Circular Dichroism - A spectroscopic technique
What is Circular Dichroism?
Circular dichroism (CD) is a spectroscopic technique which measures differences in the absorption of left-handed circularly polarised light and right-handed circularly polarised light.
To really understand circular dichroism, one must first understand the basics of polarisation.
Linear and Circular Polarised Light.
Linearly polarised light only oscillates in one specific direction. All polarised light states can be derived as a sum of two linearly polarised states at right angles to each other, usually referenced to the viewer as vertically and horizontally polarised light. As shown in the two animations below. (Move the mouse over the images to view animations.)
Vertically Polarised Light
Horizontally Polarised Light
If for instance we take equal parts horizontal and vertically polarised that are in phase with each other. As shown in the animation below the resulting light wave (the blue curve) is linearly polarised at 45 degrees.
45 Degree Polarised Light
If the phase of the two-polarisation planes is out of phase, the propagating ray ceases to be linearly polarised. For example, when one of the polarisation planes is out of phase by a quarter-wave from the other, the propagating ray will form a spiral; this is called circularly polarised light. This spiral, like all helices, has left-handed and a mirror image right-handed forms, which are mirror images (and therefore non-superimposable).
The optical element that converts between linear polarised light and circularly polarised light is termed a quarter-wave plate. A quarter-wave plate is a birefringent material, a material where the two planes of linear polarised light have different speeds. The plate will convert a 45 degree linearly polarised beam into circularly polarised light by slowing one of the linearly polarised components of the light beam so it is a quarter-wave out of phase. This will produce a beam of either left or right circularly polarised light.
Left Circularly Polarised (LCP) Light
Right Circularly Polarised (RCP) Light
Animations created from the Emanim program.
Chiral Molecules
Chiral molecules are molecules that have non-superimposable mirror image isomers. The mirror image isomers are almost chemically identical apart from two distinct features, how they interact with polarized light, and how they interact with other chiral molecules.
In the case of linearly polarised light, chiral molecules exhibit circular birefringence. This causes the plane of linearly polarised light to rotate in a specific direction as it passes through a solution of the chiral molecule. This effect is measured using a polarimeter, which measures the “optical rotation” of the linearly polarised beam as it passes through a chiral sample. Measurement of the optical rotation as a function of wavelength is termed Optical Rotatory Dispersion (ORD) spectroscopy.
In a related effect, for light at wavelengths that can be absorbed by a chiral molecule, left- and right-circular polarised light will be absorbed by differing amounts. For instance, a chiral chromophore may absorb 90% of right-circularly polarised light and 88% of left-circularly polarised light. This effect is called Circular Dichroism, and is the difference in absorbance between left- and right-circularly polarised light. Circularly dichroism as a function of wavelength is the primary signal recorded by a Circular Dichroism spectrophotometer such as the Chirascan.
Optical Rotation and Circular Dichroism stem from the same quantum mechanical phenomena, and one can be derived from the other mathematically if all spectral information is provided. The relationship between Optical Rotatory Dispersion, Circular Dichroism, Absorbance spectra and chirality are shown below, with a comparison of the two enantiomers of camphor sulfonic acid.
CD, ORD and Absorbance spectra of R
and S forms of camphor sulfonic acid.
Although ORD spectra and CD spectra can theoretically provide equivalent information, each technique has been used for very distinct applications. Optical rotation at a single wavelength is used as a general measurement tool for chiral molecules, to determine concentration and as a determinate of chiral purity compared to a known standard. The simplicity and low-cost of the experiment and instrumentation makes it ideal for this application. Circular dichroism spectra on the other hand are more spectrally resolved than ORD spectra, and consequently more suitable for advanced spectral analysis.
Chirality and Biology
The majority of all biological molecules are chiral molecules, for instance 19 of the 20 common amino acids are chiral, as well as DNA, RNA and a host of other biologically important molecules. The highly chiral chemistry of biological molecules lend themselves well to analysis by circular dichroism, and are the main application of the technique.
One of the largest applications of Circular Dichroism in biochemistry is in the understanding of the higher order structures of chiral macromolecules such as proteins and DNA. The reason for this is that the CD spectrum of a protein is not a sum of the individual amino acid circular dichroism spectra, but is greatly influenced by the 3-dimension structure of the macromolecule itself. Each structure has a specific Circular Dichroism signature, and this can be used to identify structural elements and to follow changes in the structure of chiral macromolecules.
