CD spectroscopy

Circular dichroism (CD) spectroscopy is extremely valuable for the structural identification and analysis of chiral molecules. Two chemical compounds can exhibit identical physicochemical properties but showcase a distinct ability to rotate the plane of vibration of plane-polarized light. Such molecules are called chiral. These chiral compounds are usually very difficult to analyze and identify. But now it is quite possible as the scientific world is getting more familiar with CD spectroscopy. Thus, we have compiled you for this article so that you can learn all there is to know about CD spectroscopy as a beginner.

What is circular dichroism spectroscopy

Circular dichroism (CD) spectroscopy is a type of absorption spectroscopy. It is based on measuring the differential absorption of left and right circularly plane-polarized light. Different optically active chemical compounds such as chiral molecules absorb light circularly polarized in a specific direction. The difference in absorption of left and right circularly polarized light can then be measured by CD spectroscopy to identify and quantify different chemical constituents of a complex sample mixture.

Circular dichroism spectroscopy was introduced in the early 19^th century by Jean-Baptiste Biot, Augustin Fresnel, and Aime Cotton. Therefore, circular dichroism is also called the cotton effect.

What are chiral compounds

Chiral molecules are asymmetric molecules that cannot be superimposed on their mirror images. Enantiomers are optically active chiral compounds that rotate plane-polarized light in opposite directions. One enantiomer can be levorotatory which rotates the plane of vibration of plane-polarized light towards the left and another dextrorotatory which does the opposite.

If a chemical compound is not chiral itself, chirality can be induced in it via covalent bonding. Chiral chromophores (such as aromatic amino acids) can be attached by chemical reaction and/or the target molecules can be placed in an asymmetric environment.

What is meant by circularly polarized light

Electromagnetic radiations (simply referred to as light here) consist of electric and magnetic fields, oscillating perpendicular to the direction of propagation. As both the electric and magnetic fields have magnitude and a specific direction, so they are known as vector quantities. Unpolarized light has no specific direction of oscillation which means it oscillates in all directions. On the other hand, when this light is circularly polarized, its electric field propagates around (in a circle) the direction of propagation, at a constant magnitude.

As light propagates, its electric field vector appears as a helix. If both the electric and magnetic fields are traced together, the vectors appear as a double helix, just like the structure of DNA.

The electric field vector can either rotate clockwise or anticlockwise to the direction of propagation. If it rotates clockwise, it appears leftwards to the observer’s eye, so it is called left circularly polarized (LCP) light. Contrarily, if the electric field vector rotates anticlockwise, it appears to be moving rightwards thus it is known as right circularly polarized (RCP) light. Superimposition of LCP and RCP results in linearly plane-polarized light.

What is the working principle of circular dichroism spectroscopy

Let us discuss how the different components of a circular dichroism spectrophotometer function to carry out CD spectroscopy.

Step I: Light irradiation

• Different regions of electromagnetic spectrum can be used as the light source in CD spectroscopy.
• It includes the far ultraviolet (UV) region (250-300 nm), ultraviolet-visible (UV-Vis) region (200-800 nm) as well as the near-infrared (IR) region. (800- 2500 nm). The chosen radiation source depends on the purpose that CD spectroscopy is being used for.
• In this way, CD spectroscopy is further categorized into UV CD spectroscopy, UV-Vis CD spectroscopy, vibrational circular dichroism spectroscopy, etc.
• The most popular light source in CD spectroscopy is a sodium D-line lamp that supplies 580 nm radiations.

Step II: Circular polarization of light

• The light beams first pass through a polarizer that converts unpolarized light into linearly plane-polarized light.
• This linearly polarized light is transformed into circularly polarized light by passing it through a piezoelectric element such as a photoelastic modulator (PEM).
• PEM is made up of a piece of quartz. When 50kHz frequency is applied to the quartz piece, it induces birefringence.
• The incident ray is split into two rays, an ordinary and an extraordinary ray, circularly polarized in opposite directions (i.e., left, and right directions respectively).

Step III: Interaction of light with sample molecules

• The sample is placed in quartz cuvettes of path lengths 1 to 10 mm. Small sample volumes are taken that do not usually require further pre-treatment.
• The targeted optically active chiral compound present in the sample absorbs left and right circularly polarized light to different extents.
• A particular compound absorbs one direction in preference to the other.
• The sample molecules thus undergo electronic transitions (such as n to π* or π to π*) or vibrational energy changes, as per the wavelengths absorbed.  

