Editing Spectrophotometry (section)

==Design==
[[File:Spetrophotometer-en.svg|thumb|upright=2.0|Single-beam scanning spectrophotometer|alt=]]
There are two major classes of devices: single-beam and double-beam. A double-beam spectrophotometer<ref>{{Cite web|url=http://www.labindia-analytical.com/fully-automatic-atomic-absorption-spectrophotometer-aa8000.html|title=Fully Automatic Double Beam - Atomic Absorption Spectrophotometer (AA 8000)|website=Laboratory Equipment|publisher=Labindia Analytical Instruments Pvt. Ltd.|access-date=2018-01-31|archive-date=2018-12-02|archive-url=https://web.archive.org/web/20181202171041/http://www.labindia-analytical.com/fully-automatic-atomic-absorption-spectrophotometer-aa8000.html|url-status=dead}}</ref> 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 [[Ultraviolet-visible spectroscopy#Microspectrophotometry|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 meter squared usually) of the transmitted or reflected light is measured with a photodiode, CCD or other [[photodetector|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:
# 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.
# Fractions of the incident wavelengths are transmitted through, or reflected from, the sample and the reference.
# The resultant light strikes the [[photodetector]] device, which compares the relative intensity of the two beams.
# Electronic circuits convert the relative currents into linear transmission percentages or absorbance or concentration values.

In an array spectrophotometer, the sequence is as follows:<ref>{{Cite web|url=https://www.mt.com/us/en/home/library/guides/laboratory-division/1/uvvis-spectrophotometry-guide-applications-fundamentals.html|title=Spectrophotometry Applications and Fundamentals|website=www.mt.com|publisher=Mettler-Toledo International Inc.|language=en-US|access-date=Jul 4, 2018}}</ref>

# The light source is shone into the sample and focused into a slit
# The transmitted light is refracted into a rainbow with the reflection grating
# The resulting light strikes the photodetector device which compares the intensity of the beam
# 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 is 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.<ref name=":3" />

===Types of spectrophotometers===
Some common types of spectrophotometers include the following:<ref>{{cite web | url=https://www.dnatestingexperts.com/spectrometer-vs-spectrophotometer-whats-the-difference/ | title=Spectrometer vs Spectrophotometer: What's the Difference? | date=14 January 2024 }}</ref>
* [[Ultraviolet–visible spectroscopy|'''UV-Vis spectrophotometer''']]: Measures light absorption in UV and visible ranges (200-800&nbsp;nm). Used for quantification of many inorganic and organic compounds.
* [[Infrared spectroscopy|'''Infrared spectrophotometer''']]: Measures infrared light absorption, allowing identification of chemical bonds and functional groups.
* [[Atomic absorption spectroscopy|'''Atomic absorption spectrophotometer (AAS)''']]: Uses absorption of light by vaporized analyte atoms to determine concentrations of metals and metalloids.
* [[Fluorescence spectroscopy|'''Fluorescence spectrophotometer''']]: Measures intensity of fluorescent light emitted from samples after excitation. Allows highly sensitive analysis of samples with native or induced fluorescence.
* [[Colorimeter (chemistry)|'''Colorimeter''']]: Simple spectrophotometers used to measure light absorption for colorimetric assays and tests.

