Editing Spectrophotometry (section)

==UV-visible spectrophotometry==
{{main|Ultraviolet-visible spectroscopy}}

Most spectrophotometers are used in the [[UV]] and [[Visible spectrum|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&nbsp;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&nbsp;nm. Nucleic acid contamination can also interfere. This method requires a spectrophotometer capable of measuring in the UV region with quartz cuvettes.<ref name=":0" />{{Rp|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.<ref name=":2">{{Cite book|title=Fundamental laboratory approaches for biochemistry and biotechnology|last1=Ninfa|first1=Alexander J.|last2=Ballou|first2=David P.|date=2004|publisher=Wiley|isbn=9781891786006|location=Hoboken|pages=66|oclc=633862582|name-list-style=vanc}}</ref>

Visible region 400–700&nbsp;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.
[[File:Mettler Toledo UV5Nano micro-volume.jpg|thumb|METTLER TOLEDO UV5Nano Micro-Volume Spectrophotometer]]
Samples are usually prepared in [[cuvette]]s; depending on the region of interest, they may be constructed of [[glass]], [[plastic]] (visible spectrum region of interest), or [[quartz glass|quartz]] (Far UV spectrum region of interest). Some applications require small volume measurements which can be performed with micro-volume platforms.

===Applications===
* Estimating [[dissolved organic carbon]] concentration
* [[Specific ultraviolet absorbance]] for metric of aromaticity
* [[Bial's test]] for concentration of pentoses

===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.<ref name=":2" /> 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 [[Ortho-Nitrophenyl-β-galactoside]] (ONPG) and determining if the sample turns yellow.<ref name=":0" />{{Rp|21–119}} Following this testing the sample at 420&nbsp;nm for specific interaction with ONPG and at 595 for a Bradford Assay the amount of purification can be assessed quantitatively.<ref name=":0" />{{Rp|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.