Editing Ultraviolet–visible spectroscopy (section)

==Applications==
[[File:Bis(triphenylphosphine) nickel (II) chloride UV-vis.JPG|thumb|right|300px|An example of a UV-Vis readout]]

UV-Vis can be used to monitor structural changes in DNA.<ref name=":0">{{Cite journal |last1=Carroll |first1=Gregory T. |last2=Dowling |first2=Reed C. |last3=Kirschman |first3=David L. |last4=Masthay |first4=Mark B. |last5=Mammana |first5=Angela |date=2023 |title=Intrinsic fluorescence of UV-irradiated DNA |url=https://linkinghub.elsevier.com/retrieve/pii/S1010603022007079 |journal=Journal of Photochemistry and Photobiology A: Chemistry |language=en |volume=437 |pages=114484 |doi=10.1016/j.jphotochem.2022.114484 |s2cid=254622477|url-access=subscription }}</ref> 

UV-Vis spectroscopy is routinely used in [[analytical chemistry]] for the [[quantitative analysis (chemistry)|quantitative]] determination of diverse analytes or sample, such as [[transition metal]] ions, highly [[Conjugated system|conjugated]] [[organic compound]]s, and biological macromolecules. Spectroscopic analysis is commonly carried out in solutions but solids and gases may also be studied.
*[[Organic compound]]s, especially those with a high degree of [[conjugated system|conjugation]], also absorb light in the UV or visible regions of the [[electromagnetic spectrum]]. The solvents for these determinations are often water for water-soluble compounds, or [[ethanol]] for organic-soluble compounds. (Organic solvents may have significant UV absorption; not all solvents are suitable for use in UV spectroscopy. Ethanol absorbs very weakly at most wavelengths.) Solvent polarity and pH can affect the absorption spectrum of an organic compound. [[Tyrosine]], for example, increases in absorption maxima and molar extinction coefficient when pH increases from 6 to 13 or when solvent polarity decreases.
*While [[charge transfer complexes]] also give rise to colours, the colours are often too intense to be used for quantitative measurement.

The [[Beer–Lambert law]] states that the absorbance of a solution is directly proportional to the concentration of the absorbing species in the solution and the path length.<ref>{{cite web |last= Metha |first= Akul |title= Derivation of Beer–Lambert Law |website= PharmaXChange.info |date= 22 Apr 2012 |url= http://pharmaxchange.info/press/2012/04/ultraviolet-visible-uv-vis-spectroscopy-%e2%80%93-derivation-of-beer-lambert-law/}}</ref> Thus, for a fixed path length, UV-Vis spectroscopy can be used to determine the concentration of the absorber in a solution. It is necessary to know how quickly the absorbance changes with concentration. This can be taken from references (tables of [[molar extinction coefficients]]), or more accurately, determined from a [[calibration curve]].

A UV-Vis spectrophotometer may be used as a detector for [[High-performance liquid chromatography|'''HPLC''']]. The presence of an analyte gives a response assumed to be proportional to the concentration. For accurate results, the instrument's response to the analyte in the unknown should be compared with the response to a standard; this is very similar to the use of calibration curves. The response (e.g., peak height) for a particular concentration is known as the [[response factor]].

The wavelengths of absorption peaks can be correlated with the types of bonds in a given molecule and are valuable in determining the functional groups within a molecule. The [[Woodward–Fieser rules]], for instance, are a set of empirical observations used to predict λ<sub>max</sub>, the wavelength of the most intense UV-Vis absorption, for conjugated organic compounds such as [[diene]]s and [[ketone]]s. The spectrum alone is not, however, a specific test for any given sample. The nature of the solvent, the pH of the solution, temperature, high electrolyte concentrations, and the presence of interfering substances can influence the absorption spectrum. Experimental variations such as the slit width (effective bandwidth) of the spectrophotometer will also alter the spectrum. To apply UV-Vis spectroscopy to analysis, these variables must be controlled or accounted for in order to identify the substances present.<ref>{{cite book |title=Ultraviolet Spectroscopy and UV Lasers |editor-link=Prabhakar Misra |editor-first=Prabhakar |editor-last=Misra |editor2-first=Mark |editor2-last=Dubinskii |publisher=[[Marcel Dekker]] |location=New York |year=2002 |isbn=978-0-8247-0668-5 }}{{pn|date=March 2020}}</ref>

