1) Circular dichroism arises from the differential absorption of left and right circularly polarized light by chiral molecules.
2) CD spectra are more sensitive to conformational changes in proteins and nucleic acids than absorption spectra.
3) CD spectra can provide information about secondary structure in proteins.
Circular dichroism is the difference in absorption of left and right circularly polarized light by a chiral molecule. It occurs due to interactions between the molecule's chiral chromophores and polarized light. CD spectroscopy is used to analyze the secondary structure of proteins and monitor structural changes. The technique provides structural signatures for alpha helices, beta sheets, and random coils. It is a powerful tool for studying protein folding and structural changes under various conditions.
This document provides an overview of circular dichroism (CD) spectroscopy. It begins with a brief history and definitions. The key principles are that CD measures the differential absorption of left and right circularly polarized light, providing structural information. Instrumentation and experimental procedures are described. Applications include determining protein secondary structure and monitoring structural changes. Software for CD analysis is also discussed. Examples are provided to illustrate how CD can be used to analyze protein secondary structure and monitor DNA structural changes.
Circular dichroism spectroscopy measures the differential absorption of left and right circularly polarized light by chiral molecules. When light passes through an optically active substance, the left and right circular polarizations are absorbed to different extents. A CD spectrometer contains a light source, monochromator, polarizer, photoelastic modulator and detector. It measures the CD signal as a function of wavelength, providing information about secondary structure of proteins and nucleic acids. CD spectroscopy requires minimal sample amounts and can quickly analyze secondary structure without crystallization. It is useful for studying protein folding, ligand binding and environmental effects on structure.
This document discusses the basics of infrared spectroscopy. It explains that infrared absorption spectroscopy works because infrared radiation can induce molecular vibrations and rotations if the radiation frequency matches the natural frequency of the vibration or rotation. It also describes the different types of molecular transitions that occur in infrared spectroscopy, including rotational, vibrational-rotational, and vibrational transitions. Finally, it provides an overview of common infrared radiation sources and detectors.
Molecular dissymmetry and chiroptical propertiespriyaswain27
This document discusses concepts related to stereochemistry and chiroptical properties. It defines linear and circularly polarized light and explains how circular birefringence and dichroism arise from molecular asymmetry. It describes how ORD and CD curves are used to analyze stereochemistry and determine functional groups and configurations using the Cotton effect. The axial α-haloketone rule and octant rule for assigning absolute configuration are also covered along with applications of these concepts.
The document provides an overview of photonic light sources, specifically LEDs and lasers. It discusses:
1) How LEDs work by emitting photons when electrons fall from a higher to lower energy level within a semiconductor, causing light. The color depends on the energy level difference.
2) The principle of lasers, which involves stimulating emission of radiation to achieve population inversion and optical gain, allowing for amplification of photons within the laser medium.
3) How a laser diode works by achieving population inversion through forward biasing of a p-n junction, allowing stimulated emission and optical feedback via mirrors to produce coherent, collimated light amplification.
Microwave and infrared spectroscopy of polyatomic moleculesAreebaWarraich1
Microwave and infrared spectroscopy can be used to study the rotational and vibrational states of polyatomic molecules. Microwave spectroscopy specifically probes the rotational transitions of molecules with a permanent dipole moment, in the microwave frequency range of 300MHz-300GHz. Infrared spectroscopy analyzes the vibrational transitions of molecules when exposed to infrared radiation, divided into stretching and bending vibrations. Both techniques provide information on molecular structure through analysis of absorption spectra.
Circular dichroism is the difference in absorption of left and right circularly polarized light by a chiral molecule. It occurs due to interactions between the molecule's chiral chromophores and polarized light. CD spectroscopy is used to analyze the secondary structure of proteins and monitor structural changes. The technique provides structural signatures for alpha helices, beta sheets, and random coils. It is a powerful tool for studying protein folding and structural changes under various conditions.
This document provides an overview of circular dichroism (CD) spectroscopy. It begins with a brief history and definitions. The key principles are that CD measures the differential absorption of left and right circularly polarized light, providing structural information. Instrumentation and experimental procedures are described. Applications include determining protein secondary structure and monitoring structural changes. Software for CD analysis is also discussed. Examples are provided to illustrate how CD can be used to analyze protein secondary structure and monitor DNA structural changes.
Circular dichroism spectroscopy measures the differential absorption of left and right circularly polarized light by chiral molecules. When light passes through an optically active substance, the left and right circular polarizations are absorbed to different extents. A CD spectrometer contains a light source, monochromator, polarizer, photoelastic modulator and detector. It measures the CD signal as a function of wavelength, providing information about secondary structure of proteins and nucleic acids. CD spectroscopy requires minimal sample amounts and can quickly analyze secondary structure without crystallization. It is useful for studying protein folding, ligand binding and environmental effects on structure.
This document discusses the basics of infrared spectroscopy. It explains that infrared absorption spectroscopy works because infrared radiation can induce molecular vibrations and rotations if the radiation frequency matches the natural frequency of the vibration or rotation. It also describes the different types of molecular transitions that occur in infrared spectroscopy, including rotational, vibrational-rotational, and vibrational transitions. Finally, it provides an overview of common infrared radiation sources and detectors.
Molecular dissymmetry and chiroptical propertiespriyaswain27
This document discusses concepts related to stereochemistry and chiroptical properties. It defines linear and circularly polarized light and explains how circular birefringence and dichroism arise from molecular asymmetry. It describes how ORD and CD curves are used to analyze stereochemistry and determine functional groups and configurations using the Cotton effect. The axial α-haloketone rule and octant rule for assigning absolute configuration are also covered along with applications of these concepts.
The document provides an overview of photonic light sources, specifically LEDs and lasers. It discusses:
1) How LEDs work by emitting photons when electrons fall from a higher to lower energy level within a semiconductor, causing light. The color depends on the energy level difference.
2) The principle of lasers, which involves stimulating emission of radiation to achieve population inversion and optical gain, allowing for amplification of photons within the laser medium.
3) How a laser diode works by achieving population inversion through forward biasing of a p-n junction, allowing stimulated emission and optical feedback via mirrors to produce coherent, collimated light amplification.
Microwave and infrared spectroscopy of polyatomic moleculesAreebaWarraich1
Microwave and infrared spectroscopy can be used to study the rotational and vibrational states of polyatomic molecules. Microwave spectroscopy specifically probes the rotational transitions of molecules with a permanent dipole moment, in the microwave frequency range of 300MHz-300GHz. Infrared spectroscopy analyzes the vibrational transitions of molecules when exposed to infrared radiation, divided into stretching and bending vibrations. Both techniques provide information on molecular structure through analysis of absorption spectra.
ir spectroscopy: introduction modes of vibration, selection rule, factor, influcing of vibration, scaning of ir spectroscopy(instrumentation) vibration frequency of organic and inorganic compound
Electron Spin Resonance Spectroscopy by arjuArjun kumar
Electron spin resonance (ESR) spectroscopy is a technique used to study materials with unpaired electrons. It detects transitions between spin energy levels induced by a microwave source in the presence of a strong magnetic field. The three key points are:
1. ESR detects the absorption of microwaves by unpaired electrons in a material when it is exposed to a strong magnetic field, which splits the electronic energy levels.
2. The absorbed frequency is dependent on factors like the local electron environment and applied field strength, allowing structural information to be obtained.
3. Hyperfine interactions with neighboring atomic nuclei further split the energy levels and provide details like the number and identity of interacting nuclei.
