells per milliliter).

Clinical chemistry

Clinical chemistry (also known as chemical pathology, clinical biochemistry or medical biochemistry) is the area of chemistry that is generally concerned with analysis of bodily fluids for diagnostic and therapeutic purposes. It is an applied form of biochemistry (not to be confused with medicinal chemistry, which involves basic research for drug development).

The discipline originated in the late 19th century with the use of simple chemical reaction tests for various components of blood and urine. In the many decades since, other techniques have been applied as science and technology have advanced, including the use and measurement of enzyme activities, spectrophotometry, electrophoresis, and immunoassay. There are now many blood tests and clinical urine tests with extensive diagnostic capabilities.

Most current laboratories are now highly automated to accommodate the high workload typical of a hospital laboratory. Tests performed are closely monitored and quality controlled.

All biochemical tests come under chemical pathology. These are performed on any kind of body fluid, but mostly on serum or plasma. Serum is the yellow watery part of blood that is left after blood has been allowed to clot and all blood cells have been removed. This is most easily done by centrifugation, which packs the denser blood cells and platelets to the bottom of the centrifuge tube, leaving the liquid serum fraction resting above the packed cells. This initial step before analysis has recently been included in instruments that operate on the "integrated system" principle. Plasma is in essence the same as serum, but is obtained by centrifuging the blood without clotting. Plasma is obtained by centrifugation before clotting occurs. The type of test required dictates what type of sample is used.

A large medical laboratory will accept samples for up to about 700 different kinds of tests. Even the largest of laboratories rarely do all these tests themselves, and some must be referred to other labs.

This large array of tests can be categorised into sub-specialities of:

General or routine chemistry – commonly ordered blood chemistries (e.g., liver and kidney function tests).

Special chemistry - elaborate techniques such as electrophoresis, and manual testing methods.

Clinical endocrinology – the study of hormones, and diagnosis of endocrine disorders.

Toxicology – the study of drugs of abuse and other chemicals.

Therapeutic Drug Monitoring – measurement of therapeutic medication levels to optimize dosage.

Urinalysis – chemical analysis of urine for a wide array of diseases, along with other fluids such as CSF and effusions

Fecal analysis – mostly for detection of gastrointestinal disorders.


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 tristimulus values and related quantities.


Cytochemistry is the biochemistry of cells, especially that of the macromolecules responsible for cell structure and function. The term is also used to describe a process of identification of the biochemical content of cells. Cytochemistry is a science of localizing chemical components of cells and cell organelles on thin histological sections by using several techniques like enzyme localization, micro-incineration, micro-spectrophotometry, radioautography, cryo-electron microscopy, X-ray microanalysis by energy-dispersive X-ray spectroscopy, immunohistochemistry and cytochemistry, etc.


FLiBe is a molten salt made from a mixture of lithium fluoride (LiF) and beryllium fluoride (BeF2). It is both a nuclear reactor coolant and solvent for fertile or fissile material. It served both purposes in the Molten-Salt Reactor Experiment (MSRE).

The 2:1 mixture forms a stoichiometric compound, Li2BeF4, which has a melting point of 459 °C, a boiling point of 1430 °C, and a density of 1.94 g/cm3. Its volumetric heat capacity is 4540 kJ/m3K, which is similar to that of water, more than four times that of sodium, and more than 200 times that of helium at typical reactor conditions. Its appearance is white to transparent, with crystalline grains in a solid state, morphing into a completely clear liquid upon melting. However, soluble fluorides such as UF4 and NiF2, can dramatically change the color salt in both solid and liquid state. This made spectrophotometry a viable analysis tool, and it was employed extensively during the MSRE operations.The eutectic mixture is slightly greater than 50% BeF2 and has a melting point of 360 °C. This mixture was never used in practice due to the overwhelming increase in viscosity caused by the BeF2 addition in the eutectic mixture. BeF2, which behaves as a glass, is only fluid in salt mixtures containing enough molar percent of Lewis base. Lewis bases, such as the alkali fluorides, will donate fluoride ions to the beryllium, breaking the glassy bonds which increase viscosity. In FLiBe, beryllium fluoride is able to sequester two fluoride ions from two lithium fluorides in a liquid state, converting it into the tetrafluorberyllate ion BeF4−2.


