Circular dichroism

Circular dichroism

Overview
Circular dichroism refers to the differential absorption of left and right circularly polarized
Circular polarization
In electrodynamics, circular polarization of an electromagnetic wave is a polarization in which the electric field of the passing wave does not change strength but only changes direction in a rotary type manner....

 light
Light
Light or visible light is electromagnetic radiation that is visible to the human eye, and is responsible for the sense of sight. Visible light has wavelength in a range from about 380 nanometres to about 740 nm, with a frequency range of about 405 THz to 790 THz...

. This phenomenon was discovered by Jean-Baptiste Biot
Jean-Baptiste Biot
Jean-Baptiste Biot was a French physicist, astronomer, and mathematician who established the reality of meteorites, made an early balloon flight, and studied the polarization of light.- Biography :...

, Augustin Fresnel, and Aimé Cotton
Aimé Cotton
Aimé Auguste Cotton was a French physicist known for his studies of the interaction of light with chiral molecules...

 in the first half of the 19th century. It is exhibited in the absorption bands
Absorption (electromagnetic radiation)
In physics, absorption of electromagnetic radiation is the way by which the energy of a photon is taken up by matter, typically the electrons of an atom. Thus, the electromagnetic energy is transformed to other forms of energy for example, to heat. The absorption of light during wave propagation is...

 of optically active chiral
Chirality (chemistry)
A chiral molecule is a type of molecule that lacks an internal plane of symmetry and thus has a non-superimposable mirror image. The feature that is most often the cause of chirality in molecules is the presence of an asymmetric carbon atom....

 molecules. CD spectroscopy
Spectroscopy
Spectroscopy is the study of the interaction between matter and radiated energy. Historically, spectroscopy originated through the study of visible light dispersed according to its wavelength, e.g., by a prism. Later the concept was expanded greatly to comprise any interaction with radiative...

 has a wide range of applications in many different fields. Most notably, UV
Ultraviolet
Ultraviolet light is electromagnetic radiation with a wavelength shorter than that of visible light, but longer than X-rays, in the range 10 nm to 400 nm, and energies from 3 eV to 124 eV...

 CD is used to investigate the secondary structure
Secondary structure
In biochemistry and structural biology, secondary structure is the general three-dimensional form of local segments of biopolymers such as proteins and nucleic acids...

 of proteins. UV/Vis CD is used to investigate charge-transfer transitions.
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Encyclopedia
Circular dichroism refers to the differential absorption of left and right circularly polarized
Circular polarization
In electrodynamics, circular polarization of an electromagnetic wave is a polarization in which the electric field of the passing wave does not change strength but only changes direction in a rotary type manner....

 light
Light
Light or visible light is electromagnetic radiation that is visible to the human eye, and is responsible for the sense of sight. Visible light has wavelength in a range from about 380 nanometres to about 740 nm, with a frequency range of about 405 THz to 790 THz...

. This phenomenon was discovered by Jean-Baptiste Biot
Jean-Baptiste Biot
Jean-Baptiste Biot was a French physicist, astronomer, and mathematician who established the reality of meteorites, made an early balloon flight, and studied the polarization of light.- Biography :...

, Augustin Fresnel, and Aimé Cotton
Aimé Cotton
Aimé Auguste Cotton was a French physicist known for his studies of the interaction of light with chiral molecules...

 in the first half of the 19th century. It is exhibited in the absorption bands
Absorption (electromagnetic radiation)
In physics, absorption of electromagnetic radiation is the way by which the energy of a photon is taken up by matter, typically the electrons of an atom. Thus, the electromagnetic energy is transformed to other forms of energy for example, to heat. The absorption of light during wave propagation is...

 of optically active chiral
Chirality (chemistry)
A chiral molecule is a type of molecule that lacks an internal plane of symmetry and thus has a non-superimposable mirror image. The feature that is most often the cause of chirality in molecules is the presence of an asymmetric carbon atom....

 molecules. CD spectroscopy
Spectroscopy
Spectroscopy is the study of the interaction between matter and radiated energy. Historically, spectroscopy originated through the study of visible light dispersed according to its wavelength, e.g., by a prism. Later the concept was expanded greatly to comprise any interaction with radiative...

 has a wide range of applications in many different fields. Most notably, UV
Ultraviolet
Ultraviolet light is electromagnetic radiation with a wavelength shorter than that of visible light, but longer than X-rays, in the range 10 nm to 400 nm, and energies from 3 eV to 124 eV...