The method is most often used in a quantitative way to determine concentrations of an absorbing species in solution, using the [[Beer–Lambert law]]:<ref>{{Cite web |date=2013-10-03 |title=The Beer-Lambert Law |url=https://chem.libretexts.org/Bookshelves/Physical_and_Theoretical_Chemistry_Textbook_Maps/Supplemental_Modules_(Physical_and_Theoretical_Chemistry)/Spectroscopy/Electronic_Spectroscopy/Electronic_Spectroscopy_Basics/The_Beer-Lambert_Law |access-date=2023-10-19 |website=Chemistry LibreTexts |language=en}}</ref>
:<math>A=\log_{10}(I_0/I)=\varepsilon c L</math>,

where ''A'' is the measured [[absorbance]] (formally dimensionless but generally reported in absorbance units (AU)<ref>Historically, the term "Optical Density" (OD) was used instead of AU. But it is also worth noting that what is usually measured is percent transmission (%T), a linear ratio, which is converted to the logarithm by the instrument for presentation.</ref>), <math>I_0</math> is the intensity of the incident light at a given [[wavelength]], <math>I</math> is the transmitted intensity, ''L'' the path length through the sample, and ''c'' the [[concentration]] of the absorbing species. For each species and wavelength, ε is a constant known as the [[molar absorptivity]] or extinction coefficient. This constant is a fundamental molecular property in a given solvent, at a particular temperature and pressure, and has units of <math>1/M*cm</math>.

The absorbance and extinction ''ε'' are sometimes defined in terms of the [[natural logarithm]] instead of the base-10 logarithm.

The Beer–Lambert law is useful for characterizing many compounds but does not hold as a universal relationship for the concentration and absorption of all substances. A 2nd order polynomial relationship between absorption and concentration is sometimes encountered<ref>{{Cite journal |last=Bozdoğan |first=Abdürrezzak E. |date=2022-11-01 |title=Polynomial Equations based on Bouguer–Lambert and Beer Laws for Deviations from Linearity and Absorption Flattening |url=https://doi.org/10.1134/S1061934822110028 |journal=Journal of Analytical Chemistry |language=en |volume=77 |issue=11 |pages=1426–1432 |doi=10.1134/S1061934822110028 |s2cid=253463022 |issn=1608-3199|url-access=subscription }}</ref> for very large, complex molecules such as [[organic dye]]s ([[xylenol orange]] or [[neutral red]], for example).<ref name="dev" /><ref>{{Cite journal |last1=Cinar |first1=Mehmet |last2=Coruh |first2=Ali |last3=Karabacak |first3=Mehmet |date=2014-03-25 |title=A comparative study of selected disperse azo dye derivatives based on spectroscopic (FT-IR, NMR and UV–Vis) and nonlinear optical behaviors |url=https://www.sciencedirect.com/science/article/pii/S1386142513014236 |journal=Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy |volume=122 |pages=682–689 |doi=10.1016/j.saa.2013.11.106 |pmid=24345608 |bibcode=2014AcSpA.122..682C |issn=1386-1425|url-access=subscription }}</ref>

UV–Vis spectroscopy is also used in the semiconductor industry to measure the thickness and optical properties of thin films on a wafer. UV–Vis spectrometers are used to measure the reflectance of light, and can be analyzed via the [[Forouhi–Bloomer model|Forouhi–Bloomer dispersion equations]] to determine the index of refraction (<math>n</math>) and the extinction coefficient (<math>k</math>) of a given film across the measured spectral range.<ref name = "Löper2015">{{cite journal | last1 = Löper | first1 = Philipp | last2 = Stuckelberger | first2 = Michael | last3 = Niesen | first3 = Bjoern | last4 = Werner | first4 = Jérémie | last5 = Filipič | first5 = Miha | last6 = Moon | first6 = Soo-Jin | last7 = Yum | first7 = Jun-Ho | last8 = Topič | first8 = Marko | last9 = De Wolf | first9 = Stefaan | last10 = Ballif | first10 = Christophe | title = Complex Refractive Index Spectra of CH3NH3PbI3 Perovskite Thin Films Determined by Spectroscopic Ellipsometry and Spectrophotometry | journal = The Journal of Physical Chemistry Letters | volume = 6 | issue = 1 | pages = 66–71 | year = 2015 | url = https://doi.org/10.1021/jz502471h | doi = 10.1021/jz502471h | pmid = 26263093 | access-date = 2021-11-16| url-access = subscription }}</ref>