Electron paramagnetic resonance (EPR) spectroscopy measures transitions between electron spin energy levels when molecules with unpaired electrons are exposed to microwave radiation in an applied magnetic field. The document discusses the principles of EPR, including the Zeeman effect where electron spin states split into distinct energy levels. Hyperfine interactions between unpaired electrons and neighboring atomic nuclei provide information on the local electronic structure. More complex splitting patterns can arise from interactions with multiple equivalent nuclei, known as superhyperfine splitting. EPR spectroscopy thus provides insights into electron distributions and neighboring atomic environments.
This document provides an overview of molecular spectroscopy techniques, including rotational spectroscopy, vibrational spectroscopy, and absorption and emission spectroscopy. Rotational spectroscopy uses microwave spectroscopy to study the quantized rotational energy levels of molecules. Vibrational spectroscopy uses infrared spectroscopy to analyze the quantized vibrational energy levels of bonds as they stretch, bend, and vibrate. Absorption and emission spectroscopy examines how molecules absorb and emit photons during electronic transitions between energy levels.
Electron spin resonance (ESR) spectroscopy involves applying a magnetic field to a chemical species with unpaired electrons and measuring the absorption of microwave radiation, which causes transitions between spin energy levels. ESR provides information about electron environments and can be used to study metalloproteins and incorporate spin labels to probe protein structure and dynamics. The instrumentation required includes an electromagnet, microwave source, and detector, along with components to sweep the magnetic field and modulate the signal.
UV-Visible spectroscopy involves using electromagnetic radiation in the UV-Visible range to analyze molecules based on their absorption characteristics, which are determined by electronic transitions between molecular orbitals. Different types of transitions like σ→σ*, n→π*, and π→π* occur at different wavelengths and can be used to identify functional groups in compounds. This technique provides information about the structure and bonding of molecules based on their absorption spectra.
Infrared spectroscopy (IR spectroscopy or vibrational spectroscopy) is the measurement of the interaction of infrared radiation with the matter by absorption, emission, or reflection. It is used to study and identify chemical substances or functional groups in solid, liquid, or gaseous forms.
UV spectroscopy involves absorption spectroscopy from 160 nm to 780 nm to identify inorganic and organic species. It uses ultraviolet radiation that stimulates molecular vibrations and electronic transitions. When a molecule absorbs UV radiation, it causes excitation of electrons from occupied bonding molecular orbitals to unoccupied antibonding molecular orbitals. The absorption spectrum obtained will show "gaps" at discrete energies where particular electronic transitions match the energy of bands of UV light. Factors like conjugation, substituents, and solvent can affect the absorption spectrum by causing bathochromic or hypsochromic shifts.
Nuclear Quadrupole Resonance Spectroscopy (NQR) is a chemical analysis technique that detects nuclear energy level transitions in the absence of a magnetic field through the absorption of radio frequency radiation. NQR is applicable to solids due to the quadrupole moment averaging to zero in liquids and gases. The interaction between a nucleus's quadrupole moment and the electric field gradient of its surroundings results in quantized energy levels. Transitions between these levels are detected as NQR spectra and provide information about electronic structure, hybridization, and charge distribution. NQR finds applications in studying charge transfer complexes, detecting crystal imperfections, and locating land mines.
The document discusses optical polarization and modulation. It defines key concepts like linear, circular, and elliptical polarization of light. It explains how polarization occurs through interactions with materials like polarizers. Polarization states are described by the behavior of the electric field vector over time and space. Optically anisotropic crystals like calcite exhibit birefringence, causing an incident light beam to split into two rays with orthogonal polarizations known as ordinary and extraordinary waves.
Lasers produce a very narrow, intense beam of coherent light through the process of stimulated emission of radiation. Key characteristics of laser light include high monochromaticity, directionality, intensity, and coherence. Einstein's theory of stimulated emission explains how excited atoms or molecules can emit photons when stimulated by an incoming photon, leading to amplification of the light beam. Population inversion, where more atoms are in an excited state than a lower state, must be achieved for lasing to occur. Common laser types include solid-state, gas, liquid/dye, and semiconductor lasers, which use different active media and pumping mechanisms to produce stimulated emission. A notable example is the Nd:YAG laser, which uses a neody
UV/Vis spectroscopy is routinely used in analytical chemistry for the quantitative determination of different analytes, such as transition metal ions, highly conjugated organic compounds, and biological macromolecules. Molecules containing bonding and non-bonding electrons undergo electronic transitions and absorb energy in the form of ultraviolet or visible light to excite these electrons to higher anti-bonding molecular orbitals.
Electron spin resonance (ESR) spectroscopy is a technique used to study compounds with unpaired electrons. In ESR, a sample is placed in a static magnetic field and irradiated with microwaves. This causes transitions between the electron spin energy levels. The absorption of microwave energy is detected to obtain an ESR spectrum. ESR spectra provide information about electron environments through parameters like g-values and hyperfine splitting patterns. ESR finds applications in studying transition metal complexes and unstable free radicals.
- Spectroscopy is the study of the interaction of electromagnetic radiation with matter. It probes features of a sample through light interaction to learn about its consistency or structure.
- Spectroscopy techniques employ different wavelengths of light which can interact with matter through electronic, vibrational, or rotational energy level transitions. The energy of the radiation determines what type of transition it causes.
- Absorption spectroscopy measures the absorption of radiation while emission spectroscopy measures radiation emitted from transitions between energy levels.
Light has several properties that make it useful for information processing and optical communication systems. It can be transmitted without interference from electrical signals or other light beams crossing its path. Optical signals also allow high parallelism and bandwidth exceeding 1013 bits per second. Radiation sources can be classified by their flux output and spectrum. Light behaves as an electromagnetic wave that propagates through space as oscillating electric and magnetic fields. In a material medium, the light's phase velocity decreases and is characterized by the medium's refractive index. Crystalline materials exhibit anisotropic refractive indices depending on the propagation and polarization directions.
UV-visible spectroscopy involves using ultraviolet or visible light to analyze compounds. When molecules absorb UV or visible light, their electrons are excited from the ground state to a higher energy state. There are several types of electronic transitions that can occur: n→π*, π→π*, n→σ*, and σ→σ*. The energy required for these transitions increases in the order n→π* < π→π* < n→σ* < σ→σ*. Solvents play an important role, as solvent peaks can obscure sample peaks, and polar solvents can cause bathochromic or blue shifts in transition wavelengths.
The document discusses infrared (IR) spectroscopy. It explains that IR spectroscopy analyzes molecular vibrations and rotations that are excited when molecules absorb IR radiation. The experimental setup for IR spectroscopy includes an IR source, fore optics to direct the beam at the sample, a monochromator to separate wavelengths, a detector to measure absorption, and a recorder to display the results. Molecular vibrations that can be measured include stretching and bending vibrations of bonds that change the molecule's dipole moment.
1. Electronic spectroscopy relies on quantized energy states of electrons. Absorption of photons promotes electrons to excited states, and fluorescence occurs when electrons return to lower states.
2. Molecular electronic spectra involve changes in electronic, vibrational, and rotational energies of molecules. They appear in the visible and ultraviolet regions.
3. Potential energy curves describe different electronic states of diatomic molecules. Transitions between states emit or absorb radiation and give rise to band systems consisting of vibrational and rotational transitions within those bands.
The document provides information on rotational spectroscopy and the rotational spectra of molecules. It discusses key topics like:
1) Classification of molecules as linear, symmetric top, spherical top, and asymmetric top based on their moments of inertia.
2) The rigid rotor model and how it leads to quantized rotational energy levels expressed by the rotational constant B.
3) The selection rule for rotational transitions of ΔJ = ±1, which results in a series of equally spaced spectral lines.
4) Factors that determine the intensity of rotational lines, including Boltzmann distribution of molecular populations and degeneracy of energy levels.
This document discusses key concepts in quantum mechanics including:
- Planck's quantum theory which established that atoms can only emit or absorb energy in discrete quanta.