===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.<ref>{{Cite journal|last1=Trumbo|first1=Toni A.|last2=Schultz|first2=Emeric|last3=Borland|first3=Michael G.|last4=Pugh|first4=Michael Eugene|date=April 27, 2013|title=Applied Spectrophotometry: Analysis of a Biochemical Mixture|journal=Biochemistry and Molecular Biology Education|volume=41|issue=4|pages=242–50|doi=10.1002/bmb.20694|pmid=23625877|doi-access=}}</ref> Since samples in these applications are not readily available in large quantities, they are especially suited to be 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.<ref>{{Cite web|url=https://www.mt.com/dam/Analytical/uv-vis/uv-pdf/uvbio-ds/30255608B_V11.16_UVBio_Datasheet_RedDot_EN_LR.pdf|title=FastTrack™ UV/VIS Spectroscopy|date=2016|website=www.mt.com|publisher=Mettler-Toledo AG, Analytical|access-date=Dec 23, 2018}}</ref> 
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 Coomassie Brilliant Blue G-250 dye measured at 595&nbsp;nm or by an enzymatic reaction as seen between β-galactosidase and ONPG (turns sample yellow) measured at 420&nbsp; nm.<ref name=":0" />{{Rp|21–119}} The spectrophotometer is used to measure colored compounds in the visible region of light (between 350&nbsp;nm and 800&nbsp;nm),<ref name=":0">{{Cite book|title=Fundamental Laboratory Approaches for Biochemistry and Biotechnology|last1=Ninfa|first1=Alexander J.|last2=Ballou|first2=David P.|last3=Benore|first3=Marilee|publisher=Wiley & Sons|year=2010|isbn=9780470087664|edition=2nd|location=Hoboken|oclc=488246403|name-list-style=vanc}}</ref>{{Rp|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 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.<ref name=":0" />{{Rp|21–119}} Spectrophotometry is also a helpful process for protein purification<ref>{{Cite journal|last1=Cortez|first1=C.|last2=Szepaniuk|first2=A.|last3=Gomes da Silva|first3=L.|date=May 1, 2010|title=Exploring Proteins Purification Techniques Animations as Tools for the Biochemistry Teaching.|journal=Journal of Biochemistry Education|volume=8|issue=2|pages=12|doi=10.16923/reb.v8i2.215|doi-access=free}}</ref> 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, <math display="inline">A=-\log_{10}T=\epsilon cl=OD</math>, to determine various relationships between transmittance and concentration, and absorbance and concentration.<ref name=":0" />{{Rp|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.<ref>{{Cite book|title=Biochemistry|last1=Garrett|first1=Reginald H.|last2=Grisham|first2=Charles M.|publisher=[[Cengage]]|year=2013|isbn=978-1133106296|location=Belmont, CA|pages=106|oclc=801650341|name-list-style=vanc}}</ref> 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 wavelengths and the extinction coefficients of solutions that contain the known weights of the two components.<ref>{{Cite journal|last=Holiday|first=Ensor Roslyn|date=May 27, 1936|title=Spectrophotometry of proteins|journal=Biochemical Journal|volume=30|issue=10|pages=1795–1803|doi=10.1042/bj0301795|pmc=1263262|pmid=16746224}}</ref> In addition to the traditional Beer-Lamberts law model, cuvette based label free spectroscopy can be used, which add an optical filter in the pathways of the light, enabling the spectrophotometer to quantify concentration, size and refractive index of samples following the hands law.<ref>{{Cite journal|url=https://aip.scitation.org/doi/10.1063/1.4928548|doi = 10.1063/1.4928548|title = Refractive index dispersion sensing using an array of photonic crystal resonant reflectors|year = 2015|last1 = Hermannsson|first1 = Pétur G.|last2 = Vannahme|first2 = Christoph|last3 = Smith|first3 = Cameron L. C.|last4 = Sørensen|first4 = Kristian T.|last5 = Kristensen|first5 = Anders|journal = Applied Physics Letters|volume = 107|issue = 6|page = 061101|bibcode = 2015ApPhL.107f1101H| s2cid=62897708 }}</ref> 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.<ref name=":0" />{{Rp|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.<ref>{{Cite book|url=https://babel.hathitrust.org/cgi/pt?id=mdp.39015077585209;view=1up;seq=16|title=Accuracy in Spectrophotometry and Luminescence Measurements: Proceedings|series=U.S. Department of Commerce National Bureau of Standards special publication; 378|publisher=U.S. National Bureau of Standards|year=1973|editor-last=Mavrodineanu|editor-first=Radu|location=Washington, D.C.|pages=2|oclc=920079|name-list-style=vanc|editor-last2=Schultz|editor-first2=J. I.|editor-last3=Menis|editor-first3=Oscar}}</ref> This method is also convenient for use in laboratory experiments because it is an inexpensive and relatively simple process.