View source for Circular dichroism

{{Short description|Dichroism with circularly polarized light}}
'''Circular dichroism''' ('''CD''') is [[dichroism]] involving [[circular polarization|circularly polarized]] light, i.e., the differential [[Absorption (electromagnetic radiation)|absorption]] of left- and right-handed [[light]].<ref>{{cite book|author1=P. Atkins |author2=J. de Paula |title= Elements of Physical Chemistry |edition=4th |publisher=[[Oxford University Press]]|year=2005|isbn=978-0-7167-7329-0}}</ref><ref name="E. I. Solomon and A. B. P. Lever 2006 78">{{cite book|author1=Edward I. Solomon|author2=A. B. P. Lever|title=Inorganic electronic structure and spectroscopy|url=https://books.google.com/books?id=C7KaQgAACAAJ|access-date=29 April 2011|date=3 February 2006|publisher=[[Wiley-Interscience]]|isbn=978-0-471-97124-5|page=78}}</ref> Left-hand circular (LHC) and right-hand circular (RHC) polarized light represent two possible [[spin angular momentum of light|spin angular momentum]] states for a photon, and so circular dichroism is also referred to as dichroism for spin angular momentum.<ref>Introduction to Quantum Theory 2ED David Park Sec 2.2 Pg32 "...the polarization of a beam of light is exactly the same kind of thing as the spin of a beam of electrons, the differences of terminology reflecting only the accidents of the historical order of discovery."</ref>  This phenomenon was discovered by [[Jean-Baptiste Biot]], [[Augustin Fresnel]], and [[Aimé Cotton]] in the first half of the 19th century.<ref>{{cite book|author=Gerald D. Fasman|title=Circular dichroism and the conformational analysis of biomolecules|url=https://books.google.com/books?id=ivc0FaowEYMC&pg=PA3|access-date=29 April 2011|year=1996|publisher=Springer|isbn=978-0-306-45142-3|pages=3–}}</ref> Circular dichroism and [[optical rotation|circular birefringence]] are manifestations of '''optical activity'''. It is exhibited in the [[absorption (electromagnetic radiation)|absorption bands]] of [[optical activity|optically active]] [[chirality (chemistry)|chiral]] molecules. CD [[spectroscopy]] has a wide range of applications in many different fields. Most notably, [[Ultraviolet|UV]] CD is used to investigate the [[secondary structure]] of proteins.<ref name=b1>{{cite book|author1=Kōji Nakanishi|author2=Nina Berova|author-link2=Nina Berova|author3=Robert Woody|title=Circular dichroism: principles and applications|url=https://books.google.com/books?id=aNWjQgAACAAJ|access-date=29 April 2011|year=1994|publisher=VCH|isbn=978-1-56081-618-8|page=473}}</ref> UV/Vis CD is used to investigate [[charge-transfer complex|charge-transfer transitions]].<ref>{{cite journal|first=Neidig|last=Solomon|author2=A. T. Wecksler |author3=G. Schenk |author4=T. R. Holman |title= Kinetic and Spectroscopic Studies of N694C Lipoxygenase: A Probe of the Substrate Activation Mechanism of a Non-Heme Ferric Enzyme| journal=J. Am. Chem. Soc.| volume=129|pages=7531–7537|year=2007|doi=10.1021/ja068503d|pmc=2896304|pmid=17523638|issue=24}}</ref> [[Near-infrared]] CD is used to investigate [[molecular structure|geometric]] and [[electronic structure]] by probing [[transition metal|metal]] [[D orbital|d]]→[[D orbital|d]] transitions.<ref name="E. I. Solomon and A. B. P. Lever 2006 78"/> [[Vibrational circular dichroism]], which uses light from the [[infrared]] energy region, is used for structural studies of small organic molecules, and most recently proteins and DNA.<ref name=b1/>