The most widely studied Circular Dichroism signature are the various secondary structural elements of proteins like the α-helix and the β-sheet. This is understood to the point that the CD spectra in the far-UV (below 260nm) can be used to predict the percentages of each secondary structural element in the structure of a protein. Some of the common protein secondary structural elements and the CD spectra associated with them are shown below.
The secondary structure
conformation and the CD spectra of protein structural elements. Right is
an example of the backbone conformation of a peptide in an α-helix
and left is the conformation of a peptide in a β-sheet. In
the centre are the associated CD spectra for these different
conformations.
There are many algorithms designed for fitting the Circular Dichroism spectra of Proteins to provide estimates of secondary structure. The protein CD analysis software distributed with the Chirascan is CDNN. For those interested in other software packaged used for these prediction, a web based front end for a number of different algorithms is hosted here.
Secondary structure prediction is only part of the power of Circular Dichroism spectroscopy. As changes in Circular Dichroism spectra are very good proxies for changes in structure of a molecule. Couple this with the fact that spectra can be recorded in minutes, and single wavelength kinetics can be recorded from milliseconds onwards, CD is a particularly powerful tool to follow dynamic changes in protein structure. For instance changes induced by changing temperature, pH, ligands, or denaturants are all commonly used.
An application note that describes the use of Circular Dichroism to follow the kinetics of refolding of the secondary structure of a protein using changes in denaturant concentration can be found here. Another application note that describes the use of Circular Dichroism to follow the unfolding of proteins by thermal denaturation can be found here.
A powerful application of Circular Dichroism is to compare two macromolecules, or the same molecule under different conditions, and determine if they have a similar structure. This can be used simply to ascertain if a newly purified protein is correctly folded, determine if a mutant protein has folded correctly in comparison to the wild-type, or for the analysis of biopharmaceutical products to determine that the biopharmaceutical is still in a correctly folded active conformation.
Principle of operation of a Circular Dichroism spectrophotometer
As Circular Dichroism is the difference in light absorbance between left- and right-circularly polarised light, Circular Dichroism spectrophotometers are highly specialised variations on the absorbance spectrophotometer.
A Circular Dichroism spectrophotometer is also commonly termed a Circular Dichroism spectropolarimeter or a circular dichrograph.
Most modern Circular Dichroism instruments operate on the same principles. There is a source of monochromatic linearly polarised light, this can be turned into either left- or right-circularly polarised light by passing it through a quarter-wave plate that is at 45 degrees to the linear polarisation plane as described before.
Instead of a static quarter-wave plate a Circular Dichroism spectrophotometer has a specialised optical element called a photo-elastic modulator (PEM). This is a piezoelectric element fused to a block of fused silica. The piezoelectric element oscillates at its resonance frequency (typically around 50 kHz). The alternating stress applied turns the fused silica element into a dynamic quarter-wave plate, producing left and then right circular polarised light at the drive frequency. For more information about the PEM and its various uses see the major manufacturer Hinds.
On the other side of the sample position there is a light detector, most commonly a photomultiplier tube. When there is no circular dichroic sample in the light path, the light hitting the detector is constant. If there is a circular dichroic sample in the light path, the recorded light intensity will vary with the resonance frequency of the PEM. Using a lock-in amplifier tuned to the frequency of the PEM, it is possible to measure the difference in intensity between the two circular polarisations (vAC). The average total light intensity across many PEM oscillations (vDC) can be used to scale the size of the lock-in amplifier signal to take into account variations in total light level. Both signals can be recorded and from them the Circular Dichroism signal can be calculated easily by dividing the vAC component by the vDC signal.
G is a calibration-scaling factor to provide either ellipticity or difference in absorbance.
Principle of operation of a
Circular Dichroism Spectrophotometer (click to progress through slideshow)
| Please note, this slide-show animation requires Adobe Flash Player |  |
Performance of a Circular Dichroism Spectrophotometer
The limit of detection is defined by the signal-to-noise of the Circular Dichroism spectrophotometer. The signal-to-noise performance of the Circular Dichroism spectrophotometer is limited by the noise of the detector and associated electronics. For a photomultiplier tube the major contribution is photon shot noise, which is described here.
