Spectroscopy

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Spectroscopy
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Interaction Types

• When light interacts with an object, we can
  normally see only reflected or transmitted
  radiation. Three phenomena that occur when
  electromagnetic radiation interacts with matter
  can be defined more precisely as

• Scattering

• Absorption

• Emission

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Scattering

• When electromagnetic radiation passes through
  matter, most of the radiation continues in its
  original direction but a small fraction is
  scattered in other directions.
• Light that is scattered at the same wavelength as
  the incoming light is called Rayleigh scattering.
  
• Light that is scattered in transparent solids due
  to vibrations (phonons) is called Brillouin
  scattering. Brillouin scattering is typically
  shifted by 0.1 to 1 cm-1 from the incident light.
  
• Light that is scattered due to vibrations in
  molecules or optical phonons in solids is called
  Raman scattering. Raman scattered light is
  shifted by as much as 4000 cm-1 from the incident
  light.
• The sky is blue because fluctuating particles in
  the atmosphere scatter blue light more than red
  light

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Absorption

• When atoms or molecules absorb light, the
  incoming energy excites a quantized structure to
  a higher energy level. The type of excitation
  depends on the wavelength of the light.
• electrons are promoted to higher orbitals by
  ultraviolet or visible light,
• vibrations are excited by infrared light, and
• rotations are excited by microwaves.

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Absorption

• An absorption spectrum is the absorption of light
  as a function of wavelength. The spectrum of an
  atom or molecule depends on its energy level
  structure, and absorption spectra are useful for
  identifying of compounds.

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Absorption

• Measuring the concentration of an absorbing
  species in a sample is accomplished by applying
  the Beer-Lambert Law.
• Red light absorbed by a piece of glass causes the
  transmitted light to be appear blue.

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Emission

• Atoms or molecules that are excited to high
  energy levels can decay to lower levels by
  emitting radiation (emission or luminescence).
• For atoms excited by a high-temperature energy
  source this light emission is commonly called
  atomic or optical emission (atomic-emission
  spectroscopy), and
• For atoms excited with light it is called atomic
  fluorescence (atomic-fluorescence spectroscopy)
  or molecular fluorescence (molecular fluorescence
  spectroscopy).
• For molecules it is called fluorescence if the
  transition is between states of the same spin and
  phosphorescence if the transition occurs between
  states of different spin.
• The emission intensity of an emitting substance
  is linearly proportional to analyte concentration
  at low concentrations, and is useful for
  quantitating emitting species.
• A flourescent dye may emit green light after
  absorbing blue

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Beer-Lambert Law

• The Beer-Lambert law (or Beer's law) is the
  linear relationship between absorbance and
  concentration of an absorbing species. The
  general Beer-Lambert law is usually written asA
  a(l) b cwhere A is the measured
  absorbance, a(l) is a wavelength-dependent
  absorptivity coefficient, b is the path length,
  and c is the analyte concentration.

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Instrumentation

• Experimental measurements are usually made in
  terms of transmittance (T), which is defined
  asT I / Iowhere I is the light intensity
  after it passes through the sample and Io is the
  initial light intensity. The relation between A
  and T isA -log T - log (I / Io).

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Instrumentation

• Modern absorption instruments can usually display
  the data as either transmittance,
  -transmittance, or absorbance. An unknown
  concentration of an analyte can be determined by
  measuring the amount of light that a sample
  absorbs and applying Beer's law. If the
  absorptivity coefficient is not known, the
  unknown concentration can be determined using a
  working curve of absorbance versus concentration
  derived from standards.

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Working Curve

• A working curve is a plot of the analytical
  signal (the instrument or detector response) as a
  function of analyte concentration. These working
  curves are obtained by measuring the signal from
  a series of standards of known concentration. The
  working curves are then used to determine the
  concentration of an unknown sample, or to
  calibrate the linearity of an analytical
  instrument.

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Photochemical Interaction

• Another result of the interaction of
  electromagnetic interaction with matter is
  photochemistry. This is obviously extremely
  important in biology (such as in vision and
  photosynthesis) but this aspect is not dealt with
  here.