- Einstein's explanation of the photoelectric effect using the particle nature of light (photons).
- Bohr's model of the hydrogen atom which explained its spectral lines by postulating discrete electron energy levels.
- Quantum numbers which describe the state of an electron including its orbital, orientation, and spin.
- Electron configuration which shows how electrons fill atomic orbitals according to the aufbau principle.
The document discusses interference and thin film interference. Some key points:
- Interference occurs when light waves combine, and conditions for interference require coherent light sources that maintain a constant phase relationship.
- Thin film interference is observed when light reflects or transmits through a thin film. It results from the optical path difference between light rays that undergo different numbers of reflections within the film.
- Interference patterns in thin films depend on factors like the film thickness, wavelength of light, and angle of incidence. This allows properties like wavelength and refractive index to be measured from analysis of the interference fringe patterns.
ir spectroscopy: introduction modes of vibration, selection rule, factor, influcing of vibration, scaning of ir spectroscopy(instrumentation) vibration frequency of organic and inorganic compound
Electron Spin Resonance Spectroscopy by arjuArjun kumar
Electron spin resonance (ESR) spectroscopy is a technique used to study materials with unpaired electrons. It detects transitions between spin energy levels induced by a microwave source in the presence of a strong magnetic field. The three key points are:
1. ESR detects the absorption of microwaves by unpaired electrons in a material when it is exposed to a strong magnetic field, which splits the electronic energy levels.
2. The absorbed frequency is dependent on factors like the local electron environment and applied field strength, allowing structural information to be obtained.
3. Hyperfine interactions with neighboring atomic nuclei further split the energy levels and provide details like the number and identity of interacting nuclei.
Electron paramagnetic resonance (EPR) spectroscopy measures transitions between electron spin energy levels when molecules with unpaired electrons are exposed to microwave radiation in an applied magnetic field. The document discusses the principles of EPR, including the Zeeman effect where electron spin states split into distinct energy levels. Hyperfine interactions between unpaired electrons and neighboring atomic nuclei provide information on the local electronic structure. More complex splitting patterns can arise from interactions with multiple equivalent nuclei, known as superhyperfine splitting. EPR spectroscopy thus provides insights into electron distributions and neighboring atomic environments.
This document provides an overview of molecular spectroscopy techniques, including rotational spectroscopy, vibrational spectroscopy, and absorption and emission spectroscopy. Rotational spectroscopy uses microwave spectroscopy to study the quantized rotational energy levels of molecules. Vibrational spectroscopy uses infrared spectroscopy to analyze the quantized vibrational energy levels of bonds as they stretch, bend, and vibrate. Absorption and emission spectroscopy examines how molecules absorb and emit photons during electronic transitions between energy levels.
Electron spin resonance (ESR) spectroscopy involves applying a magnetic field to a chemical species with unpaired electrons and measuring the absorption of microwave radiation, which causes transitions between spin energy levels. ESR provides information about electron environments and can be used to study metalloproteins and incorporate spin labels to probe protein structure and dynamics. The instrumentation required includes an electromagnet, microwave source, and detector, along with components to sweep the magnetic field and modulate the signal.
UV-Visible spectroscopy involves using electromagnetic radiation in the UV-Visible range to analyze molecules based on their absorption characteristics, which are determined by electronic transitions between molecular orbitals. Different types of transitions like σ→σ*, n→π*, and π→π* occur at different wavelengths and can be used to identify functional groups in compounds. This technique provides information about the structure and bonding of molecules based on their absorption spectra.
Infrared spectroscopy (IR spectroscopy or vibrational spectroscopy) is the measurement of the interaction of infrared radiation with the matter by absorption, emission, or reflection. It is used to study and identify chemical substances or functional groups in solid, liquid, or gaseous forms.
UV spectroscopy involves absorption spectroscopy from 160 nm to 780 nm to identify inorganic and organic species. It uses ultraviolet radiation that stimulates molecular vibrations and electronic transitions. When a molecule absorbs UV radiation, it causes excitation of electrons from occupied bonding molecular orbitals to unoccupied antibonding molecular orbitals. The absorption spectrum obtained will show "gaps" at discrete energies where particular electronic transitions match the energy of bands of UV light. Factors like conjugation, substituents, and solvent can affect the absorption spectrum by causing bathochromic or hypsochromic shifts.
Nuclear Quadrupole Resonance Spectroscopy (NQR) is a chemical analysis technique that detects nuclear energy level transitions in the absence of a magnetic field through the absorption of radio frequency radiation. NQR is applicable to solids due to the quadrupole moment averaging to zero in liquids and gases. The interaction between a nucleus's quadrupole moment and the electric field gradient of its surroundings results in quantized energy levels. Transitions between these levels are detected as NQR spectra and provide information about electronic structure, hybridization, and charge distribution. NQR finds applications in studying charge transfer complexes, detecting crystal imperfections, and locating land mines.
The document discusses optical polarization and modulation. It defines key concepts like linear, circular, and elliptical polarization of light. It explains how polarization occurs through interactions with materials like polarizers. Polarization states are described by the behavior of the electric field vector over time and space. Optically anisotropic crystals like calcite exhibit birefringence, causing an incident light beam to split into two rays with orthogonal polarizations known as ordinary and extraordinary waves.
Lasers produce a very narrow, intense beam of coherent light through the process of stimulated emission of radiation. Key characteristics of laser light include high monochromaticity, directionality, intensity, and coherence. Einstein's theory of stimulated emission explains how excited atoms or molecules can emit photons when stimulated by an incoming photon, leading to amplification of the light beam. Population inversion, where more atoms are in an excited state than a lower state, must be achieved for lasing to occur. Common laser types include solid-state, gas, liquid/dye, and semiconductor lasers, which use different active media and pumping mechanisms to produce stimulated emission. A notable example is the Nd:YAG laser, which uses a neody
UV/Vis spectroscopy is routinely used in analytical chemistry for the quantitative determination of different analytes, such as transition metal ions, highly conjugated organic compounds, and biological macromolecules. Molecules containing bonding and non-bonding electrons undergo electronic transitions and absorb energy in the form of ultraviolet or visible light to excite these electrons to higher anti-bonding molecular orbitals.
Electron spin resonance (ESR) spectroscopy is a technique used to study compounds with unpaired electrons. In ESR, a sample is placed in a static magnetic field and irradiated with microwaves. This causes transitions between the electron spin energy levels. The absorption of microwave energy is detected to obtain an ESR spectrum. ESR spectra provide information about electron environments through parameters like g-values and hyperfine splitting patterns. ESR finds applications in studying transition metal complexes and unstable free radicals.
- Spectroscopy is the study of the interaction of electromagnetic radiation with matter. It probes features of a sample through light interaction to learn about its consistency or structure.
- Spectroscopy techniques employ different wavelengths of light which can interact with matter through electronic, vibrational, or rotational energy level transitions. The energy of the radiation determines what type of transition it causes.
- Absorption spectroscopy measures the absorption of radiation while emission spectroscopy measures radiation emitted from transitions between energy levels.
Light has several properties that make it useful for information processing and optical communication systems. It can be transmitted without interference from electrical signals or other light beams crossing its path. Optical signals also allow high parallelism and bandwidth exceeding 1013 bits per second. Radiation sources can be classified by their flux output and spectrum. Light behaves as an electromagnetic wave that propagates through space as oscillating electric and magnetic fields. In a material medium, the light's phase velocity decreases and is characterized by the medium's refractive index. Crystalline materials exhibit anisotropic refractive indices depending on the propagation and polarization directions.