=== Practical considerations ===
The Beer–Lambert law has implicit assumptions that must be met experimentally for it to apply; otherwise there is a possibility of deviations from the law.<ref name=dev>{{cite web |last= Metha |first= Akul |title= Limitations and Deviations of Beer–Lambert Law |website= PharmaXChange.info |date= 14 May 2012 |url= http://pharmaxchange.info/press/2012/05/ultraviolet-visible-uv-vis-spectroscopy-%e2%80%93-limitations-and-deviations-of-beer-lambert-law/}}</ref> For instance, the chemical makeup and physical environment of the sample can alter its extinction coefficient. The chemical and physical conditions of a test sample therefore must match reference measurements for conclusions to be valid. Worldwide, pharmacopoeias such as the American (USP) and European (Ph. Eur.) pharmacopeias demand that spectrophotometers perform according to strict regulatory requirements encompassing factors such as [[#Stray light|stray light]]<ref>{{Cite web | url=https://www.mt.com/ch/en/home/library/white-papers/lab-analytical-instruments/stray-light-and-performance-verification.html | title=Stray Light and Performance Verification}}</ref> and wavelength accuracy.<ref>{{Cite web | url=https://www.chemeurope.com/en/whitepapers/126586/wavelength-accuracy-in-uv-vis-spectrophotometry.html | title=Wavelength Accuracy in UV/VIS Spectrophotometry}}</ref>

====Spectral bandwidth====
Spectral bandwidth of a spectrophotometer is the range of wavelengths that the instrument transmits through a sample at a given time.<ref>{{Cite web |title=Persee PG Scientific Inc. – New-UV FAQ: Spectral Band Width |url=http://www.perseena.com/index/newsinfo/c_id/37/n_id/7.html |archive-url=https://web.archive.org/web/20180921065923/http://www.perseena.com/index/newsinfo/c_id/37/n_id/7.html |url-status=usurped |archive-date=21 September 2018 |access-date= |website=www.perseena.com}}</ref> It is determined by the light source, the [[monochromator]], its physical slit-width and optical dispersion and the detector of the spectrophotometer. The spectral bandwidth affects the resolution and accuracy of the measurement. A narrower spectral bandwidth provides higher resolution and accuracy, but also requires more time and energy to scan the entire spectrum. A wider spectral bandwidth allows for faster and easier scanning, but may result in lower resolution and accuracy, especially for samples with overlapping absorption peaks. Therefore, choosing an appropriate spectral bandwidth is important for obtaining reliable and precise results.

It is important to have a monochromatic source of radiation for the light incident on the sample cell to enhance the linearity of the response.<ref name="dev" /> The closer the bandwidth is to be monochromatic (transmitting unit of wavelength) the more linear will be the response. The spectral bandwidth is measured as the number of wavelengths transmitted at half the maximum intensity of the light leaving the monochromator. 

The best spectral [[bandwidth (signal processing)#Photonics|bandwidth]] achievable is a specification of the UV spectrophotometer, and it characterizes how [[monochromatic]] the incident light can be. If this bandwidth is comparable to (or more than) the [[spectral linewidth|width]] of the absorption peak of the sample component, then the measured extinction coefficient will not be accurate. In reference measurements, the instrument bandwidth (bandwidth of the incident light) is kept below the width of the spectral peaks. When a test material is being measured, the bandwidth of the incident light should also be sufficiently narrow. Reducing the spectral bandwidth reduces the energy passed to the detector and will, therefore, require a longer measurement time to achieve the same signal to noise ratio.

====Wavelength error====
The extinction coefficient of an analyte in solution changes gradually with wavelength. A peak (a wavelength where the absorbance reaches a maximum) in the absorbance curve vs wavelength, i.e. the UV-VIS spectrum, is where the rate of change of absorbance with wavelength is the lowest.<ref name=dev /> Therefore, quantitative measurements of a solute are usually conducted, using a wavelength around the absorbance peak, to minimize inaccuracies produced by errors in wavelength, due to the change of extinction coefficient with wavelength.

====Stray light====
{{See also|Stray light}}
Stray light<ref>{{Cite web |date=2015-06-12 |title=What is Stray light and how it is monitored? |url=https://lab-training.com/what-is-stray-light-and-how-it-is-monitored/ |access-date= |language=en-US}}</ref> in a UV spectrophotometer is any light that reaches its detector that is not of the wavelength selected by the monochromator. This can be caused, for instance, by scattering of light within the instrument, or by reflections from optical surfaces.

Stray light can cause significant errors in absorbance measurements, especially at high absorbances, because the stray light will be added to the signal detected by the detector, even though it is not part of the actually selected wavelength. The result is that the measured and reported absorbance will be lower than the actual absorbance of the sample.