 CD is used to investigate the secondary structure
Secondary structure
In biochemistry and structural biology, secondary structure is the general three-dimensional form of local segments of biopolymers such as proteins and nucleic acids...

 of proteins. UV/Vis CD is used to investigate charge-transfer transitions. Near-infrared CD is used to investigate geometric
Molecular structure
The molecular structure of a substance is described by the combination of nuclei and electrons that comprise its constitute molecules. This includes the molecular geometry , the electronic properties of the...

 and electronic structure by probing metal
Transition metal
The term transition metal has two possible meanings:*The IUPAC definition states that a transition metal is "an element whose atom has an incomplete d sub-shell, or which can give rise to cations with an incomplete d sub-shell." Group 12 elements are not transition metals in this definition.*Some...

 d→d transitions. Vibrational circular dichroism
Vibrational circular dichroism
Vibrational circular dichroism is a spectroscopic technique which detects differences in attenuation of left and right circularly polarized light passing through a sample...

, which uses light from the infrared
Infrared
Infrared light is electromagnetic radiation with a wavelength longer than that of visible light, measured from the nominal edge of visible red light at 0.74 micrometres , and extending conventionally to 300 µm...

 energy region, is used for structural studies of small organic molecules, and most recently proteins and DNA.

Circular polarization of light



Electromagnetic radiation
Electromagnetic radiation
Electromagnetic radiation is a form of energy that exhibits wave-like behavior as it travels through space...

 consists of an electric (E) and magnetic (B) field that oscillate perpendicular to one another and to the propagating direction., a transverse wave
Transverse wave
A transverse wave is a moving wave that consists of oscillations occurring perpendicular to the direction of energy transfer...

. While 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 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 (k). For left circularly polarized light (LCP) with propagation towards the observer, the electric vector rotates counterclockwise. For right circularly polarized light (RCP), the electric vector rotates clockwise.


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 (cL cR) as well as their wavelength
Wavelength
In physics, the wavelength of a sinusoidal wave is the spatial period of the wave—the distance over which the wave's shape repeats.It is usually determined by considering the distance between consecutive corresponding points of the same phase, such as crests, troughs, or zero crossings, and is a...

 (LR) and the extent to which they are absorbed (LR). Circular dichroism is the difference L- R. The electric field of a light beam causes a linear displacement of charge when interacting with a molecule (electric dipole), whereas the magnetic field of it causes a circulation of charge (magnetic dipole
Magnetic dipole
A magnetic dipole is the limit of either a closed loop of electric current or a pair of poles as the dimensions of the source are reduced to zero while keeping the magnetic moment constant. It is a magnetic analogue of the electric dipole, but the analogy is not complete. In particular, a magnetic...

). These two motions combined cause an excitation of an electron
Electron
The electron is a subatomic particle with a negative elementary electric charge. It has no known components or substructure; in other words, it is generally thought to be an elementary particle. An electron has a mass that is approximately 1/1836 that of the proton...

 in a helical motion, which includes translation
Translation (physics)
In physics, translation is movement that changes the position of an object, as opposed to rotation. For example, according to Whittaker:...

 and rotation
Rotation
A rotation is a circular movement of an object around a center of rotation. A three-dimensional object rotates always around an imaginary line called a rotation axis. If the axis is within the body, and passes through its center of mass the body is said to rotate upon itself, or spin. A rotation...

 and their associated operators. The experimentally determined relationship between the rotational strength (R) of a sample and the is given by
The rotational strength has also been determined theoretically,
We see from these two equations that in order to have non-zero , the electric and magnetic dipole moment operators ( and ) must transform as the same irreducible representation
Group theory
In mathematics and abstract algebra, group theory studies the algebraic structures known as groups.The concept of a group is central to abstract algebra: other well-known algebraic structures, such as rings, fields, and vector spaces can all be seen as groups endowed with additional operations and...

