Circular Dichroism (CD) is an absorbance technique that involves generating a beam from a light source, similar to UV/vis spectroscopy. This beam then undergoes wavelength selection before finally passing through a sample and reaching a detector.
The two techniques differ in the level of polarization of the generated light (Box 5): while UV/Vis spectroscopy uses unpolarized light, CD spectroscopy utilizes circularly polarized (CP) light that is modulated between left and right circular polarizations. A CD spectrometer is used to record the differential absorbance as a function of wavelength. In other words, CD calculates the difference in absorbance between left-handed circularly polarized (L-CP) and right-handed circularly polarized (R-CP) light, as demonstrated in the formula below:
CD = ∆A(λ) = A(λ)L-CP - A(λ)R-CP
As a consequence, CD spectra can be denoted in both positive and negative values. To illustrate this, the signal is positive when the L-CP light is absorbed to a greater degree than the R-CP light at a particular wavelength. Conversely, the signal is negative when the R-CP light is absorbed more than the L-CP light (Figure 1).
Figure 1: CD spectra of Camphor sulfonic acid (CSA). CSA is a chiral molecule and exists as two enantiomers which are non-superimposable mirror-images of each other. Their corresponding CD spectra are mirrored at the x-axis. Positive CD signals are obtained at wavelengths where the left-handed component of circularly polarized light is absorbed more strongly than the right-handed component and negative when it is the other way around.
Since the recorded difference in absorbance is extremely small — in fact, several orders of magnitude smaller than a typical sample’s total absorbance — CD spectrometers must be highly sensitive. To this end, the introduction of avalanche photodiode detectors has been a key technical advancement that has significantly increased the sensitivity of CD spectrometers. This has effectively replaced conventional photomultiplier detectors and lowered the background noise, thereby facilitating the detection of minor changes even in the near-UV range where the magnitude of CD signals is typically low.
In order to generate a CD signal, a molecule needs to fulfill two basic requirements. Firstly, it must possess a chromophore (i.e. absorb light) and secondly, it must exhibit the state of chirality (i.e. independence and not superimposability on its mirror image).
Enantiomers are basically two isomers that are a chiral molecule’s ‘non-superimposable’ mirror-images. A pair of enantiomers possess identical physical and chemical properties, with two major exceptions: the first being the manner in which they interact with polarized light, and the second being the way in which they interact with other chiral molecules.
Figure 1 displays a chiral absorbing molecule – camphor sulfonic acid (CSA), which exists as two enantiomers. The CD spectrum of (1S)-(+)-CSA exhibits a positive peak at 290.5 nm and a negative peak at 191 nm. On the other hand, the CD spectrum of the other enantiomer, (1R)-(-)-CSA, has exactly the same profile, but is mirrored in the x-axis.
Box 5: Polarization Basics
Light is an electromagnetic wave, and polarization describes the orientation of its electric or magnetic vector. A good example of this is how sun light reflected at a certain angle from the sea surface is linearly polarized. Here, the electric vector oscillates along a single line, allowing the wave to propagate through space within a plane. This plane can be rotated at any angle around the wave’s propagation direction, thus achieving a number of different orientations (for instance, horizontal or vertical).
In this vein, any polarized state of light can be described as the consequence of two linearly polarized states at right angles to each other. For example, when a horizontal polarized wave combines with a vertical-linearly polarized wave of equal amplitude (and they are in phase with each other), it creates a linearly polarized light wave. Moreover, this wave is at 45° to the planes of the two combined waves.
Conversely, if the two polarization states are out of phase, the wave that results from it is elliptically polarized. However, if the phase difference Δφ of the vertical component in relation to the horizontal component is 1/4th the wavelength, resulting in circularly polarized (CP) light. In such a scenario, there is a rotation in the electric vector and thus, a helix while propagating through space is born.
This helix can be right-handed (R-CP light), which means if Δφ is negative (‑90°), the rotation is clockwise when viewed towards the light source. Alternatively, the helix can be left-handed (L-CP light), i.e. when Δφ is positive (90°) and the oscillation is counterclockwise. Such states of polarization are non-superimposable mirror images, akin to the threads of the right and left pedals of a bicycle.
About Applied Photophysics
Applied Photophysics is a leading provider of systems and accessories for the biophysical characterization of biomolecules. Headquartered in Leatherhead, Surrey, UK, the Company has been established for more than 40 years.
The SX-range of stopped-flow spectrometers, used to monitor changes in absorbance and fluorescence during fast biological reactions, is acknowledged globally as the gold standard for kinetic studies. In 2005, the Company introduced the first Chirascan™ system, using the phenomenon of circular dichroism (CD) to characterize changes in the higher order structure of proteins.
Since then, the company has continued to incorporate its in-depth knowledge and understanding of CD into a range of Chirascan products that are used in cutting-edge research and to support the development of innovator drugs and biosimilars in the biopharmaceutical industry. Compared to conventional CD instruments, the new generation of Chirascan systems ensures that every scientist gets the most from every CD analysis.
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