UV-visible spectroscopy involves using ultraviolet or visible light to analyze compounds. When molecules absorb UV or visible light, their electrons are excited from the ground state to a higher energy state. There are several types of electronic transitions that can occur: n→π*, π→π*, n→σ*, and σ→σ*. The energy required for these transitions increases in the order n→π* < π→π* < n→σ* < σ→σ*. Solvents play an important role, as solvent peaks can obscure sample peaks, and polar solvents can cause bathochromic or blue shifts in transition wavelengths.
The document discusses infrared (IR) spectroscopy. It explains that IR spectroscopy analyzes molecular vibrations and rotations that are excited when molecules absorb IR radiation. The experimental setup for IR spectroscopy includes an IR source, fore optics to direct the beam at the sample, a monochromator to separate wavelengths, a detector to measure absorption, and a recorder to display the results. Molecular vibrations that can be measured include stretching and bending vibrations of bonds that change the molecule's dipole moment.
1. Electronic spectroscopy relies on quantized energy states of electrons. Absorption of photons promotes electrons to excited states, and fluorescence occurs when electrons return to lower states.
2. Molecular electronic spectra involve changes in electronic, vibrational, and rotational energies of molecules. They appear in the visible and ultraviolet regions.
3. Potential energy curves describe different electronic states of diatomic molecules. Transitions between states emit or absorb radiation and give rise to band systems consisting of vibrational and rotational transitions within those bands.
The document provides information on rotational spectroscopy and the rotational spectra of molecules. It discusses key topics like:
1) Classification of molecules as linear, symmetric top, spherical top, and asymmetric top based on their moments of inertia.
2) The rigid rotor model and how it leads to quantized rotational energy levels expressed by the rotational constant B.
3) The selection rule for rotational transitions of ΔJ = ±1, which results in a series of equally spaced spectral lines.
4) Factors that determine the intensity of rotational lines, including Boltzmann distribution of molecular populations and degeneracy of energy levels.
This document discusses key concepts in quantum mechanics including:
- Planck's quantum theory which established that atoms can only emit or absorb energy in discrete quanta.
- Einstein's explanation of the photoelectric effect using the particle nature of light (photons).
- Bohr's model of the hydrogen atom which explained its spectral lines by postulating discrete electron energy levels.
- Quantum numbers which describe the state of an electron including its orbital, orientation, and spin.
- Electron configuration which shows how electrons fill atomic orbitals according to the aufbau principle.
The document discusses interference and thin film interference. Some key points:
- Interference occurs when light waves combine, and conditions for interference require coherent light sources that maintain a constant phase relationship.
- Thin film interference is observed when light reflects or transmits through a thin film. It results from the optical path difference between light rays that undergo different numbers of reflections within the film.
- Interference patterns in thin films depend on factors like the film thickness, wavelength of light, and angle of incidence. This allows properties like wavelength and refractive index to be measured from analysis of the interference fringe patterns.
Dielectrics are materials that contain permanently aligned electric dipoles. When an electric field is applied, the dipoles in dielectric materials can undergo several types of polarization, including electronic, ionic, orientational, and space charge polarization. This polarization leads to an increase in the electric flux density and dielectric constant within the material. The dielectric constant is the ratio of the material's permeability to the permeability of free space and determines the material's behavior in electric fields.
The document provides information about electronic spectra and terms for carbon p electrons and transition metal d electron configurations. It discusses:
1) Possible terms that arise from carbon's 2p electrons, including 1D2, 3P2, 3P1, 3P0 and 1S0 terms. Hund's rules are used to determine the ground state term.
2) Microstate tables that list all possible combinations of orbital angular momentum (L) and spin (S) for electron configurations.
3) Tanabe-Sugano diagrams that show the splitting of d electron terms in an octahedral ligand field and allow determination of transition energies.
4) Charge transfer transitions that can occur from the
The document discusses Auger electron emission and Auger electron spectroscopy. It describes how Auger electrons are emitted when an atom relaxes from an excited state following ionization. It also discusses how AES can be used to identify elements based on peak positions and obtain quantitative composition by comparing peak intensities to sensitivity factors. The technique provides chemical information through variations in peak shape and can determine properties like thickness and growth mode of thin films.
1.crystal structure using x – ray diffractionNarayan Behera
The document discusses crystal structure determination using X-ray diffraction. It describes how X-rays are used to probe interatomic distances in solids and explains key concepts like Bragg's law, reciprocal lattices, and Miller indices that are used to index diffraction patterns and determine unit cell parameters and crystal structures. Examples of common crystal structures like NaCl, CsCl are given along with methods to analyze diffraction data.
This document summarizes key concepts in wave optics, including:
1. Diffraction occurs when light bends around obstacles and into regions of geometric shadows. Diffraction can be observed at a single slit, with a central bright fringe and alternating dark and bright fringes at increasing angles.
2. The theory of diffraction is based on interference between secondary wavelets. The angles of maxima and minima depend on the slit width and wavelength.
3. Polarization of light occurs as the electric field oscillates perpendicular to the propagation direction. Polarizers and analyzers can be used to produce and detect plane polarized light according to Malus' law.
The equations are not directly applicable here because n1 ≠ n3. A more general approach is needed such as:
1) Calculate the reflection and transmission coefficients at each interface using Fresnel's equations.
2) Use the optical path length approach and phase changes to determine the net amplitude and phase for light reflecting from the front and back surfaces.
3) Combine using the principle of superposition to determine the net intensity.
The key is that the film thickness and refractive indices of all materials must be accounted for to determine the interference condition. Equations 35-36 and 35-37 are only valid when the surrounding media have the same refractive index.
Science Cafe Discovers a New Form of Alternative EnergyEngenuitySC
These are the slides from the May Science Cafe featuring Dr. MVS Chandrashekhar. During this cafe he discussed his work with graphene a new, clean energy source.
This document summarizes key concepts in wave optics, including:
1. Diffraction occurs when light bends around obstacles and into regions of geometric shadows. Diffraction patterns from a single slit include a central maximum surrounded by alternating dark and bright fringes whose angles follow mathematical formulas.
2. The theory of diffraction is based on the principle of interference of secondary wavelets emerging from different parts of a wavefront.
3. Polarization of light waves occurs as the electric field oscillates perpendicular to the direction of propagation, and polarizers and analyzers can be used to study polarized light according to Malus' Law.
This document provides an overview of basic electrochemistry concepts. It discusses the charge and current involved in electrochemical processes. It introduces Faraday's laws relating the amount of material transformed to the quantity of electricity passed. It also covers conductivity, Nernst equation, different types of electrodes, potentiometry, and various electrochemical techniques including cyclic voltammetry. The key concepts covered include electron transfer processes, Butler-Volmer equation, mass transport by diffusion and convection, and reversible cyclic voltammograms.
This document provides an overview of crystal field theory and how it can be used to explain the bonding and spectroscopic properties of transition metal complexes. It discusses how ligands arranged in octahedral, tetrahedral and square planar geometries cause the d-orbitals of the transition metal to split into different energy levels. Factors that influence the size of the crystal field splitting parameter Δo, such as oxidation state, metal identity and ligand type, are also covered.
This document discusses various topics related to dielectrics and capacitors including:
- What a capacitor and dielectric material are
- How capacitance can be increased by using a dielectric
- Different polarization mechanisms in materials including electronic, ionic, orientational and interfacial polarization
- How polarization leads to the development of bound surface charges in dielectrics
- The relationship between polarization, electric susceptibility, relative permittivity and dielectric constant
- Frequency dependence of real and imaginary parts of relative permittivity due to dielectric losses
- Derivation of the Clausius-Mossotti equation relating polarization to relative permittivity
- Calculation of relative permittivity for a material considering both electronic and
Bonding in Tranisiton Metal Compounds - Part 2Chris Sonntag
The document discusses transition metal bonding and spectroscopy. Key points include:
1. Transition metal geometries include octahedral, tetrahedral, and square planar depending on which d-orbitals interact most with ligands.