==Physical principles==

===Circular polarization of light===
{{main|Circular polarization}}
[[Electromagnetic radiation]] consists of an electric <math>\boldsymbol E</math> and magnetic <math>\boldsymbol B</math> field that oscillate perpendicular to one another and to the propagating direction,<ref>{{cite book|author1=Alison Rodger|author2=Bengt Nordén|title=Circular dichroism and linear dichroism|url=https://books.google.com/books?id=THeKGC99hJcC|access-date=29 April 2011|year=1997|publisher=Oxford University Press|isbn=978-0-19-855897-2}}</ref> a [[transverse wave]]. While [[Polarization (waves)|linearly polarized]] light occurs when the electric field vector oscillates only in one plane, circularly polarized light occurs when the direction of the electric field vector rotates about its propagation direction while the vector retains constant magnitude. At a single point in space, the circularly polarized-vector will trace out a circle over one period of the wave frequency, hence the name. The two diagrams below show the electric field vectors of linearly and circularly polarized light, at one moment of time, for a range of positions; the plot of the circularly polarized electric vector forms a helix along the direction of propagation <math>\boldsymbol k</math>. For left circularly polarized light (LCP) with propagation towards the observer, the electric vector rotates [[clockwise|counterclockwise]].<ref name="E. I. Solomon and A. B. P. Lever 2006 78"/> For right circularly polarized light (RCP), the electric vector rotates clockwise.

[[File:Linearly pol.png]]     [[File:Circularly pol.png]]

===Interaction of circularly polarized light with matter===
When circularly polarized light passes through an absorbing optically active medium, the speeds between right and left polarizations differ (<math>c_\mathrm{L} \neq c_\mathrm{R}</math>) as well as their [[wavelength]](<math>\lambda_\mathrm{L} \neq \lambda_\mathrm{R}</math>) and the extent to which they are absorbed (<math>\varepsilon_\mathrm{L} \neq \varepsilon_\mathrm{R}</math>). ''Circular dichroism'' is the difference <math>\Delta\varepsilon \equiv \varepsilon_\mathrm{L} - \varepsilon_\mathrm{R}</math>.<ref name=b1/> The electric field of a light beam causes a linear displacement of charge when interacting with a molecule ([[electric dipole]]), whereas its magnetic field causes a circulation of charge ([[magnetic dipole]]). These two motions combined cause an excitation of an [[electron]] in a helical motion, which includes [[translation (physics)|translation]] and [[rotation]] and their associated [[Operator (mathematics)|operators]]. The experimentally determined relationship between the [[rotational strength]] <math>R</math> of a sample and the <math>\Delta\varepsilon</math> is given by
:<math>R_{\mathrm{exp}} = \frac{3hc10^{3} \ln(10)}{32\pi^{3}N_\mathrm{A}} \int \frac{\Delta\varepsilon}{\nu} \mathrm d{\nu}</math>
The rotational strength has also been determined theoretically,
:<math>R_\mathrm{theo} = \frac{1}{2mc} \mathrm{Im} \int \Psi_g \widehat{M}_\mathrm{(elec. dipole)} \Psi_e \mathrm d\tau \bullet \int \Psi_g \widehat{M}_\mathrm{(mag. dipole)} \Psi_e \mathrm d\tau </math>
We see from these two equations that in order to have non-zero <math>\Delta\varepsilon</math>, the electric and magnetic dipole moment operators (<math>\widehat{M}_\mathrm{(elec. dipole)}</math> and <math>\widehat{M}_\mathrm{(mag. dipole)}</math>) must transform as the same [[group theory|irreducible representation]]. <math>\mathrm C_n</math> and <math>\mathrm D_n </math> are the only [[point groups]] where this can occur, making only chiral molecules CD active.

Simply put, since circularly polarized light itself is "chiral", it interacts differently with [[chirality (chemistry)|chiral molecules]]. That is, the two types of circularly polarized light are absorbed to different extents. In a CD experiment, equal amounts of left and right circularly polarized light of a selected wavelength are alternately radiated into a (chiral) sample. One of the two polarizations is absorbed more than the other one, and this wavelength-dependent difference of absorption is measured, yielding the CD spectrum of the sample. Due to the interaction with the molecule, the electric field vector of the light traces out an elliptical path after passing through the sample.

It is important that the chirality of the molecule can be conformational rather than structural. That is, for instance, a protein molecule with a helical [[Protein secondary structure|secondary structure]] can have a CD that changes with changes in the conformation.

====Delta absorbance====
By definition,

:<math>\Delta A=A_\mathrm L-A_\mathrm R \,</math>

where <math>\Delta A</math> (Delta Absorbance) is the difference between absorbance of left circularly polarized (LCP) and right circularly polarized (RCP) light (this is what is usually measured). <math>\Delta A</math> is a function of [[wavelength]], so for a measurement to be meaningful the wavelength at which it was performed must be known.

