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Spectroscopy 2003: 5 e kwartaal UV Circular Dichroism. Dr. Marco Tessari Afdeling Fysische Chemie This presentation can be found at the website: http://www.nmr.kun.nl/Education. Overview. Introduction: Optical Activity Polarization of EM Radiation
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Spectroscopy 2003: 5e kwartaal UV Circular Dichroism Dr. Marco Tessari Afdeling Fysische Chemie This presentation can be found at the website: http://www.nmr.kun.nl/Education
Overview Introduction: Optical Activity Polarization of EM Radiation Experimental Detection of Optical Activity: -Ellipticity -Optical Rotation -Circular Dichroism (CD) -Circular Birefringence Application of CD to conformational studies Material from Cantor and Schimmel Biophysical Chemistry ch. 8
Introduction: Optical Activity So far, our attention was focused on the energy transfer from EM radiation to matter: as a consequence of such exchange of energy, molecules are promoted to higher rotational-, vibrational- and electronic states. We are now going to examine what effects such EM-matter interaction can determine on the trasmitted radiation. I0 I [J] l
Introduction: Optical Activity Optical Activity refers to the capacity of some substances to alter the properties of transmitted light. In general, for small molecules, optically activity is a consequence of lack of symmetry. For macromolecules an important contribution to their optical activity derives from their conformation. Therefore, optical activity can provide important information on their structural properties in solution.
Polarization of EM Radiation EM radiation is a transverse wave consisting of oscillating electric- and magnetic fields, mutually perpendicular. In general, the oscillation of the electric field does NOT take place on a fixed (y,x) plane. The radiation is not polarized. l
y x z y y x z z y y x z z y y x z z y y x z z y y x z z y z Polarization of EM Radiation When EM radiation is not polarized, all types of oscillations are equally probable. As a consequence, the plane of oscillation of the EM radiation varies randomly in time.
Polarization of EM Radiation A polarizing filter can convert unpolarized into linearly polarized EM radiation. A good example of this is a Polaroid filter. This kind of filter is made up of parallel strands of long molecules. Only the light polarized along a certain direction passes through the filter, while the perpendicular component gets completely absorbed. The vertically polarized component of EM radiation passes through. Unpolarized EM radiation The horizontally polarized component of EM radiation is absorbed
Polarization of EM Radiation Linear polarized radiation correspond to the superposition of left and right circularly polarized waves of the same intensity. y x z y y x z z y x z y z
Polarization of EM Radiation Elliptical polarized radiation correspond to the superposition of left and right circularly polarized waves of different intensity. y x z y y x z z y y z x z y z
Experimental Detection of Optical Activity • There are four experimentally detectable ways that an optically active ABSORBING sample can alter the properties of transmitted radiation: • Ellipticity • Optical Rotation • Circular Dichroism • Circular Birifringence
Ellipticity In general, an optically active sample will convert linear polarized into elliptical polarized radiation. The ellipticity of the light is one measure of optical activity and it is defined as: where a and b are the major and the minor axis of the ellipse, respectively Linear polarized radiation Elliptical polarized radiation
Optical Rotation The orientation of the ellipse defines the Optical Rotation (f), a second indicator of optical activity. The optical rotation as a function of the wavelength of the radiation is called Optical Rotatory Dispersion (ORD) f Linear polarized radiation Elliptical polarized radiation
Circular Dichroism Linear polarized radiation correspond to the superposition of left and right circularly polarized waves. In an optically active medium these two components are absorbed to a different extent: This phenomenon is called Circular Dichroism. Linear polarized radiation Elliptical polarized radiation
Circular Birefringence An optically active sample has two different refraction index, nL and nR, for the two circular components of radiation. This means that one of the two components propagates more rapidly than the other through the medium. The result is a phase shift between the two components, proportional to the refraction index difference nL –nR. This phenomenon is called Circular Birefringence. Linear polarized radiation Elliptical polarized radiation
Experimental Detection of Optical Activity Circular Dichroism and Ellipticity refer to the same physical phenomenon. The same holds for Optical Rotation and Birefringence. This is expressed by the followings equations: Usually only Circular Dichroism and Optical Rotation are measured experimentally for practical reasons.