. and 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 chiral molecules
Chirality (chemistry)
A chiral molecule is a type of molecule that lacks an internal plane of symmetry and thus has a non-superimposable mirror image. The feature that is most often the cause of chirality in molecules is the presence of an asymmetric carbon atom....

. 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 secondary structure can have a CD that changes with changes in the conformation.

Delta absorbance


By definition,


where ΔA (Delta Absorbance) is the difference between absorbance of left circularly polarized (LCP) and right circularly polarized (RCP) light (this is what is usually measured). ΔA is a function of wavelength
Wavelength
In physics, the wavelength of a sinusoidal wave is the spatial period of the wave—the distance over which the wave's shape repeats.It is usually determined by considering the distance between consecutive corresponding points of the same phase, such as crests, troughs, or zero crossings, and is a...

, 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's law
Beer-Lambert law
In optics, the Beer–Lambert law, also known as Beer's law or the Lambert–Beer law or the Beer–Lambert–Bouguer law relates the absorption of light to the properties of the material through which the light is travelling.-Equations:The law states that there is a logarithmic dependence between the...

, as:


where
εL and εR are the molar extinction coefficients for LCP and RCP light,
C is the molar concentration
l is the path length in centimeters (cm).


Then


is the molar circular dichroism. This intrinsic property is what is usually meant by the circular dichroism of the substance. Since is a function of wavelength, a molar circular dichroism value () 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.

Molar ellipticity


Although ΔA 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, [θ], are readily interconverted by the equation:




This relationship is derived by defining the ellipticity of the polarization as:


where
ER and EL are the magnitudes of the electric field
Electric field
In physics, an electric field surrounds electrically charged particles and time-varying magnetic fields. The electric field depicts the force exerted on other electrically charged objects by the electrically charged particle the field is surrounding...

 vectors of the right-circularly and left-circularly polarized light, respectively.


When ER equals EL (when there is no difference in the absorbance of right- and left-circular polarized light), θ is 0° and the light is linearly polarized
Linear polarization
In electrodynamics, linear polarization or plane polarization of electromagnetic radiation is a confinement of the electric field vector or magnetic field vector to a given plane along the direction of propagation...

. When either ER or EL is equal to zero (when there is complete absorbance of the circular polarized light in one direction), θ is 45° and the light is circularly polarized
Circular polarization
In electrodynamics, circular polarization of an electromagnetic wave is a polarization in which the electric field of the passing wave does not change strength but only changes direction in a rotary type manner....

.

Generally, the circular dichroism effect is small, so tanθ is small and can be approximated as θ in radians. Since the intensity
Radiant intensity
In radiometry, radiant intensity is a measure of the intensity of electromagnetic radiation. It is defined as power per unit solid angle. The SI unit of radiant intensity is watts per steradian . Radiant intensity is distinct from irradiance and radiant exitance, which are often called intensity...

 or irradiance
Irradiance
Irradiance is the power of electromagnetic radiation per unit area incident on a surface. Radiant emittance or radiant exitance is the power per unit area radiated by a surface. The SI units for all of these quantities are watts per square meter , while the cgs units are ergs per square centimeter...

, I, of light is proportional to the square of the electric-field vector, the ellipticity becomes:


Then by substituting for I using Beer's law in natural logarithm
Natural logarithm
The natural logarithm is the logarithm to the base e, where e is an irrational and transcendental constant approximately equal to 2.718281828...

 form:


The ellipticity can now be written as:


Since ΔA << 1, this expression can be approximated by expanding the exponentials in a Taylor series
Taylor series
In mathematics, a Taylor series is a representation of a function as an infinite sum of terms that are calculated from the values of the function's derivatives at a single point....

 to first-order and then discarding terms of ΔA in comparison with unity and converting from radians to degrees:


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


Then combining the last two expression with Beer's law, molar ellipticity becomes:


The units of molar ellipticity are historically (deg·cm2/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 residual weight (average molecular weight of the amino acids it contains) is used in place of the molecular weight, essentially treating the protein as a solution of amino acids.