====Molar circular dichroism====
It can also be expressed, by applying [[Beer-Lambert law|Beer's law]], as:

:<math>\Delta A = (\varepsilon_\mathrm L - \varepsilon_\mathrm R)Cl\,</math>

where
:<math>\varepsilon_\mathrm L</math> and <math>\varepsilon_\mathrm R</math> are the molar extinction coefficients for LCP and RCP light,
:''<math>C</math>'' is the [[molar concentration]],
:''<math>l</math>'' is the path length in centimeters (cm).

Then

:<math> \Delta \varepsilon =\varepsilon_\mathrm L-\varepsilon_\mathrm R\,</math>

is the molar circular dichroism. This intrinsic property is what is usually meant by the circular dichroism of the substance. Since <math> \Delta \varepsilon</math> is a function of wavelength, a molar circular dichroism value (<math> \Delta \varepsilon</math>) must specify the wavelength at which it is valid.

====Extrinsic effects on circular dichroism====
In many practical applications of circular dichroism (CD), as discussed below, the measured CD is not simply an intrinsic property of the molecule, but rather depends on the molecular conformation. In such a case the CD may also be a function of temperature, concentration, and the chemical environment, including solvents. In this case the reported CD value must also specify these other relevant factors in order to be meaningful.

In ordered structures lacking two-fold rotational symmetry, optical activity,<ref>{{cite journal|author=R. Williams|doi=10.1103/PhysRevLett.21.342|title=Optical Rotatory Effect in the Nematic Liquid Phase of p-Azoxyanisole|journal=Physical Review Letters|volume=21|page=342|year=1968|issue=6|bibcode=1968PhRvL..21..342W}}</ref><ref>{{cite journal|author=R. Williams|doi=10.1063/1.1671194|title=Optical-rotary power and linear electro-optic effect in nematic liquid crystals of p-azoxyanisole|journal=Journal of Chemical Physics|volume=50|page=1324|year=1969|issue=3|bibcode=1969JChPh..50.1324W}}</ref> including differential transmission<ref>{{Cite journal| last = Plum| first =E.|author2=Fedotov, V. A. |author3=Zheludev, N. I.  | title =Optical activity in extrinsically chiral metamaterial| journal =Applied Physics Letters| volume =93| page =191911| year =2008| issue =19| doi =10.1063/1.3021082 | arxiv =0807.0523| bibcode =2008ApPhL..93s1911P| s2cid =117891131| url =https://eprints.soton.ac.uk/65831/1/4221.pdf}}</ref> (and reflection<ref>{{Cite journal| last = Plum| first =E.|author2=Fedotov, V. A. |author3=Zheludev, N. I.  | title =Specular optical activity of achiral metasurfaces| journal =Applied Physics Letters| volume =108| page =141905| year =2016| issue =14| doi =10.1063/1.4944775 | bibcode =2016ApPhL.108n1905P| hdl =10220/40854| url =https://eprints.soton.ac.uk/389739/1/specular%2520optical%2520activity%25207rev.pdf}}</ref>) of circularly polarized waves also depends on the propagation direction through the material. In this case, so-called [[chirality (electromagnetism)#Extrinsic 3d chirality|extrinsic 3d chirality]] is associated with the mutual orientation of light beam and structure.

====Molar ellipticity====

Although <math> \Delta A </math> is usually measured, for historical reasons most measurements are reported in degrees of ellipticity.
Molar ellipticity is circular dichroism corrected for concentration. Molar circular dichroism and molar ellipticity, <math> [\theta]</math>, are readily interconverted by the equation:

[[File:Electric Vectors 1.png|thumb|200px|right|Elliptical polarized light (violet) is composed of unequal contributions of right (blue) and left (red) circular polarized light.]]

:<math> [\theta] = 3298.2\,\Delta \varepsilon.\, </math>

This relationship is derived by defining the [[polarization (waves)|ellipticity of the polarization]] as:

:<math> \tan \theta = \frac{E_\mathrm R - E_\mathrm L}{E_\mathrm R + E_\mathrm L} \,</math>

where
:<math> E_\mathrm R</math> and <math> E_\mathrm L</math> are the magnitudes of the [[electric field]] [[vector (geometry)|vectors]] of the right-circularly and left-circularly polarized light, respectively.