2. Tetrahedral geometry is most common for early transition metals while square planar is typical for later transition metals.
3. UV-visible spectroscopy of transition metal complexes reveals information about electronic transitions between d-orbital energy levels.
4. Factors like spin and orbital angular momentum selection rules determine which transitions are allowed and affect spectral features. Jahn-Teller distortions can also influence spectra.
NANO106 is UCSD Department of NanoEngineering's core course on crystallography of materials taught by Prof Shyue Ping Ong. For more information, visit the course wiki at http://nano106.wikispaces.com.
This document provides an overview of key concepts in semiconductor physics. It begins by introducing the crystal structure of silicon and how dopants can create an excess or deficiency of electrons (N-type or P-type silicon). It then discusses the energy band model and defines important terms like the band gap, Fermi energy level, density of states, and thermal equilibrium. The document derives expressions for the concentrations of electrons and holes as a function of doping, temperature, and the Fermi level position. It also examines intrinsic carrier concentration and how doping affects charge neutrality. Overall, the document establishes fundamental principles for understanding how electrons and holes behave in semiconductors.
Mie theory describes the scattering of electromagnetic radiation by a spherical particle. It provides an exact solution to Maxwell's equations for the scattering of a plane electromagnetic wave by a homogeneous sphere. Gustav Mie provided the mathematical description for the spectral dependence of scattering by a spherical nanoparticle. Mie theory can be used to calculate the absorption and scattering cross sections of nanoparticles and provides the basis for measuring particle size through light scattering. It is valid for particles ranging from much smaller to larger than the wavelength of light.
Dielectrics are materials that have permanent electric dipole moments. All dielectrics are electrical insulators and are mainly used to store electrical energy by utilizing bound electric charges and dipoles within their molecular structure. Important properties of dielectrics include their electric intensity or field strength, electric flux density, dielectric parameters such as dielectric constant and electric dipole moment, and polarization processes including electronic, ionic, and orientation polarization. Dielectrics are characterized by their complex permittivity, which relates to their ability to transmit electric fields and is dependent on factors like frequency, temperature, and humidity that can influence dielectric losses.
1) Polymeric excipients like PEG can stabilize proteins against denaturation during freezing by increasing the transfer free energy of the protein. However, these same polymers can induce phase separation in aqueous solutions.
2) During lyophilization, the concentrating effects of freezing can cause formulations to enter the two-phase region, resulting in liquid/liquid phase separation. This subjects the protein to potential partitioning between phases with different compositions.
3) Experimental studies on hemoglobin lyophilized in PEG/dextran mixtures provide evidence that phase separation during lyophilization can damage protein structure in the dried state.
This document is the user manual for the VP-DSC MicroCalorimeter. It provides specifications for the instrument, safety information, and instructions for operation. Key details include:
- The VP-DSC allows for high sensitivity measurement of heat capacity, binding thermodynamics, and kinetics.
- Safety precautions must be followed when using hazardous or volatile solutions in the tantalum cells.
- VPViewer software interfaces with Origin for instrument control and real-time data display.
- Sections provide tutorials for common experiments, calibration procedures, troubleshooting tips, and maintenance instructions.
1. The study characterized the aggregation of recombinant human Interleukin-1 receptor type II (rhuIL-1RII) using differential scanning calorimetry (DSC) and size exclusion chromatography (SEC).
2. A scan-rate dependence in the DSC experiment and a break from linearity in initial aggregation rates near the melting temperature (Tm) suggested that protein unfolding significantly contributes to the aggregation reaction pathway.
3. A mechanistic model was developed to extract meaningful thermodynamic and kinetic parameters from the irreversibly denatured aggregation process by simulating how unfolding properties could predict aggregation rates at different temperatures above and below the Tm.
This document reviews lyophilization (freeze-drying) as a method for developing solid protein pharmaceuticals. Lyophilization generates stresses that can denature proteins, including low temperature stress, freezing stresses from increased concentration and ice formation, and drying stress from removing the hydration shell. Several studies are discussed that demonstrate denaturation of specific proteins from these stresses during lyophilization and storage. The review discusses excipient protection of proteins, lyophilization cycle design, and formulation strategies to increase stability of solid protein pharmaceuticals and overcome instability issues.
This document summarizes the calculation of translational friction and intrinsic viscosity for four globular proteins (ribonuclease A, lysozyme, myoglobin, and chymotrypsinogen A) using their detailed atomic structures. The inclusion of a 0.9 Angstrom thick hydration shell around each protein allows the calculated translational friction and intrinsic viscosity to match experimental measurements. This hydration shell thickness corresponds to a hydration level of 0.3-0.4 grams of water per gram of protein, consistent with measurements from other techniques. Using detailed protein structures thus allows hydrodynamic measurements to support a unified picture of protein hydration, in contrast to earlier models that treated proteins as ellipsoids and found widely varying hydr
This document summarizes a study that investigated how different salts screen charge interactions in proteins. Specifically, it examined the effects of NaCl, guanidinium chloride, and guanidinium thiocyanate on the stability of wild-type E. coli thioredoxin and a variant. The results suggest that more denaturing salts like guanidinium chloride are more efficient at screening charge interactions than NaCl. This efficiency correlates with the salts' position in the Hofmeister series and ability to accumulate on protein surfaces. An electrostatic model was used to estimate contributions of charge interactions to stability.
Proton euilibria in minor groove of dnamganguly123
1) The document describes an experiment testing the prediction that regions of increased hydrogen ion density exist in the grooves of DNA. Probes with variable linker lengths and a proton-sensitive carboxyl group were attached to DNA in the minor groove.
2) The apparent pKa values of the carboxyl groups were higher than in free solution, increasing with shorter linker lengths. This agrees with calculations showing higher hydrogen ion density in the grooves.
3) The experiment provides experimental evidence supporting the theoretical prediction of acidic domains with elevated hydrogen ion density in the DNA minor groove.
The document describes an experiment measuring the static light scattering of concentrated protein solutions as a function of concentration. Specifically, it measured bovine serum albumin, ovalbumin, ovomucoid, and mixtures of these proteins up to 125 g/L, as well as chymotrypsin A at different pH levels up to 70 g/L. The measured scattering was quantitatively accounted for by an effective hard particle model, in which each protein is represented as a hard sphere and interactions are treated as hard particle repulsions and association equilibria.
This document summarizes a study that investigated the effects of two disaccharides (trehalose and sucrose) and trimethylamine N-oxide (TMAO) on amyloid-beta (Aβ) aggregation and interaction with lipid membranes. The key findings were:
1) In the absence of lipid vesicles, trehalose and sucrose delayed Aβ aggregation as measured by Thioflavin T fluorescence, but TMAO did not affect aggregation.
2) In the presence of lipid vesicles, all three osmolytes (trehalose, sucrose, TMAO) significantly attenuated dye leakage from the vesicles induced by Aβ aggregates.
3) Hydrogen exchange mass spectrometry (HX-MS) and
1) The study examines how long- and short-range electrostatic interactions affect the rheology and protein-protein interactions (PPI) of highly concentrated monoclonal antibody (mAb) solutions.
2) At high concentrations, both long- and short-range interactions contribute significantly to PPI, whereas at low concentrations only long-range interactions are important.
3) The study uses high frequency rheology, dynamic light scattering, circular dichroism, and zeta potential measurements to characterize PPI over a range of pH and ionic strengths, and develops a 3D computer model of the mAb to study charge distribution.