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


In general, this phenomenon will be exhibited in absorption bands of any 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
Secondary structure
In biochemistry and structural biology, secondary structure is the general three-dimensional form of local segments of biopolymers such as proteins and nucleic acids...

 will also impart a distinct CD to its respective molecules. Therefore, the alpha helix
Alpha helix
A common motif in the secondary structure of proteins, the alpha helix is a right-handed coiled or spiral conformation, in which every backbone N-H group donates a hydrogen bond to the backbone C=O group of the amino acid four residues earlier...

 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
Optical rotatory dispersion
Optical rotatory dispersion is the variation in the optical rotation of a substance with a change in the wavelength of light. Optical rotatory dispersion can be used to find the absolute configuration of metal complexes....

 (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
Ultraviolet
Ultraviolet light is electromagnetic radiation with a wavelength shorter than that of visible light, but longer than X-rays, in the range 10 nm to 400 nm, and energies from 3 eV to 124 eV...

) CD spectrum of proteins can reveal important characteristics of their secondary structure
Secondary structure
In biochemistry and structural biology, secondary structure is the general three-dimensional form of local segments of biopolymers such as proteins and nucleic acids...

. 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
Random coil
A random coil is a polymer conformation where the monomer subunits are oriented randomly while still being bonded to adjacent units. It is not one specific shape, but a statistical distribution of shapes for all the chains in a population of macromolecules...

) conformation. 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. Guanidinium hydrochloride
Guanidine
Guanidine is a crystalline compound of strong alkalinity formed by the oxidation of guanine. It is used in the manufacture of plastics and explosives. It is found in urine as a normal product of protein metabolism. The molecule was first synthesized in 1861 by the oxidative degradation of an...

 or urea
Urea
Urea or carbamide is an organic compound with the chemical formula CO2. The molecule has two —NH2 groups joined by a carbonyl functional group....

. In this way it can reveal important thermodynamic information about the molecule (such as the enthalpy
Enthalpy
Enthalpy is a measure of the total energy of a thermodynamic system. It includes the internal energy, which is the energy required to create a system, and the amount of energy required to make room for it by displacing its environment and establishing its volume and pressure.Enthalpy is a...

 and Gibbs free energy
Gibbs free energy
In thermodynamics, the Gibbs free energy is a thermodynamic potential that measures the "useful" or process-initiating work obtainable from a thermodynamic system at a constant temperature and pressure...

 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.

The near-UV CD spectrum (>250 nm) of proteins provides information on the tertiary structure
Tertiary structure
In biochemistry and molecular biology, the tertiary structure of a protein or any other macromolecule is its three-dimensional structure, as defined by the atomic coordinates.-Relationship to primary structure:...

. The signals obtained in the 250–300 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 bridges
Disulfide bond
In chemistry, a disulfide bond is a covalent bond, usually derived by the coupling of two thiol groups. The linkage is also called an SS-bond or disulfide bridge. The overall connectivity is therefore R-S-S-R. The terminology is widely used in biochemistry...

) and tryptophan amino acid
Amino acid
Amino acids are molecules containing an amine group, a carboxylic acid group and a side-chain that varies between different amino acids. The key elements of an amino acid are carbon, hydrogen, oxygen, and nitrogen...

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
Hemoglobin
Hemoglobin is the iron-containing oxygen-transport metalloprotein in the red blood cells of all vertebrates, with the exception of the fish family Channichthyidae, as well as the tissues of some invertebrates...

 and cytochrome c
Cytochrome c
The Cytochrome complex, or cyt c is a small heme protein found loosely associated with the inner membrane of the mitochondrion. It belongs to the cytochrome c family of proteins. Cytochrome c is a highly soluble protein, unlike other cytochromes, with a solubility of about 100 g/L and is an...

.

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 Cu2+ and Ni2+ square-planar complexes involving histidine and main-chain coordination.