Step IV: Collecting circular dichroism data

• In the final step, the differential absorption of left and right circularly polarized light based on the structure of sample molecules is collected.

• The difference in the amount of RCP and LCP light absorbed (∆ A) can be measured using Beer Lambert’s law.

∆ A = A_l – A_r

where A_r = absorbance of right circularly polarized light and A_l = absorbance of left circularly polarized light.

A= (ε_l – ε_r) cl

where c= concentration of chiral molecules in the sample, l= pathlength, ε_l, and ε_r are molar absorptivity of a medium for LCP and RCP light respectively. The difference in ε_r and ε_l is known as molar circular dichroism (∆ε). The differential absorption of circularly polarized light in two different directions as a function of frequency is called dichroism.

• This helps plot the circular dichroism (CD) spectrum as a graph of CD versus wavelength that allows compound structural identification and its quantification.

How do you analyze circular dichroism data

Circular dichroism in the CD spectrum is measured in ellipticity (θ). θ is calculated as the tangent of the ratio of the minor elliptical axis to the major elliptical axis. Ellipticity represents differential absorption by the sample molecules. It can be further related to molar circular dichroism (∆ ε), as shown in the equation below.

∆ ε = ∆ A/cl= θ / cl

The unit of θ is mdeg

∆ε is characteristic of a specific chiral compound or structural feature. Additionally,  the CD spectrum obtained acts as a fingerprint of the chiral compound. The CD spectrum is usually a sum of light absorption by all the different chromophores present in a molecule. The position and strength of a CD absorbance band differ based on the compound under examination. A positive CD signal indicates a greater absorbance of LCP light and vice versa.

CD spectroscopy uses in biochemistry

• CD spectroscopy is specifically used for studying macromolecular structures and biomolecules such as proteins and nucleic acids.
• Complex biological molecules such as proteins are made up of secondary structures;α-helices and β-pleated sheets.CD spectroscopy helps in studying these different structural conformations present in a biological molecule.

• Circular dichroism spectroscopy can reveal any undesirable changes in these structural conformations. Protein structural denaturation by a change in temperature, pH, and biological environment can be analyzed through CD spectroscopy.
• Protein-ligand binding and disease-caused protein mutation can also be studied through circular dichroism spectroscopy. Similarly, it also gives structural information about the prosthetic groups present in a protein molecule such as the heme groups present in hemoglobin and cytochrome c.
• CD spectroscopy can also help study the thermal stability of a protein by giving thermodynamics information such as enthalpy change (∆H) and Gibbs free energy change (∆G) of protein denaturation. 

Advantages of circular dichroism spectroscopy

• A prominent advantage of circular dichroism spectroscopy is that it is an accurate and quick spectroscopic technique that does not require any extensive data processing.
• CD spectroscopy offers wide flexibility. It can be applied in varying solvent conditions and also under different temperatures, pH, and sample concentrations.
• It is a non-destructive technique, easy to operate, and requires a small sample volume.
• Even less than 20 μg of proteins present in a sample can be readily analyzed via circular dichroism spectroscopy.
• CD spectroscopy can be performed on a wide range of samples including solid, liquid, films, gel samples, etc.

Limitations of CD spectroscopy

• CD spectroscopy provides less specific structural information as compared to other superior methods such as NMR spectroscopy and X-ray spectroscopy.
• Therefore, CD spectroscopy is usually performed as a preliminary protein structural detection step before applying extensive proton NMR and ¹³C-NMR spectroscopic techniques.
• Also, the CD spectrophotometer is a relatively expensive instrument as compared to a simple UV-Vis spectrophotometer.

Check out this article for further information on circular dichroism spectroscopy and its application in studying protein structures.

Last but not the least, here is a video tutorial for you on CD spectroscopy.

References

1. Banerjee, B., G. Misra and M. T. Ashraf (2019). Chapter 2 – Circular dichroism. Data Processing Handbook for Complex Biological Data Sources. G. Misra, Academic Press: 21-30.

2. Corrêa, D. and C. Ramos (2009). “The use of circular dichroism spectroscopy to study protein folding, form, and function.” African Journal of Biochemistry Research 3: 164-173.

3. Greenfield, N. J. (2006). “Using circular dichroism spectra to estimate protein secondary structure.” Nature Protocols 1(6): 2876-2890.

4. Siligardi, G. and R. Hussain (2017). Circular Dichroism, Applications. Encyclopedia of Spectroscopy and Spectrometry (Third Edition). J. C. Lindon, G. E. Tranter and D. W. Koppenaal. Oxford, Academic Press: 293-298.