Experimental Detection of Optical Activity Exercise 1 A sample of 1cm path-length rotates linear polarized radiation (l=300nm) of f = 0.01 deg. Calculate its circular birefringence (defined as nL - nR).
Experimental Detection of Optical Activity Exercise 1: Solution A sample of 1cm pathlength rotates linear polarized radiation (l=300nm) of f = 0.01 deg. Calculate its circular birefringence (defined as nL-nR). Almost undetectable.
Experimental Detection of Optical Activity Exercise 2 A sample of 1cm pathlength has an ellipticity of q = 0.01 deg. Calculate its circular dichroism (defined as AL-AR).
Experimental Detection of Optical Activity Exercise 2: Solution A sample of 1cm pathlength has an ellipticity of q = 0.01 deg. Calculate its circular dichroism (defined as AL-AR).
Experimental Detection of Optical Activity The measurement of Circular Dichroism is very similar to the conventional UV absorption. Circular Dichroism is measured exposing the sample alternatively to left-hand and right-hand circularly polarized light and detecting the differential absorption. The difference, typically about 0.03% to 0.3% of the total absorption, can be accurately determined with modern instrumentation. CD is usually reported in ellipticity units, using the expression:
Experimental Detection of Optical Activity The measurement of Optical Rotation requires a rather simple instrument called POLARIMETER. Since most natural compounds are optically active, Optical Rotation is usually employed to characterize the purity of products in pharmaceutical industry, food industry, flavor industry, etc. A modern polarimeter can detect optical rotations as little as 0.002 deg.
Experimental Detection of Optical Activity To compare results from different samples, it is necessary to normalize the experimental data with respect to concentration (c), path length (l) and, for polymers, number of chromophores (Ncr ):
Experimental Detection of Optical Activity Exercise 3 A sample of a polypeptide consisting of Ncr=20 amino-acids (optical path l = 0.2 cm, concentration c = 10mM) has an ellipticity q = -9.6 mdeg at l = 222 nm. The ratio of the molar extinction coefficients for left and right circular polarized light eL/ eR = 0.964 at l= 222 nm. Determine the transmittance of this sample for linear polarized light at l= 222 nm, with an optical path l = 1 cm.
Experimental Detection of Optical Activity Exercise 3: Solution
Experimental Detection of Optical Activity Exercise 3: Solution
Application of CD to Conformational Studies A quantum-mechanical analysis reveals that ORD and CD spectra of a biopolymer are very sensitive to its geometry. The ab-initio calculation of CD spectra of macromolecules is still a task of formidable complexity. Therefore, a number of semi-empirical methods have been developed to extract structural information from CD data. For proteins and peptides the major objective has been to deduce the secondary structure from measured CD spectra. In general, the CD spectrum of a poly-peptide in the region 190-230 nm is dominated by the signals of the peptide groups. The CD spectrum in this region can then be expressed as the sum of the contribution of individual secondary structure regions:
alpha-helix beta-sheet random coil Application of CD to Conformational Studies
alpha-helix beta-sheet random coil Application of CD to Conformational Studies
Application of CD to Conformational Studies The decomposition of a CD spectrum in the sum of the contributions of the three basic conformations is performed routinely in the conformational analysis of peptides and proteins. The accuracy of this method is hardly better than 5-10%. A number of factors limit the accuracy of this approach: -the contributions from other chromophores -the choice of the three basis spectra -the packing of secondary structure elements in the tertiary structure. In summary, this analysis of CD data provides a rapid, global, but not veryaccurate picture of the secondary structure of a polypeptide.
beta-sheet random coil Application of CD to Conformational Studies The CD technique is, however, very reliable for monitoring changes in the conformation of proteins under different conditions (denaturation studies, unfolding experiments, binding experiments, etc). The advantage of CD for monitoring conformational changes is its sensitivity. Even if a detailed structural interpretation is not possible, a change in structure will almost surely show up as a change in the CD spectrum.
Application of CD to Conformational Studies The CD spectra of the three basic conformations (alpha-helix, beta-sheet and random coil) are very different from each other. Therefore, minor local conformational rearrangements are reflected in noticeably changes in the CD spectrum. alpha-helix alpha-helix beta-sheet random coil