CD gives less specific structural information than X-ray crystallography
X-ray crystallography
X-ray crystallography is a method of determining the arrangement of atoms within a crystal, in which a beam of X-rays strikes a crystal and causes the beam of light to spread into many specific directions. From the angles and intensities of these diffracted beams, a crystallographer can produce a...

 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
Solvent
A solvent is a liquid, solid, or gas that dissolves another solid, liquid, or gaseous solute, resulting in a solution that is soluble in a certain volume of solvent at a specified temperature...

 conditions, varying temperature
Temperature
Temperature is a physical property of matter that quantitatively expresses the common notions of hot and cold. Objects of low temperature are cold, while various degrees of higher temperatures are referred to as warm or hot...

, pH
PH
In chemistry, pH is a measure of the acidity or basicity of an aqueous solution. Pure water is said to be neutral, with a pH close to 7.0 at . Solutions with a pH less than 7 are said to be acidic and solutions with a pH greater than 7 are basic or alkaline...

, salinity
Salinity
Salinity is the saltiness or dissolved salt content of a body of water. It is a general term used to describe the levels of different salts such as sodium chloride, magnesium and calcium sulfates, and bicarbonates...

, and the presence of various cofactors.

CD spectroscopy
Spectroscopy
Spectroscopy is the study of the interaction between matter and radiated energy. Historically, spectroscopy originated through the study of visible light dispersed according to its wavelength, e.g., by a prism. Later the concept was expanded greatly to comprise any interaction with radiative...

 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 membranes
Biological membrane
A biological membrane or biomembrane is an enclosing or separatingmembrane that acts as a selective barrier, within or around a cell. It consists of a lipid bilayer with embedded proteins that may constitute close to 50% of membrane content...

 in their native state, and solutions containing membrane structures are often strongly scattering. CD is sometimes measured in thin films.

Experimental limitations



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 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
Phosphate
A phosphate, an inorganic chemical, is a salt of phosphoric acid. In organic chemistry, a phosphate, or organophosphate, is an ester of phosphoric acid. Organic phosphates are important in biochemistry and biogeochemistry or ecology. Inorganic phosphates are mined to obtain phosphorus for use in...

, sulfate
Sulfate
In inorganic chemistry, a sulfate is a salt of sulfuric acid.-Chemical properties:...

, carbonate
Carbonate
In chemistry, a carbonate is a salt of carbonic acid, characterized by the presence of the carbonate ion, . The name may also mean an ester of carbonic acid, an organic compound containing the carbonate group C2....

, and acetate
Acetate
An acetate is a derivative of acetic acid. This term includes salts and esters, as well as the anion found in solution. Most of the approximately 5 billion kilograms of acetic acid produced annually in industry are used in the production of acetates, which usually take the form of polymers. In...

 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
Borate
Borates are chemical compounds which contain oxoanions of boron in oxidation state +3. The simplest borate ion, BO33−, has a trigonal planar structure. Other borates are made up of trigonal BO3 or tetrahedral BO4 structural units, sharing oxygen atoms...

 and Onium compounds
Onium compounds
Onium compounds are cations derived by the protonation of mononuclear parent hydrides of elements of the nitrogen group , chalcogens , or halogens , and similar cations derived by the substitution of hydrogen atoms in the former by other groups, such as organic radicals, or halogens, for example...

 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 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
Acetonitrile
Acetonitrile is the chemical compound with formula . This colourless liquid is the simplest organic nitrile. It is produced mainly as a byproduct of acrylonitrile manufacture...

, THF
ThF
Follicular B helper T cells , are antigen-experienced CD4+ T cells found in the B cell follicles of secondary lymphoid organs such as lymph nodes, spleens and Peyer's patches, and are identified by their constitutive expression of the B cell follicle homing receptor CXCR5...

, chloroform
Chloroform
Chloroform is an organic compound with formula CHCl3. It is one of the four chloromethanes. The colorless, sweet-smelling, dense liquid is a trihalomethane, and is considered somewhat hazardous...

, dichloromethane
Dichloromethane
Dichloromethane is an organic compound with the formula CH2Cl2. This colorless, volatile liquid with a moderately sweet aroma is widely used as a solvent. Although it is not miscible with water, it is miscible with many organic solvents...