Geoarchaeology is a multi-disciplinary approach which uses the techniques and subject matter of geography, geology and other Earth sciences to examine topics which inform archaeological knowledge and thought. Geoarchaeologists study the natural physical processes that affect archaeological sites such as geomorphology, the formation of sites through geological processes and the effects on buried sites and artifacts post-deposition. Geoarchaeologists' work frequently involves studying soil and sediments as well as other geographical concepts to contribute an archaeological study. Geoarchaeologists may also use computer cartography, geographic information systems (GIS) and digital elevation models (DEM) in combination with disciplines from human and social sciences and earth sciences. Geoarchaeology is important to society because it informs archaeologists about the geomorphology of the soil, sediments and the rocks on the buried sites and artifacts they're researching on. By doing this we are able locate ancient cities and artifacts and estimate by the quality of soil how "prehistoric" they really are.

Harry Hemley Plaskett

Harry Hemley Plaskett FRS (July 5, 1893 – January 26, 1980) was a Canadian astronomer who made significant contributions to the fields of solar physics, astronomical spectroscopy and spectrophotometry. From 1932 to 1960, he served as the Savilian Professor of Astronomy at the University of Oxford, and in 1963 was awarded the Gold Medal of the Royal Astronomical Society.

Infrared spectroscopy

Infrared spectroscopy (IR spectroscopy or vibrational spectroscopy) involves the interaction of infrared radiation with matter. It covers a range of techniques, mostly based on absorption spectroscopy. As with all spectroscopic techniques, it can be used to identify and study chemicals. Samples may be solid, liquid, or gas. The method or technique of infrared spectroscopy is conducted with an instrument called an infrared spectrometer (or spectrophotometer) to produce an infrared spectrum. An IR spectrum can be visualized in a graph of infrared light absorbance (or transmittance) on the vertical axis vs. frequency or wavelength on the horizontal axis. Typical units of frequency used in IR spectra are reciprocal centimeters (sometimes called wave numbers), with the symbol cm−1. Units of IR wavelength are commonly given in micrometers (formerly called "microns"), symbol μm, which are related to wave numbers in a reciprocal way. A common laboratory instrument that uses this technique is a Fourier transform infrared (FTIR) spectrometer. Two-dimensional IR is also possible as discussed below.

The infrared portion of the electromagnetic spectrum is usually divided into three regions; the near-, mid- and far- infrared, named for their relation to the visible spectrum. The higher-energy near-IR, approximately 14000–4000 cm−1 (0.7–2.5 μm wavelength) can excite overtone or harmonic vibrations. The mid-infrared, approximately 4000–400 cm−1 (2.5–25 μm) may be used to study the fundamental vibrations and associated rotational-vibrational structure. The far-infrared, approximately 400–10 cm−1 (25–1000 μm), lying adjacent to the microwave region, has low energy and may be used for rotational spectroscopy. The names and classifications of these subregions are conventions, and are only loosely based on the relative molecular or electromagnetic properties.

Matthew Pothen Thekaekara

Rev. Dr. Matthew Pothen Thekaekara (1914–1974) was a scientist and author of many books and papers relating to spectrophotometry and the solar constant besides works on theology.He was instrumental in publishing some of the earliest AM0 spectra, which is a model spectrum of the sun in space. The historic 1973 Thekaekara spectrum was the basis for ASTM E490 (American Society for Testing and Materials Standard Solar Constant and Zero Air Mass Solar Spectral Irradiance Table) from 1974 to 2000, when it was replaced by the most recent AM0 upgrade, in ASTM E490-00.

