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Circular Dichroism (CD) is a spectroscopic technique which reveals information about a mol-
ecule's chirality or "handedness". This technique has been used for many years, along with
the complementary techniques of polarimetry and optical rotatory dispersion (ORD), for
studying and quantifying optically active compounds and their interactions. The information
content of steady state CD spectra can be used to uniquely identify chiral compounds and
their configurations, predict the secondary structure of proteins and other biological macro-
molecules, and in kinetic mode as a probe to monitor the structural changes accompanying
protein folding or unfolding. CD can be used to monitor and quantify ligand binding process-
es and is an increasingly important tool in chiral drug development. The quality of CD instru-
mentation today is better than ever and the latest technological advances will be discussed
in this article.
Chirality arises in compounds which contain at least one chiral centre com- compounds. Such differential absorption at a chiral centre arises because of a
prising a central carbon atom that is bound to four different atomic or preferential interaction of the electrons in one optical isomer with the cir-
molecular groups in a tetrahedral configuration. This means such a com- cularly polarised fields of the monitoring light in either the left or right cir-
pound can exist in two distinct geometric configurations, or stereo-isomers, cularly polarised states. The difference in the relative absorptions of the two
which are non-superimposable mirror images of each other. Members of states as a function of wavelength yields a characteristic CD spectrum. It
such a pair of stereo-isomers are called enantiomers. If a larger molecule has must be emphasised that the CD signal of each pair of enantiomers is of
several (n) chiral centres, the compound will have 2n stereo-isomers, and opposite sign and so the amplitude of a measurement is not proportional
pairs of non-superimposable stereo-isomers in this population are also to concentration where a mixture of enantiomers exists; rather it provides
termed enantiomers. an indication of enantiomeric excess. A racemic mixture (50:50) of enan-
tiomers will show no CD signal at all.
Optical activity On a larger scale, the differential interaction of circularly polarised light
Chirality is the underlying basis for optical activity. In the simplest case an with asymmetric or helical electron distributions in macromolecular sec-
optically active sample causes rotation of the plane of a linearly polarised ondary structures yields the distinctive spectral features used to identify
beam of light transmitted through it. Each member of a pair of enantiomers those structures, their integrity and their relative concentration.
will cause a rotation of opposite sign and the terms dextro-rotatory and levo-
rotatory were first used to distinguish the two chemical forms responsible. Polarimetry, though a routine analytical tool, is not generally as sensitive
This gave rise to the corresponding name prefixes 'd' and 'l', although the and versatile as CD in terms of ultimate limits of detection and delivery of
alternative prefixes '+' and '-' are now preferred so as not to conflict with information content in either steady state or kinetic modes. The remainder
absolute configuration terminology. (The convention for assigning absolute of this article will therefore focus on the latest applications and develop-
configuration, based on the molecular groups around a chiral centre, uses the ments in CD technology.
prefix L and R to distinguish enantiomers. The L and R in this case does not
imply a corresponding rotational direction.) The mirrored geometry in
enantiomers is directly analogous to a pair of hands and it is from the Greek CD spectrometer design
for hand that the word "chiral" is derived. The basic CD spectrometer design most widely adopted by the leading
manufacturers comprises a high intensity broadband light source (usually
a high pressure Xenon arc lamp), a double prism monochromator which
Circular dichroism and not only serves to disperse the wavelength but is also used to linearly
optical rotation polarise the light beam, an electro-optic modulator which transforms the
Optical rotation arises from the differences in refractive index exhibited by linearly polarised light into circularly polarised states, a sample chamber
the two chiral forms of a molecule, and results in a rotation of the plane of and photomultiplier detector. Sophisticated control and signal processing
incident linearly polarised light when it passes though a sample of the mole- electronics, all under PC control, complete a typical instrument [Figure 1].
cule. Circular dichroism, on the other hand, arises as a result of the differen-
tial absorption of left and right circularly polarised light by chiral
ANALYTICAL SPOTLIGHT As published in LPI-October 2004
Summary
The combination of advances in technology described above has led to a
significant improvement in the speed and reliability of CD measurement.
Whilst sample preparation and an understanding of the limitations
caused by sample and solvent absorption and some knowledge of the
Table 1. The results of a protein secondary structure analysis of the theory of CD measurement is unavoidable, the latest CD instruments are
lysozyme data shown in Figure 2 using the CDNN protein analysis highly sophisticated, easy-to-use and highly reliable. The cost of the
application [2]. Note the sensitivity of the results is dependant on the required technology is dropping and so the entry cost for CD instruments
wavelength range included. The sum row provides a net contribu- has come down and there are a number of manufacturers keeping compe-
tion of all underlying structures and should ideally equal 100%. tition keen. The importance of chirality in so many areas of chemistry and
biochemistry is also likely to drive further developments and improve-
ments and, given the unique capability of CD as a probe for this fascinat-
Software and data ing phenomenon, the future of CD can only be described as looking bright.
processing References
All current CD instruments are accompanied by sophisticated PC software 1. Velluz L, Legrand M, Grosjean M. Optical circular dichroism, principles,
providing data acquisition control, data visualisation and management fea- measurements and applications. 1965, Academic, New York.
tures. These software packages also provide a range of data processing func- 2. CDNN: Gerald Böhm, Institut für Biotechnologie, Martin-Luther-
tions such as spectral baseline subtraction and basic math functions Universität, Halle-Wittenberg, Germany. SELCON: Prof. Robert Woody,
including averaging, curve fitting and data filtering. The protein analysis Colorado State University, USA. CONTIN: Stephen Provencher, 48
described earlier is frequently performed using programs available in the Chancery Lane East, Oakville, Ontario L6J 5P6, Canada.
public domain such as CDNN, SELCON and CONTIN [2]. Data files and
sample concentrations must be submitted in precise formats to these pro- The author
grams and so facilities to export correctly formatted data files are also pro- Peter King, Ph.D., Technical Director,
vided. For added convenience, client-server software architecture Applied Photophysics Ltd
developed for Chirascan allows experiments to be conducted remotely and 203-205 Kingston Road
monitored over a network. Leatherhead, Surrey KT22 7PB, UK
Tel.: +44 1372 386537
Fax: +44 1372 386 477
Data smoothing and filtering Email: Petek@photophysics.com
This topic is discussed in its own right as it is a matter of considerable
importance. CD measurements are often small and frequently noisy, par-
ticularly in the far-UV. To improve their signal-to-noise ratio filtering is
usually applied, either electronically to the raw analogue signals themselves
or, with the advent of fast digital signal processing, to the raw acquired data
in the digital domain. It can-
not be over-stressed that
electronic analogue filtering
is a risky process and can
lead to the distortion of
measured spectra. The
selection of time constants
to filter real time signals
during scans is highly
Figure 3. The Chirascan CD spectrom- dependent on both spectral
eter from Applied Photophysics Ltd, complexity and required
Leatherhead, UK. scan speed and should be
considered obsolete given the speed and reversibility of digital filtering
approaches. Most importantly, if a noisy trace is filtered in the digital