Circular Dichroism. Part I. Introduction. Circular Dichroism.
Part I. Introduction
Circular dichroism (CD) spectroscopy measures differences in the absorption of left-handed polarized light versus right-handed polarized light which arise due to structural asymmetry. The absence of regular structure results in zero CD intensity, while an ordered structure results in a spectrum which can contain both positive and negative signals.
Jasco J-810 Circular Dichroism System
Optical rotation: the rotation of linearly polarized light by the sample
Optical rotary dispersion: the variation of optical rotation as a function of wavelength. The spectrum of optical rotation.
Circular Dichroism: the difference in absorption of left and right circularly light.
Linear Polarized Light
Passing plane polarized light through a birefringent plate (in the z-direction) which splits the light into two plane-polarized beams oscillating along different axes (e.g., fast along x and slow along y). When one of the beams is retarded by 90º (using a quarter-wave retarder) then the two beams which are now 90º out of phase are added together, the result is circularly polarized light of one direction. By inverting the two axes such that the alternate beam is retarded than circularly polarized light of the other direction is generated.
The result of adding the right and left circularly polarized that passes through the optically active sample is elliptically polarized light, thus circular dichroism is equivalent to ellipticity
Circularly Polarized Light
n refractive index
l wavelength of light
f angle of rotation
l path length through the sample
Linear polarized light can be viewed as a superposition of opposite circular polarized light of equal amplitude and phase
different absorption of the left- and right hand polarized component leads to ellipticity (CD) and optical rotation (OR).
Thedifference between the absorption of left and right handed circularly-polarised light and is measured as a function of wavelength. CD is measured as a quantity called mean residue ellipticity, whose units are degrees-cm2/dmol.
ORD spectra are dispersive (called a Cotton effect for a single band) whereas circular dichroism spectra are absorptive. The two phenomena are related by the so-called König-Kramers transforms.
Protein Concentration: 0.5 mg/ml
Cell Path Length: 0.5 mm
Stabilizers (Metal ions, etc.): minimum
Buffer Concentration : 5 mM or as low as possible while maintaining protein stability