This chapter reviews the oxidation of methionine residues in model peptides. It discusses how neighboring amino acids can influence the oxidation pattern of methionine. For example, when a hydroxyl radical attacks Thr-Met, the neighboring threonine residue is cleaved. It also notes that the oxidation of peptides and proteins is a complex process that depends on the nature of the oxidizing species and the peptide/protein sequence and structure. Oxidation can lead to chain reactions as oxidation products themselves can initiate further oxidation remote from the initial attack site.
The document discusses several phase diagrams generated using different experimental data visualization techniques including:
1) A phase diagram of ricin toxin A-chain created using fluorescence and circular dichroism spectroscopic data showing four protein states.
2) An empirical phase diagram of the respiratory syncytial virus determined from multiple biophysical measurements across a pH range.
3) Phase diagrams of various non-viral gene delivery vehicles and proteins mapped against pH and temperature.
Basic fibroblast growth factor (bFGF) is being investigated for its ability to accelerate wound healing. Sulfated compounds like heparin enhance the stability of bFGF against thermal denaturation. To assess the effect on bFGF shelf life, formulations containing these excipients were incubated and analyzed. In the presence of sulfated compounds, precipitates formed that dissociated back to multimers with native structure, whereas without them precipitates were unfolded protein. Disulfide-linked multimers also increased in solution with sulfated compounds. Heparin stabilized bFGF structure and prevented rearrangement of disulfide bonds, indirectly promoting multimerization. However, loss of soluble bFGF monomer still
This document summarizes the use of differential scanning calorimetry (DSC) to optimize an antibody manufacturing process. DSC was used to screen conditions for a viral inactivation step and identify increased pH storage conditions for maximum stability. Low pH treatment reduced thermal stability, indicating structure loss. DSC provided insights into instability causes and process improvements, demonstrating its role in biotherapeutic development.
This document describes a study on controlled intracranial delivery of antibodies in rats. The researchers developed polymer matrices and microspheres for long-term antibody release directly in the brain. They implanted polymer discs containing IgG antibodies in rat brains and measured IgG concentrations at the implantation site and other brain regions over 28 days, finding highest levels with the polymer implants. The polymer provided sustained antibody levels beyond what was achieved with direct injection.
This document summarizes a study that measured the enthalpy change (ΔH) associated with the α-helix to random coil transition of an alanine peptide in water using calorimetry. The researchers synthesized a 50-residue peptide containing primarily alanine residues and determined its ΔH to be between 0.9-1.3 kcal/mol per residue, providing a basic parameter for predicting thermal unfolding of peptide helices. Circular dichroism spectra and melting curves confirmed the peptide adopted an α-helical structure at low temperatures and underwent a reversible helix-coil transition. The ΔH value suggests the peptide backbone, rather than side chains, makes the dominant contribution to helix stability.
1) Aspartame degradation kinetics depend on factors like pH, temperature, buffer type and concentration, and water activity. Higher temperatures, pH, buffer concentrations and water activities increase degradation rates.
2) The activation energies for aspartame degradation decrease with increasing pH or moisture content. Phosphate buffer significantly enhances degradation more than citrate buffer.
3) In solid systems, degradation rates increase with higher initial buffer concentrations and water activities. However, the glass transition temperature does not influence degradation rates as much as water activity.
This chapter discusses the application of light scattering techniques to analyze the solution behavior of protein pharmaceuticals. It provides examples of using light scattering to characterize proteins and protein complexes, detect soluble aggregate formation, and elucidate protein-ligand interactions. The chapter also describes the theoretical background and instrumentation for light scattering measurements and analysis. It presents applications of light scattering including analyzing self-associating protein systems, selecting optimal solvent conditions, and studying the kinetics of molecular interactions.
Ion water interaction biophysical journalmganguly123
The document discusses how the charge density of ions affects their strength of hydration and interactions in biological structures. It finds that small, highly charged ions (kosmotropes) strongly bind water molecules, while large monovalent ions of low charge density (chaotropes) weakly bind water. Crystalline salts dissolve exothermically only when one ion is a kosmotrope and the other is a chaotrope. This suggests kosmotropes and chaotropes preferentially form ion pairs in solution. The major intracellular ions—phosphate and carboxylate anions and potassium/arginine cations—behave as kosmotropes and chaotropes, respectively, allowing them
This document presents a study using differential scanning calorimetry (DSC) to examine the thermal stability of S-protein and its complexes with S-peptide at pH 7.0. DSC measurements showed that S-protein denatures through a reversible two-state transition with a denaturation temperature between 38.5-40.0°C and enthalpy of 165-180 kJ/mol, demonstrating its lower stability without S-peptide. A two-dimensional nonlinear regression analysis of excess heat capacity curves at varying temperatures and S-peptide concentrations was used to determine the binding thermodynamic parameters, yielding values of Kb = 1.10 × 106 M-1, ΔbH = -185 kJ
1. 03-871 Molecular Biophysics CD Spectroscopy September 25, 2009
Optical Rotary Dispersion and Circular Dichroism
Suggested reading: Chapter 10 van Holde et al., pp. 465-496.
: The Feynman Lectures on Physics. Vol 2, Chapter 32.
Summary:
• Circular dichroism (CD) arises because of the differential
interaction of circularly polarized light with molecules which
contain a chiral center or have coupled electric dipoles that are
chiral.
• CD is not observed unless an absorption band exists.
• CD spectra of proteins and nucleic acids are much more sensitive
to conformational changes than absorption spectra.
• CD spectra of proteins can be used to obtain useful information on
secondary structure.
Introduction: Until now we have been studying the interaction of the
transition electric dipole of molecules with the electric field of light. In
addition to this interaction, the magnetic field of light can also interact
with magnetic transition dipoles in molecules. In order for this interaction
to be productive it is necessary that the molecule possess a center of
asymmetry (such as α-carbons) or to have coupled electronic dipoles.
These centers will interact with polarized light. The consequences of this
interaction are:
λ >> λABS:
• Birefringence: (nL - nR)
• Optical Rotary Dispersion (ORD): φ = 180 l (nL - nR)/ λ
λ ≈ λABS:
• Circular dichroism (CD) differential absorption of circularly
polarized light: AL-AR
• Ellipticity: θ = 2.303 (AL-AR) 180/ 4π or ∆ε = εL – εR
Linear & Circularly Polarized Light: To understand how these effects
occur when polarized light is passed through a sample we will decompose
linearly polarized light into right and left circular-polarized light. For a
wave propagating along the y axis (j), the electric field at the origin is:
ˆ
E r = k cos ωt + iˆ sin ωt ˆ ˆ
E l = k cos ωt − i sin ωt
The sum of these two fields gives linearly polarized light.
For the case of Er the electric field vector will rotate in a right hand
direction because the x-component of the electric field grows after t=0. In
contrast El will rotate in a left hand direction.
Figure 1: Generation of circular polarized light.
Figure 1: Generation of circular-polarized light
The optical activity of a molecule is due to a differential interaction of the
molecule with right or left circularly polarized light. To help understand
the origin of optical activity consider the
following two 'molecules', which are mirror images of each other:
Figure 1: Some "lock-washer" molecules
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2. 03-871 Molecular Biophysics CD Spectroscopy September 25, 2009
Origin of Optical Activity: A flat molecule has no optical activity
because it possesses a plane of symmetry. In contrast, the helical
molecules shown below have optical activity. When a molecule is placed
in an electric field the electrons involved in the transition will oscillate
according to the direction of the electric field. In the case of the helical
molecule they can be driven in a helical path. This generates a current
loop in the helix and the current loop generates a magnetic dipole in the
same way an electromagnet generates a magnetic field. The induced
magnetic dipole is parallel with the z-component of the electric field that
generated it. The induced magnetic dipole can interact with the magnetic
component of the electric field.