A general relationship between the contributing factors to the signal-to-noise in an optical spectrometer can be written as:
Q = detector performance, I = light intensity, t = time scale of the measurement.
From this it is apparent there are three ways to improve the signal to noise of a circular dichroism spectrophotometer: decrease the photon shot noise and so improve the noise characteristics of the detector, increase the intensity of linearly polarised monochromatic light out of the monochromator, or spend more time collecting and averaging data points.
The first two factors, detector performance and light intenisty are the most important in terms of the design of a high performance Circular Dichroism spectrophotometer, and work together to lower the last factor, the time required to carry out a measurement. The higher the light throughput and better the detector and electronics, the less time it takes to collect quality data or higher the quality data can be collected in time limited experiments like kinetic measurements.
High light throughput is the main avenue to increase the performance of Circular Dichroism spectrophotomers. For example there has been a growth in the use of synchrotrons as Circular Dichroism lightsources because of their very high light intensity. Two UK facilities are available, at the Diamond synchrotron and the Daresbury SRS. These facilities provide tremendous light intensity across very wide spectral ranges, but are not practical for the majority of applications for Circular Dichroism measurements due to the costs to build and operate these facilities.
Apart from the high intensity of light to improve signal to noise, the light out of the monochromator has to have very low stray-light, and with a high purity of linear polarisation to provide accurate measurements. All of these three key elements have been optimised in the Chirascan Circular Dichroism spectrophotometer, and have been achieved by key design features of the Chirascan monochromator. These design features are outlined in the slide show below.
The Chirascan Circular Dichroism
spectrophotometer, (click to progress through slideshow)
| Please note, this slide-show animation requires Adobe Flash Player | |
Chirascan uses two artificial Quartz prisms instead of the diffraction gratings that most people are familiar with from normal absorbance spectrophotometers. Quartz prism are more efficient compared to diffraction gratings for a very wide range of wavelengths, particularly in the UV. Quartz is also birefringent, and so the prisms used not only disperse light into the component wavelengths but also disperses linear polarised components. Another advantage of prisms is they do not pass second order multiples of the desired wavelength, a major source of stray-light in grating-based monochromators.
Why aren't prism monochromators more widely used? The dispersion of a grating is linear and highly customisable, while the dispersion of a prism is non-linear and is set by the materials the prism is made out of. The wavelengths separation of a prism is different across the spectral range, with a large separation at the UV end of the spectrum and a small separation at the near infrared end. Consequently the optics and mechanics of a prism monochromator have to be more complex than a grating monochromator, with the need to constantly vary the slit width to maintain a constant bandpass and a complex relationship between prism movement and wavelength. However, the large separation in the UV means that wider slits can be used even at a small spectral bandpass, which means a greater light collection efficiency in the UV to Far-UV region. The large majority of Circular Dichroism applications are carried out in the UV to Far-UV, and the very high light throughput characteristics of a prism are a major advantage.
There are several other commercial Circular Dichroism instruments using quartz prisms monochromators. These instruments are based on legacy optical designs from over 30 years ago. The Chirascan's optical bench is a very recent design, completed in 2004. This allowed us to use modern optical and mechanical CAD design tools to fully optimize the optical performance of the monochromator in the ways described above, and so achieve significant signal-to-noise improvements.
For a more detailed overview of the Chirascan Circular Dichroism Spectrophotometer, please have have a read of the Technical Description.
Further reading
Wikipedia has an article on Circular Dichroism, and searching Google for Circular Dichroism will lead to many articles on the subject.
Some useful books: Circular Dichroism and the Conformational Analysis of Biomolecules, editor Gerald D. Fasman; Circular Dichroism: Principles and Applications, editors Nina Berova, Koji Nakanishi, and Robert W. Woody; Organic Conformational Analysis and Stereochemistry from Circular Dichroism, by David A. Lightner and Jerome E. Gurst. Circular Dichroism and Linear Dichroism by Alison Rodger and Bengt Nordén.
A list of Publications using the Chirascan can be found here.
Chirascan -
the new standard in circular dichroism
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