Circular Dichroism

Introduction

Circular dichroism (CD) is form of light absorption spectroscopy that measures the difference in absorbance of right- and left-circularly polarized light (rather than the commonly used absorbance of isotropic light) by a substance. It is applicable for molecules have one or more chiral chromophores [1].

Circular dichroism = ΔA(λ) = A(λ)LCPL - A(λ)RCPL, where λ is the wavelength

This technique measured a molecule over a range of wavelengths. All chiral molecules can be studied, particularly in study of large biological molecules. A primary application is in the analysing the conformation of macromolecules or secondary structure (particularly proteins). Circular dichroism is used to measure the changes of secondary structure

It is also known as birefringent (the refractive indexes seen by horizontally and vertically polarised light are different). Slowing one of the linear components of the beam, oriented plate will convert linearly polarised light into circularly polarised light. A beam (left- or right-CPL) will produced [1].

The basis of circular dichroism is the difference in the absorbance of left- and right-CPL. A molecule that absorbs LCP and RCP differently is considered as optically active or chiral molecule.

At light wavelengths that can be absorbed by a chiral molecule, Circular dichroism is occur. Absorption may occur at different extents (e.g., 90% of R-CPL and 88% of L-CPL may absorbed by chiral chromophore). The primary spectroscopic property measured by CD spectrometer is the Chirascan. Thus, Circular dichroism measured as a function of wavelength.

Optical rotation (ORD) or circular dichroism (CD) can be calculated from the other if spectral information of ORD or CD is available. CD spectra is better resolved spectrally than optical rotation

Better the S/N is means better in limit of detection.

A measurement of long period of time must be considered to determine the true average. The time is inversely proportional to S/N. thus, maximum S/N required for designing optimum circular dichroism spectrophotometer.

S/N can be enhanced by increasing the light intensity of incident linearly-polarised, increasing the efficiency of the detector, or averaging and collecting data points in long time.

Sample preparation and measurement

A buffer or detergent or other chemical should not be used unless it will not mask the signal of protein. In addition, compound that could be absorbed in the desired region (190 - 250 nm) should not be used. However, Protein solution should contain only chemicals (with its lowest concentrations) that maintain protein stability. Pure protein should be used if possible, hence any additional peptide or protein will interfere with the CD signal. Noise of signal will appear in the presence of Unfolded protein, peptides, scattering particles . Filtering of the solutions by 0.02 um filters may improve signal to noise