When <math> E_\mathrm R</math> equals <math> E_\mathrm L</math> (when there is no difference in the absorbance of right- and left-circular polarized light), <math> \theta</math> is 0° and the light is [[linear polarization|linearly polarized]]. When either <math> E_\mathrm R</math> or <math> E_\mathrm L</math> is equal to zero (when there is complete absorbance of the circular polarized light in one direction), <math> \theta</math> is 45° and the light is [[circular polarization|circularly polarized]].

Generally, the circular dichroism effect is small, so <math> \tan\theta</math> is small and can be approximated as <math> \theta</math> in [[radian]]s. Since the [[Radiant intensity|intensity]] or [[irradiance]], <math> I</math>, of light is proportional to the square of the electric-field vector, the ellipticity becomes:

:<math> \theta (\text{radians}) = \frac{(I_\mathrm R^{1/2} - I_\mathrm L^{1/2})}{(I_\mathrm R^{1/2} + I_\mathrm L^{1/2})}\,</math>

Then by substituting for I using [[Beer–Lambert law|Beer's law]] in [[natural logarithm]] form:

:<math> I = I_0 \mathrm e^{-A\ln 10}\,</math>

The ellipticity can now be written as:

:<math> \theta (\text{radians}) = \frac{(\mathrm e^{\frac{-A_\mathrm R}{2}\ln 10} - \mathrm e^{\frac{-A_\mathrm L}{2}\ln 10})}{(\mathrm e^{\frac{-A_\mathrm R}{2}\ln 10} + \mathrm e^{\frac{-A_\mathrm L}{2}\ln 10})} = \frac{\mathrm e^{\Delta A \frac{\ln 10}{2}} - 1}{\mathrm e^{\Delta A \frac{\ln 10}{2}} + 1} \,</math>

Since <math> \Delta A \ll 1</math>, this expression can be approximated by expanding the exponentials in a [[Taylor series]] to first-order and then discarding terms of <math> \Delta A </math> in comparison with unity and converting from [[radian]]s to degrees:

:<math> \theta (\text{degrees}) = \Delta A \left( \frac {\ln 10}{4} \right) \left( \frac {180}{\pi} \right)\, </math>

The linear dependence of solute concentration and pathlength is removed by defining molar ellipticity as,

:<math> [\theta] = \frac {100\theta}{Cl}\, </math>

Then combining the last two expression with [[Beer–Lambert law|Beer's law]], molar ellipticity becomes:

:<math> [\theta]= 100 \,\Delta \varepsilon \left( \frac {\ln 10}{4} \right) \left( \frac {180}{\pi} \right) = 3298.2\,\Delta \varepsilon \,</math>

The units of molar ellipticity are historically (deg·cm<sup>2</sup>/dmol). To calculate molar ellipticity, the sample concentration (g/L), cell pathlength (cm), and the molecular weight (g/mol) must be known.

If the sample is a protein, the mean residue weight (average molecular weight of the amino acid residues it contains) is often used in place of the molecular weight, essentially treating the protein as a solution of amino acids. Using mean residue ellipticity facilitates comparing the CD of proteins of different molecular weight; use of this normalized CD is important in studies of protein structure.

====Mean residue ellipticity====
Methods for estimating secondary structure in polymers, proteins and polypeptides in particular, often require that the measured molar ellipticity spectrum be converted to a normalized value, specifically a value independent of the polymer length. Mean residue ellipticity is used for this purpose; it is simply the measured molar ellipticity of the molecule divided by the number of monomer units (residues) in the molecule.