Recent publications such as a 2007 paper authored by fellow Malayali scientist P. Shahmugan made extensive reference to the Thekaekara spectrum. In 2008 a paper by authors from NASA Goddard Space Flight Center and UC Laboratory for Atmospheric and Space Physics also made extensive application of the Thekaekara spectrum.


Microspectrophotometry is the measure of the spectra of microscopic samples using different wavelengths of electromagnetic radiation (e.g. ultraviolet, visible and near infrared, etc.) It is accomplished with microspectrophotometers, cytospectrophotometers, microfluorometers, Raman microspectrophotometers, etc. A microspectrophotometer can be configured to measure transmittance, absorbance, reflectance, light polarization, fluorescence (or other types of luminescence such as photoluminescence) of sample areas less than a micrometer in diameter through a modified optical microscope.

Nuclear Institute for Food and Agriculture

The Nuclear Institute for Food and Agriculture, known as NIFA, is one of four agriculture and food irradiation research institute managed by the Pakistan Atomic Energy Commission. The institute is tasked to carry out research in Crop production and protection, soil fertility, water management and conservation and value addition of food resources, employing nuclear and other contemporary techniques.

NIFA was the brainchild of Ishrat Hussain Usmani, bureaucrat and chairman of the Pakistan Atomic Energy Commission, however due to economic difficulties, the plans were not carried out until the 1980s. In 1982, Munir Ahmad Khan led the establishment of the institute and its first director was Abdul Rashid who revolutionized the institute.

The NIFA administers cobalt-60 radiation source, Laser absorption spectrometer and Atomic Absorption Spectrophotometry, Near-infrared spectrometer and Ultraviolet–visible spectroscopy.

A library was opened in 1990, and recently, the institute has acquired 75 acres of land at CHASNUPP-I site.


Photometry can refer to:

Photometry (optics), the science of measurement of visible light in terms of its perceived brightness to human vision

Photometry (astronomy), the measurement of the flux or intensity of an astronomical object's electromagnetic radiation

Spectrophotometry, the measurement of spectral distribution along with the flux or intensity

A photometric study, sometimes also referred to as a lighting "layout" or "point by point"

Photometric stereo, a computer vision technique for estimating 3D shape from one or more images.

Photometry (astronomy)

Photometry is a technique of astronomy concerned with measuring the flux, or intensity of an astronomical object's electromagnetic radiation. When photometry is performed over broad wavelength bands of radiation, where not only the amount of radiation but also its spectral distribution is measured, the term spectrophotometry is used.

The word is composed of the Greek affixes photo- ("light") and -metry ("measure").

Ultraviolet–visible spectroscopy

Ultraviolet–visible spectroscopy or ultraviolet–visible spectrophotometry (UV–Vis or UV/Vis) refers to absorption spectroscopy or reflectance spectroscopy in part of the ultraviolet and the full, adjacent visible spectral regions. This means it uses light in the visible and adjacent ranges. The absorption or reflectance in the visible range directly affects the perceived color of the chemicals involved. In this region of the electromagnetic spectrum, atoms and molecules undergo electronic transitions. Absorption spectroscopy is complementary to fluorescence spectroscopy, in that fluorescence deals with transitions from the excited state to the ground state, while absorption measures transitions from the ground state to the excited state.

William Michael Herbert Greaves

Prof William Michael Herbert Greaves FRS FREng FRSE (10 September 1897 – 24 December 1955) was a British astronomer. He is most noted for his work on stellar spectrophotometry.


Xanthochromia, from the Greek xanthos (ξανθός) "yellow" and chroma (χρώμα) "colour", is the yellowish appearance of cerebrospinal fluid that occurs several hours after bleeding into the subarachnoid space caused by certain medical conditions, most commonly subarachnoid hemorrhage. Its presence can be determined by either spectrophotometry (measuring the absorption of particular wavelengths of light) or simple visual examination. It is unclear which method is superior.

Science instruments on satellites and spacecraft
Radio science

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