The stray light is an important factor, as it determines the ''purity'' of the light used for the analysis. The most important factor affecting it is the [[Monochromator#Stray light|''stray light'' level of the monochromator]].<ref name="dev" />

Typically a detector used in a UV-VIS spectrophotometer is broadband; it responds to all the light that reaches it. If a significant amount of the light passed through the sample contains wavelengths that have much lower extinction coefficients than the nominal one, the instrument will report an incorrectly low absorbance. Any instrument will reach a point where an increase in sample concentration will not result in an increase in the reported absorbance, because the detector is simply responding to the stray light. In practice the concentration of the sample or the optical path length must be adjusted to place the unknown absorbance within a range that is valid for the instrument. Sometimes an empirical calibration function is developed, using known concentrations of the sample, to allow measurements into the region where the instrument is becoming non-linear.

As a rough guide, an instrument with a single monochromator would typically have a stray light level corresponding to about 3 Absorbance Units (AU), which would make measurements above about 2 AU problematic. A more complex instrument with a [[Monochromator#Double monochromators|double monochromator]] would have a stray light level corresponding to about 6 AU, which would therefore allow measuring a much wider absorbance range.

====Deviations from the Beer–Lambert law====
At sufficiently high concentrations, the absorption bands will saturate and show absorption flattening. The absorption peak appears to flatten because close to 100% of the light is already being absorbed. The concentration at which this occurs depends on the particular compound being measured. One test that can be used to test for this effect is to vary the path length of the measurement. In the Beer–Lambert law, varying concentration and path length has an equivalent effect—diluting a solution by a factor of 10 has the same effect as shortening the path length by a factor of 10. If cells of different path lengths are available, testing if this relationship holds true is one way to judge if absorption flattening is occurring.

Solutions that are not homogeneous can show deviations from the Beer–Lambert law because of the phenomenon of absorption flattening. This can happen, for instance, where the absorbing substance is located within suspended particles.<ref>{{cite journal |last1=Berberan-Santos |first1=M. N. |title=Beer's law revisited |journal=Journal of Chemical Education |date=September 1990 |volume=67 |issue=9 |pages=757 |doi=10.1021/ed067p757 |bibcode=1990JChEd..67..757B }}</ref><ref>{{cite journal |last1=Wittung |first1=Pernilla |last2=Kajanus |first2=Johan |last3=Kubista |first3=Mikael |last4=Malmström |first4=Bo G. |title=Absorption flattening in the optical spectra of liposome-entrapped substances |journal=FEBS Letters |date=19 September 1994 |volume=352 |issue=1 |pages=37–40 |doi=10.1016/0014-5793(94)00912-0 |pmid=7925937 |s2cid=11419856 |doi-access= }}</ref> The deviations will be most noticeable under conditions of low concentration and high absorbance. The last reference describes a way to correct for this deviation.

Some solutions, like copper(II) chloride in water, change visually at a certain concentration because of changed conditions around the coloured ion (the divalent copper ion). For copper(II) chloride it means a shift from blue to green,<ref>{{cite journal |last1=Ansell |first1=S |last2=Tromp |first2=R H |last3=Neilson |first3=G W |title=The solute and aquaion structure in a concentrated aqueous solution of copper(II) chloride |journal=Journal of Physics: Condensed Matter |date=20 February 1995 |volume=7 |issue=8 |pages=1513–1524 |doi=10.1088/0953-8984/7/8/002 |bibcode=1995JPCM....7.1513A |s2cid=250898349 }}</ref> which would mean that monochromatic measurements would deviate from the Beer–Lambert law.

====Measurement uncertainty sources====
The above factors contribute to the [[measurement uncertainty]] of the results obtained with UV-Vis [[spectrophotometry]]. If UV-Vis spectrophotometry is used in quantitative chemical analysis then the results are additionally affected by uncertainty sources arising from the nature of the compounds and/or solutions that are measured. These include spectral interferences caused by absorption band overlap, fading of the color of the absorbing species (caused by decomposition or reaction) and possible composition mismatch between the sample and the calibration solution.<ref>{{cite journal |last1= Sooväli |first1= L. |last2= Rõõm |first2= E.-I. |last3= Kütt |first3= A. |last4= Kaljurand |first4= I. |last5= Leito |first5= I. |year= 2006 |title= Uncertainty sources in UV–Vis spectrophotometric measurement |journal= Accreditation and Quality Assurance |volume= 11 |issue= 5 |pages= 246–255 |doi= 10.1007/s00769-006-0124-x |s2cid= 94520012 |display-authors= 3}}</ref>