==Application to biological molecules==
[[File:Circular dichroism and structures calculation-protein in detergents.svg|thumb|400px|Upper panel: Circular dichroism spectroscopy in the ultraviolet wavelength region (UV-CD) of MBP-cytochrome b<sub>6</sub> fusion protein in different detergent solutions. It shows that the protein in DM, as well as in Triton X-100 solution, recovered its structure. However the spectra obtained from SDS solution shows decreased ellipticity in the range between 200 and 210&nbsp;nm, which indicates incomplete secondary structure recovery. <br/>
Lower panel: The content of secondary structures predicted from the CD spectra using the CDSSTR algorithm. The protein in SDS solution shows increased content of unordered structures and decreased helices content.<ref>{{cite journal|author=Surma M.A.|author2=Szczepaniak A.|author3=Króliczewski J.|title=Comparative Studies on Detergent-Assisted Apocytochrome b6 Reconstitution into Liposomal Bilayers Monitored by Zetasizer Instruments|journal=PLOS ONE|volume=9|issue=11|year=2014|pages=e111341|issn=1932-6203|doi=10.1371/journal.pone.0111341|bibcode = 2014PLoSO...9k1341S|pmid=25423011|pmc=4244035|doi-access=free}}</ref>]]
In general, this phenomenon will be exhibited in absorption bands of any [[optical activity|optically active]] molecule. As a consequence, circular dichroism is exhibited by biological molecules, because of their [[dextrorotary]] and [[levorotary]] components. Even more important is that a [[secondary structure]] will also impart a distinct CD to its respective molecules. Therefore, the [[alpha helix]] of proteins and the [[double helix]] of [[nucleic acids]] have CD spectral signatures representative of their structures.  The capacity of CD to give a representative structural signature makes it a powerful tool in modern biochemistry with applications that can be found in virtually every field of study.

CD is closely related to the [[optical rotatory dispersion]] (ORD) technique, and is generally considered to be more advanced. CD is measured in or near the absorption bands of the molecule of interest, while ORD can be measured far from these bands. CD's advantage is apparent in the data analysis. Structural elements are more clearly distinguished since their recorded bands do not overlap extensively at particular wavelengths as they do in ORD. In principle, these two spectral measurements can be interconverted through an integral transform ([[Kramers–Kronig relation]]), if all the absorptions are included in the measurements.

The far-UV ([[ultraviolet]]) CD spectrum of proteins can reveal important characteristics of their [[secondary structure]]. CD spectra can be readily used to estimate the fraction of a molecule that is in the [[alpha-helix]] conformation, the [[beta-sheet]] conformation, the [[beta-turn]] conformation, or some other (e.g. [[random coil]]) conformation.<ref>{{cite journal |vauthors=Hall V, Nash A, Rodger A |title=SSNN, a method for neural network protein secondary structure fitting using circular dichroism data |journal=Analytical Methods |volume=6 |issue=17 |pages=6721–26 |year=2014|doi=10.1039/C3AY41831F |url=http://wrap.warwick.ac.uk/75654/1/WRAP_9471544-ch-040116-140618_ssnn_a_method_for_neural_network_protein_secondary_structure_fitting_using_circular_dichroism_data_main.pdf |archive-url=https://ghostarchive.org/archive/20221009/http://wrap.warwick.ac.uk/75654/1/WRAP_9471544-ch-040116-140618_ssnn_a_method_for_neural_network_protein_secondary_structure_fitting_using_circular_dichroism_data_main.pdf |archive-date=2022-10-09 |url-status=live }}</ref><ref>{{cite journal |vauthors=Hall V, Nash A, Hines E, Rodger A |title=Elucidating protein secondary structure with circular dichroism and a neural network |journal=Journal of Computational Chemistry |volume=34 |issue=32 |pages=2774–86 |year=2013|pmid=24122928 |doi=10.1002/jcc.23456 |s2cid=19685126 }}</ref><ref name="pmid17896349">{{cite journal |vauthors=Whitmore L, Wallace BA |title=Protein secondary structure analyses from circular dichroism spectroscopy: methods and reference databases |journal=Biopolymers |volume=89 |issue=5 |pages=392–400 |year=2008|pmid=17896349 |doi=10.1002/bip.20853|doi-access= }}</ref><ref name="pmid17406547">{{cite journal |author=Greenfield NJ |title=Using circular dichroism spectra to estimate protein secondary structure |journal=Nature Protocols |volume=1 |issue=6 |pages=2876–90 |year=2006 |pmid=17406547 |doi=10.1038/nprot.2006.202 |pmc=2728378}}</ref> These fractional assignments place important constraints on the possible secondary conformations that the protein can be in. CD cannot, in general, say where the alpha helices that are detected are located within the molecule or even completely predict how many there are. Despite this, CD is a valuable tool, especially for showing changes in conformation. It can, for instance, be used to study how the secondary structure of a molecule changes as a function of temperature or of the concentration of denaturing agents, e.g. [[Guanidine|Guanidinium chloride]] or [[urea]]. In this way it can reveal important thermodynamic information about the molecule (such as the [[enthalpy]] and [[Gibbs free energy]] of denaturation) that cannot otherwise be easily obtained. Anyone attempting to study a protein will find CD a valuable tool for verifying that the protein is in its native conformation before undertaking extensive and/or expensive experiments with it. Also, there are a number of other uses for CD spectroscopy in protein chemistry not related to alpha-helix fraction estimation. Moreover, CD spectroscopy has been used in bioinorganic interface studies. Specifically it has been used to analyze the differences in secondary structure of an  engineered protein before and after titration with a reagent.<ref name="pubs.acs.org">Bioinorganic Interface: Mechanistic Studies of Protein-Directed Nanomaterial Synthesis. (2016, May 5). Retrieved March 1, 2019, from https://pubs.acs.org/doi/pdf/10.1021/acs.jpcc.6b02569</ref>

