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Linear dichroism (LD) 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 . LD measurements are based on the interaction between matter and light and thus are a form of electromagnetic spectroscopy. This effect has been applied across the EM spectrum, where different wavelengths of light can probe a host of chemical systems. The predominant use of LD currently is in the study of bio-macromolecules (e.g. DNA) as well as synthetic polymers.
Contents
1 Basic information
2 UV linear dichroism
3 Alignment methods
4 Associated techniques
5 References
6 External links
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Basic information
Linear polarization
LD uses linearly polarized light, which is light that has been polarized in one direction only. This produces a wave, the electric field vector, which oscillates in only one plane, giving rise to a classic sinusoidal wave shape as the light travels through space. By using light parallel and perpendicular to the orientation direction it is possible to measure how much more energy is absorbed in one dimension of the molecule relative to the other, providing information to the experimentalist.
As light interacts with the molecule being investigated, should the molecule start absorbing the light then electron density inside the molecule will be shifted as the electron becomes photo-excited. This movement of charge is known as an electronic transition, the direction of which is called the electric transition polarisation. It is this property for which LD is a measurement.
The LD of an orientated molecule can be calculated using the following equation:-
LD = A?- A?
Where A? is the absorbance parallel to the orientation axis and A? is the absorbance perpendicular to the orientation axis.
Note that light of any wavelength can be used to generate an LD signal.
The LD signal generated therefore has two limits upon the signal that can be generated. For a chemical system whose electric transition is parallel to the orientation axis, the following equation can be written:
LD = A?- A? = A? > 0
For most chemical systems this represents an electric transition polarised across the length of the molecule (i.e. parallel to the orientation axis).
Alternatively, the electric transition polarisation can be found to be perfectly perpendicular to the orientation of the molecule, giving rise to the following equation:
LD = A?- A? = – A? < 0
This equation represents the LD signal recorded if the electric transition is polarised across the width of the molecule (i.e. perpendicular to the orientation axis) , which in the case of LD is the smaller of the two investigable axes.
LD can therefore be used in two ways. If the orientation of the molecules in flow is known, then the experimentalist can look at the direction of polarisation in the molecule (which gives an insight into the chemical structure of a molecule), or if the polarisation direction is known it can be used as a means of working out how orientated in flow a molecule is.
UV linear dichroism
UV LD is typically employed in the analysis of biological molecules, especially large, flexible, long molecules that prove difficult to structurally determine by such methods as NMR and X-ray diffraction.
DNA
DNA is almost ideally suited for UV LD detection. The molecule is very long and very thin, making it very easy to orientate in flow. This gives rise to a strong LD signal. DNA systems that have been studied using UV LD include DNA-enzyme complexes and DNA-ligand complexes, the formation of the latter being easily observable through kinetic experiments.
Fibrous protein
Fibrous proteins, such as proteins involved in Alzheimer disease and prion proteins fulfil the requirements for UV LD in that they are a class of long, thin molecules. In addition, cyto-skeletal proteins can also be measured using LD.
Membrane proteins
The insertion of membrane proteins into a lipid membrane has been monitored using LD, supplying the experimentalist with information about the orientation of the protein relative to the lipid membrane at different time points.
In addition, other types of molecule have been analysed by UV LD, including carbon nano-tubes and their associated ligand complexes.
Alignment methods
Couette flow
The Couette flow orientation system is the most widely used method of sample orientation for UV LD. It has a number of characteristics which make it highly suitable as a method of sample alignment. Couette flow is currently the only established means of orientating molecules in the solution phase. This method also requires only very small amounts of analysis sample ( 20 – 40 ?l) in…(and so on)
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