Editing Circular dichroism (section)

== Additional applications and related techniques ==

=== Fluorescence ===
Fluorescence measurements in CD spectroscopy, recorded at a 90 degree angle to the incident light, often serve as a complementary data channel, providing additional insights into protein stability and conformational changes. By detecting intrinsic fluorescence from aromatic residues such as tryptophan and tyrosine, researchers can assess environmental shifts that accompany structural transitions. Fluorescence data can be acquired alongside CD signals, particularly in temperature ramp experiments, where it helps monitor unfolding events by tracking changes in emission intensity or wavelength shifts. This process could be followed by recording CD spectra at intervals of 1&nbsp;°C, for example, while increasing the temperature continuously at 1&nbsp;°C per minute. The excitation bandwidth required for the fluorescence spectra is usually larger than what would be used as spectral bandwidth for CD measurements, so sometimes the use of different spectral bandwidths for CD and fluorescence measurements is necessary. This dual approach enhances the interpretation of protein behavior under varying conditions, improving confidence in structural and stability assessments.

There are multiple approaches to collecting fluorescence data, including the use of a [[photomultiplier tube]] (PMT) to record total fluorescence, the use of a [[Charge-coupled device|CCD]] fluorometer to record full-spectrum fluorescence, or the use of a scanning emission monochromator (SEM) to allow for scanning the fluorescence spectrum at a fixed excitation wavelength.

=== Optical rotatory dispersion (ORD) ===
CD is closely related to the technique of [[optical rotatory dispersion]] (ORD). Optical rotation is the rotation of linearly polarized light as a result of it passing through an optically active material, and is a consequence of a difference in refractive index between the left and right circularly polarized components of the light (linearly polarized light can be considered to be an equal combination of right and left circularly polarized light). Optical rotatory dispersion is the variation in optical rotation with wavelength. ORD and CD spectra both derive from the interaction of polarized (circularly or linearly) light with chiral molecules and are directly related: each can be derived from the other using the [[Kramers-Kronig transformation|Kramers-Kronig transform]]. Measurements of ORD and CD give essentially the same information, but experimentally each technique has been favored for particular applications. CD is the higher resolution technique since it is only observed at wavelengths where the chiral molecule absorbs light. This makes complicated spectra involving multiple absorbance bands easier to interpret. Alternatively, ORD measurements can be obtained at wavelengths where the chiral molecule being measured does not necessarily absorb light. This means that ORD measurements can be made on materials with high concentrations or where absorbance bands are obscured by solvent or buffer salt absorbance. ORD is particularly useful for unsubstituted sugars as this class of biomolecule does not possess chromophores that absorb in the UV-Vis regions accessible via commercial CD instruments.

=== Linear dichroism (LD) ===
With the appropriate accessory, it is possible to perform [[linear dichroism]] (LD) measurements on CD spectrophotometers. Linear dichroism is the difference in the absorbance of light polarized parallel and perpendicular to an orientation axis:

'''ΔA = A<sub>para</sub> - A<sub>perp</sub>'''

where '''A''' is the absorbance of the linearly polarized light, and the subscripts indicate parallel and perpendicular, respectively. For a sample to have non-zero LD it must be anisotropic; the anisotropy may be intrinsic, as in liquid crystals for example, or it may be induced, for example, by stretching a polymer film, by the application of an electrical field, or by alignment or deformation in a shear field (e.g., nucleic acids such as DNA).

An LD device uses a [[Couette flow|Couette]] cell to generate a shear field. The sample is contained in the annular gap between two concentric quartz cylinders, the outer of which, the rotor, is rotated about its cylindrical axis, while the inner, the stator, is stationary. This arrangement is named after its originator, [[Maurice Couette]].

Shearing can cause large particles in the liquid to align or deform. For example, rigid, rod-like particles, such as carbon nanotubes and glass fibers, will align, whereas vesicles, micelles, and flexible polymers will deform from their equilibrium conformations, extending in the direction of shear. Linear dichroism is then used to determine the direction of net electron transfer in an absorbed chromophore relative to the shear direction, and thereby to the alignment direction of the absorbing species or the deformation direction of its environment.

For more information on LD and its measurement, consult this book.<ref>{{Citation |last1=Rodger |first1=Alison |title=Circular Dichroism and Linear Dichroism |date=2023 |encyclopedia=Encyclopedia of Analytical Chemistry |pages=1–42 |url=https://onlinelibrary.wiley.com/doi/abs/10.1002/9780470027318.a5402.pub3 |access-date=2025-02-17 |publisher=John Wiley & Sons, Ltd |language=en |doi=10.1002/9780470027318.a5402.pub3 |isbn=978-0-470-02731-8 |last2=Chubb |first2=Joel J.}}</ref>

=== Magnetic circular dichroism (MCD) ===
[[Magnetic circular dichroism]] (MCD) is the differential absorption of left and right circularly polarized light in the presence of a magnetic field oriented parallel to the direction of light propagation. MCD differs from natural CD in that it does not require a chiral sample; the origins of CD and MCD are quite different.

There are three contributions to an MCD spectrum, known as the A-, B-, and C-terms. At room temperature, the A- and B-terms dominate, especially for non-magnetic chromophores, and form the majority of what can be observed in a standard MCD experiment.

The A-term is due to [[Zeeman splitting]] of degenerate excited states by a difference in spin. The result is a small shift apart in the wavelengths of the UV-Visible spectrum for each spin state, with each state preferentially absorbing left or right circularly polarized light. The A-term MCD spectrum is observed as a derivative of an absorbance transition with a sharp transition around the absorbance peak. Because of the A-term bands, the MCD spectrum is more structured than the absorbance or natural CD spectrum, multiple overlapping chromophores can be quantified much more easily, and changes in individual chromophores can be tracked.