The near-UV CD spectrum (>250&nbsp;nm) of proteins provides information on the [[tertiary structure]]. The signals obtained in the 250–300&nbsp;nm region are due to the absorption, dipole orientation and the nature of the surrounding environment of the phenylalanine, tyrosine, cysteine (or S-S [[disulfide bond|disulfide bridges]]) and tryptophan [[amino acid]]s. Unlike in far-UV CD, the near-UV CD spectrum cannot be assigned to any particular 3D structure. Rather, near-UV CD spectra provide structural information on the nature of the prosthetic groups in proteins, e.g., the heme groups in [[hemoglobin]] and [[cytochrome c]].

Visible CD spectroscopy is a very powerful technique to study metal–protein interactions and can resolve individual d–d electronic transitions as separate bands. CD spectra in the visible light region are only produced when a metal ion is in a chiral environment, thus, free metal ions in solution are not detected. This has the advantage of only observing the protein-bound metal, so pH dependence and stoichiometries are readily obtained. Optical activity in transition metal ion complexes have been attributed to configurational, conformational and the vicinal effects. Klewpatinond and Viles (2007) have produced a set of empirical rules for predicting the appearance of visible CD spectra for Cu<sup>2+</sup> and Ni<sup>2+</sup> square-planar complexes involving histidine and main-chain coordination.

CD gives less specific structural information than [[X-ray crystallography]] and [[protein NMR]] spectroscopy, for example, which both give atomic resolution data. However, CD spectroscopy is a quick method that does not require large amounts of proteins or extensive data processing. Thus CD can be used to survey a large number of [[solvent]] conditions, varying [[temperature]], [[pH]], [[salinity]], and the presence of various cofactors.

CD [[spectroscopy]] is usually used to study proteins in solution, and thus it complements methods that study the solid state. This is also a limitation, in that many proteins are embedded in [[biological membrane|membranes]] in their native state, and solutions containing membrane structures are often strongly scattering. CD is sometimes measured in thin films.

CD [[spectroscopy]] has also been done using semiconducting materials such as TiO<sub>2</sub> to obtain large signals in the UV range of wavelengths, where the electronic transitions for biomolecules often occur.<ref>Sarkar, Sumant, Ryan Behunin, and John G. Gibbs. "Shape-Dependent, Chiro-Optical Response of UV-Active, Nanohelix Metamaterials." Nano letters (2019). https://pubs.acs.org/doi/10.1021/acs.nanolett.9b03274</ref>

==Experimental limitations==
{{More citations needed|date=March 2010}}

CD has also been studied in [[carbohydrates]], but with limited success due to the experimental difficulties associated with measurement of CD spectra in the vacuum ultraviolet (VUV) region of the spectrum (100–200&nbsp;nm), where the corresponding CD bands of unsubstituted carbohydrates lie. Substituted carbohydrates with bands above the VUV region have been successfully measured.

Measurement of CD is also complicated by the fact that typical aqueous buffer systems often absorb in the range where structural features exhibit differential absorption of circularly polarized light. [[Phosphate]], [[sulfate]], [[carbonate]], and [[acetate]] buffers are generally incompatible with CD unless made extremely dilute e.g. in the 10–50 mM range. The TRIS buffer system should be completely avoided when performing far-UV CD. [[Borate]] and [[Onium compounds]] are often used to establish the appropriate pH range for CD experiments. Some experimenters have substituted fluoride for chloride ion because fluoride absorbs less in the far UV, and some have worked in pure water. Another, almost universal, technique is to minimize solvent absorption by using shorter path length cells when working in the far UV, 0.1&nbsp;mm path lengths are not uncommon in this work.