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Spectroscopy

• The study of the interaction of Electromagnetic
  radiation with matter, excluding chemical effects.

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Spectroscopic Techniques

• Irradiation of a sample with some form of
  electromagnetic radiation
• Measurement of the scattering, absorption, or
  emission in terms of some measured parameters
  (e.g., scattering intensity at some angle q,
  extinction coefficient at a particular
  wavelength, or fluorescent lifetime)

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Spectroscopic Techniques

• The interpretation of these measured parameters
  to give useful biological information.
• This last stage requires some understanding of
  the physical basis of the interaction, whether it
  is scattering by electrons or nuclei, absorption
  by excitation to a higher vibrational level, or
  emission from a triplet state.

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Information Available From Spect.

• Detailed study of scattering, absorption, and
  emission yields biological information of various
  kinds. This information can be broadly classified
  as
• structural,
• dynamic,
• energetic,
• analytical.

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Information Available From Spect.

• The information available depends on the
  instrument used to make the measurements.
• While the eye is exceptionally powerful and
  versatile, instruments such as the microscope or
  the spectrometer can enhance and quantify the
  information discernible.

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Each technique has different advantages and
disadvantages, both experimentally and in the
interpretation of the measurements.
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Information Available From Spectroscopy

• The best techniques for determining structure or
  the coordinates of a biological system are
  microscopy and diffraction.

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Microscopy

• Light microscopy is a technique that can give
  structural information directly and
  non-invasively about living systems, but the
  resolution that can be achieved ( 1 mm) is not
  sufficient to study individual molecules.

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Electron Microscopy

• Electron microscopy can achieve higher resolution
  ( 2 nm), but the sample must be studied in a
  vacuum and is normally covered with a metallic
  stain, which causes a problem with regard to the
  integrity of the structure determined.

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Diffraction Studies

• Diffraction studies of crystals of pure
  macromoleculcs can give structural information to
  the atomic level (0.15 nm), but this technique
  requires crystals, and the structure is no longer
  obtained directly but must be interpreted from an
  observed diffraction pattern.

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Other Techniques

• Some techniques that can be used to study
  macromolecular structure in solution include
• optical activity measurements, which give
  information on secondary structure of proteins
• fluorescence and nuclear magnetic resonance
  (NMR), which can give information about
  interactions between pairs of centers
• electron paramagnetic resonance (EPR), and
  resonance Raman, which can "fingerprint" certain
  types of structure around a metal ion or
  chromophore and solution scattering, which can
  give information about the overall shape of a
  molecule in solution.
• The structural information available from these
  methods is nearly always equivocal, although it
  is often very useful and important because it can
  be obtained in solution and can be combined with
  other information on energetics and dynamics.

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Dymamic Information

• Dynamic information about biological systems can
  be obtained in a variety of ways. The best
  methods are those that give information in
  solution.
• Examples are
• dynamic light scattering studies of chemotaxis by
  bacteria,
• fluorescence depolarization studies of the
  rotational diffusion of macromolecules,
• EPR spin-label studies of lipid fluidity, and
• studies of the movement of fluorescent labels
  with the fluorescence microscope.

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Information About Energetics

• Information about energetics can be obtained by
  studying
• the influence of environment, such as
  temperature, ligand concentration, pH, and ionic
  strength of the system.
• The techniques of UV / visible spectroscopy,
  fluorescence, optical activity, and NMR are all
  good for distinguishing between bound and unbound
  forms of a ligand or a macromolecule, different
  ionization states, and different structural forms
  of a macromolecule.

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Analytical Information

• The best methods to obtain analytical
  information, by which we mean the identification
  of a particular compound and the determination of
  its concentration, are
• UV/visible Spectroscopy,
• NMR, and
• atomic absorption Spectroscopy (AAS).
• It should also be noted that different methods
  operate best in different concentration ranges.
  NMR is best for studying changes in the
  millimolar range, while flourescence is used to
  study much lower concentrations ( 1 mM).