 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 bonds linking the amino acids. These absorption bands lie partly in the so-called vacuum ultraviolet (wavelengths less than about 200 nm). The wavelength region of interest is actually inaccessible in air because of the strong absorption of light by oxygen
Oxygen
Oxygen is the element with atomic number 8 and represented by the symbol O. Its name derives from the Greek roots ὀξύς and -γενής , because at the time of naming, it was mistakenly thought that all acids required oxygen in their composition...

 at these wavelengths. In practice these spectra are measured not in vacuum but in an oxygen-free instrument (filled with pure nitrogen
Nitrogen
Nitrogen is a chemical element that has the symbol N, atomic number of 7 and atomic mass 14.00674 u. Elemental nitrogen is a colorless, odorless, tasteless, and mostly inert diatomic gas at standard conditions, constituting 78.08% by volume of Earth's atmosphere...

 gas).

Once oxygen has been eliminated, perhaps the second most important technical factor in working below 200 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 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 lamp
Xenon arc lamp
A xenon arc lamp is a specialized type of gas discharge lamp, an electric light that produces light by passing electricity through ionized xenon gas at high pressure to produce a bright white light that closely mimics natural sunlight...

. 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
Synchrotron
A synchrotron is a particular type of cyclic particle accelerator in which the magnetic field and the electric field are carefully synchronised with the travelling particle beam. The proton synchrotron was originally conceived by Sir Marcus Oliphant...

 sources has a much higher flux at short wavelengths, and has been used to record CD down to 160 nm. Recently the CD spectrometer at the electron storage ring facility ISA at the University of Aarhus
Aarhus
Aarhus or Århus is the second-largest city in Denmark. The principal port of Denmark, Aarhus is on the east side of the peninsula of Jutland in the geographical center of Denmark...

 in Denmark was used to record solid state CD spectra down to 120 nm.
At the quantum mechanical
Quantum mechanics
Quantum mechanics, also known as quantum physics or quantum theory, is a branch of physics providing a mathematical description of much of the dual particle-like and wave-like behavior and interactions of energy and matter. It departs from classical mechanics primarily at the atomic and subatomic...

 level, the information content of circular dichroism and optical rotation
Optical rotation
Optical rotation is the turning of the plane of linearly polarized light about the direction of motion as the light travels through certain materials. It occurs in solutions of chiral molecules such as sucrose , solids with rotated crystal planes such as quartz, and spin-polarized gases of atoms...

 are identical.

See also


  • Circular polarization in nature
  • Dichroism
    Dichroism
    Dichroism has two related but distinct meanings in optics. A dichroic material is either one which causes visible light to be split up into distinct beams of different wavelengths , or one in which light rays having different polarizations are absorbed by different amounts.The original meaning of...

  • Linear dichroism
    Linear dichroism
    Linear dichroism is a spectroscopic technique that is primarily used to study the functionality and structure of molecules. LD can be defined as the difference in absorption of light polarized parallel and perpendicular to an orientation axis...

  • Magnetic circular dichroism
    Magnetic circular dichroism
    Magnetic circular dichroism is the differential absorption of left and right circularly polarized light, induced in a sample by a strong magnetic field oriented parallel to the direction of light propagation...

  • Optical activity
  • Optical isomerism
  • Optical rotation
    Optical rotation
    Optical rotation is the turning of the plane of linearly polarized light about the direction of motion as the light travels through certain materials. It occurs in solutions of chiral molecules such as sucrose , solids with rotated crystal planes such as quartz, and spin-polarized gases of atoms...

  • Optical rotatory dispersion
    Optical rotatory dispersion
    Optical rotatory dispersion is the variation in the optical rotation of a substance with a change in the wavelength of light. Optical rotatory dispersion can be used to find the absolute configuration of metal complexes....

  • Vibrational circular dichroism
    Vibrational circular dichroism
    Vibrational circular dichroism is a spectroscopic technique which detects differences in attenuation of left and right circularly polarized light passing through a sample...



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