• No interaction is possible with the magnetic field associated from
the z-component of the electric field. Why?
• An interaction between the induced magnetic dipole and the
electric field in the x-y plane is possible. Why?
The phase of the electric field oscillation affects the coupling between the
induced magnetic dipole and the magnetic field. For a productive
interaction between the induced magnetic dipole and the magnetic field
from the x-y electric vector the two fields must oscillate with the same
phase. Only one direction of the circularly polarized light (right or left)
will possess the correct phase, hence the reason why one of the
components of the polarized light (Er or El) interacts more strongly with
the chromophore.
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3. 03-871 Molecular Biophysics CD Spectroscopy September 25, 2009
Circular Birefringence & Optical Rotary Dispersion (ORD)
Optically active material will show a different refractive index for right
and left circular polarized light. The index of refraction of a material is a
consequence of the generation of an electric field by the dipoles within the
material which oscillate due to the applied light.
• At frequencies far removed from the absorption band the
oscillations of the applied field induce oscillations of charges in
the material which are in phase with the applied field.
• As the frequency of the applied light approaches the absorption
maximum there is an increasingly larger phase shift in the induced
oscillations.
• At higher frequencies, the induced oscillations change sign,
reversing the effects seen at low frequencies.
The oscillating electrons generate an electric field that contributes to the
field of the transmitted light. The induced field generates a difference in
refractive index for the two forms of light. The difference in refractive
index is called circular birefringence: nl - nr
Optical Rotary Dispersion (ORD) arises because of a difference in the
index of refraction for left and right circularly polarized light. Due to the
difference in refractive index the wavelength of the light for each direction
of circularly polarized light is different while the light is in the media.
Consequently, a phase shift will develop between the two circular
components. This phase shift will cause a rotation of the linearly polarized
light when it leaves the media.
180l (nl − nr )
φ= [degrees]
λ
The optical rotation as a function of wavelength is referred to the optical
rotary dispersion. The ORD curve changes sign at the absorption
maximum. This occurs because the phase of the oscillations of the
electrons becomes 180 degrees out of phase from the incident light. This
implies that the polarized light (right or left) which was retarded by the
material at longer wavelength now becomes advanced with respect to the
other polarized direction at shorter wavelengths.
Circular Dichroism occurs as the wavelength of the incident light
approaches that of the absorption band. In this case the oscillation of
charges in the material is damped as energy is removed from the field by
the absorption process. If the absorption is different for right and left
handed circular-polarized light then the linearly polarized light will
become elliptically polarized. The ellipticity (θ) of the light is defined by
the arc tangent of the ratio of the major axis to the minor axis of the
transmitted light. Usually, the actual absorption of each component of
light is measured and the difference in absorption is called the circular
dichroism (CD):
CD = Al – Ar
Which is related to the ellipticity by:
2.303( Al − Ar )180
θ= = 32.98 × ( Al − Ar )
4π
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4. 03-871 Molecular Biophysics CD Spectroscopy September 25, 2009
ER − EL
tan θ =
ER + EL
I R/ 2 − I L/ 2
1 1 Small angle approximation.
θ = 1/ 2 1/ 2
IR + IL
I = I 0 e − A ln10 Beer’s law A=-log(I/Io)
− AR − AL ∆A
ln x = ln10 log10 x
ln 10 ln 10 ln 10
e 2
−e 2
e 2
−1
θ= − AR − AL
= ∆A
+ AL ln 10
ln10 ln10 ln 10 2
+1 Multiply by e
e 2
+e 2
e 2
∆A
ln 10 ∆A
e2 ≈1+ ln 10
2 Series expansion of ex ≈1 + x
∆A Apply series expansion
ln 10
θ≈ 2
∆A
ln 10 + 2
2
∆A Assume ∆A <<1
ln 10
θ≈ 2
2
Convert to degrees.
∆A 360
θ≈ ln 10 ×
4 2π
The molar ellipticity of the sample is given by:
[θ ] = 100θ
Cl
Where C is the concentration in moles/liter and l is the path-length in cm,
and θ is the measured ellipticity. The historical units of [θ] are deg cm2
dmole-1 instead of deg M-1 cm-1. Although these units seem odd, they can
be easily derived:
L mol 1000cm 3 mol
deg M −1cm −1 = deg × cm −1 = deg × = deg100cm 2 dmol −1
mol 10dmol mol 10dmol
The molar circular dichroism can also be related to the difference in the
extinction coefficient for right and left circularly polarized light:
θ = 32.98 × (ε l − ε r ) deg M −1cm
[θ ] = 3,298∆ε = 3,298 (ε l − ε r ) deg cm2 dmol-1
It is becoming more common to express CD spectra in terms of the much
more straight-forward difference in molar extinction coefficient:
∆ε = ε l − ε r
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5. 03-871 Molecular Biophysics CD Spectroscopy September 25, 2009
Applications of Circular Dichroism:
Determining Secondary Structure by CD: Various secondary
structures have characteristic CD spectra (Figure 3).
helix
beta
Figure 3: CD spectra of polypeptides (left) and CD spectra extracted from proteins.
The scale on the left has been multiplied by 10-3, i.e. 80 = 80,000.
Note that the CD spectrum (or optical activity) of α-helical residues is
significantly greater than either random coil or β-sheet. This is a
consequence of the interaction between transition dipoles oriented by the
helical structure of peptide. This interaction is weaker in β-sheet and
random coil. A similar interaction will also occur with nucleic acids (see
below).
The determination of secondary structure by CD is based on the fact that
the CD spectra of a protein is well approximated by the CD of each
peptide linkage:
N
θ λ = ∑θ iλ
i =1
Where θiλ is the CD of a single peptide bond.
There are three methods are currently used to determine the secondary
structure of a protein from it's CD spectrum.
Method 1: The simplest method is to use the CD spectra of polyamino
acids. The CD spectrum of a protein can be taken to be the linear sum of
the CD spectra from these various secondary structures:
θ λ = fαθ λ + f βθ λβ + f cθ λc
α
Where fα are the fraction of the number of residues in the protein that are
in an α-helical conformation. fβ is the fraction in the β-sheet, etc. The
experimental data are fit to the reference spectra to determine the amounts
of each secondary structure in the protein. A significant problem with this
approach is that the model compounds are usually infinite in length, thus
they do not mimic secondary structures in proteins, which are of finite
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6. 03-871 Molecular Biophysics CD Spectroscopy September 25, 2009
length. In addition, the following problems also occur with any method of
using CD to obtain secondary structure:
• random coils are seldom random
• Phe, Tyr, His, and Trp can contribute to peptide CD spectra
• Left handed helical structures can occur
• Disulfide bonds are very active
• Prosthetic groups are also very active
Method 2: A more reliable approach is to use proteins of known structure
(and CD spectra) to define the basis set. For each of the known proteins
the following is assumed:
3
θ λ = ∑ χ jθ λj
j =1
For each of the known proteins, the fractions of residues in various
secondary structures are known ( e.g. χ α ). From these data it is possible
to obtain basis spectra for the three types of secondary structure. These
basis spectra should now reflect the influence of the protein structure of
the CD spectra of residues in various secondary structures. As with the
first method, the CD spectra of the unknown protein is the weighted sum
of the reference CD spectra:
θ λ = fαθ λ + f βθ λβ + f cθ λc
α
The hope here is that the new reference spectra will be 'more accurate' than
those based on homo-polymers.
A flaw in the above approach is the assumption that there are only three
classes of secondary structure and that each class has a unique CD
spectrum.