In addition to measuring in aqueous systems, CD, particularly far-UV CD, can be measured in organic solvents e.g. ethanol, methanol, [[trifluoroethanol]] (TFE). The latter has the advantage to induce structure formation of proteins, inducing beta-sheets in some and alpha helices in others, which they would not show under normal aqueous conditions. Most common organic solvents such as [[acetonitrile]], [[THF]], [[chloroform]], [[dichloromethane]] are however, incompatible with far-UV CD.

It may be of interest to note that the protein CD spectra used in secondary structure estimation are related to the π to π* orbital absorptions of the [[amide bond]]s linking the amino acids. These absorption bands lie partly in the ''so-called'' [[Ultraviolet#Vacuum UV|vacuum ultraviolet]] (wavelengths less than about 200&nbsp;nm). The wavelength region of interest is actually inaccessible in air because of the strong absorption of light by [[oxygen]] at these wavelengths. In practice these spectra are measured not in vacuum but in an oxygen-free instrument (filled with pure [[nitrogen]] gas).

Once oxygen has been eliminated, perhaps the second most important technical factor in working below 200&nbsp;nm is to design the rest of the optical system to have low losses in this region. Critical in this regard is the use of [[Mirror#Instruments|aluminized mirrors]] whose coatings have been optimized for low loss in this region of the spectrum.

The usual light source in these instruments is a high pressure, short-arc [[Xenon arc lamp|xenon lamp]]. Ordinary xenon arc lamps are unsuitable for use in the low UV. Instead, specially constructed lamps with envelopes made from high-purity synthetic [[fused silica]] must be used.

Light from [[synchrotron]] sources has a much higher flux at short wavelengths, and has been used to record CD down to 160&nbsp;nm. In 2010 the CD spectrophotometer at the electron storage ring facility ISA at the University of [[Aarhus]] in Denmark was used to record solid state CD spectra down to 120&nbsp;nm.<ref>{{cite journal |author1=U. Meierhenrich |author2=J.J. Filippi |author3=C. Meinert |author4=J. H. Bredehöft |author5=J. Takahashi |author6=L. Nahon |author7=N. C. Jones |author8=S. V. Hoffmann |title=Circular Dichroism of Amino Acids in the Vacuum-Ultraviolet Region |journal=Angew. Chem. Int. Ed. |volume=49 |pages=7799–7802 |year=2010 |pmid=20845349 |doi=10.1002/anie.201003877 |issue=42 |url=https://zenodo.org/record/3409037 }}{{Dead link|date=December 2023 |bot=InternetArchiveBot |fix-attempted=yes }}</ref>
At the [[quantum mechanics|quantum mechanical]] level, the feature density of circular dichroism and [[optical rotation]] are identical. [[Optical rotary dispersion]] and circular dichroism share the same [[quantum information]] content.

==See also==
{{div col|colwidth=22em}}
*[[Chirality-induced spin selectivity]]
*[[Hyper–Rayleigh scattering|Hyper Rayleigh scattering optical activity]]
*[[Linear dichroism]]
*[[Magnetic circular dichroism]]
*[[Optical activity]]
*[[Optical isomerism]]
*[[Optical rotation]]
*[[Optical rotatory dispersion]]
*[[Protein Circular Dichroism Data Bank]]
*[[Synchrotron radiation circular dichroism spectroscopy]]
*[[Two-photon circular dichroism]]
*[[Vibrational circular dichroism]]
{{div col end}}

==References==
{{Reflist|30em}}

==External links==
* [http://www.ap-lab.com/circular_dichroism.htm Circular Dichroism spectroscopy] by Alliance Protein Laboratories, a commercial service provider
* [https://www.photophysics.com/circular-dichroism/chirascan-technology/chirality-and-cd/ An Introduction to Circular Dichroism Spectroscopy] by Applied Photophysics, an equipment supplier
* [https://people.bath.ac.uk/vkv23/English/RechercheT0.htm An animated, step-by-step tutorial on Circular Dichroism and Optical Rotation] by Prof Valev.

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[[Category:Polarization (waves)]]
[[Category:Physical quantities]]
[[Category:Protein structure]]