The B-term is due to mixing of non-degenerate ground states. It is normally observed as a single absorption band type peak, which may be either positive or negative.

The C-term is due to changes in populations of molecules over the Zeeman sublevels due to the response to the field of a paramagnetic ground state. They become significant in the MCD spectrum only at very low temperatures, and the instrumentation requirements of high field strengths and cryogenic temperatures are very similar to those of the related technique of [[electron paramagnetic resonance]] (EPR). This is an expensive and dedicated system, and access to the C-terms is therefore beyond the scope of an easily interchangeable MCD accessory on a benchtop CD instrument.

Collection of MCD data is similar to that of natural CD, except that there are two possible orientations of the magnet, with either the north or south pole closer to the detector. Normally a spectrum is acquired with the magnet in each orientation; since the MCD contribution to the spectrum changes sign when the magnetic field is reversed, whereas the natural CD contribution does not, both contributions can be obtained by averaging the two spectra.

For further information on MCD see this book.<ref>{{Cite book |last=Mason |first=W. Roy |url=https://onlinelibrary.wiley.com/doi/book/10.1002/9780470139233 |title=A Practical Guide to Magnetic Circular Dichroism Spectroscopy |date=2006-11-08 |publisher=Wiley |isbn=978-0-470-06978-3 |language=en |doi=10.1002/9780470139233}}</ref>

=== Circularly polarized luminescence (CPL) ===
Circularly polarized luminescence (CPL) is the emission analog of CD. While CD examines the geometry of molecules in their ground state, CPL, typically observed perpendicular to the incident light beam, provides insight into the geometry of molecules in their excited or luminescent state. In a CPL experiment, the emitted light is analyzed for its contents of left and right circularly polarized light.

The difference in intensity between left and right circularly polarized emitted light, '''Δ''I''''', is given by:

'''Δ''I'' = ''I''<sub>L</sub> – ''I''<sub>R</sub>'''

where '''''I''<sub>L</sub>''' and '''''I''<sub>R</sub>''' are the intensity of left and right circularly polarized emitted light, respectively. The average luminescence intensity is given by:

'''''I'' = (''I''<sub>L</sub> + ''I''<sub>R</sub>) / 2'''

Both '''Δ''I''''' and '''''I''''' are in relative units because in luminescence experiments not all the luminescent light is detected, only that in a restricted solid angle. Moreover, an absolute value for the concentration of luminescent molecules may be hard to obtain. For these reasons, a dissymmetry factor in the luminescence is defined as:

'''''g''<sub>lum</sub> = Δ''I'' / ''I'''''

with '''''g''<sub>lum</sub>''' being an absolute number that is unique to each CPL-active molecule under specific conditions.

Many commercially available CD spectrophotometers can be adapted with an accessory to measure the circularly polarized luminescence of a sample.

=== Analysis of solid samples ===
It is possible to analyze solid-state samples by CD spectroscopy. There are a few options to accomplish this.

Samples are often prepared in the form of discs, either by compression with an inert dispersant such as potassium bromide or potassium chloride or by deposition of the sample as a thin film on top of the disc. This technique is well established in IR absorbance spectroscopy, and it has also been used in UV/visible spectroscopy. The sample is measured in transmission mode, with the light passing through the sample to a detector mounted in the spectrometer transmission port. To eliminate the effect of [[anisotropy]], the sample is usually mounted in a wheel and can be manually rotated to allow measurements to be made at several angular positions. The spectra of all positions can then be averaged.

If the solid samples exist in the form of a powder, they can be measured via diffuse reflectance by using a device known as an [[integrating sphere]].

Integrating spheres were developed early in the twentieth century and were originally used to measure the total output of a light source without reference to the original direction of the light. They are now commonly used when measuring the UV/visible or IR absorption spectra of solid samples, and their use can be extended to the UV/visible CD spectroscopy of solids.

Light enters the sphere through an inlet port, and the internal surfaces of the sphere are usually white, reflective, and diffuse, so that the light becomes equally distributed within the sphere through multiple scattering reflections. A detector is placed at an outlet port and the intensity of the light is measured. For CD spectroscopy, the sample can be placed either at the inlet port, so that the light passes through it before entering the sphere, or at a point on the sphere opposite the inlet port, so that light is diffusely reflected from the sample after entering the sphere. These two operating modes are called transmission and diffuse reflectance, respectively. In both cases the sample is prepared in powder form. For transmission mode, it must then be dispersed in either potassium bromide or potassium chloride and compressed into a disc. For diffuse reflectance mode, it remains as a free powder, if necessary dispersed in a diluent such as barium sulphate or polytetrafluoroethylene (PTFE).

=== Studying fast reactions ===
Many commercially available CD spectrophotometers are designed to be outfitted with a [[stopped-flow]] device for studying the kinetics of fast reactions in solution. In the simplest form of the technique, the solutions of two reactants are rapidly mixed by being forced through a mixing chamber, on emerging from which the mixed fluid passes through an optical observation cell. At some point in time, the flow is suddenly stopped, and the reaction is monitored using a suitable spectroscopic probe, such as absorbance, fluorescence, [[fluorescence polarization]], or circular dichroism. The change in spectroscopic signal as a function of time is recorded, and the rate constants that define the reaction kinetics can then be obtained by fitting the data using a suitable model.