Method 3 [Hennessey & Johnson, Biochemistry, 20, 1085.]: A more
unbiased method of approaching this problem is to extract, using
mathematical methods, a generalized basis set from the CD spectra of
proteins with known structures without regard to the actual secondary
structure of the protein. That is, we now assume the CD spectra of a
protein is:
N
θ λ = ∑ aiθ λi
i
Where N are the total number of basis spectra, ai is the weight of the ith
spectra to the total CD and θλi is the CD of the ith basis spectra at λ.
Determining the basis spectra and the coefficients, ai is accomplished by a
general technique called singular value decomposition.
SVD: Singular Value Decomposition: This is a general mathematical
technique that can be used to extract out the principle components of a
complex mixture of different spectra. Principle components are analogous
to the three basis vectors that can be used to define any vector in a three-
dimensional space.
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7. 03-871 Molecular Biophysics CD Spectroscopy September 25, 2009
SVD can be used in many different situations. As an example, say CD
spectra (or IR spectra, or UV-Vis spectra...) were acquired at 3 different
ligand concentrations and at five different wavelengths and we wanted to
extract out the 'part' of the spectra that was affected by ligand binding.
The raw experimental data can be written in matrix form (each column
represents a different ligand concentration):
aλ 1 aλ 1 aλ 1
a aλ 2 aλ 2
λ2
A = aλ 3 aλ 3 aλ 3
aλ 4 aλ 4 aλ 4
aλ 5
aλ 5 aλ 5
This matrix can be decomposed into a product of three matrices:
A = USV T
U: The columns of this matrix are the 'basis spectra', ui. A weighted linear
sum of these spectra can be used to generate the original raw data.
u1 1
λ
2
uλ 1 uλ1
3
1 2 3
uλ 2 uλ 2 uλ 2
U = uλ 3
1 2
uλ 3 uλ 3
3
1 2 3
uλ 4 uλ 4 uλ 4
u1 2
uλ 5 uλ 5
3
λ5
S: This is a diagonal matrix:
s1 0 0
S = 0
s2 0
0
0 s3
Each entry in this matrix is associated with a basis spectrum in the U
matrix (e.g. s2 is associated with u2. The size of each si determines the
importance, or size of contribution, of this basis to the experimental data.
This can be seen by taking the product of U and S:
uλ 1 u λ 1
1 2
uλ 1
3
s1uλ1
1 2
s 2 uλ 1 s3uλ1
3
1 3 1 3
2
uλ 2 uλ 2 uλ 2 s1 0 0 s1uλ 2 2
s 2 uλ 2 s3uλ 2
U × S = uλ 3 u λ 3 0 = s1uλ 3
1 2
uλ 3 × 0
3
s2
1 2
s 2 uλ 3 s3uλ 3
3
1 3 3
2
uλ 4 uλ 4 uλ 4 0
0 s3 s1uλ 4
1 2
s 2 uλ 4 s3uλ 4
u1 u 2 3
uλ 5 s u1 2
s 2 uλ 5 s3uλ 5
3
λ5 λ5 1 λ5
VT Is a square matrix, each entry gives the contribution of each basis
vector to the signal at a particular wavelength:
s1uλ1
1
s2uλ12
s3uλ1
3
1 3
s1uλ 2
2
s 2 uλ 2 s3uλ 2 V11 V12 V13
U × S × V = s1uλ 3
s3uλ 3 × V21 V22 V23
T 1 2 3
s 2 uλ 3
1 3
s1uλ 4
2
s 2 uλ 4 s3uλ 4 V31 V32 V33
s u1 2
s2uλ 5 3
s3uλ 5
1 λ5
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8. 03-871 Molecular Biophysics CD Spectroscopy September 25, 2009
For example, the measured spectra at λ1 for the 2nd ligand concentration is:
•
A C2 = s1uλ1V12 + s2uλ1V22 + s3uλ1V33
λ1
1 2 31
Figure 4: Basis sets used to fit CD data (left). Fit to CD spectrum of papain (right).
SVD of Protein CD spectra:
Determining Secondary Structure by SVD:
We would like to obtain the secondary structure of a protein from its CD
spectrum. In matrix form, this relationship is:
F= XA
where F is a vector that is the fraction of each secondary structure, A is the
CD spectrum of the unknown protein, and X relates the two. Since we
know from SVD that there are only five significant components to the CD
spectrum of a protein we will assume that we can represent a protein in
terms of five secondary structures:
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9. 03-871 Molecular Biophysics CD Spectroscopy September 25, 2009
θ λ 1
f H X 11 . . . . .
f θλ 2
⊥β . . . . . .
θ
λ3
f||β = . . . . . . ×
θλ4
fT . . . . . .
θ λ 5
fO .
. . . . .
θ λ 6
If we can determine X, then the fraction of each secondary structure for an
unknown protein can be found.
If we begin with a large set of proteins with known secondary structure (F)
and CD spectra (A) we can obtain X, using SVD. For a set of 3 proteins
and CD data collected at six wavelengths, the matrices would look like:
f1 θ 1 θ 2 θ 3
fH 2
fH X
3
. . . . . λ 1 λ1 λ1
3
H
1 3
11 1 2
2
f⊥ β f⊥ β f ⊥ β . . . . . . θ λ 2 θ λ 2 θ λ 2
1 2 3
θ 1 θ 2 θ 3
f ||β f f = . . . . . . × λ 31
λ3
2
λ3
3
|| β ||β θ θλ 4 θ λ 4
1 3 . . . . . . λ 4
θ 1 θλ 5 θ λ 5
2
fT fT fT 2 3
fO 1 2 3 .
. . . . . λ 5
fO fO 1 2 3
θ λ 6 θ λ 6 θ λ 6
For this set of known proteins:
F = XA = X (USV T ) , where A has been subject to SVD, i.e.
A=USVT
X can be solved by multiplying F by V, S-1, and U:
F × V × S −1 × U T = X (USV T ) × V × S −1 × U T
= X (US ) × S −1 × U T = X (U ) × U T = X
Note:
U×UT = 1 since all U vectors are orthonormal.
V×VT = 1 from the theory of SVD.
1 / s 0
S × S-1 =1 if we define S-1 = 1
0 1 / s2
Secondary Structure of Membrane Proteins:
CD can also be used to determine the secondary structure of
membrane proteins (or other aggregated systems). However, the
analysis is far from simple. Two optical effects occur in
suspensions of particles which distort CD spectra. First, the
overall absorption of the system decreases because proteins in
the particle can be "shaded" by others in the same particle.
Second, the scattering of right and left circularly polarized light
is not equal. The former problem affects the overall amplitude of
the spectrum, while the latter affects the shape of the CD
Figure 5: Effect of particle size on CD Spectrum
spectrum. These effects can be corrected to produce rather
9
10. 03-871 Molecular Biophysics CD Spectroscopy September 25, 2009
reasonable CD spectra of membrane bound proteins. However, it is clear
that IR is the preferred technique for obtaining information on the
secondary structure membrane proteins.
Other Application of CD Spectroscopy to Proteins.
CD can also be used in an empirical manner to determine protein
unfolding, ligand binding, etc. CD is much more sensitive to changes in
secondary structure than absorption spectroscopy and is thus an excellent
method of following protein denaturation.
Application of CD to Nucleic acids:
The major application of CD to the study of nucleic acids is to determine
the degree of base stacking. The CD of a dimer is very dependent on the
interaction of the monomers. For example: poly C has the following
spectral properties:
Solvent Ellipticity A260
Water 35,000 1.0
Ethylene glycol 7,000 1.3
In this case both the CD and the hyper-chromicity show that polyC is a
helix in water and that this helix is due to base stacking.
DNA-Protein Interactions:
Since proteins have weak CD bands at 250nm, CD is well suited for
following protein induced changes in nucleic acid structure. These changes
can be convenient for monitoring binding of proteins to nucleic acids.
10