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Properties of EM Radiation

• Electromagnetic radiation is made up of two wave
  motions perpendicular to each other. One is a
  magnetic (M) wave, the other an electric (E)
  wave. The waves are propagated along the
  z-direction.

Electromagnetic waves are generated by
oscillating electric or magnetic dipoles and are
propagated through a vacuum at the velocity of
light (c). The energies associated with E. and M
are equal, but most optical effects are concerned
with the electric wave, E.
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Polarization

• Since the E- and the M-components are always
  perpendicular to each other, it is sufficient, in
  many cases, to consider only the E-component in
  describing the wave.

Directions of the electric vector In polarized
and unpolarized light. In unpolarized light (a),
or partly polarized light (c), the oscillations
take place at all angles perpendicular to the
direction of travel In polarized light (a) they
are restricted to one angle.
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Degree of Polarization

• It is convenient to introduce a parameter called
  the degree of polarization (P) to describe
  situations where the radiation is partially
  polarized

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Degree of Polarization

• Illustration of (a) how two beams polarized along
  xz and yz, 90phase shifted with respect to each
  other, generate a circularly polarized beam (b)
  when they are superimposed.

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Frequency, Wavelength, Energy, and Wavenumber

• u c / l
• E hu h 6.63 10-34 J.s
• Expression of the radiation as a frequency (Hz)
  gives results with very large numbers therefore
  it is common to find, particularly for EM
  radiation in the microwave to X-ray range, the
  frequency expressed as a wavenumber (cm-1). The
  wavenumber (u') is defined as the inverse of the
  wavelength in centimeters.

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EM and Scales
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What is Matter?

• There are, two concepts arising from wave
  mechanics that are very important
• The distribution of a particle in space is given
  by the square of its wave-function. This leads to
  an understanding of orbitals.
• Energy states are quantized thus any system has
  certain characteristic energy values or levels.

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What is Matter?

• An understanding of the properties of matter,
  whether they are describable by wave mechanics or
  not, depends on knowledge about its energy and
  its coordinates in space and time (that is, its
  shape and dynamic properties), it is important to
  realize that the three properties of energy,
  shape, and dynamics are closely interdependent.

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energy, shape, and dynamics

• the angle of 105 between the H atoms and the O
  atom of the H20 molecule, which arises because
  this gives an energy minimum to the molecule
• the folding of a polypeptide chain in solution to
  give a well-defined globular protein, which also
  depends on the energy of the system

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Brownian Motion

• Molecules in solution have kinetic energy because
  they undergo Brownian motion, that is, they
  rotate and diffuse laterally (translate). In
  addition, the molecules vibrate because of their
  thermal energy. Thus, dynamic and energetic
  properties are also related.

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Interparticle Forces and Energies

• The forces between various particles, such as a
  nucleus and an electron or between molecules,
  often result in a characteristic behavior for the
  energy of the system.
• If two particles are far apart, there is no
  interaction between them. As the particles
  approach, there may be direct attraction of
  positive and negative charges or there may be a
  change in charge distribution on the particle,
  resulting in a net attraction.

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Interparticle Forces and Energies

• The energy of the system decreases until an
  equilibrium value of the distance between the
  particles is reached. As the particles get closer
  than this, they begin to repel each other and the
  energy increases.

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Interparticle Forces and Energies

• This sort of behavior is typical of many
  situations. Examples include
• an electron orbiting a nucleus,
• the interaction between atoms in the formation of
  chemical bonds and crystals,
• the interaction between molecules in gases and
  liquids, and
• the specific binding of a substrate to an enzyme.
  

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Energy Levels

• The energy levels represent the characteristic
  states of the molecule.

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Energy Levels

• Although every type of energy is quantized, the
  separation between neighboring translational
  energy levels is so small that for practical
  purposes we can disregard the quantization of the
  translational energy.

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The Dependence of the Population of Energy Levels
on Temperature

• The exact distribution will depend on the
  temperature (thermal energy) and on the
  separation between energy levels (DE) in the
  energy ladder. At a given temperature the number
  of molecules in an upper state (hupper) relative
  to the number in a lower stale (hlower) is given
  by Boltzmann distribution law